discovery and development of apaf-1 inhibitors:...
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DISCOVERY AND DEVELOPMENT OF APAF-1 INHIBITORS: ADVANCING TOWARDS UNWANTED
CELL DEATH TREATMENTS
Thesis Project presented by
Laura Mondragón Martínez
in order to obtain the European Doctorate in Biochemistry
Thesis supervisors
Dr. Enrique Pérez Payá and
Dr. María J. Vicent Docón
Departament de Bioquímica i Biologia Molecular
Departament de Bioquímica i Biologia Molecular
Enrique Pérez Payá, Ph.D. in Chemistry, Consejo Superior de Investigaciones Científicas (C.S.I.C) Research Professor and Dr. María J. Vicent Docón, Ph.D. in Chemistry CERTIFY, that the work “DISCOVERY AND DEVELOPMENT OF APAF-1 INHIBITORS: ADVANCING TOWARDS UNWANTED CELL DEATH TREATMENTS” has been developed by Laura Mondragón Martínez under their supervision in the Centro de Investigación Príncipe Felipe in Valencia, as a thesis project in order to obtain a Ph.D. degree in Biochemistry from the University of Valencia, Department of Biochemistry and Molecular Biology. Prof. Enrique Pérez Payá Dr. María J. Vicent Docon
To my parents and brother,
A mis padres y a mi hermano,
“There is no pillow softer than a clear conscience”
“No hay mejor almohada que una conciencia limpia”
Acknowledgements:
It is a bit difficult to try to thank all the people that have taken part in this thesis project,
but due to their importance I would like to thank my parents for all their support during
these years.
I would also like to thank Enrique Pérez Payá for allowing me to work in his lab, the
Spanish Ministry of Science and Innovation and the European Social Funds for the
economical support for and the Centro de Investigación Príncipe Felipe for providing
the necessary infrastructure to develop this thesis project. I have really enjoyed the
opportunity to work in the field of apoptosis in Spain and in the laboratories that I have
been able to visit. Thanks a million, Quique!
I would like to say thank you to all the people from the I-12 laboratory Mar, Ana, Anna,
Eliana, Ally, Susana, Mónica, Tatiana, Yadira, Andrés, Laura C., David, Puig, Silvia,
Rebeca, Inma, Guillermo. I have really enjoyed spending these four years with all of
you and the races through the lab trying to find a place in the hood in our cell culture
room. Specially I would like to thank Mar (the third thesis supervisor) for teaching me
almost all the techniques that I have learned in the lab, for listening to me no matter
how busy she was and for that coffee under the rain at the terrace of the hotel at the
ECDO meeting in 2007.
I should also show my gratitude to María Jesús Vicent for being my thesis supervisor
together with Quique and all the people in the I-36 lab. In particular, I would like to
express my gratitude to Lucille for her help with the characterization of polymer-
conjugates and Vanessa for her support and useful talks while we were in Cardiff.
I want to express my gratitude to Pino and the Proteomics team for providing us all the
western-blot tools when we run out of them.
In addition, I would like to thank Àngel Messeguer and his team for their help and for
designing together with Quique the compound QM31 the most useful compound in my
thesis. I also want to thank Glòria, Sandra and Dani for synthesizing all the necessary
peptoid 1 derivatives employed in the project and Jordi Bujons for his in silico studies
of the interaction among Apaf-1 and QM31.
I cannot forget to say thank you to Laboratorios Salvat S.L. for their economical support
during the last two years and for confiding in the project created by Quique and Àngel.
Thanks a lot for believing in this project.
Although, I did not have the pleasure to meet Antonio Alcaraz and his team I am very
grateful for their help developing the in vivo hot ischemia experiments.
From the six months spent at the Apoptosis laboratory at the Kraefte Bekaempelse I
would like to thank Marja Jäätellä for allowing me to stay in her lab and helping me to
find a nice apartment to live. I would also like to say thank you to the people in her lab
Anna, Anne, Maria, Mads, Mikel, Thomas, Oleg, Piotr, Nicole among others. I am
really happy to have met Griselda Herrero during my stay in Denmark. I am really
grateful for all your help with the experiments and the funny chats at the end of the day.
Finally I would like to thank the Spanish Erasmus group (María, Cristina, Flor, Pedro,
Diego...) for being so helpful and teaching me how to ride a bike again.
I would also like to thank Dr. Guido Kroemer for allowing me to visit his laboratory and
helping me with my second article, I really appreciate it. I would like to specially thank
Laura Senovilla (and her boyfriend Nacho) for all her help during my stay and the good
pieces of advice received. I want to thank Lorenzo and Ilio for teaching me so many
techniques in my stay and the rest of the lab Didier, Nazanine, María, Amina, Eugenia,
“masters” José Miguel and Alfredo, Max, Oliver, Ezgi, Chiara, Sonia, Claire, Emilie,
Gwenola, Jean-Luc, Nick, Nicolas for making my stay in Paris so comfortable. I cannot
forget Santi Rello for his optimistic point of view and for carrying my suitcase while I
chased a bus the day I left Paris. Finally, I cannot forget Rubén and the funny
conversations while having dinner at the Colegio de España.
I want to show my gratitude to the Prof. Douglas R. Green for letting me to visit his lab
at St. Jude Children’s Research Hospital in Memphis. Especially I want to express my
gratitude to Lisa for her help with the confocal microscope and my experiments and to
Fabien for his patience with my doubts. I am also very grateful to the people in the lab
Stephen, Valère, Laura, Tudor, Ruoning, Jerry, Pat, Sandra, Melissa, Andrew, Sam,
Gavin, Chris, Sebo, Diana, Nora for making so easy to work with them. Of course, I
would like to thank to the Spanish colony (Guillermo, Víctor, Amanda, Blas, Adolfo,
Lorraine, Blanca, Gaëlle, Patricia, Diego, baby Sofía ...) that take care all the new
Spanish people that visits Memphis every year. I will always remember Miguel
Sanjuán for making me feel just like family and solving all my problems in the lab. I
would like to thank Alfonso for his “croquetas de jamón” and for taking us to the NBA
matches. I would like to thank Mar for their happiness despite studying 24 hours/day
and for that unforgettable shopping day. I had the time of my life! Finally, I would like
to thank my roommate Yingdi for making possible that we travel in a golf car with her
new mattress at 10 p.m. I really enjoyed that experience.
Finally, I would like to thank all the friends that have supported me while working in
my thesis and I would like to express my gratitude to Fernando for visiting me in Paris
and Copenhagen and the small trips to the labs at weekends.
I hope not forgetting anyone, but just in case… Thanks a million to all of you!
Agradecimientos:
Resulta un poco difícil conseguir agradecer a todas las personas que han participado en
este proyecto, pero creo que por “orden de aparición e importancia” en primer lugar
quisiera agradecer a mis padres por hacer posible que llegara hasta aquí y por ayudarme
durante todos estos años tuviera el problema que tuviera.
También quisiera agradecer a Quique por concederme esa beca FPI que me ha
permitido hacer esta tesis doctoral, así como al Ministerio de Ciencia e Innovación y a
los Fondos Sociales de la Unión Europea por financiar dichas becas y al Centro de
Investigación Príncipe Felipe por facilitarnos la infraestructura necesaria para llevar a
cabo este proyecto. La verdad es que conseguir esta beca fue una gran alegría y poder
trabajar dentro del campo de la apoptosis mucho más, tanto en España como en los
diferentes laboratorios en los que he podido realizar estancias. ¡Quique, muchísimas
gracias, lo he pasado genial este tiempo!
Deseo agradecer también a todas las personas que han ido pasando por el laboratorio I-
12 Mar, Ana, Anna, Eliana, Ally, Susana, Mónica, Tatiana, Yadira, Andrés, Laura C.,
David, Puig, Silvia, Rebeca, Inma, Guillermo (espero no olvidar a nadie) y que han
hecho que estos cuatro años se pasaran volando entre experimento y experimento y
carreras por el laboratorio para conseguir reservar en la campana de cultivos. En
especial quisiera agradecer a Mar (la tercera codirectora) haberme enseñado
prácticamente todo lo que he aprendido en el I-12, y por atender cualquier duda o crisis
que tuviera con los experimentos y con lo que no fueran experimentos no importa lo
ocupada que estuviera. También quisiera agradecerle aquel café en la terraza del hotel
en el congreso de la ECDO de 2007 (mojarnos nos mojamos, pero mantuvimos el tipo
como nadie). Y aunque resulte raro, mención especial se merecen las células SF9 (mis
chicas) por lo entretenida que me tuvieron los primeros tres meses de doctorado
intentando ver cómo podía hacerlas crecer.
Debo agradecer también a María Jesús Vicent como codirectora de tesis y a toda la
gente del I-36. Asimismo, quisiera agradecer especialmente a Lucille por su ayuda en
los experimentos de degradación y caracterización de polímeros y a Vanessa por sus
charlas y ánimos en el congreso de Cardiff.
Estoy también muy agradecida a Pino y a todo el equipo de Proteómica por prestarnos
todo tipo de cubetas y fuentes para westerns y correr geles, así como a Virginia por
todos los experimentos de fraccionamiento de extractos (¡Espero que el experimento
salga ya de una vez!).
Asimismo, deseo agradecer a Àngel Messeguer y a todo su grupo la ayuda prestada y
por diseñar junto a Quique al inhibidor de Apaf-1 QM31 que tantas alegrías ha dado a
este proyecto. Quisiera dar las gracias a Glòria, Sandra y Dani que se han encargado de
sintetizar todos los derivados del peptoide 1 que hemos utilizado en la realización de
esta tesis. También quisiera agradecer a Jordi Bujons el estupendo estudio in silico de
interacción QM31/Apaf-1 realizado y que se ha incluido en esta tesis.
Dentro de los ensayos de caracterización de la interacción QM31/Apaf-1 Rodrigo
Carbajo también ha jugado un papel importante ayudándonos a realizar los
experimentos de RMN. ¡Muchas gracias, Rodri!
Igualmente, quisiera agradecer a los Laboratorios Salvat S.L. por su inestimable apoyo
económico en estos dos últimos años, por creer en el proyecto iniciado por Quique y
Àngel y por confiar en nuestro trabajo en este tiempo.
Aunque todavía no he tenido oportunidad de conocerlo, estoy muy agradecida a Dr.
Antonio Alcaraz y a su grupo por llevar a cabo los experimentos con los modelos
animales de daño isquémico en rata.
De mi estancia en Copenhague dentro del laboratorio de Apoptosis de la Kraefte
Bekaempelse quisiera agradecer a Marja Jäätellä por acceder a que realizara una
estancia de seis meses y preocuparse de buscarme el alojamiento necesario para llevarla
a cabo. Quisiera agradecer también dentro de su grupo a Anna, Anne, Mads, Maria,
Mikel, Thomas, Oleg, Piotr, Nicole y a otros muchos su ayuda durante mi estancia.
Aunque no salieron los resultados esperados fue mucho lo que aprendí durante este
tiempo en su laboratorio. Especialmente quisiera agradecer a Anna Ritter por su ayuda.
También quisiera mencionar a la mayor alegría que tuve en Copenhague como fue
conocer a Griselda Herrero. Griselda, de no ser por ti hubiera tirado la toalla a las
primeras de cambio, y aunque algunas tardes nos las pasáramos llorando (es el problema
de las estancias cortas con pocos resultados y lo de ahogarse en un vaso de agua)
también pasamos muy buenos ratos cotilleando en español. Por último quisiera
agradecer a todos los chicos de Erasmus que conocí (María, Cristina, Flor, Pedro,
Diego,...) por estar siempre disponibles para ayudar en cualquier cosa y por enseñarme
los trucos necesarios para conseguir tarjetas de transporte y bicis a buen precio, así
como “obligarme” a reaprender a ir en bici y conseguir frenar con el contrapedal (no me
maté de milagro).
De mi estancia en París quiero agradecer enormemente a Guido Kroemer la cantidad de
medios que poner al alcance de todos los que trabajan en su laboratorio dentro del
Institut Gustave Roussy con el fin de conseguir una buena publicación. En especial
quisiera agradecer a Laura Senovilla (y a su novio Nacho) los buenos momentos
pasados en París, por adoptarme en su casa hasta que me admitieron en el Colegio de
España, por enseñarme a manejarme dentro del laboratorio, así como los cotilleos
mientras trabajábamos en la campana de cultivos (o mientras mirábamos a los bomberos
que venían cada dos por tres a apagar algún fuego al hospital). Asimismo, quisiera
agradecer a Lorenzo y a Ilio todas las técnicas nuevas que me enseñaron, a Didier por su
ayuda con el citómetro y a también a Nazanine, María, Amina, Eugenia, los maestros
José Miguel y Alfredo, Max, Oliver, Ezgi, Chiara, Sonia, Claire, Emilie, Gwenola,
Jean-Luc, Nick, Nicolas y otros muchos por hacer que esta estancia fuera tan agradable.
No se me olvida Santi Rello por su optimismo y su ayuda cargando con mi maleta
mientras yo corría detrás del autobús el día de vuelta para España (espero que ya no te
duela la espalda). Por último quisiera agradecer a Rubén del Colegio de España las
conversaciones todas las noches en la cocina de la residencia.
Quisiera agradecer también al profesor Douglas R. Green por permitirme pasar dos
meses y medio dentro de su laboratorio en el St. Jude Children’s Research Hospital en
Memphis. Quisiera agradecer especialmente a Lisa su ayuda con el microscopio
confocal y orientar mis experimentos y a Fabien su paciencia con todas las cosas que
me pasaban por el laboratorio (Fabs, aunque mis botas aparecieron en casa de Melissa
como tú decías yo sigo pensando que me las dejé en el patio de mi casa y que llegaron
solas allí). También quisiera agradecer a Stephen, Valère, Laura, Tudor, Ruoning,
Jerry, Pat, Sandra, Melissa, Andrew, Sam, Gavin, Chris, Sebo, Diana, Nora por toda su
ayuda durante este tiempo y por ayudarme a integrarme tan rápidamente dentro del
laboratorio. Por supuesto, también quisiera dar las gracias a toda la colonia de
españoles y casi españoles en Memphis (Guillermo, Víctor, Amanda, Blas, Adolfo,
Lorraine, Blanca, Gaëlle, Patricia, Diego, la pequeña Sofía,...) que adoptan cada año a
todos los nuevos que vamos llegando al St. Jude. En especial quisiera agradecer
enormemente a Miguel Sanjuán por tratarme como si fuera una más de la familia y dejar
que me pegara a su chepa durante toda la estancia en Memphis y aunque le destrozara
su estrategia del “low profile” dentro del laboratorio de Doug. A Alfonso me gustaría
agradecerle todas las croquetas de jamón y pollo que nos cocinó y la paciencia para
cargar con todas las chicas en su coche siempre que se lo pidiéramos para ir a ver un
partido de la NBA. También a Mar por su alegría aunque estuviera agobiadísima por
los 20 exámenes a la semana que tenía. Mar, pasarán más de mil años pero esa tarde de
compras en los “moles” fundiendo la tarjeta de crédito no la olvidaré en la vida, ¡Qué
bien me lo pasé! Por último quisiera agradecer a mi compañera de piso Yingdi hacer
que la estancia fuera diferente. Nunca pensé que acabaría montada en un coche de golf
dando vueltas a las diez de la noche por la urbanización donde vivíamos y cargando con
un colchón viscoelástico (como el de Miguel y Mar, que aunque saltes no se mueve).
Por supuesto, me gustaría agradecer a todos los amigos en Soneja que me han dado
ánimos con la tesis y en especial a Susi y Santa, compañeras de juergas. Y a Santa,
decirle que me encantó su visita con Jimmy a Copenhague, y reírme viendo cómo
fueron las únicas personas que volvieron quemadas por el sol de Dinamarca. A Raquel,
María, Sonia y Dolores por las comidas en el piso de Burjassot. También me gustaría
agradecer a Fernando por sus visitas a París y a Copenhague así como los viajes al
laboratorio algunos fines de semana.
Espero que no se me haya olvidado nadie y que disfrutéis el día de la tesis tanto como
yo, que al final habrá picoteo y se tiene que cumplir el lema del laboratorio... ¡Reventar
antes que sobre!
I N D E X:
Abbreviations
CHAPTER 1 – Introduction ...................................................................... 1
1.1 Apoptosis .............................................................................................................. 1 1.2 Apoptosis activation and apoptotic cell death machinery ............................... 3 1.3 The apoptosome, components and cellular regulation..................................... 8 1.4 Apoptosome implication in disease .................................................................. 13 1.5 Chemical modulation of the apoptosome ........................................................ 14 1.6 Identification of an Apaf-1 inhibitor, peptoid 1, through the screening of an
N-alkylglycine chemical library .......................................................................... 18
CHAPTER 2 – Objetives ..........................................................................21
CHAPTER 3 – Synthesis of peptoid 1 and peptoid 1 derivatives.........23 3.1 Synthesis of peptoid 1 ........................................................................................ 33 3.2 Cell-penetrating peptides and cell-penetrating peptide/peptoid hybrids
synthesis PEN-1 and TAT-1 ................................................................................ 33 3.3 Synthesis and characterization of nanoconjugates......................................... 34
3.3.1 Synthesis and characterization of QM56 (peptoid 1-GG-PGA conjugate).. 34 3.3.2 Synthesis and characterization of QM56-OG (peptoid 1-GG-PGA-oregon
green).................................................................................................................. 41 3.4 Synthesis of conformationally constrained peptoid 1 mimetics .................... 42
3.4.1 Synthesis of QM31........................................................................................ 42 3.4.2 Synthesis of QM31 derivatives ..................................................................... 42 3.4.3 Synthesis of QM57........................................................................................ 43 3.4.4 Synthesis of QM57 analogues ...................................................................... 43
3.5 Synthesis of QM56 analogues and polymeric derivatives of QM31 and QM57 ..................................................................................................................... 45
CHAPTER 4 – Systematic analysis of inhibition of apoptosis by
peptoid 1 derivatives .................................................................................51 4.1 Apoptotic inhibition activity of CPP-peptoid 1 hybrids, PEN-1 and TAT-1 57
4.1.1 PEN-1 and TAT-1 antiapoptotic activity in cell extracts ............................. 57 4.1.2 Evaluation of cell toxicity and biological activity of PEN-1 and TAT-1...... 58 4.1.3 Cellular internalization studies and structural characterization of PEN-1
and TAT-1........................................................................................................... 60 4.2 Apoptosis inhibitory activity of the polymer-peptoid 1 nanoconjugate,
QM56 ..................................................................................................................... 68 4.2.1 Evaluation of cellular internalization of QM56 ........................................... 68 4.2.2 Evaluation of cell toxicity and biological activity of polymer-peptoid 1
conjugates, QM56 and derivatives ..................................................................... 70 4.3 Apoptotic inhibitory activity of conformationally constrained peptoid 1
derivatives ............................................................................................................. 73
4.3.1 Conformationally constrained peptoid 1 derivatives inhibit apoptosome assembly in an in vitro apoptosome reconstitution assay .................................. 73
4.3.2 Evaluation of cell toxicity and biological activity of conformationally constrained peptoid 1 derivatives, QM31 and QM57 derivatives...................... 75
4.4 Apoptotic inhibition activity of the synthesized QM31 and QM57 polymeric derivatives ............................................................................................................. 84
CHAPTER 5 – Peptoid 1 derivatives as inhibitors of the apoptosome,
molecular mechanism of action................................................................87 5.1 Characterization of peptoid 1 binding site in different in vitro systems....... 87
5.1.1 Fluorescence polarization spectroscopy ...................................................... 88 5.1.2 NMR 15N-rCARDApaf-1 peptoid 1 interaction assays..................................... 89 5.1.3 Yeast two-hybrid system ............................................................................... 91 5.1.4 In silico modelization analysis ..................................................................... 95
5.2 Characterization of peptoid 1 binding site in whole cell systems.................. 98 5.2.1 Cell extracts fractionation studies................................................................ 98 5.2.2 Inhibition of apoptosome-dependent apoptosis induction............................ 99
CHAPTER 6 – Characterization of the cytoprotective effect of peptoid
1 derivative QM31 ...................................................................................101 6.1 Characterization of QM31 cytoprotective effect in cellular models ........... 103
6.1.1 Cytoprotective effects of QM31 and Z-VAD-fmk in A549 cells.................. 104 6.1.2 Cytoprotective effect of QM31 and q-VD-OPH in Heltog cell line............ 108 6.1.3 Comparison of the clonogenic cell survival effect of QM31 and q-VD-OPH
in Heltog cell line ............................................................................................. 111
CHAPTER 7 – Apaf-1 non-apoptotic functions in cell........................113 7.1 Inhibition of the intra-S-phase DNA damage checkpoint by QM31 .......... 113 7.2 Influence of high doses of L-glutamine in cell survival after peptoid 1...... 116
CHAPTER 8 – Primary culture and in vivo hypoxia experiments ....125 8.1 Analysis of apoptosis inhibition in primary culture cells, cardiomyocytes
under hypoxia conditions................................................................................... 126 8.2 Analysis in animal models............................................................................... 129
8.2.1 Safety and toxicology evaluation of peptoid 1 derivatives in mice............. 130 8.2.2 Analysis of apoptosis inhibition in an animal model of kidney hot ischemia
.......................................................................................................................... 131
CHAPTER 9 – General discussion ........................................................139
CHAPTER 10 – Conclusions..................................................................143
CHAPTER 11 – Materials and methods...............................................145 11.1 Equipment and material ............................................................................... 145
11.1.1 Equipment and material employed in the synthesis of peptoid 1 and peptoid 1 derivatives...................................................................................................... 145
11.1.2 Materials and equipment employed in the biological assays ................... 146 11.2 Peptoid 1 and peptoid 1 derivatives synthesis............................................. 149
11.2.1 Synthesis of peptoid 1 ................................................................................ 149 11.2.2 Cell-penetrating peptides and cell-penetrating peptide/peptoid hybrids
synthesis............................................................................................................ 149 11.2.3 Synthesis of QM56 and QM56-OG........................................................... 150 11.2.4 Synthesis of conformationally constrained peptoid 1 mimetics, QM31 ... 155
11.3 E. coli manipulation....................................................................................... 156 11.3.1 E. coli strains and plasmid constructs...................................................... 156 11.3.2 Culture conditions and manipulation of E. coli ....................................... 156 11.3.3 Obtention of recombinant proteins........................................................... 157
11.4 S. cerevisiae manipulation............................................................................. 160 11.4.1 S. cerevisiae strain.................................................................................... 160 11.4.2 Culture conditions and manipulation of EGY048 strain .......................... 160 11.4.3 Yeast two-hybrid system assay ................................................................. 161
11.5 Insect cell manipulation ................................................................................ 161 11.5.1 Insect cell lines ......................................................................................... 161 11.5.2 Culture conditions and manipulation of insect cells ................................ 161 11.5.3 Baculovirus manipulation and recombinant Apaf-1 production.............. 162
11.6 Mammal cell lines manipulation .................................................................. 164 11.6.1 Mammal cell lines..................................................................................... 164 11.6.2 Culture conditions .................................................................................... 164 11.6.3 HEK 293 cell extracts preparation and cell-free caspase activation assay
(DEVDase activity)........................................................................................... 164 11.6.4 In vivo reconstitution assay of the apoptosome........................................ 165 11.6.5 MTT cell viability assays .......................................................................... 166 11.6.6 Caspase actvation assays in cellular models (DEVDase activity) ........... 167 11.6.7 Cellular internalization studies by flow cytometry and confocal
fluorescence microscopy .................................................................................. 167 11.6.8 Circular Dichroism spectroscopy............................................................. 169 11.6.9 Fluorescence polarization assay .............................................................. 169 11.6.10 NMR 15N-CARD peptoid 1 interaction assays........................................ 169 11.6.11 In silico modelization analysis ............................................................... 169 11.6.12 Gel filtration studies in whole cells ........................................................ 170 11.6.13 Apoptosis induction experiments in A549 cell lines ............................... 170 11.6.14 Apoptosis induction experiments in Heltog cell lines............................. 171 11.6.15 Cell cycle experiments ............................................................................ 173
11.7 Cardiomyocyte primary culture .................................................................. 175 11.7.1 Culture of neonatal rat cardiomyocytes in hypoxic conditions................ 175 11.7.2 Immunohistochemistry studies in cardiomyocytes ................................... 175
11.8 In vivo kidney hot ischemia models ............................................................. 176 11.8.1 Kidney hot ischemia induction ................................................................. 176 11.8.2 Pathological studies ................................................................................. 176 11.8.3 TUNEL analysis........................................................................................ 177 11.8.4 Caspase 3 activity in kidney extracts........................................................ 177
CHAPTER 12 – Bibliography................................................................179
Appendix: Summary of the thesis project in Spanish
FIGURE INDEX
CHAPTER 1 Figure 1.1 The apoptotic machinery ....................................................................... 1 Figure 1.2 Structural characteristics of caspases ................................................... 4 Figure 1.3 Structural characteristics of Bcl-2 family of proteins ........................... 5 Figure 1.4 Models for the functional interactions between the different Bcl-2
family members..................................................................................................... 6 Figure 1.5 Apaf-1-XL structure ............................................................................... 8 Figure 1.6 Cryo-EM structure of the apoptosome .................................................. 9 Figure 1.7 Apaf-1 interacting proteins.................................................................. 13 Figure 1.8 Chemical structure of the different Apaf-1 modulators....................... 17 Figure 1.9 Chemical structure of peptoid 1 and its derivatives ............................ 19
CHAPTER 3
Figure 3.1 Chemical structure of the 2nd generation of peptoid 1 derivatives...... 23 Figure 3.2 Cellular internalization pathways of peptoid 1 derivatives................. 24 Figure 3.3 Examples of nanopharmaceutics ......................................................... 27 Figure 3.4 EPR effect and lysosomotrophyc intracellular polymer-conjugates drug
delivery ............................................................................................................... 29 Figure 3.5 Scheme of peptoid 1 chemical synthesis .............................................. 33 Figure 3.6 Chemical structure of PEN-1 and TAT-1 ............................................ 34 Figure 3.7 Synthesis of peptoid 1-GG ................................................................... 35 Figure 3.8 Scheme of conjugation of the peptoid 1-GG to the polymeric carrier 35 Figure 3.9 Polymer-conjugate total drug content quantification by UV
spectroscopy ....................................................................................................... 36 Figure 3.10 Calibration curve for polymer conjugate peptoid 1-GG content by
HPLC.................................................................................................................. 37 Figure 3.11 1D 1H NMR spectrum of NH2-GG-peptoid 1, PGA and QM56........ 39 Figure 3.12 Expansion of 2D TOCSY NMR spectra of PGA vs. QM56................ 39 Figure 3.13 Kinetic study of QM56 degradation in the presence of cathepsin B . 40 Figure 3.14 Scheme of QM56-OG synthesis ......................................................... 41 Figure 3.15 Determination of OG content on the nanoconjugate by fluorimetry. 41 Figure 3.16 QM31 synthesis scheme..................................................................... 42 Figure 3.17 Chemical structure of QM31 and its derivatives............................... 43 Figure 3.18 Influence of polymer-drug linkers on drug release kinetics .............. 48 Figure 3.19 GPC-HPLC-based analysis of released QM57 from QM57-P2 in
serum and blood ................................................................................................. 49 CHAPTER 4
Figure 4.1 Scheme of in vitro and cell-free extract apoptosome reconstitution assay ................................................................................................................... 53
Figure 4.2 Chemical structure of doxorubicin ...................................................... 54 Figure 4.3 Chemical structure of doxycycline....................................................... 54 Figure 4.4 Schematic development of MTT assay................................................. 55 Figure 4.5 Scheme of caspase 3 activity fluorogenic assay .................................. 56 Figure 4.6 Chemical structure of fluorescein and derivatives .............................. 57 Figure 4.7 Concentration dependent inhibition of Apaf-1 mediated activation of
caspases by peptoid 1, PEN-1 and TAT-1 .......................................................... 58 Figure 4.8 Evaluation of cell toxicity in different cell lines by PEN-1 and TAT-1 59
Figure 4.9 Evaluation of cell-viability recovery in different cell lines by PEN-1. 59 Figure 4.10 Evaluation of caspase 3 activity inhibition in different cell lines after
apoptotic insult by PEN-1 .................................................................................. 60 Figure 4.11 Time-dependent uptake of PEN-1 and TAT-1 by U937 cells............. 61 Figure 4.12 Confocal microscopy live cell images of PEN-1 internalization by
Saos-2 cells ......................................................................................................... 62 Figure 4.13 CD spectra of TAT-1 and PEN-1 in the presence of TFE ................. 63 Figure 4.14 CD spectra of PEN-1 and TAT-1 in the presence of SDS.................. 64 Figure 4.15 CD spectra of PEN-1 and TAT-1 in the presence of LPC ................. 65 Figure 4.16 Cellular uptake of QM56-OG in established cell lines ..................... 69 Figure 4.17 Evaluation of cell toxicity in different cell lines by QM56 ................ 70 Figure 4.18 Evaluation of cell-viability recovery in different cell lines by QM56 71 Figure 4.19 Caspase 3 activity inhibition after an apoptotic insult in different cell
lines by QM56...........71 Figure 4.20 Caspase 9 activity inhibition by QM31 and derivatives........................................................................................................... 74
Figure 4.21 Caspase 9 activity inhibition by QM57 and derivatives.................... 74 Figure 4.22 Evaluation of cell toxicity in different cell lines by QM31 ................. 76 Figure 4.23 Evaluation of cell-viability recovery in different cell lines in QM31 76 Figure 4.24 Caspase 3 activity inhibition after an apoptotic insult in different cell
lines by QM31..................................................................................................... 81 CHAPTER 5
Figure 5.1 Fluorescence polarization studies of CF-peptoid 1 and rCARDApaf-1 interaction........................................................................................................... 89
Figure 5.2 PEN-1 bidns to 15N-rCARDApaf-1 domain............................................. 90 Figure 5.3 Yeast two-hybrid system ...................................................................... 92 Figure 5.4 pGilda and pJG4-5 plasmids structure ............................................... 92 Figure 5.5 Two-hybrid yeast system....................................................................... 94 Figure 5.6 Yeast two-hybrid system OD values after QM31 treatment ................ 95 Figure 5.7 In silico model of the complex CARD-NODApaf-1/QM31 ..................... 97 Figure 5.8 Diagram of QM31 and Apaf-1 interaction determined by docking..... 97 Figure 5.9 Gel filtration results for HeLa cells after doxorubicin treatment and
QM56 addition.................................................................................................... 99 Figure 5.10 Chemical structure of compound 2.................................................. 100
CHAPTER 6
Figure 6.1 CDDP chemical structure.................................................................. 104 Figure 6.2 Z-Val-Ala-Asp-fmk chemical structure .............................................. 104 Figure 6.3 Apoptotic cell death inhibition in A549 after CDDP treatment by
QM31................................................................................................................ 106 Figure 6.4 Cytoprotective effect exerted by peptoide 1 derivative QM31 in A549
after CDDP treatment ...................................................................................... 107 Figure 6.5 Chemical structure of STS ................................................................. 108 Figure 6.6 Chemical structure of q-VD-OPH ..................................................... 108 Figure 6.7 Apoptotic cell death inhibition by QM31 and q-VD-OPH in Heltog cell
line after STS treatment .................................................................................... 109 Figure 6.8 Study of cyt c release kinetics in Heltog cell line after an apoptotic
insult in the presence of QM31 and q-VD-OPH............................................... 110 Figure 6.9 Clonogenic survival assay in Heltog cell line after an apoptotic insult
in the presence of QM31 and q-VD-OPH ........................................................ 112
CHAPTER 7 Figure 7.1 Inhibition of the intra-S-phase cell cycle arrest in A549 after CDDP
treatment in the presence of QM31 .................................................................. 115 Figure 7.2 Inhibition of Chk1 phosphorilation in A549 after DNA-damage
induction in the presence of QM31 .................................................................. 116 Figure 7.3 Influence of GlutaMAX and L-glutamine on QM56 cell toxicity in
different cell lines ............................................................................................. 118 Figure 7.4 Influence of GlutaMAX and L-glutamine on QM31 cell toxicity in
different cell lines ............................................................................................. 119 Figure 7.5 LC-3 GFP translocation to the autolysosomes induction in the
presence of GlutaMAX plus peptoid 1 derivatives ........................................... 122 Figure 7.6 In vitro apoptosome reconstitution inhibition by GFAT2 ................. 124
CHAPTER 8
Figure 8.1 Immunohistochemical caspase 3 detection in neonatal cardiomyocyte primary culture ................................................................................................. 127
Figure 8.2 Cellular uptake of the polymer conjugate QM56-OG in cardiomyocyte primary culture cells......................................................................................... 128
Figure 8.3 Pathological analysis of rat kidney tissue subjected to hot ischemia after peptoid 1 derivatives treatment................................................................ 134
Figure 8.4 TUNEL assay fo the hot ischemia animal model after peptoid 1 derivatives treatment ........................................................................................ 135
Figure 8.5 Analysis of caspase 3 activity in kidney extracts subjected to hot ischemia after peptoid 1 derivatives treatment................................................. 136
TABLE INDEX
CHAPTER 1 Table 1.1 Different types of cell death..................................................................... 2
CHAPTER 3
Table 3.1 Polymer-drug conjugates in clinics....................................................... 31 Table 3.2 Chemical structure for the substituents at R1 position in QM31 .......... 44 Table 3.3 Schematic structure of QM56 and heterocyclic polymeric derivatives. 45 Table 3.4 Loading parameters for PGA-conjugates ............................................. 46 Table 3.5 Degradation studies for PGA-based derivatives................................... 47
CHAPTER 4
Table 4.1 Percentage of secondary structure obtained from the CD data interpreted with the program provided by Jasco ............................................... 66
Table 4.2 Percentage of secondary structure obtained from the CD data interpreted with the K2D program of the Dichroweb ........................................ 67
Table 4.4 Percentage of caspase 3 inhibition after an apoptotic insult by QM56 derivatives in Saos-2........................................................................................... 72
Table 4.5 Evaluation of QM31 derivatives cell toxicity in U937 and Saos-2 cell lines..................................................................................................................... 77
Table 4.6 Evaluation of QM57 derivatives cell toxicity in U937 and Saos-2 cell lines..................................................................................................................... 78
Table 4.7 Evaluation of QM31 derivatives cell recovery in U937 and Saos-2 cell lines..................................................................................................................... 79
Table 4.8 Evaluation of QM57 derivatives cell recovery in U937 and Saos-2 cell lines..................................................................................................................... 80
Table 4.9 Caspase 3 activity inhibition in U937 and Saos-2 cell lines by QM31 and derivatives.................................................................................................... 82
Table 4.10 Caspase 3 activity inhibition in U937 and Saos-2 cell lines by QM57 and derivatives.................................................................................................... 83
Table 4.11 Caspase 3 activity inhibition in U937 and Saos-2 cell lines by QM31 and QM57 polymeric derivatives ....................................................................... 85
CHAPTER 5
Table 5.1 Plasmid constructs employed in the yeast two-hybrid system............... 93 Table 5.2 Percentage of caspase 3 activity inhibition in U937 and Saos-2 cell lines
by QM56 and QM31-P2 after compound 2 treatment...................................... 100 CHAPTER 8
Table 8.1 Percentage of cell recovery and caspase 3 inhibition in cardiomyocytes after hypoxia-induced apoptosis in the presence of QM31 and QM56............ 129
Table 8.2 Evaluation of peptoid 1 derivatives toxicity in mice ........................... 130 Table 8.3 Mice blood analysis parameter results after peptoid 1 derivatives
treatment........................................................................................................... 131 Table 8.4 Description of the groups of animals and treatments selected for the in
vivo hot-ischemia model ................................................................................... 130 Table 8.5 Analysis of caspase 3 activity in kidney extracts after peptoid 1
derivatives treatment ........................................................................................ 134
CHAPTER 11 Table 11.1 Internal control of the yeast two-hybrid assay.................................. 166
ABBREVIATIONS: A. victoria: Aequorea victoria A1: B-cell leukemia/lymphoma 2 related protein A1a Ac-LEHD-afc: Ac-Leucine-glutamate-histidine-aspartate-afc AD: Transcriptional activation domain ADP: adenosine diphosphate afc: 7-amino-4-trifluoromethyl coumarine AIF: Apoptosis inducing factor AML: Amyotrophic lateral sclerosis Amp: Ampicilin Ann V: Annexin V ANOVA: Analysis of variance ANT: Adenine nucleotide transporter AOBS: Acoustic optical beam splitter Apaf-1: Apoptotic peptidase activating factor 1 ATP: Adenosine triphosphate AUC: Analytical ultracentrifugation AUC: Area under the curve Aven: Protein whose name is derived form the Roman emperor Aventine. It is a caspase activator inhibitor Bad: Bcl-2 associated death promoter Bak: Bcl-2-antagonist/killer 1 Bax: Bcl-2 associated X protein Bcl-2: B-cell lymphoma 2 B-CLL: B-cell chronic lymphocytic leukemia Bcl-XL: B-cell lymphoma extra large Bcr-Abl: Breakpoint cluster region-Abl tyrosine kinase BH1-4: Bcl-2 homology domains from 1 to 4 Bid: BH3 interacting domain death agonist Bik: BCL2-interacting killer (apoptosis-inducing) BimL: BCL2-like 11 (apoptosis facilitator) BIR: Baculovirus IAP repeat Bmf: Bcl-2 modifying factor Bok: Bcl-2 related ovarian killer C. elegans: Caernohabditis elegans CARD: Caspase recruitment domain CAS: Cellular apoptosis susceptibility protein Caspase: Cysteine aspartic serine protease CD: Circular dichroism spectroscopy CDDP: Cis-diamminedichloridoplatinum (II) CED-3,4,9: Cell death abnormalities 3, 4 and 9 CF: Carboxyfluoresceine CF-peptoid 1: Carboxyfluorescein-peptoid 1 conjugate Chk1: Checkpoint kinase 1 Chlo: Chloramphenicol Cladribine: 2-chlorodeoxyadenosine or 2CdA cmc: Critical micellar concentration CML: chronic myelogenous leukemia CPK: Creatin phosphokinase
CPP: Cell penetrating peptide cyt c: Cytochrome c D. melanogaster: Drosophila melanogaster dADP: deoxyadenosine diphosphate DARK: Drosophila Apaf-1 related killer dATP: Deoxyadenosine triphosphate DB: DNA-binding domain DIC: diisopropylcarbodiimide DIEA: diisopropylethylamine DiOC6(3): 3,3'- Dihexyloxacarbocyanine iodide DIVA/Boo: Death inducer binding to vBcl-2 and Apaf-1/Bcl-2 homologue of ovary DMF: dimethylformamide DMSO: Dimethylsulfoxide DRONC: Drosophila initiator caspase DTT: Dithiotreitol EDTA: Ethylenediaminetetraacetic acid EGF: Epidermal growth factor from murine submaxillary gland EGL-1: Egg laying defective 1 EPR: Enhanced permeability and retention effect ERK: Extra-cellular signal regulated kinase) EtOH: Ethanol FBS: Foetal bovine serum FC: Flow-cytometry FITC: Fluorescein isothiocyanate Fludarabine: 9-b-d-arabinofuranosyl-2-fluoradenine or F-Ara-A FT: Flow-through FU: Fluorescence unit GFAT: Glutamine-fructose-6-phosphate aminotransferase GFP: Green fluorescent protein GlutaMAX: L-alanyl-L-glutamine dipeptide GPC: Gel permeation chromatography GPT: Glutamate pyruvate transaminase HBXIP: Hepatitis B X-interacting protein HIF: Hypoxia-inducible factor HIV-1: Human immunodeficiency virus type 1 HObt: 1-hydroxybenzotriazole HP: Hexosamine pathway HPLC: High-performance liquid chromatography HPMA: N-(2-hydroxypropylmethacrylamide) Hrk: harakiri, BCL2 interacting protein HSP (Hsp): heat-shock protein HSQC: Heteronuclear Single-Quantum Correlation HTS: High throughput screening IAP: Inhibitor of apoptosis protein IPTG: Isopropyl β-D-1-thiogalactopyranoside LB: Luria Bertani LC-3: Microtubule-associated protein 1 light chain 3 alpha LHRH: human luteinizing hormone-releasing hormone LPC: 1-palmitoyl-2-hydroxy-snglycero-3-phosphocoline MALDI-TOF: Matrix Assisted Laser Desorption/Ionization-Time of Flight
Mcl-1: myeloid cell leukemia sequence 1 (BCL2-related) MeOH: Methanol MOI: Multiplicity of infection MOMP: Mitochondrial outer membrane permeabilization MPTP: Mitochondrial permeability transition pore mTOR: Mammalian target of rapamacyn MTT: 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazoliumbromide bromide Mw,a: Average molecular weight Mw/Mn: Polymer polydispersity Mw: Molecular weight NHBD: Non-heart-beating donors NMR: Nuclear magnetic resonance NOESY: Nuclear Overhauser effect spectroscopy Noxa: also known as Phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) NSCLC: Non-small cell lung carcinoma OD: Optical density OG: Oregon Green Omi/Htra2: Serine Protease OMI/High temperature required protein A2 Puma: also known as Bcl-2 binding component 3 (BBC3) PARCS: Pro-apoptotic protein required for cell survival PBS: Phosphate buffered saline PEG: Poly(ethyleneglycol) PEN: Penetratin (3rd helix of D. melanogaster Antennapedia protein) PEN-1: Penetratin-GG-peptoid 1 conjugate PETCM: α-(trichloromethyl)-4-pyridineethanol PGA: Poly-L-glutamic acid PHAPI: Potent heat-stable protein phosphatase 2A inhibitor I1PP2A PI: Propidium iodide PKA: Protein kinase A PKB: Protein kinase B PKCzeta: Protein kinase C zeta PPII-like: Poly(proline) type II-like ProT: Prothymosin-α PS: Phosphatidylserine q-VD-OPH: Quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone RA: Reumatoid arthritis RES: Reticulum endothelial system ROS: Reactive oxygen stress RT: Room temperature S. staruosporeus: Streptomyces staurosporeus S: Sedimentation coefficient s: Standard deviation SC-drop out: Synthetic-Complete drop out medium SD: Standard error deviation SDS: Sodium dodecyl sulphate SE: Standard error SEC: Size exclusion chromatography Smac/DIABLO: Supramolecular adhesion complex / direct inhibitor of apoptosis protein-binding protein with low pI STS: Staurosporine
TAT: TAT protein (transcription factor of HIV-1) TAT-1: TAT-GG-peptoid 1 conjugate TBS: Tris-HCl buffer solution TBST: Tris-HCl buffer solution plus tween 20 TCA: Trichloroacetic acid TFA: Trifluoroacetic acid TFE: Trifluoroethanol TOCSY: Total correlation spectroscopy tr: Retention time TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling vbar: partial specific volume VDAC: Voltage-dependent anion channel XIAP: X chromosome-linked inhibitor of apoptosis Y2H: Yeast-two hybrid system Z-VAD-fmk: Z-Val-Ala-Asp(OMe)fluoromethylketone ΔΨm: Mitochondrial transmembrane potential
1
CHAPTER 1. INTRODUCTION
1.1 Apoptosis
In the late 19th century, the anatomist Walter Flemming described ovarian follicle cells
that died spontaneously. He termed this process chromatolysis, referring to the break
and disappearance of the nucleus. Such observation was followed by early attempts to
distinguish between spontaneous and other forms of cell death. In 1972 Kerr and
coworkers introduced apoptosis (Greek: ‘dropping of leaves from a tree’) to describe a
common observed morphology to most spontaneous cell deaths and also this study
confirmed the widespread of the process [1].
DNA
Ub
MDM2p53
Proteasome
EGLN2
ROS(Hypoxia/
Reperfusion)
UbHIF-α
DNA damage
Cyt c
TNF receptor
Ligands (FasL, Apo2L/TRAIL…)
DISCFADD
Procaspases 8,10
Caspases 8,10
Caspases 3,7
Procaspases 3,7
Bid tBid
Procaspase 9
Apaf-1
IAPs
Bcl-XLBcl-2
Apoptosome
Smac/DIABLO
Bax/Bak
EndoG
p53
Bcl-XLBcl-2
PumaNoxa
BNIP3
Transcription
Piddosome
?
Caspase 2
HtrA2/Omi
RAIDD
PIDD
Procaspase 2
p53
Bax/BakMOMP
“BH3 only”Proteins
HIF-α
DNA
Ub
MDM2p53
Proteasome
EGLN2
ROS(Hypoxia/
Reperfusion)
UbHIF-α
DNA damage
Cyt c
TNF receptor
Ligands (FasL, Apo2L/TRAIL…)
DISCFADD
Procaspases 8,10
Caspases 8,10
Caspases 3,7
Procaspases 3,7
Bid tBid
Procaspase 9
Apaf-1
IAPs
Bcl-XLBcl-2
Apoptosome
Smac/DIABLO
Bax/Bak
EndoG
p53
Bcl-XLBcl-2
PumaNoxa
BNIP3
Transcription
Piddosome
?
Caspase 2
HtrA2/Omi
RAIDD
PIDD
Procaspase 2
p53
Bax/BakMOMP
“BH3 only”Proteins
HIF-α
DNADNA
Ub
MDM2p53 Ub
MDM2p53
MDM2p53
Proteasome
EGLN2
ROS(Hypoxia/
Reperfusion)
UbHIF-α
EGLN2
ROS(Hypoxia/
Reperfusion)
UbHIF-α
DNA damage
Cyt c
TNF receptor
Ligands (FasL, Apo2L/TRAIL…)
DISCFADD
Procaspases 8,10
Caspases 8,10
Caspases 3,7
Procaspases 3,7
Bid tBid
Procaspase 9
Apaf-1
IAPsIAPs
Bcl-XLBcl-2
Apoptosome
Smac/DIABLO
Bax/Bak
EndoG
p53
Bcl-XLBcl-2
PumaNoxa
BNIP3
Transcription
Piddosome
?
Caspase 2
HtrA2/Omi
RAIDD
PIDD
Procaspase 2
p53
Bax/BakMOMP
“BH3 only”Proteins
HIF-α
Figure 1.1. The apoptotic machinery.
However, the genetic nature of the process remained unknown till Horvitz and Sulston
[2] introduced Caenorhabditis elegans (C. elegans) as a perfect model for the study of
the genetic basis of apoptosis. Later on, successful studies in Drosophila melanogaster
(D. melanogaster) elegantly complemented those discoveries performed in C. elegans
(see review [3] for further information). As a result a core of essential proteins that
2
control this process was defined and it has been demonstrated that the encoding genes
are highly conserved from nematodes to humans. As an example in C. elegans, the
inactive CED-3 zymogen (Cell death abnormalities 3, homologous to the mammalian
caspases) is activated by CED-4 (homologous to the Apoptosis protease activating
factor 1, Apaf-1) that is retained in an inactive form in the mitochondrial membrane by
CED-9 (homologous to the B-cell lymphoma 2, Bcl-2). Upon cell death induction,
EGL-1 (Egg lying defective 1, a nematode protein containing the Bcl-2 homology
domain 3-only, “BH3-only”) is up-regulated and binds to CED-9 inducing the
translocation of CED-4 to the perinuclear membrane, where it oligomerizes forming an
active apoptosome that activates CED-3. A similar apoptotic pathway is found in
Drosophila (e.g. the CED-3 homolog DRONC is activated by the CED-4 homologue
Apaf-1 related killer (DARK), however, the two CED-9 homologues that have been
found in the fly do not appear to play major roles in the control of apoptosis in this
organism) (Figure 1.1).
Table 1.1. Different types of cell death (modified from [4]). Type of cell death Morphological characteristics Apoptosis Rounding-up of the cell
Retraction of pseudopodes Reduction of cellular and nuclear volume Nuclear fragmentation Plasma membrane blebbing Engulfment by resident phagocytes, in vivo
Autophagy related cell death
Lack of chromatin condensation Massive vacuolization of the cytoplasm Accumulation of (double-membrane) autophagic vesicles Little or no uptake by phagocytic cells, in vivo
Necrosis Cytoplasmic swelling Rupture of plasma membrane Swelling of cytoplasmic organelles Moderate chromatin condensation
Cornification Elimination of cytosolic organelles Modifications of plasma membrane Accumulation of lipids in F and L granules Extrusion of lipids in the extracellular space Desquamation (loss of corneocytes) by protease activation
Nowadays apoptosis is defined as a highly regulated cellular pathway responsible for
the elimination of cells in the organism that are no longer needed or extensively
damaged. In multicellular organisms, apoptosis is not the only type of cell death.
Necrosis, cornification and macroautophagy associated cell death processes occur in the
3
cell, some of them simultaneously. Several revisions of the main features of these cell
death processes have been now reported and these different types of cell death have
been redefined according to the characteristics depicted in Table 1.1 [4].
1.2 Apoptosis activation and apoptotic cell death machinery
Apoptosis can be initiated by different apoptotic stimuli, including infectious agents,
anticancer agents, toxic agents, irradiation, growth factor withdrawal, heat shock,
ischemia and degenerative processes [5-8]. Depending on the nature of these stimuli
two different apoptotic pathways can be activated: the extrinsic and the intrinsic
pathways; although they are connected in different steps of the process [9].
The extrinsic apoptotic pathway is initiated by apoptotic stimuli from the outside of the
cell. This apoptosis induction signal is transduced into the cell by specific apoptotic cell
death receptors. Once these cell death receptors are activated, they oligomerize and
recruit and activate initiator caspases 8 and 10 via interactions with adaptor proteins.
As a consequence, initiator caspases spread the apoptotic signal by activating effector
caspases 3 and 7. By contrast, the intrinsic apoptotic pathway is activated by internal
cell death signals such as DNA damage or the presence of reactive oxygen stress (ROS),
among others. Different proteins take part in this process, but two main events should
be highlighted: the activation of caspases and the induction of the mitochondrial outer
membrane permeabilization (MOMP) by Bcl-2 family members.
Caspases are a family of cystein aspartyl proteases that constitute the executioner
machinery in apoptotic processes. Caspases are present in the cell as inactive zymogens
(30-50 kDa) essentially containing three domains: N-terminal prodomain, a large
subunit and a small subunit [10] (Figure 1.2). The caspase family of proteins can be
divided in two groups: initiator and effector caspases. Initiator caspases (e.g., caspases
8, 9 and 10) are the first to be activated upon apoptosis induction; and subsequently they
are in charge of the signal transduction that activates effector caspases (e.g., caspases 3
and 7) that are responsible for the disassembly of cellular components. The molecular
mechanism of caspase activation differs from initiator and effector caspases. The
dimerization process of initiator caspases induces their auto activation by the cleavage
of the large-small subunit connecting linker, besides their enzymatic activity depends on
4
their ability to bind to an allosteric regulator [11-17]. By contrast, effector caspases can
be found as dimers in normal conditions and the subsequent cleavage of the inter-
subunit linker by initiator caspases is the cause of their activation [11, 13]. Once
effector caspases are activated they process their target proteins. The target amino acid
sequence in substrate proteins is defined by an aspartic acid residue in the P1 position
[10], a glutamate residue in P3 and different and certain preference in the P2 position
for threonine, histidine and valine residues [18]. Despite its cell dismantling role recent
data published by Mahrus et al. [19] demonstrate that caspases target a limited group of
pathways (cytoskeleton maintenance and organization, caspase activated DNases,
metabolism maintenance) and have special affinity for protein complexes. This
specificity for substrates would be explained through its dimeric structure, rare in
proteases, that allows them targeting multiple proteins contained in a protein complex at
the same time.
DED
L S
L S
L S
L S
L S
L S
L S
L S
L S
L S
L S
CARD
CARD
CARD
CARD
CARD
DED
DEDDED
Caspase 1
Caspase 2
Caspase 3
Caspase 4
Caspase 5
Caspase 6
Caspase 8
Caspase 9
Caspase 10
Caspase 14
Caspase 7
DED
L S
L S
L S
L S
L S
L S
L S
L S
L S
L S
L S
CARD
CARD
CARD
CARD
CARD
DED
DEDDED
Caspase 1
Caspase 2
Caspase 3
Caspase 4
Caspase 5
Caspase 6
Caspase 8
Caspase 9
Caspase 10
Caspase 14
Caspase 7
Figure 1.2. Structural characteristics of caspases [20]. Procaspases contain in their structure a large (L) and a small (S) subunit and in some cases they can also contain a caspase recruitment domain (CARD) or death effector domains (DEDs).
The mechanisms by which caspases are activated differ from one organism to another,
but in mammals, despite the activation of caspase 8, massive activation of caspases
occurs mainly after the induction of MOMP. This process is regulated by the Bcl-2
family proteins, whose members can play a pro or an antiapoptotic role. All the
5
members of this family contain in their structure till four Bcl-2 homology (BH) domains
which define their role in apoptosis, being the BH3 the one that seems to determine the
proapoptotic role of the protein and the rest related to the antiapoptotic function. The
Bcl-2 family can be divided in three groups according to the BH domains that compose
their structure: the antiapoptotic members (Bcl-2, Bcl-XL, A1 and Mcl-1) contain the
fourth domains (BH1 to BH4) with the exception of Mcl-1 that contains just three;
proapoptotic BH3-only members, that contain just one of the BH3 domains in their
structure (Bid, Bik, BimL, Bad, Noxa, Puma, Bmf and Hrk) and proapoptotic
multidomain members (Bax, Bak and Bok) that contain BH1, BH2 and BH3 domains
(Figure 1.3). The formation of homo- or heterodimers among the members of this
family through selective BH-BH domain interactions controls the activation or
inactivation of apoptosis.
Proapoptotic
Antiapoptotic
Proapoptotic
Antiapoptotic
Figure 1.3. Structural characteristics of Bcl-2 family of proteins. Schematic representation of members of Bcl-2 family. Example of anti-apoptotic members include Bcl-2, Bcl-xl and A1. Their multiple BH3 domains are highlighted in grey. Examples of pro-apoptotic members of the Bcl-2 family are presented. They include the multiple BH3 domain containing Bax and Bak and the BH3-only Bid, Bim, Bad, Noxa and Puma (modified from [21]).
At present, there is some controversy about the type of interactions among the different
members of the Bcl-2 family that originates MOMP-induction and two models have
been proposed (Figure 1.4). Both models agree that BH3-only members act as initiators
of the apoptotic process by selective activation of the BH multi-domain proapoptotic
6
members Bak and Bax (Bcl-2-antagonist-killer 1 and Bcl-2 associated X protein,
respectively). Bak/Bax are direct effectors of MOMP-induction [22] (Figure 1.4 A).
However, the activation models differ in the proposed mechanism of protein-protein
interaction that drives the process. In one of the models a direct interaction among the
BH3-only members and Bak/Bax that is inhibited by the antiapoptotic members of the
family is proposed [23-25]. However, the second model suggests that the antiapoptotic
members of the family inhibit Bak/Bax and is the BH3-only proteins-dependent release
of such inhibition which activates MOMP-induction process [26-28] (Figure 1.4 B).
Bcl-2
Bax/Bak
BH3BH3
Bax/Bak
Bcl-2
A B
Bcl-2
Bax/Bak
BH3BH3
Bax/Bak
Bcl-2
BH3
Bax/Bak
Bcl-2
A B
Figure 1.4. Models for the functional interactions between the three subclasses of Bcl-2 family proteins. (A) Bcl-2 inhibitory effect on BH3-only proteins (BH3 in the figure) impedes Bax/Bak activation. (B) BH3 inhibits Bcl-2 in order to stop the blockage exerted by Bcl-2 over Bax/Bak.
Nevertheless, cells lacking Bak/Bax are unable to undergo apoptosis [29]. Bak and Bax
have a different localization in the cell as Bax is considered as a cytosolic protein while
Bak is attached to the mitochondrial membrane [30]. However, it has been reported that
apoptosis-activation induces a conformational change in Bax protein and its subsequent
translocation to the mitochondrial membrane, where it oligomerizes like Bak [31]. The
mechanism of Bak/Bax-mediated release of mitochondrial intermembrane space
proteins is also controversial and three model mechanisms have been proposed. In the
first model it was suggested that the oligomerization of Bax in the mitochondrial
membrane would lead to the formation of a pore able to cause MOMP. This model is
based on the structural similarity among Bax and certain pore-forming bacterial toxins
[31]. The second model proposed relies on the ability of Bcl-2 family proteins to
modulate the mitochondrial permeability transition pore (MPTP) [32]. This pore is
formed by the voltage-dependent anion channel (VDAC) and the adenine nucleotide
transporter (ANT) and it is implicated in the regulation of pH, Ca+2 homeostasis,
mitochondrial volume and it behaves as an anion channel. It has been postulated that
7
Bax could interact with ANT keeping the pore open which would cause mitochondrial
membrane shrinkage due to a massive entrance of water to the mitochondria [33].
Finally, MOMP induction would be also explained by the ability of Bax to destabilize
and permeabilize the mitochondrial phospholipidic membrane, as it has been shown in
different assays [34, 35].
Nevertheless, what it is important is the post MOMP events. A series of proteins are
released from the mitochondrial intermembrane space to the cytosol such as Omi/Htra2,
apoptosis inductor factor (AIF), supramolecular adhesion complex (Smac)/Direct
inhibitor of apoptosis-binding protein with low pI (DIABLO) and cytochrome c (cyt c).
The mechanism of cyt c release is not fully understood, but it happens in the early
stages of apoptosis and it happens in a quick and unique step [36, 37]. Once in the
cytosol this protein binds to the cytosolic protein Apaf-1 to constitute the protein
complex called apoptosome. Once assembled, this complex will recruit and activate
procaspase 9 [38-41]. The caspase 9 active form will process procaspase 3 that, in the
end, will initiate the process of cell dismantling. Then the apoptosome complex plays a
crucial role connecting MOMP processes and caspase activation that will
sophisticatedly tear down the cell. The discovery of the implication of cyt c, a heme
protein implicated in the mitochondrial respiratory chain, in apoptosis increased the
cellular importance of mitochondria not only as the energy generator organelle but also
as a crucial organelle in the equilibrium life/death of the cell.
However, the role of the Bcl-2 family members in apoptosis does not seem to finish
here. Some amplification loops of the apoptotic signal have been described and one of
them involves the proapoptotic BH-multidomain protein Bid (BH3 interacting domain
death agonist) [42]. This protein is the direct activator of Bax/Bak after an activation
process in which the protein is cleaved into its active form. Different apoptotic stimuli
can induce the processing of the protein, but also after MOMP induction Bid can be
processed by caspase 3. As a consequence, there is an increase in Bax/Bak activation
and an increase in MOMP induction.
8
1.3 The apoptosome: components and cellular regulation
The apoptosome complex was first described by Xiaodong Wang and coworkers using
a cell-free system based on cytosolic extracts from HeLa cells [39, 40]. Having first
identified a role for mitochondrial cyt c in activating caspases, Wang and colleagues
then purified the factors that assisted cyt c in its deadly mission. Their search led them
to Apaf-1, the first identified mammalian homolog of CED-4, a protein known to
participate in programmed cell death in C. elegans.
Apaf-1 (130 kDa) contains three functional domains: an N-terminal CARD, a central
nucleotide-binding oligomerization domain (NOD) similar to that found in the C.
elegans protein CED-4, and twelve to thirteen WD40 repeats at the carboxy terminal
half that are responsible for both, binding to cyt c and regulating Apaf-1 function [43,
44]. Apaf-1 is cytoplasmic by nature and it is found in most tissues with highest
expression in adult spleen and peripheral blood leukocytes, fetal brain, kidney, and
lung. Apaf-1 is alternatively spliced up to five forms that vary in length but only two of
them are active: Apaf-1-XL (Apaf-1) and Apaf-1-LC (Figure 1.5). These two isoforms
differ in an 11 amino acids sequence between the CARD and NOD domain contained
by the Apaf-1-XL
NODCARD
WD4011ACN NODCARD
WD4011ANODCARD
WD4011ACN
Figure 1.5. Apaf-1-XL structure.
The exact composition, stechiometry and structure of the apoptosome remain unknown.
However, electron cryomicroscopy data suggest that the apoptosome complex is a
wheel-like structure comprising a central hub composed of seven Apaf-1 CARD
domains, connected to seven radial spokes that end with a Y-shaped tail formed by a
bundle of WD-40 repeats (Figure 1.6) [41, 45]. This structure allowed determining one
necessary step for the apoptosome formation that is the activation of Apaf-1 through
dATP/ATP (deoxyadenosine and adenosine triphosphate, respectively) binding. Apaf-1
is present in the cytosol in an inactive or closed conformation in which the WD40
blocks the CARD domain as a negative auto regulation mechanism. Upon MOMP, cyt
9
c binds to Apaf-1 and induces a conformational change that makes the N-terminal
CARD domain accessible to interact with procaspase 9 CARD domain [41, 45-47]. The
disposition of the procaspase 9 in the apoptosome allows its dimerization and the
subsequent cleavage of the subunit linker. However, the molecular mechanism of
apoptosome-mediated caspase 9 activation is controversial. The two currently accepted
models are the induced proximity model [13, 14] and the allosteric model [48, 49].
Also, how does active caspase 9 activate caspase 3/7? Either Apaf-1 can release the
activated caspase 9 from the complex and caspase 3/7 is activated in the cytosol, or it
could bring procaspase 3/7 into the complex where it gets activated by bound caspase 9
and then is released into the cytosol. Recent results suggest the later as the most
possible option [50, 51] although it was also postulated that caspase 3 cleavage of
caspase 9 is required for full activation of the apoptosome [52, 53].
Cyt c
CARD
WD40
NOD
A B
Cyt c
CARD
WD40
NOD
Cyt c
CARD
WD40
NOD
A B
Figure 1.6. (A) the 12.8 Å cryo-EM structure of the apoptosome. (B) Localization of the different Apaf-1 domains inside the apoptosome (modified from [54]).
The key Apaf-1 conformational change induced by cyt c is only possible in the presence
of dATP/ATP bound to the Apaf-1 NOD domain. Once cyt c is bound, Apaf-1 is able
to oligomerize and apoptosome complexes ranging from 700 to 1400 kDa have been
isolated [55]. These complexes differ in their ability to recruit procaspase 9, being the
complex of 700 kDa the most active to develop this task, while the 1400 kDa seems to
be composed by two 700 kDa brought face to face bound through procaspase 9
dependent interactions.
10
The importance of the apoptosome has been demonstrated in the experiments developed
employing Apaf-1-deficient mice. Apaf-1 turned out to be crucial in the development
of the central nervous system and these mice present embryonic lethality with severe
craniofacial malformations, brain overgrowth, and other phenotypic features of severe
apoptosis impairment [56, 57]. In fact, Apaf-1 and procaspase 9 are essential as tumor
suppressor genes and for the induction of apoptosis dependent on the tumor suppressor
protein p53 [58]. For these reason, there are several mechanisms of apoptosome control
in the cell in order to rule the apoptosome activity through direct interaction with their
individual components. In case of procaspase 9, the most important form of regulation
relies on the family of the inhibitor of apoptosis proteins (IAPs): XIAP, cIAP1 and
cIAP2, NAIP, survivin, ILP2, BRUCE and LIVIN. This is a group of anti-apoptotic
proteins that were first described as products of baculovirus genes which allow virus
propagation by inhibiting the apoptotic defense mechanism of the host insect cell [59-
61]. All IAP proteins have one or three tandem baculovirus IAP repeat (BIR) domains
of 70–80 amino acids. BIR domains are essential for the anti-apoptotic activity of IAPs
[62]. From this family of proteins XIAP (X-linked inhibitor of apoptosis protein),
cIAP1 and cIAP2 (cellular IAPs 1 and 2) are able to bind to caspase 9 and inhibit their
cleavage and subsequent activation. The presence of IAPs proteins is counteracted by
the release of the mitochondrial protein Smac/DIABLO to the cytosol after MOMP.
Smac binds to XIAP more efficiently than procaspase 9 and inhibits its activity [63-65].
Another way of regulation of procaspase 9 is its phosphorilation through different
proteins such as ERK (extra-cellular signal regulated kinase), PKB (protein kinase B) ,
PKCzeta (protein kinase C zeta) [66-68] or preventing its recruitment by Apaf-1 which
is the case of PKA (protein kinase A) and HBXIP (hepatitis B X-interacting protein)
[52, 69].
The apoptosome is targeted by a large number of proteins that upon interaction will
influence in its regulation. In particular, a series of proteins have been described to bind
to Apaf-1. From these proteins one can identify well defined apoptosis-related proteins,
that will act as an apoptosis self-regulatory system and non-classical apoptosis proteins
that will probably account for relationships between apoptosis and other cell signaling
pathways. It has been reported that the heat-shock proteins family (HSP), whose
expression is induced in response to cellular stress, modulates the activity of the
apoptosome. Hsp27 is able to bind to cyt c preventing its interaction with Apaf-1 [70,
11
71]. Besides, the overexpression of Hsp70 protected cells from certain death stimulus
through direct binding to Apaf-1 and inhibition of the formation of the apoptosome [72-
75]. By contrast, Hsp70, PHAPI (Potent heat-stable protein phosphatase 2A inhibitor
I1PP2A) (see below) and cellular apoptosis susceptibility protein (CAS) were found to
stimulate apoptosome formation by facilitating Apaf-1 conformational changes related
to nucleotide exchange [76]. Another HSP protein, Hsp90β was shown to have affinity
to bind to Apaf-1 WD40 domain and inhibit cyt c-mediated oligomerization of Apaf-1
[77, 78]. How can this dual role of HSP proteins be explained? Perhaps the different
‘sensing’ conditions of the cell and the relative intracellular protein concentration will
have the answer. This and some other controversies related to the cellular role of Apaf-
1 and Apaf-1-binding proteins present interesting questions to be solved.
Probably influenced by the well documented pathway of cell death in C. elegans, it was
originally proposed that Bcl-XL bound directly to Apaf-1 and inhibited its function [43,
79, 80] (Figure 1.7). However, other studies have suggested that Apaf-1 does not bind
to prosurvival members of Bcl-2 family of proteins [81, 82]. Another Bcl-2 protein,
Diva/Boo (death inducer binding to vBcl-2 and Apaf-1/Bcl-2 homologue of ovary), was
reported as Apaf-1 interacting protein [83-85] and Aven (caspase activator inhibitor)
was identified as a new Bcl-XL interacting protein and was also postulated to bind to
and prevent Apaf-1 oligomerization [86]. Also, apo-cytochrome c is able to bind to
Apaf-1 and compete with the holoform, but it is unable to form an active apoptosome
[87].
In addition, several cell cycle-related proteins have been reported as Apaf-1 interacting
proteins. Nucling, has an increased expression shortly after apoptosis activation and
influences both, the synthesis of apoptosome-related proteins and a putative
translocation of the Apaf-1/procaspase 9 complex to the nucleus [88]. Also the histone
H1.2 has been found to interact with Apaf-1 [89]. PARCS (pro-apoptotic protein
required for cell survival) was found to bind to a GST-Apaf-1 fusion protein containing
the CARD and NOD domains [90]. PARCS induces cell cycle arrest in G1 phase [90-
92] and has been implicated in the role of Apaf-1 in cell cycle [93, 94].
Apoptosis has been connected to both acute and chronic phases of ischemia-related
pathologies as heart failure, stroke and those derived from organ transplantation [95,
12
96]. Hypoxia-inducible factor (HIF) is the principal transcription factor involved in the
regulation of transcriptional responses to hypoxia [97] and some HIF-regulated proteins
showed putative Apaf-1 binding properties [98, 99]. Moreover, Apaf-1 has also been
described as target for both protein kinases and phosphatases [100-104] and the
phosphorylation state of Apaf-1 could have relevance on apoptosome activity
regulation. The non-essential sulfur-containing amino acid taurine seems to decrease
the amount of caspase 9 associated to Apaf-1, which would explain the cardio protective
role of this molecule. However, to date the interaction between Apaf-1 or caspase 9 and
taurine has not been demonstrated. Nevertheless, the increased PKB activity observed
after taurine treatment has been suggested as the negative regulator of the Apaf-
1/caspase 9 interaction [105-108].
In the search of Apaf-1 protein interactors, new proteins have been described. This is
the case of NAC [109] (nucleotide binding domain and CARD) that stimulates
procaspase 9 recruitment by Apaf-1 and HCA66 [110] and its spliced variant HCA66S
that bind to the NOD domain of Apaf-1 and can activate or inhibit its activity,
respectively.
Finally, additional regulation of the apoptosome has been found by by intracellular level
of ATP [111] and ions such as nitric oxide donors and K+ [112, 113]. In fact, it was
early demonstrated that in the in vitro reconstituted apoptosome, Apaf-1 binds and
hydrolyzes ATP or dATP to ADP or dADP, respectively [38]. However, the high
millimolar concentrations of nucleotides in the cell opened discussions on how such
physiological nucleotide levels affect the apoptosome. A dose-dependent inhibitory
effect of dATP on the cyt c-initiated caspase activation was observed [111]. It was
shown that the nucleotides directly interact with some lysine residues of cyt c
preventing its association with Apaf-1 and therefore the apoptosome formation. These
studies confirmed that the intracellular nucleotide concentration represents an additional
critical prosurvival factor by functioning as a natural inhibitor of apoptosome formation
probably to provide a physiological barrier to unwanted apoptosis.
13
H1.2NuclingPPI
124898
NOD WD40CARDCARD4121
Apo cyt cDiva/BooHsp 70Hsp 90
Hsp70/CAS/PHAPIJNK
ATP/dATPHCA66PARCS
ADPBcl-XL
Diva/BooHCA66S
APIPAIP Aven
Bcr-Abl
Procaspase 9NAC
Cytochrome c
H1.2NuclingPPI
H1.2Nucling
H1.2NuclingPPIPPI
124898
NOD WD40CARDCARD4121
Apo cyt cDiva/BooHsp 70Hsp 90
Hsp70/CAS/PHAPIJNK
ATP/dATPHCA66PARCS
ADPBcl-XL
Diva/BooHCA66S
APIPAIP Aven
Bcr-Abl
Procaspase 9NAC
Cytochrome c
124898
NOD WD40CARDCARD4121 124898
NOD WD40CARDCARD4121
Apo cyt cDiva/BooHsp 70Hsp 90
Hsp70/CAS/PHAPIJNK
ATP/dATPHCA66PARCS
ADPBcl-XL
Diva/BooHCA66S
APIPAIP Aven
Bcr-Abl
Procaspase 9NAC
Cytochrome c
Figure 1.7. Apaf-1 interacting proteins. Inhibitory proteins are shown in black and Apaf-1 activator proteins in white. Proteins with an unknown function are represented as grey boxes. The different proteins are located according to the Apaf-1 domain of interaction.
1.4 Apoptosome implication in disease
Due to the importance of apoptosis in development, normal cell turnover, and immune
system function, any defect in the apoptotic pathways induces the appearance of a series
of pathologies. Defects in appropriate suppression of apoptosis are observed in cancer
pathogenesis and autoimmune disorders, while anomalous induced apoptotic signaling
that induces unwanted cell death plays an important role in pathological conditions as
tissue infarctation, ischemia-reperfusion damage, degenerative diseases and AIDS [114-
121]. A deeper knowledge of the apoptotic machinery has determined the proteins
implicated in the development of the different diseases associated to apoptosis and the
apoptosome has been one of protein complexes implicated in development of disease
[122-125].
Specifically, it is broadly accepted the implication of Apaf-1 in the development of
certain types of cancer. The inactivation of apaf-1 gene through methylation-induced
transcriptional silencing is directly connected to the appearance of metastatic melanoma
[126]. Downregulation of Apaf-1 expression is associated with the appearance of
14
bladder cancer [127] or LOH-induced Apaf-1 inactivation causes gliobastomas [128].
Moreover, it has been shown that the apoptotic response driven by chemotherapy can be
significantly improved by the restoration of Apaf-1 expression in cancer cells or by
reintroduction of a splicing variant that enables a correct apoptosome formation [126-
132]. Apaf-1 is also inactive in cervical carcinomas, colorectal cancer and in B-cell
chronic lymphocytic leukemia (B-CLL) [133-136]. In fibrosarcoma cells, both Apaf-1
and caspase 9 are downregulated and in ovarian cancer caspase 9 is unable to bound to
the oligomerized Apaf-1 [129, 130].
However, defects on the components of the apoptosome might not be the direct cause of
a cancer, but on the molecules that modulate this apoptosome. As an example, it has
been related to the appearance of non-small cell lung carcinoma (NSCLC); in this type
of cancer the overexpression of XIAP inhibits apoptosome activation [137]. Or in case
of breast cancer, a resistance to cyt c release induced by chemotherapeutic agents has
been reported [138]. ProT overexpression inhibits apoptosome formation in lung,
breast, hepatocellular and colon cancers [139, 140]. Finally, the phosphorilation of
Apaf-1 by Bcr-Abl kinase (Breakpoint cluster region-Abl tyrosine kinase) causes
chronic myelogenous leukemia (CML) as it inhibits Apaf-1 and procaspase 9 interaction
[101].
In the other side, massive apoptosome activation is the cause of the appearance of
certain neurodegenerative disorders [124]. Studies using D. melanogaster have
demonstrated that the lack of Apaf-1 dramatically decreases the formation of protein
aggregates due to defective Huntington protein [141]. The lack of Apaf-1 gives
resistance to apoptosis in neural precursor cells or in amyotrophic lateral sclerosis
(ALS) [142, 143]. An increase in cyt c levels have been connected with cardiomyocyte
age-degeneration [144, 145]. Finally, massive tubular cell apoptosis is observed after
ischemia renal failure. However, this apoptotic cell death was significative reduced
after the inhibition of apoptosome formation by depleting cells from ATP [146].
1.5 Chemical modulation of the apoptosome
As stated before, apoptosome formation, which depends on oligomerization of Apaf-1 is
a crucial step in the apoptosis signaling and alterations in the function of proteins that
15
form the apoptosome could be related to diseases [126-132, 137]. For this reason,
chemical modulation of the apoptosome components such as Apaf-1 has proved to be an
interesting approach for treating these diseases and the development of new therapeutic
strategies. For this purpose, the development of in vitro assays for the study of the
apoptosome activity paved the way for the advance in the search of Apaf-1 modulators
[52, 147]. However, it should be also mentioned the reverse approach: chemicals that
were used in the preclinical setting as ‘orphan drugs’ and later identified as modulators
of the apoptosome. The most representative example can be found in the use of
different deoxynucleoside analogs such as cladribine (2-chlorodeoxyadenosine or
2CdA) or fludarabine (9-b-d-arabinofuranosyl-2-fluoradenine or F-Ara-A) in the
treatment of indolent lymphoid malignancies [148, 149]. The proposed mechanism of
action involves the direct binding of the metabolites of these purine nucleoside analogs
(such as dATP, 2CdATP and F-araATP) to Apaf-1 [150] (Figure 1.7).
Apaf-1 has to be considered as a new target for the development of chemical
modulators. However, there are no drug-like precedents, no good initial molecules.
New methods to discovery such modulators were required. To design such methods the
molecular mechanism of apoptosome formation should be taken into account. Then, the
pharmacological target Apaf-1 can be dually seen as a ‘classical’ ATP hydrolase or
alternatively as a protein-protein interaction-based target. To the best of our knowledge
no successful attempts to inhibit the hydrolase activity with small molecules have been
reported. The studies so far described aiming to discovery chemical modulators of
Apaf-1 have addressed the protein-protein interaction-based formation of the
apoptosome. Then two different assays can be envisioned. Direct assays of in vitro
apoptosome reconstitution with recombinant proteins or alternatively it is possible, from
cell extracts, to stimulate apoptosome formation and evaluate the modulating activity of
chemicals. As mention before, Apaf-1 is a new target then the discovery process has to
be initiated with the screening of large collections of compound libraries and a suitable
high throughput screening (HTS) format. Large scale production and purification of the
essential components required for apoptosome reconstitution-based assays (Apaf-1,
procaspase 9 and procaspase 3) is laborious and expensive. Thus, most of the initial
assays searching for apoptosome modulators were developed using cell extracts.
However, cell extracts-based HTS is indirect so it is possible to engage many different
points in the pathway. Thus, follow-up assays, also referred to as ‘secondary screens’
16
or ‘counter screens’, should be planned that will validate compounds targeting the
intended biological interaction(s) and assist in the elimination of compounds that
generate a positive signal via other mechanisms.
Two simultaneous studies both in time and objectives appeared in 2003 [139, 151] with
the interesting goal of identifying compounds that could induce a chemical activation of
the apoptosis machinery. As mentioned above, cancer cells have developed mechanism
to inhibit cell apoptosis and drug discovery efforts early targeted inhibition of events
upstream of cyt c release (inhibitors of the antiapoptotic members of the Bcl-2 family
[63, 152, 153]. However, studies on small molecule as direct activators of apoptosis are
less abundant. In their study, Nguyen and Wells [151] used a in vitro mimic of the in
vivo activation of the apoptosome obtained when cyt c and dATP were added into a
HeLa cell cytoplasmic extract [154]. The screening of a library of 3,500 compounds
allowed the identification of hit compounds that promoted caspase 3 activity. The most
active named compound 1 was chemically optimized and some derivatives (compounds
2 and 4) were active at low micro molar concentration on cell extracts inducing
apoptosis by enhancing the oligomerization of Apaf-1. Importantly, when tested in a
large number of normal and tumor cell lines, compound 2 induced apoptosis in tumor
cells expressing components of the caspase 9 apoptosis pathway (i.e. Apaf-1 and
caspase 3). The detailed molecular mechanism of action of compound 2 was not totally
described however, it was reported that the compound required for bioactivity a
decreased concentration of cyt c but it was inactive in its absence. Thus, these
compounds seemed to enhance the ability of cyt c to activate the apoptosome.
It was possible that the compounds identified in the Nguyen and Wells’ study share
some characteristics with those compounds identified by Xiadong Wang and co-
workers which were simultaneously reported [139]. Wang’s group screened a library of
184000 compounds for caspase 3 activators with HeLa cell extracts (S-100 fraction) in
the presence of 1 mM exogenous dATP and discovered a small molecule α-
(trichloromethyl)-4-pyridineethanol (PETCM) which does not act directly on Apaf-1
but actively promotes apoptosome formation (Figure 1.7). The molecular mechanism of
PETCM action is based on suppression of the negative regulation of Prothymosin-α
(ProT) over PHAPI that promoted caspase 9 activation after apoptosome formation.
17
Further studies on this system, suggested that PHAPI, in concert with CAS and Hsp70,
control the rate of the nucleotide exchange on Apaf-1 [76].
The discovery of drug candidates directed to inhibit unwanted apoptosis has also
targeted the apoptosome. Jäättelä and coworkers described the first chemical inhibitor
of Apaf-1. They also use cytosolic extracts from HeLa cervix carcinoma, carefully
obtained in order to avoid mitochondrial damage, where the apoptosome was activated
by further addition of dATP and cyt c and a library of 5,000 non peptide small
molecules was screened to identify apoptosis inhibitors [155]. A diarylurea derivative
compound NS3694 showed the highest inhibitory activity (Figure 1.8). NS3694 alters
the cyt c- and dATP-dependent association between Apaf-1 and procaspase 9 to form
the apoptosome complex. Instead, Apaf-1 was found as a component of an inactive 1.4
MDa complex. However, neither the molecular mechanism of action nor follow up
optimization studies on these interesting compound have been reported.
N
OO
Cl
Compound 2
HN
HN
O
Cl
COOH
F3C
N
CCl3
OH
O
HOHO
N
N
NN
NH2
Cl
O
HOO
N
N
NN
NH2
FP
O
HO
OH
PETCM
Cladribine Fludarabine
APAF-1 ACTIVATORS
NS3694
APAF-1 INHIBITOR
Cl
Figure 1.8. Chemical structure of the different Apaf-1 modulators described till present.
18
1.6 Identification of an Apaf-1 inhibitor, peptoid 1, through the screening of an N-
alkylglycine chemical library
As stated before, in the absence of detailed structural information, the conventional
methods used to identify modulators of apoptosome have been based on indirect
measurements of the cyt c- and dATP-induced activation of caspase 3-like activity on
defined cytosolic extracts. However, it would be of interest to identify apoptosome
inhibitors using an in vitro reconstituted apoptosome. This was addressed in previous
studies in Pérez-Payá’s laboratory. A medium-throughput assay with purified
recombinant Apaf-1, cyt c, dATP and [35S]-Met procaspase 9 was developed and as a
source of chemical diversity a positional scanning diversity-oriented library of trimeric
alkylglycines (peptoids) was explored [156-158]. The library consisted in 52 controlled
mixtures and a total of 5120 compounds. The mixtures making up each sub library
were screened for their ability to prevent the apoptosome-dependent activation of
procaspase 9 and the compound named peptoid 1 was discovered as inhibitor of the
activity of the apoptosome [158]. Further improvements suggested the incorporation of
two additional N-alkyl amine residues at the N-terminus end to improve water solubility
of peptoid 1, rendering peptoids 1a and 1b (Figure 1.9). Fluorescence polarization
spectroscopy assays showed that a fluorescent derivative of this peptoid binds to rApaf-
1 but no to cyt c [158].
Despite the in vitro activity of peptoid 1, this hit compound exhibited low membrane
permeability and a modest efficiency in cell assays. Then, a chemical biology-based
approach was designed in order to improve the biological activity of peptoid 1 and to
analyze the mechanism of action as well the cellular implications of selective Apaf-1
inhibition. Furthermore, a series of cell models of apoptosis were studied to investigate
the potential as drug candidates of the Apaf-1 inhibitors.
19
Cl
Cl
HN N
ON
ON
O
Cl
Cl
O
N
O NH2
N
Cl
Cl
N N
ON
ONH2
O
Cl
ClO
N O
NH
N
Cl
Cl
HN N
ON
ONH2
O
Cl
Peptoid 1bPeptoid 1a
Peptoid 1
Cl
Figure 1.9. Chemical structure of peptoid 1 and its derivatives: peptoid 1a and peptoid 1b.Figure 1.9 Chemical structure of peptoid 1 and its derivatives”
20
21
CHAPTER 2. OBJECTIVES
In my thesis I want to discuss on the discovery and development of a first-in-class series
of selective inhibitors of the apoptosome through inhibition of Apaf-1. The work here
presented follows previous discoveries in our laboratory on a program on chemical
inhibition of apoptosis. Peptoid 1, an N-alkylglycine trimer, was the hit compound
from the screening of a chemical library employing an in vitro apoptosome
reconstitution assay. However, the low membrane permeability and modest efficiency
arresting apoptosis in cells led to the design of three new drug delivery strategies by
means of adequate carriers or based on structural modifications that could diminish the
conformational mobility of peptoid 1.
The objectives were defined as:
1. To synthesize peptoid 1 derivatives by: a) conjugation of peptoid 1 to cell-
penetrating peptides; b) conjugation of peptoid 1 or derivatives to a water
soluble polymeric carrier such as poly-L-glutamic acid (PGA), obtaining PGA-
peptoid conjugates; c) by means of peptoid 1 backbone cyclization.
2. To carry out a systematic study in different in vitro and cellular models in order
to determine cellular internalization capabilities, toxicity profile and the ability
to inhibit apoptosis of the new peptoid 1 derivatives.
3. Compounds showing no cellular toxicity and significant biological activity
inhibiting apoptosis would be move forward towards structure-activity
relationship studies that will direct the design of improved series.
4. Characterization of the binding site of peptoid 1 derivatives on Apaf-1.
5. Detailed characterization of the molecular mechanism of action of Apaf-1
inhibitors and the in cell consequences of selective Apaf-1 inhibition including
the analysis of non-apoptotic roles for Apaf-1.
22
6. To develop an in vivo rat model of kidney hot-ischemia and the subsequent
study of the therapeutic effect of the compounds synthesized in the amelioration
of kidney tissue damage caused by oxygen deprivation.
23
CHAPTER 3. SYNTHESIS OF PEPTOID 1 AND
PEPTOID 1 DERIVATIVES
To accomplish the full therapeutic potential of an identified bioactive agent is required a
specific molecular delivery. It is crucial to localize therapeutics to the diseased tissue or
cell and once there, promote their efficient delivery to the required intracellular
compartment ensuring their availability for the appropriate time. The chemical features
needed for a high affinity binding of the lead molecule to the molecular target many
times differs from those that allow an efficient cellular trafficking; this was the case of
peptoid 1 inhibitor. This N-alkylglycine trimer showed a high effectiveness inhibiting
in vitro Apaf-1 dependent apoptosome formation, but resulted toxic when tested in
cellular models. For this reason, structural modifications of this lead compound were
required to overcome its poor solubility and low membrane permeability and three new
series of peptoid 1 analogues were designed covering different approaches that could
facilitate cellular drug internalization: (a) hybrid cell-penetrating peptide-peptoid 1, (b)
conjugation of peptoid 1 to a polymeric carrier and (c) synthesis of a conformationally
constrained peptoid 1 mimetic. These three system presented different cellular
internalization pathways (Figures 3.1 and 3.2).
Cl
Cl
HN
N
ON
ONH2
O
Cl
Cl
N
N
O
O O
NH
Cl
Cl Cl
Cl
Cl
NN
ON
ONH2
O
Cl
Cl
O
NH
HN
O
O
n
+Na-O
HN
O
m
O
HN O
Cl
Cl
N
NO
NO
NH2O
Cl
Cl
RQIKIWFQNRRMKWKKGG
Peptoid 1
Hybrid cell-penetrating peptide-peptoid 1
Conformationally constrained peptoid 1Polymer-peptoid 1 conjugate
PEN-1 QM31QM56 Figure 3.1. Scheme of the three different peptoid 1 derivatives synthesized and structure of peptoid 1 derivatives leading compounds.
24
ReceptorReceptor
pH 6pH 6.5.5
Endosomes
Enzymatic or pH-dependent liberation of drug which diffuses out of lysosome to access the therapeutic targetpH 5pH 5
Lysosomes
pH 7.4pH 7.4
Passive transportationLow Mw molecule(QM31)
Internalisation by endocytosis(QM56)
Trafficking via the endosomalcompartments to lysosomes
Membrane traslocationor/and endocytosis
(CPP-peptoid)
N
NN
NH2
O
O O
O
Cl
ClCl
Cl
Cl
Cl
N
NO
NO
NH2O
Cl
Cl
RQIKIWFQNRRMKWKKGG
Cl
Cl
NN
ON
ONH2
O
Cl
Cl
O
NH
HN
O
O
80
+Na-O
HN
O
20
O
HN O
ReceptorReceptor
pH 6pH 6.5.5
Endosomes
Enzymatic or pH-dependent liberation of drug which diffuses out of lysosome to access the therapeutic targetpH 5pH 5
Lysosomes
pH 7.4pH 7.4
Passive transportationLow Mw molecule(QM31)
Internalisation by endocytosis(QM56)
Trafficking via the endosomalcompartments to lysosomes
Membrane traslocationor/and endocytosis
(CPP-peptoid)
N
NN
NH2
O
O O
O
Cl
ClCl
Cl
Cl
Cl
N
NO
NO
NH2O
Cl
Cl
RQIKIWFQNRRMKWKKGG
Cl
Cl
NN
ON
ONH2
O
Cl
Cl
O
NH
HN
O
O
80
+Na-O
HN
O
20
O
HN O
Figure 3.2. Cellular internalization pathways of the different peptoid 1 derivatives synthesized.
a) Cell penetrating peptides (CPPs)
Cell penetrating peptides (CPPs) were first described fifteen years ago after a new class
of proteins able to penetrate cell membrane were discovered [159-161]. Mutation
and/or deletion analysis of these proteins or some of their domains have revealed that, in
fact, small peptide sequences of no more than 30 amino acids within these proteins are
often responsible for cellular uptake [161]. The most outstanding characteristic of these
CPPs is their ability to internalize a wide variety of cargos always covalently attached to
their sequence (low molecular weight (Mw) drugs, nucleic acids, plasmids, whole
proteins or even nanoparticles). So, CPPs have become an excellent strategy for the
improvement of pharmacological properties of different drug candidates whose size or
chemical nature limits its proper cellular internalization [162-165].
These CPPs can be classified as either cationic or amphipathic in nature, being arginine
and lysine residues the most abundant in their sequences [162]. Some of the most used
and best characterized CPPs are polybasic peptides derived from the transduction
25
domains of certain proteins, such as Tat protein TAT-(47-57) (YGRKKRRQRRR)
encoded by HIV-1, and the third helix of the Drosophila Antennapedia protein,
penetratin (PEN) (43-58) (RQIKIWFQNRRMKWKK) [164, 166, 167]. Both sequences
have proven to be efficient methods for intracellular delivery, among others, of
peptides, proteins, antisense oligonucleotides and adenoviruses [165-169].
Nevertheless, differences between TAT and PEN membrane transport capabilities have
also been observed, mainly due to the influence of the cargo. Fusion to PEN appears to
be favored when using proteins with less than 100 residues, whereas TAT has been
reported to be able to transduce larger molecules, including β-glycosidase (>1000
amino acids) [170]. The carrier efficiency and the influence of small molecule cargo to
CPPs have not been deeply analyzed [167]. The mechanism employed by CPPs to cross
the plasma membrane in cells is an active area of investigation. For many years,
microscopy and flow-cytometry studies developed employing fixed cells, postulated, as
a possible way of internalization of CPPs, a receptor-independent translocation through
the plasma membrane [169, 171-173]. However, recent data shown by Richard et al.
[174] point out the implication of endocytosis in the internalization of these peptides.
According to Richard’s study, the inaccuracies of the techniques employed for
measuring CPPs internalization will explain the assumed mechanism based on
translocation. CPPs bind so heavily to the plasma membrane that even mild cell
fixation leads to the artifactual redistribution of CPP into the nucleus. Furthermore,
flow-cytometry (FC) analysis without a trypsin-digestion process will not provide the
real amount of peptides that are really entering the cell. The amount and the mechanism
of internalization will vary depending on both, the charge of the peptide and the
characteristics of the cargo molecule. For these reasons, protocols currently accepted
are based on fluorescence microscopy studies of live cells or FC analysis on trypsin-
treated cells. In addition, spectroscopy-based techniques as circular dichroism
spectroscopy (CD) are also valuable tools to study the influenza of cargo molecules in
the structure of CPPs.
With the aim to increase the membrane permeability properties of peptoid 1, two hybrid
peptide/peptoid conjugates where design. Peptoid 1 was fused to the well characterized
sequences of penetratin (penetratin-peptoid 1, PEN-1) and Tat protein (TAT-peptoid 1,
TAT-1) by means of a Gly-Gly linker.
26
b) Conjugation of peptoid 1 to a polymeric carrier: Polymer-peptoid 1 conjugate
A polymer can be defined as a large molecule (macromolecule) composed of repeating
structural units connected by covalent chemical bonds. Looking at applications on the
biomedical field, synthetic and natural polymers have a long established role as
biomedical materials including their use to fabricate prostheses and soft contact lenses
[175]. They are also widely used as the pharmaceutical excipients in drug formulations
[175] and more recently, the use of polymeric drug delivery systems in oncology has
grown exponentially with the advent of biodegradable polymeric implants. As
examples, we can mention here the subcutaneous depot to slowly release the human
luteinizing hormone-releasing hormone (LHRH) analogues for treatment of prostate and
other hormone-dependent cancers (e.g. Zoladex®; Leupron Depot®) [176], or post-
surgery implantations for local delivery of chemotherapeutics for the treatment of brain
cancers (e.g. Gliadel®) [177].
The so-called “Polymer Therapeutics” constitutes a versatile technological platform for
drug delivery with completely different properties if compared with polymeric implants
or depots. Polymer Therapeutics are considered the first nano-sized (5-100 nm)
polymer-based pharmaceuticals. In all cases, the water-soluble polymer can develop a
function as a bioactive agent or can act as an inert part or carrier of the bioactive agent.
The main objective is to improve drug, protein or gene delivery into de organism taking
profit of advanced polymer chemistry. Therefore, the definition involves rationally
designed macromolecular drugs and encompasses five types of systems: (i) polymeric
drugs (polymeric molecules that are biologically active in their own right); (ii) polymer-
drug conjugates; (iii) polymer-protein conjugates; (iv) polymeric micelles to which drug
is covalently bound, and (v) multi-component polyplexes being developed as non-viral
vectors for gene delivery. From the industrial standpoint, these nanosized medicines are
more like new chemical entities than conventional 'drug delivery systems or
formulations' which simply entrap, solubilize or control drug release without resorting
to chemical conjugation (Figure 3.3) [175].
27
Antibodies and its conjugatesLiposomes
80-100 nm Nanoparticles
Viral vectors
Polyplexes Polymeric Drugs
Polymer-drug conjugates
5-15 nm
Polymeric Micelles
Polymer-protein conjugates
20 nm
Polymer Therapeutics
Antibodies and its conjugatesLiposomes
80-100 nm Nanoparticles
Viral vectors
Polyplexes Polymeric Drugs
Polymer-drug conjugates
5-15 nm
Polymeric Micelles
Polymer-protein conjugates
20 nm
Polymer Therapeutics
Figure 3.3. Examples of nanopharmaceutics.
Polymer-drug and polymer-protein conjugates constitute two of the most developed
Polymer Therapeutics families [178]. In general, these nanoconjugates are mainly
constituted by three parts: (a) a hydrophilic polymer backbone that acts as a carrier; (b)
a linker or spacer to attach the polymer backbone to the protein or drug of interest; and
(c) the bioactive agent (drug, peptide or protein) to be delivered. Despite the
similarities in their structure, the design of polymer-drug and polymer-protein
conjugates is performed using different biological rationales. Polymer-proteins
conjugates have a reduced immunogenicity, an increased plasma half-life and stability
due to both, a decreased elimination by the reticulum endothelial system (RES) and a
decreased exposition to blood proteases [179]. By contrast, in the case of polymer-drug
conjugates the main objective pursued apart from a solubility enhancement, as most
active low Mw drugs are hydrophobic, it is a change in drug pharmacokinetics not only
at systemic but also at cellular level. In fact, it is now well established that polymer-
drug conjugation promotes tumor targeting by the enhanced permeability and retention
effect (EPR) [177, 180, 181] and, at the cellular level, by an endocytic capture that
allows lysosomotropic drug delivery [180]. The EPR effect was firstly defined by
Maeda and col. [182]. This is a passive targeting phenomenon that arises due to two
main factors, the hyper permeability or ‘leakiness’ of angiogenic vasculature in
28
inflammation or tumor sites and the lack of an effective lymphatic drainage. The
formert allows selective extravasation of the conjugate in the damaged tissue; the later
promotes conjugate retention. EPR-mediated tumor targeting is driven by the plasma
concentration of circulating polymer conjugate (Figure 3.4).
The seminal work of De Duve's on the realization that the endocytic pathway might be
useful for ‘lysosomotropic drug delivery’ [183] and of Ringsdorf's on the vision of an
idealized polymer chemistry for drug conjugation [184] gave birth to the concept of
targetable polymer-drug conjugates. Two key parameters have to be carefully
considered during the rational design of such polymer-drug conjugates: (i) the polymer
to be used as carrier and (ii) the bioresponsive polymer-drug linker(s). It has become
clear that the Mw and physico-chemical properties of the polymer is frequently the most
important driver governing biodistribution, elimination, and metabolism of the
conjugate as a whole, so choice of a suitable polymer is vital. In particular, the polymer
backbone employed can be biodegradable or biostable, but in the last case its Mw
should be lower than 40000 g·mol-1 in order to allow its excretion through the porous
glomerular kidney membrane [180]. In all cases, the water-soluble polymeric carrier
used must be non-toxic, non-immunogenic and suitable for repeated administration.
The biostable Poly(ethyleneglycol) (PEG), N-(2-hydroxypropylmethacrylamide)
(HPMA) copolymers, and the biodegradable poly-L-glutamic acid (PGA) have been the
most widely tested clinically (see reviews [180, 185] for further information). Adequate
drug carrying capacity in relation to the potency of the agent being carried is essential,
and ability to target by an active (receptor-ligand) or a passive (patho-physiological)
mechanism are also important design features. In the case of biodegradable polymers
the polymer-drug linker is always the most important factor. In case of the spacer, after
intravenous administration (i.v.) the ideal linker should be stable in blood stream but
able to release drug at an optimum rate upon arrival to the selected cellular target.
29
Administration of the conjugate
Renal elimination of conjugated drug
Minimum extravasation into normal tissue
Tumour tissue
Immunostimulation
Extravasation of polymer conjugates into the tumour tissue through the EPR due to the disorganized, leaky tumourvessels
A
Administration of the conjugate
Renal elimination of conjugated drug
Minimum extravasation into normal tissue
Tumour tissue
Immunostimulation
Extravasation of polymer conjugates into the tumour tissue through the EPR due to the disorganized, leaky tumourvessels
Administration of the conjugate
Renal elimination of conjugated drug
Administration of the conjugate
Renal elimination of conjugated drug
Minimum extravasation into normal tissue
Tumour tissue
Immunostimulation
Extravasation of polymer conjugates into the tumour tissue through the EPR due to the disorganized, leaky tumourvessels
A
MDR1; MRP
Drug release trigerred by pH or lysosomalenzymes
Bypasses P-gp and MRP
Pinocytosis Receptor mediated pynocitosis
Endosome
Lysosome
Exocytosisand receptor recycling
Exocytosis of non-degradable polymer
Recycling
B
MDR1; MRP
Drug release trigerred by pH or lysosomalenzymes
Bypasses P-gp and MRP
Pinocytosis Receptor mediated pynocitosis
Endosome
Lysosome
Exocytosisand receptor recycling
Exocytosis of non-degradable polymer
RecyclingMDR1; MRP
Drug release trigerred by pH or lysosomalenzymes
Bypasses P-gp and MRP
Pinocytosis Receptor mediated pynocitosis
Endosome
Lysosome
Exocytosisand receptor recycling
Exocytosis of non-degradable polymer
Recycling
B
Figure 3.4. (A) Tumor targeting EPR effect; (B) Lysosomotrophyc intracellular drug delivery (modified from [175]).
Based on endocytic pathway characteristics, mainly, two types of linkers are used to
achieve an efficient lysosomotropic drug delivery. The first category encompasses acid
labile or pH-dependent linkers that are hydrolyzed in the acidic lysosomal environment
(pH 4.5-5.5 vs. physiological pH 7.4) (e. g. acetal, hidrazone,…) [186, 187]. The
second category includes the peptidyl linkers susceptible to be degraded by lysosomal
enzymes, such as the serine-thiol proteases cathepsins that recognize certain specific
peptidic sequences [175, 178, 187].
30
In the 80s and early 90s a vast number of preclinical studies were carried out to
optimize the characteristics of the polymeric carriers, of the linker and also to prove the
safety of this novel constructs. These studies were primarily the result of collaborative
work between Ruth Duncan and Jindrich Kopecek and resulted in the clinical evaluation
of HPMA copolymer-doxorubicin (FCE28068, PK1) in 1994. This was the first
synthetic polymer-anticancer drug conjugate to be tested in humans; PK1 represented a
milestone in this field [178, 188]. Since then, there has been an exponential growth in
this area with more than fourteen polymer-drug conjugates already in the clinics (Table
3.1). A PGA-paclitaxel conjugate, Opaxio(R) (CT-2103, named previously as
XyotaxTM) from Cell Therapeutics Inc. [175, 189] is expected to reach the market in a
very near future.
Clinical proof of concept for polymer conjugates has been already achieved mainly as
efficient anticancer therapy, however, many challenges and opportunities still lay ahead
providing scope to develop this platform technology further. Delivery of new
anticancer agents focusing on novel molecular targets and their combination,
development of both new and exciting polymeric materials with defined architectures
and treatment of diseases other than cancer (e.g. rheumatoid arthritis (RA), diabetes or
ischemia) are the most exciting and promising areas [185, 187]. The later is one of the
most interesting advances in the field of polymer-drug conjugates and in particular the
design of macromolecular systems to promote tissue regeneration [190, 191].
Within this context, and in particular tissue regeneration and ischemic diseases our work
with Apaf-1 inhibitors could be enclosed. In fact, as we suggest the use of a PGA-based
conjugates for the delivery of a first-in-class family of apoptosis inhibitors. It was
hypothesized that the conjugation of peptoid 1 to a polymeric carrier could offer a more
specific intracellular trafficking that coupled to an efficient lysosomotropic drug release
on the cytosol would highly enhance the antiapoptotic activity of peptoid 1 as at the
same time would definitively solved peptoid 1 low-solubility problems. So, our PGA-
peptoid conjugates were obtained by attaching peptoid 1 to PGA polymer employing a
peptidic linker and obtaining by this way the first antiapoptotic nanomedicine [192].
31
Table 3.1. Polymer-drug conjugates in clinics.
Polymer-drug conjugates Name Status
Indication
Polyglutamate (PGA)-paclitaxel
CT-2103 XyotaxTM Paclitaxel
Polyglumex (PPX)
Phase III
Various cancer, particularly non small cell lung
cancer and ovarian cancer
HPMA copolymer-doxorubicin
PK1; FCE28068 Phase II
Various, particularly lung and breast cancer
HPMA copolymer-doxorubicin-
galactosamine
PK2; FCE28069 Phase I/II Hepatocellular
carcinoma
Hyaluronic acid -doxorubicin ONCOFID-D Phase I/II Superficial bladder
cancer Oxidised dextran-
doxorubicin AD-70,
DOX-OXD Phase I
(discontinued) Various cancer
HPMA copolymer- Paclitaxel PNU166945 Phase I
(discontinued) Various cancer
HPMA copolymer- malonato-platinate AP5280 Phase I/II Various cancer
HPMA copolymer-DACH-platinate
AP5346, ProLindacTM Phase II Various cancer
PEG-camptothecin Pegamotecan, ProthecanTM
Phase II (discontinued) Various cancer
HPMA copolymer-camptothecin MAG-CPT Phase I
(discontinued) Various cancer
Polyglutamate-camptothecin CT-2106 Phase I/II Various cancer
Carboxymethyldextran-exatecan DE-310 Phase I Various cancer
β-Cyclodextrin-camptothecin IT-101 Phase I Various cancer
PHF-camptothecin XMT-1001 Phase I Various cancer
PEG-naloxol (oral administration) NKTR-118 Phase I
Opioid-induced bowel dysfunction
(OBD)
c) Conformationally constrained peptoid 1 QM31
Most small peptides or peptidomimetics are highly flexible, conformationally labile
molecules in aqueous and other environments. Its low size allows them to cross cellular
membrane through a passive non-energy consuming process regulated by concentration
gradients. As a result, internalization of a small molecule will depend on their intrinsic
32
properties such as solubility, lipofilicity and its capacity to interact with the different
components of the cellular membrane [193, 194].
The introduction of the appropriate conformational constraints in a lineal molecule can
reduce the number of conformations in solution to only one due to the high activation
energy or free energy required to change that conformation. One effective way to
reduce the number of conformations has been the introduction of covalent bonds on the
side chain functional groups present on the amino acid residues in peptide or
peptidomimetic molecules [194, 195]. It is considered that, these changes on the
peptidomimetic architecture can be helpful in its internalization process as it will reduce
the number of possible interactions of the peptoid with different molecules present in
the plasma membrane diminishing, therefore, membrane disruption.
However, these are not the only advantages of this approach as the reduction in the
molecule flexibility can also help in the specificity of the interaction with its target.
Moreover, when there is no deep knowledge of the structure of the selected protein or
there is no 3D structure of its active site, the strategies followed in order to improve the
specificity of the molecules for their target are based on different substitutions and
deletion of several functional groups of the molecule. This approach could provide an
important amount of information, although it is difficult to determine with a flexible
peptidomimetic if the differences among biological activities can be due to changes in
the conformational properties of the analogs or to the intended changes in polarity or
bulkiness. Finally, an increase in compound stability on biological environments could
be also gained by constraining its chemical conformation; this will therefore prolong its
biological activity.
Peptoid 1 presents a highly flexible linear structure and as a consequence high number
of conformations can be achieved with a slight change in the environmental conditions.
This fact could explain the cellular toxicity observed when tested in cellular models
since its labile structure could interact with the different molecules that compose the
plasma membrane. For this reason, a cyclic analogue of peptoid 1 was designed in
order to obtain a more rigid structure able to reduce the possible unspecific interactions.
At the same time, a good strategy to improve peptoid 1 biological activity would be the
33
implementation of several modifications on the different functional groups that
compose peptoid 1 structure.
3.1 Synthesis of peptoid 1
The lead compound, peptoid 1, required to synthesize the different antiapoptotic
derivatives was produced in the department of Chemical and Biomolecular
Nanotechnology, IQAC-CSIC in Barcelona supervised by Dr. Ángel Messeguer. A
scheme of this synthesis is shown in Figure 3.5.
NH2
1. BrCH2COOH, DIPCDI, DMF
NH2
HN
ONH
2. TEA, DMF,
1. BrCH2COOH, DIPCDI, DMF
2. TEA, DMF,
NH2
Cl
1. TFA:DCM:H2O (60:40:2)
Cl
ClCl
Cl
1. BrCH2COOH, DIPCDI, DMF
2. TEA, DMF,
NH2
HN
ON
Cl
Cl
NHO
Cl
HN
ON
Cl
Cl
NO
NHO
Cl Cl
H2N
ON
Cl
Cl
NO
NHO
Cl Cl
Figure 3.5. Scheme of peptoid 1 chemical synthesis.
3.2 Cell-penetrating peptides and cell-penetrating peptide/peptoid hybrids
synthesis: PEN-1 and TAT-1
a) Synthesis of PEN, TAT, PEN-1 and TAT-1
As stated before, the synthesis of hybrids peptide-peptoid was performed by fusing
peptoid 1 to the peptide carriers penetratin (PEN) and TAT peptides through a two
glycine linker [196]. The hybrids penetratin-GG-peptoid (PEN-1) and TAT-GG-
peptoid (TAT-1) molecules (Figure 3.6) identity and purity were confirmed by high-
performance liquid chromatography (HPLC) and Matrix Assisted Laser
34
Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Stock
solutions of control peptides and peptide/peptoid hybrids were prepared in PBS buffer
and the concentrations were determined by UV spectroscopy and quantitative amino
acid analysis.
Cl
Cl
N
NO
NO
NH2O
Cl
Cl
RQIKIWFQNRRMKWKKGG
A
Cl
Cl
N
NO
NO
NH2O
Cl
Cl
YGRKKRRQRRRGGG
B
Figure 3.6. Chemical structure of PEN-1 (A) and TAT-1 (B).
b) Synthesis of PEN-1-CF and TAT-1-CF
For the synthesis of carboxyfluoresceine (CF) labeled PEN-1 (PEN-1-CF) and TAT-1
(TAT-1-CF) conjugates, CF was attached to peptoid 1 structure and then it was coupled
to PEN and TAT peptides through the two Gly linker. The identity and purity were
confirmed by HPLC and MALDI-TOF mass spectrometry. Stock solutions of control
peptides and peptide/peptoid hybrids were prepared in phosphate buffered saline (PBS)
buffer and the concentrations were determined by fluorescence spectroscopy and
quantitative amino acid analysis.
3.3 Synthesis and characterization of nanoconjugates
3.3.1 Synthesis and characterization of QM56 (peptoid 1-GG-PGA conjugate)
A summary of the synthesis protocol is portrayed in the Figures 3.7 and 3.8. Prior to
peptoid 1 conjugation, our lead compound was derivatized as described in Matherials
and Methods section by introduction of a diglycil spacer to yield peptoid 1-GG. The
identity and purity of peptoid 1-GG (95 %) was confirmed by HPLC and MALDI-TOF.
35
Figure 3.7. Synthesis of peptoid 1-GG. R1=R2=H.
Poly(L-glutamic acid)-peptoid 1 conjugate (PGA-peptoid, QM56) was then synthesized
using a N,N-diisopropylcarbodiimide (DIC)-mediated coupling through a diglycil
spacer (using as reagent GG-peptoid 1 (Figure 3.8)) to contain peptoid 1 up to a 12
mol% loading (free drug content <1 %wt of total drug) as determined by UV-Vis
spectrophotometry and HPLC analysis (Figure 3.9). QM56 was then characterized.
Cl
Cl
N N
ON
ONH2
O
Cl
ClO
NH
HN
O
O
n
+Na-O
HN
O
m
O
HN O
HN
O
O
n
+Na-O
Cl
Cl
N N
ON
ONH2
O
Cl
ClO
NH2
HN O
HCl 2N
pH 2
HN
O
O
n
HO
(i) DIC/HOBt, DIEA/DMFanhrt, 36h
(ii) NaHCO3, pH 8
GG-peptoid 1
PGA
QM56 Figure 3.8. Scheme of conjugation of the peptoid 1-GG to the polymeric carrier.
36
a) Determination of total drug content by UV spectroscopy
A stock solution of peptoid 1-GG in MeOH was prepared (1 mg/mL). To obtain a
calibration curve, samples were diluted using MeOH to give a concentration range of
0–1 mg/mL. The total drug loading of the conjugates was determined by measuring the
optical density (OD) at 272 nm and 281 nm in H2O and comparing the absorbance
values to the calibration slopes obtained for the measurements at 272 and 281 nm. PGA
in the same concentration range as the analyzed conjugate (0–5 mg/mL) in H2O was
used as blank. The extinction coefficient found for peptoid 1-GG at 281 nm in MeOH
at RT was ε = 713 L mol-1 cm (Figure 3.9). The total drug content in QM56 was 28 ± 2
wt % (10.2 ± 0.6 mol % drug loading). Results are expressed as mean ± SD (n > 3).
220 270 3200
0,2
0,4
0,6
0,8
1
1,2
1,4
0 0,2 0,4 0,6 0,8 1 1,2
[Peptoid-GG-NH2] (mg/mL)
A U
BA
220 270 320220 270 320220 270 3200
0,2
0,4
0,6
0,8
1
1,2
1,4
0 0,2 0,4 0,6 0,8 1 1,2
[Peptoid-GG-NH2] (mg/mL)
A U
BA
Wavelength (nm)
Figure 3.9. Total drug content quantification by UV spectroscopy. (A) Calibration curve for peptoid 1-GG at 270 nm (■) and 282 nm (○). (B) UV spectra of PGA, PGA-GG-peptoid 1 and –NH2-GG-peptoid 1. Data are expressed as the mean ± SD (n > 3).
b) Determination of free drug content by HPLC
Even after careful purification of the nanoconjugates by dialysis and/or size exclusion
chromatography (SEC) and due to their intrinsic hydrophilic/hydrophobic behaviour,
free drug molecules could be still entrapped within the macromolecule. To ensure an
adequate biological evaluation the quantification of this free drug is very important, and
it should be controlled to be always lower than 2 wt% of the total drug content
determined for the conjugate synthesized. One aliquot of a QM56 solution was
prepared in order to analyze its free dug content through HPLC at 220 nm (see
Materials and Method section for further information). The results obtained were
37
compared to a calibration curve obtained for peptoid 1-GG at concentrations ranging
from 0.1-0.6 mg/mL (Figure 3.10). The retention time (tr) obtained for peptoid 1-GG (λ
= 220 nm) was 14.3 min (Figure 3.7) and the percentage of free drug content was 0.2 ±
0.1 wt % of total drug. Results are expressed as mean ± SD (n > 3).
020406080
100120140160180
0 0,1 0,2 0,3 0,4 0,5 0,6
Peptoid 1-GG (mg/mL)
Are
a at
220
nm
/106
Figure 3.10. Calibration curve for peptoid 1-GG content by HPLC. Data are expressed as the mean ± SD (n > 3).
c) Determination of average molecular weight (Mw), polydispersity (Mw/Mn) and
conformational behavior in solution for QM56
The average molecular weight (Mw), polydispersity (Mw/Mn) and the conformational
behavior of both QM56 and control PGA in solution were analyzed by SEC and AUC
(experimental details fully described in Matherials and Methods section). Both
techniques showed QM56 as a more compact conformational structure with a smaller
hydrodynamic volume and a greater sedimentation coefficient (S) than PGA (tr = 12.8
min and S= 3.5 ± 0.4 s vs. tr = 11.6 min and S= 1.9 ± 0.1 s for QM56 and PGA,
respectively). By sedimentation equilibrium, the apparent Mw was determined as
26700 Da for PGA sodium salt and 60300 Da for QM56. However, Mw of 35000 Da
(Mw/Mn = 1.8) and 29000 Da (Mw/Mn= 1.6) were determined for PGA sodium salt
and QM56, respectively, by SEC using PEG standards. This correlates with previous
studies that showed how polymer-drug conjugates exist in solution as unimolecular
micelles, and also how conformation is influenced by both drug loading and drug nature
[197].
38
d) Nuclear Magnetic Resonance analysis of QM56
Nuclear magnetic resonance spectroscopy (NMR) confirmed the integrity of peptoid 1
and its covalent attachment to PGA. 1D 1H NMR spectrum of QM56 showed extra
resonances arising from the peptoid moiety plus a general broadening of the signals
when compared to that spectrum from PGA (Figure 3.11). The most affected signals
are those corresponding to the NH-amide protons (at 8.5 ppm), changing from sharp J-
coupled resonances in the PGA spectrum (unstructured polymer) to broad signals in the
PGA-peptoid spectrum, as a consequence of both the NH averaging of functionalized
and non-functionalized side chains, and conformational changes. In addition, 2D
Nuclear Overshauser effect spectrum (NOESY) of QM56 showed cross peaks
correlating the aromatic groups from the peptoid moiety (6.7-7.4 ppm) with the
glutamic acid side chain (1.8-2.2 ppm) and Hα (4.0-4.3 ppm) from the polymer.
Furthermore, whereas the 2D total correlation spectrum (TOCSY) of PGA (Figure 3.12)
showed two glutamic acid Hα signals (corresponding to polymer main chain (1) and
termini (2)) that correlate with the glutamic side chain protons, the QM56 TOCSY
spectrum showed an additional Hα connected to a new glutamic side chain arising from
functionalized termini (2''). Therefore, drug-polymer connectivity was confirmed
(Figure 3.12).
e) Degradation study of PGA and QM56 upon incubation with Cathepsin B. Evaluation
of Mw loss and peptoid 1 release profile from QM56
As already stated above, effective biological activity of polymer-drug conjugates relies
on (i) stability in blood circulation and, (ii) lysosomal enzyme cleavage to release active
drug in place after cellular uptake by endocytosis. Conjugate degradation kinetics were
determined by mimicking lysosomal conditions. QM56 was dissolved in a pH 6
solution containing cathepsin B, one of the member of the lysosomal proteases
cathepsins (see experimental details in Matherials and Methods section). In case of
cathepsin B the specificity has not been fully described, but Gly, Arg and hydrophobic
amino acids are present in the position P2 of the cleavage site of cathepsin B. In our
study, a solution of QM56 was incubated at 37 ºC and samples were taken at different
time-points and storage at -80ºC until analysis by HPLC (drug release) and/or gel
39
permeation chromatography (GPC) (polymer Mw loss).
Peptoid 1-GG
PGA
QM56
Peptoid 1-GG
PGA
QM56
Figure 3.11. 1D 1H NMR spectrum of NH2-GG-peptoid 1, PGA and QM56.
Figure 3.12. Expansion of 2D TOCSY NMR spectra of PGA (a) vs. QM56 (b).
HPLC evaluation of released peptoid 1. Drug release kinetics studies were performed
per triplicate and the % of drug release quantified with time. Peptoid 1 release began
after addition of enzyme reaching a plateau after 24 h. MALTI TOF TOF analysis of
the released compounds showed that Gly-peptoid was the most abundant product
(MALDI - m/z 786 (M) (Figure 3.13 A).
40
GPC Evaluation of Polymer Mw loss. In case of GPC analysis the Mw was determined
by using PEG standards. Generally degradation profiles for biomedical polymers are
obtained under constant sink conditions and the results correlated with mass loss of the
polymer. Since PGA polymers are water soluble, the relative peak areas used in the
GPC derived Mw calculations, provided a suitable alternative to indicate the relative
amount of the polymer that remained at each time-point. As the polymer degraded there
was a notable loss of conjugate Mw. Therefore, the loss with time of the area under the
curve (AUC) for nanoconjugate and PGA control was considered to evaluate the rate of
backbone degradation (Figure 3.13 B). The lowered rate of QM56 degradation when
compared to PGA (Figure 3.13 C) is in agreement with the more compact conformation
observed by AUC that could limit enzyme accessibility. For QM56, the 50 % Mw loss
was achieved after 26 h of incubation in presence of cathepsin B while the 50 % peptoid
1 release occurs 22 h after enzyme addition.
A B
0102030405060708090
100
0 10 20 30 40 50
Time (h)
Perc
ent o
f tot
al d
rug
rele
ase
(%)
0
20
40
60
80
100
0 10 20 30 40 50 6 70 80time (h)
Mw loss (
% PGA control% QM56
Mw
loss
%
A B
0102030405060708090
100
0 10 20 30 40 50
Time (h)
Perc
ent o
f tot
al d
rug
rele
ase
(%)
0
20
40
60
80
100
0 10 20 30 40 50 6 70 80time (h)
Mw loss (
% PGA control% QM56
Mw
loss
%
0102030405060708090
100
0 10 20 30 40 50
Time (h)
Perc
ent o
f tot
al d
rug
rele
ase
(%)
0
20
40
60
80
100
0 10 20 30 40 50 6 70 80time (h)
Mw loss (
% PGA control% QM56
Mw
loss
%
Figure 3.13. Kinetic study of QM56 degradation in the presence of cathepsin B. (A) HPLC evaluation of released peptoid 1. (B) GPC evaluation of the percentage of Mw loss for QM56 in order to clearly determine the required time for a 50% Mw loss (C) Comparison of %Mw loss of QM56 and PGA, measured by GPC. Data are expressed as the mean ± SD (n > 3).
f) Stability in plasma
QM56 degradation profile was determined by the incubation of the polymer in a
solution containing PBS plus 20 % foetal bovine serum (FBS) at pH 7.4 for 1 h. QM56
showed 100 % stability in these conditions and non-significant drug release from the
polymer or polymer Mw loss was detected. The study was extended to blood, plasma or
serum freshly extracted from Wistar rats. QM56 was stable up to 24 hours.
41
Summarizing, QM56 was completely stable in plasma and in buffers but was degraded in
the presence of lysosomal enzymes (cathepsin B) releasing the drug in a time-dependent
manner
3.3.2 Synthesis and characterization of QM56-OG (peptoid 1-GG-PGA-oregon green)
The fluorescent QM56 analogue (QM56-OG) was synthesized following the same
protocol described for the conjugation of PGA to the peptoid 1-GG (Figure 3.14). The
total drug content was determined by UV (peptoid 1 content, see above section 3.3.1)
and fluorescence spectroscopy (OG content, λexc = 490 nm and λem = 520 nm) (Figure
3.15).
+ DIC/HOBt
DIEA/DMFanh
Cl
Cl
NN
ON
ONH2
O
Cl
Cl
O
NH
HN
O
1
HN
O
20
O
HN O
NH
O
O
79
+Na-O O
NH
O
HN
O
HOF
OF
OHO
Cl
Cl
NN
ON
ONH2
O
Cl
Cl
O
NH
HN
O
O
80
+Na-O
HN
O
20
O
HN ONHO
H2N
OHO
F
O
F
OH
O
Figure 3.14. Scheme of QM56-OG synthesis.
0
100
200
300
400
500
600
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Fractions
U A
Figure 3.15. Determination of OG content on the nanoconjugate by fluorimetry after conjugate purification through a PD10 column. In this case, pure QM56-OG appeared from fraction 4 to 7 giving a 70% functionalization yield in this case, therefore, 0.7 mol % OG loading in the conjugate.
42
3.4 Synthesis of conformationally constrained peptoid 1 mimetics
3.4.1 Synthesis of QM31
The synthesis of this heterocyclic derivative was carried out following a solid-phase
general procedure with microwave activation developed at Messeguer’s laboratory
(unpublished results). A scheme of the synthesis is depicted in Figure 3.16.
NH2
1. BrCH2COOH, DIPCDI, DMF
NH2
HN
ONH
OO
HOO
2. TEA, DMF,
HN
ON
O
HN
HN
ON
OO O
1. TEA, DMF
1. BrCH2COOH, DIPCDI, DMF
2. TEA, DMF,
O O
1. PhSiH3, Pd(PPh3)4 DCM 1. PyBOP, HOBt, DIEA,
DMF
2. TFA:DCM:H2O (60:40:2) N
N
O
O
Cl
N
OH2N
O
Cl
Cl
Cl
Cl
Cl
Cl
NH2
Cl
Cl
NH2Cl
Cl
HN
ON
O
N
O O
NHO
Cl
Cl
Cl
Cl
HN
ON
O
N
O OH
NHO
Cl
Cl
Cl
Cl
ClCl
ClQM31
Figure 3.16. QM31 synthesis scheme.
3.4.2 Synthesis of QM31 derivatives
Furthermore, a medicinal chemistry-based structure-activity relationship was proposed
in order to optimize QM31. For this purpose, several structural modifications of QM31
were carried out. Initially attention was focused on the 2-(2’,4’- dichlorophenyl)ethyl
43
substituent attached to the perhydro-1,4-diazepine-2,5-dione ring ( GROUP I, Scheme
3.17).
R1 NOO
O
NN
O
Cl
Cl
NH2
GROUP I
Figure 3.17. Chemical structure of QM31 and its derivatives.
This substituent turned out to be essential for activity and in our hands, any attempt of
modification resulted in a loss of antiapoptotic activity. Similar results were found in
early attempts of modification of the 3,3-diphenylpropyl moiety [158]. Then attention
was addressed to the R1 moiety. As shown in Table 3.2, a series of eleven derivatives
QM31a-QM31l were initially designed. These derivatives where synthesized
employing the same strategy than for the QM31, but introducing different substituents
at R1.
3.4.3 Synthesis of QM57
Compound QM57 was synthesized employing a chemical strategy similar to than
reported for QM31. Due to patent pending issues, no chemical structure or synthesis
strategy of QM57 or its derivatives will be displayed in this section.
3.4.4 Synthesis of QM57 analogues
The compounds were synthesized following the procedure for QM57 but by coupling
the corresponding N-alkylglycine fragment to the common intermediate. A total of
twelve QM57 derivatives were synthesized (QM57a, QM57b, QM57c, QM57d,
QM57e, QM57f, QM57g, QM57h, QM57i, QM57j, QM57k, QM57l and QM57m).
44
Table 3.2. Chemical structure for the substituents at R1 position in QM31.
QM31 analogues R1
QM31 Cl
Cl
QM31a F
QM31b
QM31c
QM31d O
QM31e F
QM31f Cl
QM31g and QM31h
(mixture) and O
QM31 i NH
O
QM31j O
QM31k Cl
QM31l
45
3.5 Synthesis of QM56 analogues and polymeric derivatives of QM31 and QM57
A series of QM56 analogues and QM31 and QM57 polymeric derivatives were
synthesized. The objective was to modulate drug release kinetics by different polymer-
drug linkers, substrates for cathepsin B in order to have access to different drug release
kinetics (see Table 3.3 for further detail).
Table 3.3. Schematic structure of QM56 and heterocyclic polymeric derivatives.
PGA derivatives Schematic structure
QM56 Peptoid 1-GG-PGA
QM56-P1 Peptoid 1-R1R2-PGA
QM56-P2 Peptoid 1-R1R3-PGA
QM31-P1 QM31-R4-PGA
QM31-P2 QM31-R5R3R6R4-PGA
QM31-P3 QM31-R1R3R6R4-PGA
QM31-P4 QM31-R5R5R4-PGA
QM57-P1 QM57-R5R3R6R4-PGA
QM57-P2 QM57-R5R5R4-PGA
Full physico-chemical characterization of the compounds was also carried out with the
new derivatives using the techniques and the strategies described for QM56. The total
drug loading and the free drug content values obtained for the different polymer
conjugates are depicted in Table 3.4.
46
Table 3.4. Loading parameters for PGA-conjugates.
PGA derivatives Total drug loading (mol %)a
Total drug loading (wt %)a
Free drug loading (wt %)b
QM56 11.8 40.0 ≤ 1.01
QM56-P1 12.0 43.2 ≤ 0.47
QM56-P2 11.4 42.2 ≤ 0.58
QM31-P1 10.2 36.0 ≤ 1.69
QM31-P2 7.6 31.9 ≤ 1.57
QM31-P3 7.5 31.9 ≤ 1.56
QM31-P4 10.2 38.3 ≤ 0.35
QM57-P1 11.6 49.1 ≤ 0.26
QM57-P2 8.5 32.1 ≤ 1.40 a determined by UV spectroscopy after conjugate purification with PD10 column (SEC, sephadex G25) and/or determined by HPLC by indirect quantification of the peptoid, which was not conjugated at the end of reaction; b determined by HPLC after extraction with chloroform. a) Determination of average molecular weight, polydispersity and behavior in solution
of QM56 analogues, QM31 and QM57 polymer derivatives
No data will be shown due to patent pending issues.
b) Nuclear Magnetic Resonance analysis
No data will be shown due to patent pending issues. c) Evaluation of Mw loss and peptoid 1, QM31 an QM57 release profile from the
different PGA nanoconjugates in the presence of cathepsin B
In order to test the different linkers introduced into the polymer peptoid 1 derivative
conjugates, similar polymer degradation kinetics studies to the ones developed with
QM56 were performed. Results are shown in Table 3.5 and Figure 3.18.
47
Table 3.5. Degradation studies for PGA-based derivatives.
Compound 50 % Mw loss (h)a,b 50 % drug release (h)b,d
QM56-P1 32 ± 2 24 ± 1 QM56-P2 5 ± 1 0.75 ± 0.3 QM31-P1 34 ± 3 > 48 ± 4 QM31-P2 12 ± 2 1 ± 1 QM31-P3 0.5 ± 0.2 0.5 ± 0.3 QM31-P4 15 ± 2 7 ± 2 QM57-P1 20 ± 2 2 ± 1 QM57-P2 3 ± 1 0.5 ± 0.3
a determined by GPC. b determined by HPLC after extraction with chloroform.
As demonstrated in Table 3.5, the use of different peptide linkers between the polymeric
support and the bioactive drug has an influence on drug release kinetics. f) Stability in plasma
PGA-based derivatives stability in plasma was firstly determined by the incubation of
the polymer in a solution containing PBS plus 20 % foetal bovine serum (FBS) at pH
7.4 for 1 and 24 h. All the polymer conjugates showed 100 % stability in these
conditions and non-significant drug release from the polymer or polymer Mw loss was
observed. Further stability studies where carried out in blood and fresh serum extracted
from Wistar rats and similar results were obtained. Data from QM57-P2 is shown as an
example (Figure 3.19).
48
QM56
QM56-P2QM56-P1
Time (hours)
Dru
g re
leas
e (%
)
A
QM56
QM56-P2QM56-P1QM56
QM56-P2QM56-P1
Time (hours)
Dru
g re
leas
e (%
)
A
QM56
QM56-P2QM56-P1
Time (hours)
Mw
loss
(%)
BQM56
QM56-P2QM56-P1
Time (hours)
Mw
loss
(%)
QM56
QM56-P2QM56-P1QM56
QM56-P2QM56-P1
Time (hours)
Mw
loss
(%)
B
Figure 3.18. Influence of polymer-drug linker on (A) drug release kinetics, measured by HPLC and (B) conjugate molecular weight (Mw) loss, measured by GPC in presence of lysosomal thiol protease cathepsin B (see Materials and Methods for further information). Data are expressed as the mean ± SD (n > 3).
49
Figure 3.19. GPC-HPLC-based analysis of released QM57 from QM57-P2 upon incubation in the presence of serum (♦) and blood (■). In all cases, SE < 5 %.
50
51
CHAPTER 4. SYSTEMATIC ANALYSIS OF INHIBITION
OF APOPTOSIS BY PEPTOID 1 DERIVATIVES
As it was mentioned in chapters 1 and 3, peptoid 1 cell toxicity, probably associated to
a putative lytic effect on the plasma membrane due to the amphipathic nature of the
compound [198, 199] precluded cell biology assays to explore the applicability of
peptoid 1 as inhibitor of cellular apoptosis. Several synthetic strategies were designed
with the aim of obtaining peptoid 1 derivatives with improved cellular internalization
properties. Such strategies were based in:
(i) synthesis of hybrid peptide/peptoid conjugates where the parent peptoid 1 was fused
to well characterized cell penetrating peptides (CPPs) such as PEN and TAT peptides,
yielding compounds PEN-1 and TAT-1 [196], respectively;
(ii) conjugation of peptoid 1 to a water soluble polymeric carrier such as poly-L-
glutamic acid (PGA), obtaining polymer-peptoid conjugates [192, 200];
(iii) synthesis of backbone conformational-restricted derivatives aiming to introduce
chemical modifications in the molecule that could diminish the conformational mobility
of peptoid 1 favoring membrane permeation. The parent compound of this series was
the peptoid 1-derivative QM31 [200].
A further description of the internalization strategies followed and the synthetic
methodologies can be found in Chapter 3 and Materials and Methods section.
Once the compounds were synthesized the first objective was to determine the
efficiency of the new drug delivery systems to be internalized and, at the same time, the
maintenance of the biological properties exhibited by the parent compound, peptoid 1.
This study was performed at three different levels: a) in vitro analysis of peptoid 1
derivatives as inhibitors of the apoptosome formation, b) evaluation as inhibitors of
apoptosis in different cellular models, c) cellular internalization studies of peptoid 1
derivatives in living cells.
52
a) In vitro reconstitution of the apoptosome and cell-free system assays
The use of cell-free systems has been crucial in the identification of important
components of the cell death machinery (reviewed in Cullen et al. [201]). These
systems reduce the complexity of the particular steps of the biological pathway under
study increasing the reproducibility of these experiments. Besides, they can be easily
modified according to the particular aims of the experiments by adding or removing the
different constituents of the assay. Cell-free systems have played an important role in
the discovery of the cross-talk pathways between mitochondria and apoptosis. Among
others, the identification of Apaf-1 and the different components of the apoptosome
were performed by using HeLa cell-free extracts [39, 40]. Similar strategies were used
by Fearnhead et al. [202] to develop a series of cell-free systems in which Apaf-1 was
separated from other components of the cell-extract through chromatography
fractionation. Then, appropriate fractions can be used in conjunction to interrogate the
selected pathway. With this methodology the fraction containing Apaf-1 can be
analyzed when combined with the fraction containing the caspases and the addition of
the exogenous dATP to induce the formation of an active apoptosome [87, 147, 202].
For the objectives of our research, two different in vitro systems were employed. We
use a recombinant protein-based reconstitution of the apoptosome [203] and a HEK
293-based cell extract depleted from endogenous Apaf-1 [147, 158]. In both cases, we
correlated apoptosome formation with apoptosome activity measuring caspase 9 and/or
3 activation employing the corresponding fluorogenic substrate (Figure 4.2) (see
Materials and Methods section for further detail).
53
ArApaf-1 + Peptoid 1 derivative
15 min, RT
ATP/Mg + Cyt c
5 min, RT
Procaspase 9
5 min, RT
Ac-LEDH-afcΔFU/min (λexc = 400 nm, λem = 505 nm)
B
rApaf-1 + Peptoid 1 derivative
15 min, 30 ºC
FT HEK 293 + ATP/Mg
30 min, 30 ºC
Ac-DEVD-afcΔFU/min (λexc = 400 nm, λem = 505 nm)
ArApaf-1 + Peptoid 1 derivative
15 min, RT
ATP/Mg + Cyt c
5 min, RT
Procaspase 9
5 min, RT
Ac-LEDH-afcΔFU/min (λexc = 400 nm, λem = 505 nm)
rApaf-1 + Peptoid 1 derivative
15 min, RT
ATP/Mg + Cyt c
5 min, RT
Procaspase 9
5 min, RT
Ac-LEDH-afcΔFU/min (λexc = 400 nm, λem = 505 nm)
15 min, RT
ATP/Mg + Cyt c
5 min, RT
Procaspase 9
5 min, RT
Ac-LEDH-afcΔFU/min (λexc = 400 nm, λem = 505 nm)
B
rApaf-1 + Peptoid 1 derivative
15 min, 30 ºC
FT HEK 293 + ATP/Mg
30 min, 30 ºC
Ac-DEVD-afcΔFU/min (λexc = 400 nm, λem = 505 nm)
rApaf-1 + Peptoid 1 derivative
15 min, 30 ºC
FT HEK 293 + ATP/Mg
30 min, 30 ºC
Ac-DEVD-afc
rApaf-1 + Peptoid 1 derivative
15 min, 30 ºC
FT HEK 293 + ATP/Mg
30 min, 30 ºC
Ac-DEVD-afc
15 min, 30 ºC
FT HEK 293 + ATP/Mg
30 min, 30 ºC
Ac-DEVD-afcΔFU/min (λexc = 400 nm, λem = 505 nm)
Figure 4.1. Scheme of in vitro assays developed (A) In vitro apoptosome reconstitution assay; (B) Cell-free extract apoptosome reconstitution assay. In both cases, rApaf-1 is incubated with peptoid 1 derivatives and after the addition of ATP and the necessary components to reconstitute the apoptosome, caspase 9 and 3 activities are measured employing the corresponding fluorogenic caspase substrates (afc, 7-amino-4-trifluoromethyl coumarine). The kinetics of the afc release will allow us to determine the degree of apoptosome formation achieved in all the samples.
b) Evaluation of peptoid 1 derivatives as inhibitors of apoptosis in different
cellular models.
The biological activity of peptoid 1 derivatives was also determined by employing
different cell lines. This study was divided in two steps: the evaluation of the intrinsic
cell toxicity and the determination of their ability to inhibit apoptosis. The cell lines
employed for this purpose were A549 human lung carcinoma, HeLa human cervix
adenocarcinoma, U937 human histiocytic lymphoma and U2-OS and Saos-2 two human
osteosarcoma cell lines.
Cell seeding settings were established in order to keep cells growing in the exponential
phase during the development of the experiments and the optimal apoptosis-inducing
agent concentration was determined. In general, percentages of cell death from 40-80%
were pursued for the different time-point measurements. Doxorubicin (dox) was the
drug chosen for the induction of cell death in U937, HeLa, A549 and U2-OS. In case of
54
Saos-2 transfected with the doxycycline (doxy)-dependent TET-ON system, (doxy) was
employed as inducer of cell death as it will be explain below.
Dox is a chemotherapeutic drug and it has been broadly used in the study of apoptosis
[204-206]. Although its mechanism of action is still a matter of concern, it seems to be
able to intercalate into the DNA, inhibiting topoisomerase II and as a consequence DNA
transcription and replication processes [207]. Other effects of dox are the induction of
reactive oxygen species (ROS) and the modification of Ca+2 homeostasis. Then, both
extrinsic and intrinsic apoptotic pathways are activated upon dox treatment [208]
(Figure 4.2).
COCH2OH
OH
O
O
NH2
OH
CH3
OHO
O OHOCH3
Figure 4.2. Chemical structure of dox.
A more close view on the influence of Apaf-1 inhibition on the intrinsic pathway of
apoptosis was obtained using the Bax Tet-On Saos-2 cell line. In this cell-based model,
apoptosis is induced by the conditional expression of the proapoptotic protein Bax
through the doxy-dependent Tet-ON system. Bax is a pro-apoptotic member of the Bcl-
2 family that induces apoptosis by directly targeting mitochondria; thus, the activation
of apoptosis solely relies on the mitochondrial or intrinsic pathway (Figure 4.3).
NH2
O
N
OOH
HOH
OH
HCH3
OOH
Figure 4.3. Chemical structure of doxy.
Cell viability and cell-viability recovery experiments were performed employing an
MTT assay (Figure 4.4) based on the ability of viable cells to reduce 3-(4,5-
dimethylthiazolyl-2)-2, 5-diphenyltetrazoliumbromide (MTT). The quantification of
55
the reduced compound allows determining the amount of viable cells in the well (see
Materials and Methods for further detail).
Living cellsLiving cells
NADH/NADPH NAD+/NADP+
CNNH
N NS
N
CH3
CH3
N
NN
NS
N
CH3
CH3Br-
+
Dead cellsDead cells
Absorbance
λ = 562 nmLiving cellsLiving cells
NADH/NADPH NAD+/NADP+
CNNH
N NS
N
CH3
CH3
N
NN
NS
N
CH3
CH3Br-
+
Dead cellsDead cells
Absorbance
λ = 562 nm
Figure 4.4. Schematic development of MTT assay. 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazoliumbromide is a yellowish water soluble compound and is converted to water-insoluble MTT formazan by mitochondrial dehydrogenase of viable cells. The addition of dimethylsulfoxide (DMSO) solubilizes the dark blue MTT formazan crystals and the intensity is measured colorimetrically at λ = 562 nm.
MTT presents the disadvantage of only determining the percentage of viable cells, but
this assay does not provide any detail about the type of cell death undergoing in the
sample. The cell extract-based caspase 3 fluorometric protease assay is a more specific
way to determine programmed cell death or apoptosis and, therefore, the Apaf-1
activation and the apoptosome formation, as caspase 9 is one of the initiator caspases
that activates procaspase 3 inactive zymogen (Figure 4.5) (see Materials and Methods
section for further information).
56
Cell extracts
Ac-DEVD-afc + PBS + 10% glicerol + DTT
Kinetics 37ºC
λexc = 400 nm
λem = 505 nm
Cell extracts
Ac-DEVD-afc + PBS + 10% glicerol + DTT
Kinetics 37ºC
λexc = 400 nm
λem = 505 nm
Figure 4.5. Scheme of caspase 3 activity fluorogenic assay. Procaspase 3 is synthesized as an inactive proenzyme that is processed in cells undergoing apoptosis by cleavage by other upstream proteases (e.g. Caspases 8, 9 and 10) into its two subunits p17 and p19 (see chapter 1 section 1.2 for further information). Once activated, caspase 3 cleaves a variety of cellular proteins that contain the amino acid motif Asp-Glu-Val-Asp (DEVD). To develop the fluorogenic assay the cell extract is supplemented with the fluorogenic caspase 3 substrate peptide Ac-DEVD-afc that upon caspase 3 cleavage will produce the free fluorophore ‘afc’ (see Materials and Methods section for further information). Then the fluorescence signal is proportional to the level of active caspase 3 in the cell lysated.
c) Cellular internalization of the compounds in living cells
One efficient and relatively simple technique to determine the cellular uptake of a
molecule is its covalent conjugation to a fluorophore and the subsequent study of the
fluorescent signal through fluorescence microscopy. Fluorescein is a fluorophore
broadly employed in fluorescence microscopy studies synthesized for Adolf von Baeyer
over one hundred years ago [209]. This molecule presents a high fluorescence (λexc =
494 nm and λem = 521 nm) pattern and perfect chemical characteristic for its
solubilization in aqueous solvents. Several derivatives have been developed by
modifying some functional groups of the molecule but keeping its fluorogenic
properties. This is the case of carboxyfluoresceine (CF) and Oregon green 488 nm
cadaverine (OG), the fluorescein derivatives that were covalently bounded to TAT-1
and PEN-1 (CF-TAT-1 and CF-PEN-1) and QM56 (QM56-OG) (see Materials and
Methods and chapter 3 for further information) to perform different compound
internalization studies.
57
O O
FCOOH
OH
HOOC
O O
FCOOH
CO NH(CH2)5NH2
OH
O O
FCOOH
OH
Fluorescein
Carboxyfluoresceine Oregon green cadaverine
Figure 4.6. Chemical structure of fluorescein and its chemical derivatives carboxyfluoresceine and Oregon green cadaverine.
4.1 Apoptotic inhibition activity of CPP-peptoid 1 hybrids: PEN-1 and TAT-1
Once peptide/peptoid 1 hybrids PEN-1 and TAT-1 were synthesized, the influence of
the fusion of peptoid 1 to CPPs on the cell membrane permeation capabilities was
analyzed. Their influence on cellular uptake, biological activity of the cargo and the
correlation with the conformational properties of these molecules in the presence of
synthetic lipid membranes and membrane mimetics were studied [196] and results are
depicted in this section.
4.1.1 PEN-1 and TAT-1 antiapoptotic activity in cell extracts
Initially, the influence of the fused PEN and TAT peptides on peptoid 1a ability to act
as an Apaf-1 inhibitor was evaluated employing HEK 293 cell extracts depleted from
endogenous Apaf-1 (Flow-through or FT) (see Materials and Methods for further
information). When recombinant Apaf-1 was pre-incubated with peptoid 1a and added
to this FT fraction, a peptoid 1a concentration-dependent inhibition of caspase 3 activity
was found (Figure 4.7) [158, 196]. Using the same procedure for both, PEN-1 and
TAT-1, a concentration-dependent profile of caspase 3 inhibition was also obtained.
The synthetic control peptides PEN and TAT did not show inhibitory activity. These
results suggest that the presence of the CPPs does not modify the capability of peptoid 1
58
to bind to Apaf-1 and, in turn, to inhibit the apoptosome-dependent activation of
caspases.
Figure 4.7. Concentration dependent inhibition of Apaf-1 mediated activation of caspases by peptoid 1, PEN-1 and TAT-1. The different peptide-peptoid hybrids and peptide controls (x axis) were analyzed at 1, 5, 10 and 20 μM (black, stripped, grey and white bars, respectively). Data are expressed as the mean ± SE (n > 3).
4.1.2 Evaluation of cell toxicity and biological activity of PEN-1 and TAT-1
a) MTT assays: PEN-1 and TAT-1 cell toxicity and cell-viability recovery assays
TAT-1 was more toxic than PEN-1 when analyzed by MTT in the different cell lines
studied (Figure 4.8). Consequently, TAT-1 showed no cell-viability recovery after
challenging the cells (data not shown). On the other hand, the addition of 1 μM PEN-1
to cells treated with dox increased cell survival significantly in different cell lines, being
U937 a good example of the effect of PEN-1 (Figure 4.9). The unspecific cell toxicity
of PEN-1 could preclude the protective effect of the compound at higher concentrations.
PEN and TAT control peptides were also evaluated as control using the same
experimental setting. Control peptides showed no cell toxicity at the concentrations
tested, and no cell-viability recovery after dox or doxy treatments (data not shown)
[196, 200].
Peptoid 1a PEN-1 PEN TAT-1 TAT
59
24H48H
72HU-2 OSHeLaA549U937Saos-2
0
20
40
60
80
100
% C
ellv
iabi
lity
(% T
otal
cel
ls)
A
24H48H
72HU-2 OSHeLaA549U937Saos-2
0
20
40
60
80
100
% C
ellv
iabi
lity
(% T
otal
cel
ls)
A
24H48H
72HU-2 OSHeLaA549U937Saos-2
0
20
40
60
80
100
% C
ellv
iabi
lity
(% T
otal
cel
ls)
B
24H48H
72HU-2 OSHeLaA549U937Saos-2
0
20
40
60
80
100
% C
ellv
iabi
lity
(% T
otal
cel
ls)
B
Figure 4.8. Evaluation of cell toxicity in different cell lines by (A) PEN-1 and (B) TAT-1. Compounds were tested at 10, 5, and 1 μM (black, gray, and white bars, respectively) after 24, 48, and 72 h of incubation. Data are expressed as the mean SE (n > 3). In all cases, SE < 10%.
24H48H
72HSaos-2A549HeLaU-2 OSU937
0
10
20
30
40
50
60
% C
ellr
ecov
ery
(% T
otal
cel
ls)
24H48H
72HSaos-2A549HeLaU-2 OSU937
0
10
20
30
40
50
60
% C
ellr
ecov
ery
(% T
otal
cel
ls)
Figure 4.9. Evaluation of cell-viability recovery in different cell lines by PEN-1. The compound was evaluated at 10, 5, and 1 μM (black, gray, and white bars, respectively) after 24, 48, and 72 h of incubation. Data are expressed as the mean ± SE (n > 3). In all cases, SE < 10%.
60
b) Caspase 3 activity measurements employing PEN-1 and TAT-1
As a more direct indicator of apoptotic cell death, the caspase 3 activity of cells treated
with dox/doxy or dox/doxy plus PEN-1 and TAT-1 (at a concentration of 5 μM) was
also analyzed using the same cellular models. PEN-1 inhibited caspase 3 activity in the
different cellular models employed. It was also able to inhibit caspase 3 activation in
Saos-2 (Figure 4.10). However, no inhibitory activity was observed with TAT-1 (data
not shown). The synthetic peptides PEN and TAT were also analyzed as controls, and
no inhibition of caspase 3 activity was observed (data not shown) [196, 200].
010
2030
4050
6070
80
Saos-2 HeLa U-2 OS U937
% C
aspa
se 3
act
ivity
inhi
bitio
n
Figure 4.10. Evaluation of caspase 3 enzymatic activity inhibition by PEN-1 in different cell lines. PEN-1 was tested at 5 μM in all cell lines with the exception of Saos-2 (2 μM). Measurements were performed after 24, 48, and 72 h of incubation (black, gray, and white bars, respectively). Data are expressed as the mean ± SE (n> 3).
4.1.3 Cellular internalization studies and structural characterization of PEN-1 and
TAT-1
In order to prove that conjugation to CPP enhances the cellular internalization of
peptoid 1, two carboxyfluoresceine (CF) labeled derivatives (CF-PEN-1 and CF-TAT-
1) were synthesized to be used as fluorescence probes in flow cytometry and live-cell
confocal fluorescence microscopy studies.
61
a) CF-TAT-1 and CF-PEN-1 cellular internalization studies
Both conjugates were rapidly internalized by the cells in a time dependent manner (with
a plateau after 30 min probably due to fluorescence quenching (Figure 4.11), as
observed by flow cytometry studies). It should be noticed that TAT-1 showed an
enhanced membrane binding propensity when compare to PEN-1 (fluorescence output
at t = 0 min) (Figure 4.12). In addition, the internalization mechanism showed to be
different for PEN-1 and TAT-1 as deduced from confocal fluorescence microscopy
observation; whereas PEN-1 showed a vesicular labeling profile at times as short as 15-
min incubation, cells treated with TAT-1 showed a more diffused cytosolic location
(Figure 4.12). Moreover, after 1-h incubation, the cell damage induced by TAT-1 was
much higher than that observed for those cells treated with PEN-1. These data are in
good agreement with TAT-1 observed cytotoxicity.
TAT-1 PEN-1A
B
Cou
nts
Cou
nts
Time (min) Time (min)
Time (min)
PEN-1TAT-1
TAT-1 PEN-1A
B
Cou
nts
Cou
nts
Time (min) Time (min)
Time (min)
PEN-1TAT-1
Figure 4.11. Time-dependent uptake of PEN-1 and TAT-1 by U-937 cells; (A) raw data collected after incubation with PEN-1 or TAT-1 and (B) cell-associated fluorescence after incubation with PEN-1 and TAT-1. Flow cytometry analysis at 37 ºC. The values represent mean ± S.E.M.; n > 3.
62
PEN-1 TAT-1
t = 0 min
t = 15 min
t = 1 h
PEN-1 TAT-1
t = 0 min
t = 15 min
t = 1 h
Figure 4.12. Confocal microscopy live cell images of Saos-2 cells incubated at 37 ºC with: PEN-1 for (A) 0 min, (B) 15 min, (C) 1 h and TAT-1 for (D) 0 min, (E) 15min, and (F) 1 h. Concentration 1 μM.
Although PEN and TAT could be initially considered as equally efficient cell
transporters for a wide variety of cargoes, the cargo could possibly influence any of the
intrinsic properties of CPPs, having a direct effect on their behavior. Thus, if the cargo
modifies the membrane-induced conformational transition capabilities of CPPs, these
could modify cell permeability. To explore such a possibility, we analyzed the
conformational behavior of both TAT-1 and PEN-1 in membrane-mimetic
environments and their respective control peptides, TAT and PEN. The CD spectra of
PEN, PEN-1, TAT and TAT-1 were initially acquired in PBS buffer at 5 ºC (Figure
4.13).
63
Figure 4.13. CD spectra in the presence of different TFE–water mixtures (—0% TFE, 20% TFE, Δ 50% TFE,O100% TFE). (A) PEN control. (B) PEN-1. (C) TAT control. (D) TAT-1.
Peptides in aqueous solution tend to be a random coil, which is a dynamic structure that
changes constantly [210]. In agreement with this statement, the spectra of PEN and
TAT are representative of a predominantly random structure (Figure 4.13 A and C,
respectively) while a comparison of the CD spectra obtained for the PEN-1 and the
TAT-1 suggested an influence of the cargo on the general structure of the peptide. In
fact, in the absence of trifluoroethanol (TFE) the CD spectrum obtained for PEN-1 was
representative of a mixed random/β-sheet conformation (Figure 4.13 B) and the
spectrum for the TAT-1 was representative of a poly(proline) type II-like (PPII-like)
conformation with a CD spectrum exhibiting a weak positive band at 223 nm and a
strong negative band at 196 nm [168] (Figure 4.13 D). TFE has been used as a solvent
additive to explore the intrinsic ability of peptides to adopt secondary structure
conformations [211]. TFE titration of PEN and PEN-1 solutions resulted in a
substantial increase of helical content (Figure 4.13 A and B), which is in good
agreement with previously reported data [212]. TFE titration of the TAT peptide also
induced the increase in the α-helix content (Figure 4.13 C). For TAT-1 however, a
TFE-induced conformational transition towards the partial stabilization of a mixed α/β
secondary structure was observed. This is characterized by a CD spectrum with
64
negative ellipticity bands at 220 and 205 nm and with marked intensity differences
[213] (Figure 4.13 D).
Figure 4.14. CD spectra in the presence of different concentrations of SDS (— 0 mM SDS, 0.5 mM, Δ 1 mM SDS, O 3 mM SDS, ◊ 11 mM SDS). (A) PEN control. (B) PEN-1. (C) TAT control.
The conformational behavior of all four compounds was also analyzed in the presence
of either negatively charged (sodium dodecyl sulfate, SDS) or zwitterionic (1-palmitoyl-
2-hydroxy-snglycero-3-phosphocoline, LPC) membrane-mimetic environments. SDS
titration of PEN induced a conformational transition towards the stabilization of a β-
sheet conformation at submicellar (< 3 mM SDS concentration) and of an α-helix above
the critical micellar concentration (cmc) where SDS forms micellar aggregates (Figure
4.14 A). The PEN-1 showed similar trends; however, an SDS-dependent induction
towards helical conformations had already occurred at submicellar concentrations.
Above the cmc, PEN-1 adopted a mixed α/β conformation with a very intense
minimum at 208 nm (Figure 4.14 B). The TAT peptide was induced into a β-sheet
conformation at submicellar SDS concentrations (Figure 4.14 C). In the presence of
micellar concentrations of SDS, the resulting CD spectrum was more difficult to
analyze due to an increased tendency of the complex to precipitate out from the
65
solution. Nevertheless, TAT-1 could not be analyzed in the presence of SDS due to a
non-specific precipitation.
The conformational behavior of the peptides and peptide/peptoid hybrids was then
studied using LPC micelles. As shown in Figure 4.15 (A,B), both the PEN peptide and
PEN-1 hybrid were induced into an α-helical conformation. However, both the TAT
peptide and TAT-1 were induced to fold into a PPII-like structure, although the
differences in the absolute intensity of the positive ellipticity band at 220 nm and the
negative ellipticity band at 196 nm suggested differences in the LPC-induced
conformation (Figure 4.15 C and D, respectively). See also Tables 4.1 and 4.2 for more
detailed information.
Figure 4.15. CD spectra in the presence of different concentrations LPC micelles (— 0 mM LPC, 100 mM LPC,Δ 400 mM LPC, O 1000 mM LPC). (A) PEN control. (B) PEN-1. (C) TAT control. (D) TAT-1.
66
Table 4.1. Percentage of secondary structure obtained from the CD data interpreted with the program provided by Jasco.
Secondary structure (%) Peptide Inductor
Alpha Beta Turn Random 0 0 20.2 32.2 47.6
0.5 16 41.3 32.3 10.4 1 40.6 34.3 25.1 0
PEN
11 45.3 16.4 0 38.4 0 0 64 13 22.9
0.5 9.7 37.2 25.7 27.4 1 14.1 42.1 17.7 26.2
PEN-1
11 7.4 66.5 0 26.1 0 0 16.2 35.3 48.4
0.5 4.2 50.9 18.9 26.1 1 5.6 57.5 16.1 20.9
TAT
3 0 42.6 16.6 40.8 0 0 0 24.4 75.6
0.5 1
TAT-1
SDS (mM)
3 20 0 20.2 32.2 47.6 50 51.1 0 0 48.9 PEN
100 61.4 0 0 38.6 20 2.6 61.5 0 35.9 50 14.6 48.8 0 36.6 PEN-1
100 28.4 48 0 23.6 20 0 8.7 39.3 51.9 50 0 20.4 25.7 53.9 TAT
100 38.8 6.8 0 54.5 20 0 0 24.4 75.6 50 0 0 11.7 88.3 TAT-1
TFE (%)
100 43.7 0 0 56.3 10 0 29.2 19.7 51.1
100 0 21.6 12.9 65.6 PEN 1000 23.2 32.1 0 44.6
10 0 66 10.7 23.4 100 4.7 61.9 7.6 25.7 PEN_15
1000 14.2 55.2 2.9 27.7 10 0 0 35.4 64.6
100 0 5 34.5 60.5 TAT 400 0 18.2 34.5 47.3 10 0 0 24.4 75.6
100 0 0 11.6 88.4 TAT_15
LPC (μM)
1000 0 0 0 100
67
Table 4.2. Percentage of secondary structure obtained from the CD data interpreted with the K2D program of the Dichroweb (http://www.cryst.bbk.ac.uk/cdweb).
Secondary structure (%) Peptide Inductor Alpha Beta Turn Random
0 0.04 0.29 0.68 0.04 0.5 0.02 0.51 0.47 0.02 1 0.25 0.4 0.35 0.25
PEN
11 0.52 0.1 0.37 0.52 0 0.09 0.48 0.44 0.09
0.5 0.08 0.44 0.48 0.08 1 0.18 0.3 0.52 0.18
PEN-1
11 0.12 0.33 0.54 0.12 0 0.11 0.42 0.48 0.11
0.5 0.09 0.43 0.48 0.09 1 0.04 0.48 0.48 0.04
TAT
3 0.05 0.47 0.48 0.05 0 0.08 0.48 0.44 0.08
0.5 1
TAT-1
SDS (mM)
3 20 0.28 0.31 0.41 0.28 50 1 0 0 1 PEN
100 1 0 0 1 20 0.08 0.4 0.52 0.08 50 0.22 0.23 0.55 0.22 PEN-1
100 0.33 0.16 0.51 0.33 20 0.09 0.47 0.44 0.09 50 0.04 0.48 0.48 0.04 TAT
100 0.03 0.49 0.47 0.03 20 0.08 0.48 0.44 0.08 50 0.08 0.49 0.43 0.08 TAT-1
TFE (%)
100 0.1 0.35 0.55 0.1 10 0.07 0.5 0.43 0.07
100 0.09 0.28 0.63 0.09 PEN 1000 0.28 0.21 0.5 0.28
10 0.09 0.47 0.44 0.09 100 0.1 0.4 0.51 0.1 PEN_15
1000 0.21 0.25 0.54 0.21 10 0.08 0.46 0.47 0.08
100 0.25 0.32 0.43 0.25 TAT 400 0.27 0.33 0.4 0.27 10 0.08 0.48 0.44 0.08
100 0.07 0.5 0.43 0.07 TAT_15
LPC (μM)
1000 0.08 0.48 0.44 0.08
68
4.2. Apoptosis inhibitory activity of the polymer-peptoid 1 nanoconjugate QM56
The positive results obtained using peptide-based cell internalization strategies for the
cellular delivery of peptoid 1 and derivatives, prompted us to explore other strategies
reported to show improved cellular internalization capabilities. Therefore, the aim of
this part was to design, synthesize and characterize polymer-drug conjugates which by
carrying the bioactive peptoid 1, would effectively rescue cells from inappropriate
apoptosis. PGA [214, 215] was chosen as a polymeric carrier since it is internalized by
cells by endocytosis and in the lysosome it can be degraded by specific enzymes that
would release peptoid 1; it is stable in plasma and it contains functional groups for drug
attachment. In addition, PGA has excellent pharmacological properties, as
demonstrated with Opaxio® [214, 215] that could represent the first polymer-anticancer
drug conjugate to be commercialized as already explained in chapter 3.
4.2.1 Evaluation of cellular internalization of QM56
Due to the intrinsic nature of the compounds, the ability of polymer-drug conjugates to
inhibit apoptosome assembly in the in vitro assays described before could not be
determined. Peptoid 1 is shielded with PGA and the presence of cathepsin B is
necessary to trigger the release the inhibitor (or derivatives) from the polymeric carrier.
Nevertheless, alternative QM56 live-cell internalization studies were performed in order
to determine the ability of this compound to reach its intracellular target.
As mentioned before it was hypothesized that the conjugation of peptoid 1 to a
polymeric carrier could offer a more specific intracellular trafficking. In order to prove
our hypothesis, QM56 was conjugated to the fluorescent dye OG (QM56-OG) and its
cellular uptake analyzed by flow cytometry (at 4ºC (cell binding) and 37ºC (cell
uptake)) and live-cell confocal fluorescence microscopy (exploring endocytic pathway).
The internalization studies were carried out in Saos-2 and U937 cells. As shown in
Figure 4.21, QM56-OG was rapidly internalized by the cells in a time-dependent
manner (reaching a plateau after 30 min) and through an energy-dependent mechanism
as could be observed by the cell-associated fluorescence differences at 4ºC and 37ºC
69
(Figure 4.21 B). As expected a vesicular labeling and co-localization with lysosomal
markers was observed (Figure 4.21) demonstrating a lysosomotropic drug delivery
mechanism.
Time0 300 60 90 120 150 180
A
B
Time0 300 60 90 120 150 180
Time0 300 60 90 120 150 180
Time0 300 60 90 120 150 180
A
B
Figure 4.16. Cellular uptake of QM56-OG in established cell lines. (A) Live-cell fluorescence confocal images in Saos-2 cells at different incubation times (from a to d, 0, 15, 30 and 120 minutes respectively). Images e and f, correspond to the polymer internalization in the presence of Texas red-Dextran as lysosomal marker. (B) Internalization of QM56-OG in U937 cell line analyzed by flow cytometry at 4 (left panel) or 37 ºC (right panel). Graphical view of the geometric mean of the flow cytometry internalization experiments.
70
4.2.2 Evaluation of cell toxicity and biological activity of polymer-peptoid 1
conjugates: QM56 and derivatives
Tested the ability of the PGA peptoid 1 conjugate QM56 to be internalized by cells,
compound cell toxicity and biological activity were evaluated by means of analyzing
cell-viability recovery using MTT assays and caspase 3 activity inhibition experiments.
a) MTT assays: QM56 cell toxicity and cell-viability recovery assays
QM56 was shown to be devoid of unspecific toxicity (Figure 4.17) and it proved to be
active inhibiting both dox- and doxy-induced cell death in different cellular models
(Figure 4.18).
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Figure 4.17. Evaluation of cell toxicity in different cell lines by QM56. This compounds was tested at 100, 50, and 20 μM drug-equivalents (black, gray, and white bars, respectively) after 24, 48, and 72 h of incubation. Data are expressed as the mean SE (n > 3). In all cases, SE < 10%.
71
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Figure 4.18. Evaluation of cell-viability recovery in different cell lines by QM56. The compound was evaluated at 100, 50, and 20 μM drug-equivalent (black, gray, and white bars, respectively) after 24, 48, and 72 h of incubation. Data are expressed as the mean ± SE (n > 3). In all cases, SE < 10%.
c) Caspase 3 activity measurements employing QM56 and its derivatives
Dox induces apoptosis through DNA damage that is transduced to the mitochondria by
disturbing the mitochondrial membrane potential (MMP) and by activating executioner
caspases (caspase 3) through the involvement of apoptosome. Doxy-induced Bax
overexpression also leads ultimately to activation of caspase 3. Then, to further analyze
the antiapoptotic activity of the nanoconjugate QM56, it was evaluated in the cell
extract-based fluorogenic caspase 3 assay (Figure 4.19).
0102030405060708090
100
Saos-2 HeLa U-2 OS U937
% C
aspa
se 3
act
ivity
inhi
bitio
n
Figure 4.19. Percentage of caspase 3 inhibition in cell extracts that where subjected to apoptotic insult and treated with QM56 50 μM drug-equivalents. Caspase 3 activity inhibition was measured after 24, 48 and 72 h (black, grey and white bars, respectively). Data are expressed as the mean ± SE (n > 3).
72
QM56 proved to be highly effective inhibiting caspase 3 activity in different cell lines
even after long-incubation times. As stated in chapter 3, QM56 employs a diglycyl
sequence as a linker between the peptoid 1 moiety and the carrier PGA. In the cellular
environment, the PGA-based compounds are substrates of cathepsin B [192, 216], also
peptidic sequences that preferentially possess peptide bonds that involve hydrophobic
residues at P1 and P2 positions. However, Gly is tolerated and offers higher structural
simplicity. In fact, Gly at P1 has been used all through the present study. Nonetheless,
as already shown in chapter 3, experimental results have shown that for some
applications the drug release rate for compounds comprising a Gly residue at P1 is too
slow to achieve the effective concentrations in the desired time range; therefore, linker
optimization was carried out. By changing Gly at P1 position by a non polar or positive
charged amino acid, it was possible to markedly modulate drug release rate. Thus, a
series of QM56 analogues with different peptide linkers were synthesized (see chapter 3
Section 3.3 for further information) and their ability to inhibit caspase 3 activation in
Saos-2 cell line was tested. The results are depicted in Table 4.4.
Table 4.4. Percentage of inhibition of apoptosis as determined by inhibition of caspase 3 activity by the different QM56 derivatives.
Saos-2 (%) Compound
24 h 48 h
QM56 54±3 (n=20) 93±5 (n=10)
QM56-P1 75±12 (n=3) 93±23 (n=3)
QM56-P2 67±4 (n=3) 34±5 (n=3)
Both QM56 derivatives were able to inhibit caspase 3 activation in a similar degree than
QM56. QM56-P1 seems to show a higher activity at shorter times due to a more rapid
drug release kinetics (see chapter 3, Figure 3.18). However, it could be considered that
in general no significative improvement in the biological activity of QM56 derivatives
was achieved when compared to the lead compound.
73
4.3 Apoptotic inhibitory activity of conformationally constrained peptoid 1
derivatives
As explained in chapter 3, it is admitted that peptoids could hardly be considered as lead
candidates due to the risks of interaction with undesired targets as consequence of their
great conformational freedom. Fortunately, they are molecules simple enough to design
second generation analogues addressed to restrain the conformational freedom. Among
other possibilities, the generation of heterocyclic derivatives in which the ring could
play the scaffold role where the chemical diversity already present in the peptoid could
be inserted, constitutes an attractive strategy. In addition, it is conceivable that upon
such backbone restriction some physicochemical features related to bioavailability and
cellular permeability of the original peptoids could be maintained or even improved,
thus leading to compounds with a better pharmacological profile.
Peptoid 1 cyclization was performed and a seven member ring peptoid 1 derivative,
QM31, was obtained as leading hit for this series (see chapter 4 for further information).
QM31 derivatives were further synthesized in order to identify the role of the different
functional groups in the development of the biological activity of the compound.
Furthermore the size of the scaffold ring was also evaluated by the synthesis of related
derivatives which leading hit was named QM57. However, due to patent pending issues
no further structural details of QM57 and QM57-derivatives can be released at this time.
4.3.1 Conformationally constrained peptoid 1 derivatives inhibit apoptosome assembly
in an in vitro apoptosome reconstitution assay
As it was detailed in the introduction, the hit peptoid 1 was discovered from the
screening of a chemical library using a partially in vitro reconstituted apoptosome [158].
However, in the present study an improved in vitro reconstituted apoptosome assay with
purified recombinant proteins was developed. Then, rApaf-1 was incubated with dATP
and cyt c in the presence of recombinant procaspase 9 (rproCasp-9) and caspase 9
activity was analyzed by the ability of the enzyme to cleave the fluorogenic substrate
Ac-Leu-Glu-His-Asp-afc (Ac-LEHD-afc). To evaluate the inhibitory activity of QM31
74
series, rApaf-1 was incubated with the selected compounds before the addition of the
other components of the apoptosome. Results are depicted in Figures 4.20 and 4.21.
02
46
810
1214
1618
20
QM31
QM31a
QM31b
QM31c
QM31d
QM31e
QM31f
QM31g,h
QM31i
QM31j
QM31k
QM31l
% C
aspa
se 9
act
ivity
inhi
bitio
n
Figure 4.20. Caspase 9 activity inhibition by QM31 and derivatives. Compounds were tested at 10 μM in all cases.
0
5
10
15
20
25
30
QM57
QM57a
QM57b
QM57c
QM57d
QM57e
QM57f
QM57g
QM57h
QM57i
QM57j
QM57k
QM57l
QM57m
% C
aspa
se 9
act
ivity
inhi
bitio
n
Figure 4.21. Caspase 9 activity inhibition by QM57 and derivatives. Compounds were tested at 10 μM in all cases.
The results obtained showed certain ability to inhibit apoptosome assembly of some
compounds. Although these results were not very promising, an important issue of
compound solubility was detected. QM31 and derivatives are soluble in DMSO, but in
75
this system the total amount of DMSO in the reaction mixture is critical for the
assembly of the apoptosome. Methods to adjust the compound solubility to the
requirements of the assay are currently under evaluation in the laboratory.
4.3.2 Evaluation of cell toxicity and biological activity of conformationally constrained
peptoid 1 derivatives: QM31 and QM57 derivatives
a) MTT assays: QM31 and derivatives cell toxicity and cell-viability recovery assays
Initially, QM31 was tested in different preliminary cell toxicity and cell-viability
recovery MTT assays. QM31 showed no cell toxicity (Figure 4.22) in the different cell
lines tested and a significant biological activity (Figure 4.23).
As explained above, the cyclic derivative QM31 was active as inhibitor of apoptosis. In
order to seek for further optimization, several structural modifications of QM31 were
carried out. As mentioned in chapter 3 we focused our attention to the 2’,4’-
dichlorophenylethyl substituent attached to the perhydro-1,4-diazepine-2,5-dione ring.
This substituent turned out to be essential for activity and, any attempt of modification
resulted in a loss of antiapoptotic activity. Similar results were found in early attempts
of modification of the 3,3-diphenylpropyl moiety [158]. Then we addressed our
attention to the R1 moiety. Initially a series of QM31 derivatives (QM31a-QM31l) were
synthesized and their cell toxicity and cell-viability recovery checked (Table 4.5). See
chapter 3 for detailed information.
76
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Figure 4.22. Evaluation of cell toxicity in different cell lines by QM31. This compounds was tested at 10, 5, and 1 μM (black, gray, and white bars, respectively) after 24, 48, and 72 h of incubation. Data are expressed as the mean SE (n > 3). In all cases, SE < 10%.
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Figure 4.23. Evaluation of cell-viability recovery in different cell lines by QM31. The compound was evaluated at 10, 5, and 1 μM (black, gray, and white bars, respectively) after 24, 48, and 72 h of incubation. Data are expressed as the mean ± SE (n > 3). In all cases, SE < 10%.
77
Table 4.5. Evaluation of QM31 derivatives cell toxicity in U937 and Saos-2 cell lines. Compounds were tested at 10, 5, and 1 μM (black, gray, and white bars, respectively) and cell viability measured after 24 h of incubation. Data are expressed as the mean SE (n > 3). In all cases, SE < 10%.
Cell line U937 Saos-2
Compound 10 μM 5 μM 1 μM 10 μM 5 μM 1 μM
QM31 87±2a (n = 3)
90±7a (n = 3)
100±1a (n = 3)
85±2 (n = 5)
100±1 (n = 5)
99±1 (n = 5)
QM31a 90±4 (n = 3)
98±2 (n = 3)
98±2 (n = 3)
91±5 (n = 6)
100±1 (n = 6)
76±1 (n = 6)
QM31b 89±6 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
99±1 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
QM31c 81±5 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
82±2 (n = 3)
98±2 (n = 3)
100±1 (n = 3)
QM31d 73±9 (n = 3)
100±1 (n = 2)
95±5 (n = 3)
100±1 (n = 3)
97±3 (n = 3)
100±1 (n = 3)
QM31e 82±12 (n = 3)
93±6 (n = 3)
96±4 (n = 3)
82±8 (n = 3)
96±4 (n = 3)
100±1 (n = 3)
QM31f 91±8 (n = 3)
97±3 (n = 3)
97±3 (n = 3) ND ND ND
QM31g,h ND ND ND ND ND ND
QM31i 89±5 (n = 2)
98±3 (n = 2)
79±12b
(n = 3) 100±1 (n = 3)
93±3 (n = 3)
97±3 (n = 3)
QM31j 50±5 (n = 2)
74±1 (n = 2)
87±2 b (n = 3)
98±2 (n = 3)
99±1 (n = 3)
95±5 (n = 3)
QM31k 51±10 (n = 3)
92±4 (n = 3)
99±1 (n = 3)
99±1 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
QM31l ND ND ND ND ND ND
Seeding density 4000 cells/well a Seeding density 2500 cells/well, b Cell viability at concentration of 3,26 μM
A second generation of QM31 derivatives was also synthesized by modifying a
different functional group in the molecule. The lead compound of this new series is
QM57. Different QM57 derivatives were also synthesized and their cell toxicity was
tested in Saos-2 and U937 cell lines (Table 4.6).
78
Table 4.6. Evaluation of QM57 derivatives cell toxicity in U937 and Saos-2 cell lines. Compounds were tested at 10, 5, and 1 μM (black, gray, and white bars, respectively) and cell viability measured after 24 h of incubation. Data are expressed as the mean SE (n > 3). In all cases, SE < 10%.
Cell line U937 Saos-2
Compound 10 μM 5 μM 1 μM 10 μM 5 μM 1 μM
QM57 84±5 (n = 3)
96±2 (n = 3)
93±7 (n = 3)
100±1 (n = 6)
99±1 (n = 6)
100±1 (n = 6)
QM57a 75±8 (n = 3)
97±3 (n = 3)
94±6 (n = 3)
85±2 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
QM57b 97±3 (n = 3)
96±4 (n = 3)
100±1 (n = 3)
84±2 (n = 3)
96±1 (n = 3)
100±1 (n = 3)
QM57c 96±2 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
95±3 (n = 3)
95±2 (n = 3)
98±1 (n = 3)
QM57d 88±6 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
94±4 (n = 3)
93±1 (n = 3)
99±1 (n = 3)
QM57e 100±1 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
QM57f 94±3 (n = 3)
89±4 (n = 3)
92±8 (n = 2)
100±1 (n = 3)
100±1 (n = 3)
100±1 (n = 3)
None of the QM31 or QM57 derivatives synthesized showed cell toxicity at the
concentrations tested. The following step was to determine its possible biological
activity and a possible improvement of QM31 biological activity by selectively
modifying some of its functional groups. For these experiments, U937 and Saos-2 cell
lines were employed and the results obtained are depicted in Tables 4.7 and 4.8.
79
Table 4.7. Evaluation of QM31 derivatives cell recovery in U937 and Saos-2 cell lines. Compounds were tested at 10, 5, and 1 μM (black, gray, and white bars, respectively) and cell viability measured after 24 h of incubation. Data are expressed as the mean SE (n > 3). In all cases, SE < 10%.
Cell line U937 Saos-2
Compound 10 μM 5 μM 1 μM 10 μM 5 μM 1 μM
QM31 27 ± 4 (n = 7)
49 ± 5 (n = 8)
32 ± 4 (n = 7)
30 ± 7 (n = 9)
36 ± 12 (n = 9)
46 ± 12 (n = 9)
QM31a 16 ± 7 (n = 6)
20 ± 5 (n = 6)
20 ± 8 (n = 6)
13 ± 7 (n = 3)
58 ± 20 (n = 3)
0 (n = 3)
QM31b 1 ± 1 (n = 6)
12 ± 4 (n = 6)
14 ± 4 (n = 6)
3 ± 3 (n = 3)
10 ± 10 (n = 3)
41 ± 11 (n = 3)
QM31c 4 ± 3 (n = 6)
5 ± 3 (n = 6)
14 ± 6 (n = 6)
0 (n = 3)
0 (n = 3)
22 ± 17 (n = 3)
QM31d 3 ± 3 (n = 6)
7 ± 2 (n = 6)
16 ± 6 (n = 6)
9 ± 6 (n = 3)
9 ± 5 (n = 3)
34 ± 3 (n = 3)
QM31e 2 ± 1 (n = 6)
5 ± 2 (n = 6)
3 ± 1 (n = 6)
0 (n = 3)
16 ± 12 (n = 3)
36 ± 13 (n = 3)
QM31f 0 (n = 6)
0 (n = 6)
0 (n = 6)
0 (n = 3)
0 (n = 3)
0 (n = 3)
QM31g,h 0 (n = 3)
0 (n = 3)
0 (n = 3)
0 (n = 3)
0 (n = 3)
0 (n = 3)
QM31i ND ND ND 23 ± 12 (n = 3)
12 ± 6 (n = 3)
18 ± 9 (n = 3)
QM31j ND ND ND 9 ± 9 (n = 3)
41 ± 22 (n = 3)
64 ± 17 (n = 3)
QM31k ND ND ND 29 ± 5 (n = 3)
23 ± 12 (n = 3)
57 ±30 (n = 3)
QM31l ND ND ND ND ND ND
80
Table 4.8. Evaluation of QM57 derivatives cell recovery in U937 and Saos-2 cell lines. Compounds were tested at 10, 5, and 1 μM (black, gray, and white bars, respectively) and cell viability measured after 24 h of incubation. Data are expressed as the mean SE (n > 3). In all cases, SE < 10%.
Cell line U937 Saos-2
Compound 10 μM 5 μM 1 μM 10 μM 5 μM 1 μM
QM57 0 (n = 9)
2 ± 1 (n = 9)
2 ± 2 (n = 9)
28 ± 12 (n = 13)
44 ± 15 (n = 9)
46 ± 13 (n = 9)
QM57a 3 ± 1 (n = 6)
8 ± 1 (n = 6)
22 ± 2 (n = 6)
0 (n = 3)
9 ± 7 (n = 3)
8 ± 4 (n = 3)
QM57b 0 (n = 6)
1 ± 8 (n = 6)
5 ± 10 (n = 6)
14 ± 6 (n = 3)
4 ± 2 (n = 3)
12 ± 4 (n = 3)
QM57c 2 ± 1 (n = 6)
2 ± 1 (n = 6)
4 ± 2 (n = 6)
0 (n = 3)
0 (n = 3)
0 (n = 3)
QM57d 0 (n = 6)
0 (n = 6)
3 ± 2 (n = 6)
0 (n = 3)
0 (n = 3)
0 (n = 3)
QM57e 4 ± 1 (n = 6)
1 ± 1 (n = 6)
3 ± 1 (n = 6)
0 (n = 3)
2 ± 2 (n = 3)
10 ± 6 (n = 3)
QM57f 0 (n = 6)
0 (n = 6)
1 ± 2 (n = 6)
2 ± 2 (n = 3)
2 ± 2 (n = 3)
7 ± 6 (n = 3)
Some of QM31 and QM57 derivatives showed certain biological activity, but in any of
the cases their activity was significantly higher than QM31 or QM57. In order to
further confirm the data obtained in cell recovery experiments, caspase 3 activity
measurements assays were carried out.
b) Caspase 3 activity measurements employing QM31 and its derivatives
Caspase 3 activity inhibition assays were performed as explained before. Cells were
subjected to apoptotic insult and treated with QM31 (or QM31-derivatives) at 5 μM.
Then caspase 3 activity measurements on cell extracts were performed at 24, 48 and 72
h after the addition of the treatments (Figure 4.24).
81
0
10
20
30
40
50
60
70
Saos-2 HeLa U2-OS U937
% C
aspa
se 3
act
ivity
inhi
bitio
n
Figure 4.24. Percentage of caspase 3 inhibition in cell extracts that where subjected to apoptotic insult and treated with QM31 5 μM. Caspase 3 activity inhibition was measured after 24, 48 and 72 h (black, grey and white bars, respectively). Data are expressed as the mean ± SE (n > 3).
QM31 proved to be active inhibiting caspase 3 assay in different cell lines. Then, the
next step was to test the ability of the different QM31 derivatives and QM57 derivatives
to inhibit caspase 3 activity after the induction of apoptosis. U937 and Saos-2 cell lines
were chosen for this purpose as examples of unspecific induction of apoptotic cell death
(U937 plus dox) and mitochondrial specific apoptotic cell death (Saos-2 over expressing
Bax protein). Results are depicted in Tables 4.9 and 4.10.
82
Table 4.9. Percentage inhibition of caspase 3 activity obtained with QM31 analogues in U937 and Saos-2 cell lines after 24 h treatment.
Cell line U937 Saos-2
Compounds 24 h 48 h 24 h 48 h
QM31 19 ± 6 (n = 8)
19 ± 11 (n = 4)
37 ± 9 (n = 10)
12 ± 4 (n = 3)
QM31a 0
(n = 3) 5 ± 3
(n = 3) 14 ± 9 (n = 5)
4 ± 4 (n = 3)
QM31b 0
(n = 3) 3 ± 3
(n = 3) 10 ± 9 (n = 5)
0 (n = 3)
QM31c 11 ± 9 (n = 3)
0 (n = 3)
17 ± 5 (n = 5)
3 ± 3 (n = 3)
QM31d 0
(n = 3) 12 ± 3 (n = 3)
57 ± 12b (n = 3)
8 ± 5 (n = 3)
QM31e 3 ± 3
(n = 3) 4 ± 4
(n = 3) 51 ± 7 (n = 6) ND
QM31f ND ND ND ND
QM31g,h ND ND ND ND
QM31i ND ND 30 ± 7 (n = 6)
15 ± 1 (n = 3)
QM31j ND ND 0 (n = 3)
25 ± 14 (n = 3)
QM31k ND ND 0 (n = 3)
0 (n = 3)
QM31l ND ND ND ND
83
Table 4.10. Percentage inhibition of caspase 3 activity obtained with QM57 analogues in U937 and Saos-2 cell lines after 24 h treatment.
Cell line U937 Saos-2
Compounds 24 h 48 h 24 h 48 h
QM57 17±7 (n=6)
38 ± 13 (n = 4)
28 ± 8 (n = 10)
15 ± 11 (n = 6)
QM57a 4±3 (n = 6)
16 ± 8 (n = 6)
11 ± 10 (n = 6)
0 (n = 3)
QM57b 9±6 (n = 3)
29 ± 3 (n = 3)
6 ± 3 (n = 6)
1 ± 1 (n = 3)
QM57c 16±8 (n = 4)
48 ± 7 (n = 4)
23 ± 13 (n = 3)
57 ± 16 (n = 3)
QM57d 4±3 (n = 4)
27 ± 12 (n = 6)
0 (n = 6)
0 (n = 3)
QM57e 9±4 (n = 6)
5 ± 3 (n = 5)
0 (n = 6)
6 ± 4 (n = 3)
QM57f 3±2 (n = 5)
57 ± 4 (n = 3)
0 (n = 6)
0 (n = 3)
QM57g 2 ± 1 (n = 3) ND ND ND
QM57h 33 ± 3 (n = 2) ND ND ND
QM57i 18 ± 5 (n = 2) ND ND ND
QM57j 2 ± 0 (n = 3) ND ND ND
QM57k 27 ± 12 (n = 6) ND ND ND
QM57l 58 ± 5a
(n = 5) ND ND ND
QM57m 69 ± 6a (n = 3) ND ND ND
ND: not determined. n refers to the number of samples used for quantification. Each sample refers to a plate-well containing a defined number of cells. a Compound was tested in this case at 10 μM.
As expected from previous results, replacement of the 2’,4’-dichlorophenylethyl moiety
at R1 by a non-aromatic moiety resulted in a decrease of potency (compounds QM31c,
QM31d, QM31e, QM31i). However, the behavior of compound QM31c was a bit aside
provided that the inhibition of caspase 3 activity was systematically close to 20 % while
the cell recovery ability was insignificant. Also, the activity in the cell recovery
experiment for this compound was slightly higher at 1 μM than at 5 μM. It should be
mentioned that in certain occasions, biological assays developed with cell extracts and
84
whole cell systems provide results which are more difficult to rationalize when
compared to those obtained from in vitro experiments.
Furthermore, parallel studies addressed to analyze the unspecific toxicity of the
compounds, suggested that only compound QM31d was toxic. Different
electronegative atoms (such as O or F) in several positions (p-, m- or o-) were also used
in order to evaluate the effect of the electronic density changes on this family of
apoptosome inhibitors (namely, QM31a, QM31e, QM31i and QM31j). In general,
although all these analogues exhibited capability to reduce the apoptosis induced in
Saos-2 cell line, none of them showed a significant improvement when compared to
compound QM31. Similar results were obtained in the other models of our cell panel
mentioned above (HeLa, A549, U-2 OS and U-937 cells, data not shown). Taken all
together, it is not easy to make an appropriate SAR as no clear trend was obtained.
However, it would be possible to suggest that the presence of a second 2’,4’-
dichlorophenylethyl substituent is not essential but important for the activity, probably
due to the presence of the two electronegative atoms and, certainly, of the aromatic ring.
4.4 Apoptotic inhibition activity of the QM31 and QM57 polymeric derivatives
As stated in section 4.2, it is possible to modulate drug release rate by modifying the
number and the nature of the amino acids that constitute the linker between the polymer
(PGA) and the bioactive compound (peptoid 1) in presence of the lysosomal enzyme
cathepsin B. Different peptide linkers were assayed in order to obtain nanoconjugates
with different drug release kinetic profiles, thus permitting its application to broader
spectra of future treatments (see detailed information in chapter 3). At the same time,
the strategy of fusion to a polymeric carrier was also applied to the parental cyclic
compounds QM31 and QM57 in order to take advantage of the increased activity of
these compounds when compare to peptoid 1. All these new polymeric derivatives
were tested for caspase 3 activity inhibition after induction of cell death at different
time-points in U937 and Saos-2 cell lines (Table 4.11).
85
Table 4.11. Inhibition of apoptosis as determined by inhibition of caspase 3 activity by the different heterocycles QM31 and QM57 polymeric derivatives.
U937 Saos-2 Compound
24 h 48 h 24 h 48 h
QM31-P2 74±3 (n=3) 85±1 (n=3) 67±4 (n=3) ND
QM31-P3 ND ND 61±7 (n=9) 84±9 (n=6)
QM31-P4 ND ND 53±3 (n=9) 100±3 (n=6)
QM57-P1 ND ND 58±5 (n=6) 100±10 (n=6)
QM57-P2 ND ND 27±9 (n=6) 95±20 (n=6)
It is important to note that QM31-P1 was not evaluated on this biological setting as drug
release was very low even after 48 h and preliminary cell toxicity experiments showed
its disadvantage in front of the other derivatives. The overall data obtained from the rest
of QM31 polymer derivatives suggest that the strategies chosen in order to better
internalize peptoid 1 have been proved to be effective. Not only did the peptoid 1
derivatives designed result non toxic for the cells, but also exerted significative
biological activity preventing caspase 3 activation in the different assays performed and
in the Saos-2 intrinsic apoptosis induction cell model. The subsequent conjugation of
QM31 and QM57 to PGA also represents an attractive strategy to improve the
biological activity of the compounds taking profit of the lysosomotropic drug release of
polymer conjugates and the higher specificity of conformationally constrained peptoid 1
drug-candidates for their protein target.
86
87
CHAPTER 5. PEPTOID 1 DERIVATIVES AS
INHIBITORS OF THE APOPTOSOME: MOLECULAR
MECHANISM OF ACTION
In previous chapters it has been described the chemical optimization of peptoid 1
rendering different families of peptoid 1 derivatives with improved biological activity
and decreased toxicity. In the present chapter the molecular mechanism of action
behind the antiapoptotic properties of peptoid 1 and its derivatives is analyzed. The
main objective was the determination of the binding site of these compounds on Apaf-1
and to analyze the in cell molecular mechanism of action.
5.1 Characterization of peptoid 1 binding site in different in vitro systems
As early mentioned, the apoptosome is assembled when seven Apaf-1:cyt c
heterodimers oligomerize to form a ‘wheel’ structure that has the ability to recruit
procaspase 9 [17, 39, 41, 44]. To initially characterize the binding site of peptoid 1 to
the apoptosome a carboxyfluoresceine-based peptoid 1-analogue (CF-peptoid 1) was
synthesized. CF-peptoid 1 bound to rApaf-1, but not to cyt c and other control proteins
with dissociation constant (Kd) 57 ± 12 nM as determined in a fluorescence polarization
spectroscopy assay [158]. Nevertheless the binding constant value contrasted with the
concentration of peptoid 1 and peptoid 1 derivatives that was needed to inhibit the
apoptosome activity in the in vitro reconstitution assays (Figures 4.20 and 4.21). A
putative explanation to reconcile these data is currently under investigation as a part of
the pharmacological development of this class of compounds. The data suggests that
peptoid 1 derivatives can be absorbed with different affinities onto the 96-well plates
used in the assays (data not shown). Furthermore, it was also shown that cyt c did not
interfered with the inhibitory activity of peptoid 1 on the apoptosome-dependent
procaspase 9 cleavage, suggesting that peptoid 1 directly binds to rApaf-1 in a binding
site different from that of the cyt c recruitment site [158]. In principle, the peptoid may
have prevented any of the steps leading to caspase 9 activation including Apaf-1
oligomerization and caspase recruitment. It was shown that peptoid 1 precluded
recruitment of procaspase 9 by the apoptosome in a rApaf-1-mediated pull-down of
non-cleavable [35S]-Met procaspase 9 [158].
88
As mentioned before, Apaf-1 binds to and recruits procaspase 9 to the apoptosome
through a CARD(Apaf-1) - CARD(procaspase 9) interaction [39]. Then it was reasoned
that if the Apaf-1 inhibitors inhibit procaspase 9 recruitment, the Apaf-1 CARD domain
could participate in the binding site of peptoid 1 and its derivatives to Apaf-1. In order
to evaluate this hypothesis a series of assays that included fluorescence polarization
spectroscopy [217], NMR [218], yeast two hybrid system (Y2H) [219, 220] and
molecular modeling were used. To this end recombinant Apaf-1 CARD (rCARDApaf-1)
domain was produced both in normal and minimal media to have access to 15N-enriched
Apaf-1 CARD domain (15N-rCARDApaf-1 see materials and method section for further
information).
5.1.1 Fluorescence polarization spectroscopy
There is a time delay (nano seconds) between exciting a molecule and the molecule
emitting fluorescence. Motion of the molecule during this time period can be detected
as a loss in polarization of fluorescence. Binding of a fluorophore to a larger protein
will result in an increase in effective size and so a reduction in motion, detected as an
increase in fluorescence polarization [217]. Fluorescence polarization spectroscopy was
helpful to identify Apaf-1 as the target of peptoid 1 among the proteins that constitute
the apoptosome [7]. As mentioned before, Apaf-1 binds to and recruits procaspase 9 to
the apoptosome through a CARD(Apaf-1) - CARD(procaspase 9) interaction [39].
Then it was reasoned that if peptoid 1 was able to bind to Apaf-1 its inhibitory effect
could be exerted by inhibiting procaspase 9 recruitment, therefore the Apaf-1 CARD
domain could participate in the binding site of peptoid 1 and its derivatives to Apaf-1.
For this purpose, rCARDApaf-1 was obtained from Escherichia coli (E. coli) and the
ability of CF-peptoid 1 to interact with rCARDApaf-1domain was measured by
fluorescence polarization (Figure 5.1). The output of the experiment suggest that
peptoid 1 binds to the CARD domain of Apaf-1 with an affinity similar to that obtained
when the full length Apaf-1 protein was used suggesting that the CARD domain defines
the binding site of peptoid 1 to Apaf-1.
89
405060708090
100110
10 30 50 70 90 110[CARD] nM
mP
O COOH
FO
FHO
O
HN
O
NH
NN
NN
NNH2
O
N
O
O
Cl
Cl
O
O
O
Cl
Cl
A
B
405060708090
100110
10 30 50 70 90 110[CARD] nM
mP
O COOH
FO
FHO
O
HN
O
NH
NN
NN
NNH2
O
N
O
O
Cl
Cl
O
O
O
Cl
Cl
405060708090
100110
10 30 50 70 90 110[CARD] nM
mP
O COOH
FO
FHO
O
HN
O
NH
NN
NN
NNH2
O
N
O
O
Cl
Cl
O
O
O
Cl
Cl
A
B
Figure 5.1. CF-peptoid 1 binds to rCARDApaf-1. (A) Chemical structure of CF-peptoid 1 (B) A 60 nM solution CF-peptoid 1 in buffer A was titrated with increasing concentrations of rCARDApaf-1 (■) and fluorescence polarization was measured at 30 ºC. The data (mean ± s.d.; n=3) were recorded in millipolarization units (mP) and adjusted to a two-state binding model.
5.1.2 NMR 15N-rCARDApaf-1 peptoid 1 interaction assays
Nuclear magnetic resonance is defined as a physical resonance phenomenon involving
the observation of specific quantum mechanical magnetic properties of an atomic
nucleus in the presence of an applied, external magnetic field. This phenomenon only
appears in atoms that have an odd number of nucleons, which implies the existence of
an intrinsic magnetic moment and angular momentum, that translates into a spin number
larger than 0 [221]. Initially, the most commonly used nuclei were 1H and 13C in order
to develop 1D NMR spectra. However, the introduction of more complex molecules
aroused overlapping resonances and increased line-widths problems. These facts led to
the development of 2D NMR spectra making use of other isotopes such as 2H, 10B, 11B, 14N, 15N, 17O and 19F. Among the most used 2D NMR experiments, 2D HSQC
(Heteronuclear Single-Quantum Correlation) experiments employing 15N-labelled
proteins are very informative. This spectrum allows the obtention of 2D heteronuclear
chemical shift correlation maps between directly-bonded 1H-15N atoms with a higher
sensitivity when compared with 13C experiments [222]. NMR spectra can be considered
as protein fingerprints, any change in the structure of the protein will be translated in a
change of the NMR spectra pattern. Taking profit of this property and our previous
90
experience in the study of protein-small molecule interaction [223], the ability of PEN-1
to interact with Apaf-1 CARD domain was studied employing 2D HSQC experiments.
15N- rCARDApaf-1 was recombinantly produced in E. coli in minimum media with 15NH4Cl as only source of nitrogen (see materials and methods for further information)
[48]. The amount of compound needed for the experimental setting and solubility
matters, precluded the use of small molecule derivatives of peptoid 1 then PEN-1
derivative was used to investigate the binding of peptoid 1 derivatives to 15N-
rCARDApaf-1. A 15N-HSQC spectroscopy procedure was implemented. As shown in
Figure 5.2 a progressive disappearance of signals from unbound 15N- rCARDApaf-1 was
observed, along with concomitant appearance of a new set of signals. These new
signals should arise from the stabilization of a 15N-rCARDApaf-1/PEN-1 complex
although the output of the experiment also suggested the appearance of PEN-1-induced
partial denaturation of the protein. As a continuation of this work new strategies are
being explored in the lab in order to have access to perform this experiment with less
interfering peptoid 1 derivatives that in turn will allow a detailed mapping of the
interaction surface.
Figure 5.2 PEN-1 binds to 15N-rCARDApaf-1 domain [48].
91
5.1.3 Yeast two-hybrid system
Fluorescent polarization and 2D HSQC spectroscopy assays pointed out the existence of
a possible interaction between Apaf-1 CARD domain and peptoid 1. In order to
confirm this fact in a more biological setting a yeast two-hybrid system (Y2H) was
employed.
Y2H was implemented by Stanley Fields and coworkers [219, 224] twenty years ago
and this strategy has completely transformed the study of proteins. This technique takes
profit of the modular nature of many site-specific transcriptional activators, which are
composed of a DNA-binding domain (BD) and a transcriptional activation domain
(AD). The BD targets the AD to the specific genes that will be expressed and once
together the transcriptional machinery can bind to the gene promoter and start
transcription. The basis of the yeast two-hybrid system is the fact that BD and AD do
not need to be covalently bond to activate transcription. However, they can be brought
together if they are fused to two proteins X and Y and these proteins interact. Then, BD
and AD will be in proximity and can interact and activate the corresponding promoter
(Figure 5.3) [225].
In practice, the two plasmids containing BD-X and AD-Y fusions (bait and prey
plasmids, respectively) are introduced in a cell containing one or more reporter genes.
Normally, an auxotrophy marker is used as reporter gene which allows yeast cells to
grow on a selective medium lacking from this specific gene product. The interaction X-
Y makes possible the activation of the expression of the auxotrophy marker and yeast
cell survival as BD and AD can bind together to the promoter (Figure 5.3). If
something (e.g. inhibitor) impedes the interaction among X and Y proteins, cells will
not be able to grow as the auxotrophy marker promoter will not be activated.
There are a large number of Y2H versions involving different BD and AD. In the
experiments performed in this section, pGilda and pJG4-5 plasmids were employed as
bait and prey, respectively (see Figure 5.4 and Table 5.1 for plasmid and constructs
description). Genetically modified yeast strain EGY048 unable to produce tryptophan
and histidine and containing the leucine reporter gene (LEU2, activable by LexA and
92
B42 binding) [226, 227] was grown in a restrictive medium and transformed according
to Figure 5.5.
promoter reporter gene
X Y
ADBD
Transcriptionmachinery
ON
I
promoter reporter gene
X
Y
AD
BD OFF
XAD
BD
Z
OFF
promoter reporter gene
A
B
C
promoter reporter gene
X Y
ADBD
Transcriptionmachinery
ON
promoter reporter gene
X Y
ADBD
X Y
ADADBD
Transcriptionmachinery
ON
I
promoter reporter gene
X
Y
AD
BD OFF
I
promoter reporter gene
X
Y
AD
Y
ADAD
BD OFF
XAD
BD
Z
OFF
promoter reporter gene
XADAD
BD
Z
OFF
promoter reporter gene
A
B
C
Figure 5.3. Yeast two-hybrid system (A) Binding domain (BD) is fused to protein X. Unless a protein Y is able to interact with X, the activation domain (AD) will not be in close proximity to BD and transcription machinery will not be recruited. If cells are growing in a restrictive medium and the reporter gene is an auxotrophic marker no cell growing will be observed in this condition. (B) Interaction among X and Y proteins results in a functional activator of transcription. Transcription machinery is recruited and the reporter gene is transcripted. Cell growth will be observed. (C) The introduction of molecules that impede interaction among X and Y proteins (I) leads to the inhibition of reporter gene transcription. No cell growth will be obtained.
pGilda6570 nt
pJG4-56449 nt
pGilda6570 nt
pJG4-56449 nt
Figure 5.4. pGilda and pJG4-5 plasmids structure (modified from [228, 229]).
93
Table 5.1. Plasmid constructs employed in the experiments. Plasmid Selection gene Promoter BD AD Fusion proteins
pGilda His3 GAL1* LexA -
Empty
Apaf-1 (95-530)
Apaf-1 CARD
Procaspase 9 CARD
pGJ4-5 Trp1 GAL1* - B42
Empty
Apaf-1 (95-530)
Apaf-1 CARD
Procaspase 9 CARD *Galactose inducible expression. No expression in presence of glucose.
Transformed cells were selected and the good performance of the system was checked
in different mediums (Figure 5.5). In a restrictive medium lacking from glucose, His,
Trp and Leu and containing galactose, only yeast cells transfected with a bait and prey
plasmids able to interact (constructs 1, 6, 12 and 15 in Figure 5.5) were able to grow.
Proved the selectivity of the system, the ability of QM31 to inhibit Apaf-1 was tested.
The aim of this experiment was to determine if the ability of peptoid 1 and derivatives
to inhibit apoptosome function was caused by inhibiting Apaf-1 oligomerization or
CARD(Apaf-1) - CARD(procaspase 9) interaction. With this objective, cell systems 1,
6, 12 and 15 were treated with different concentrations of the compound and incubated
for 96 h. After this time, no significative differences in the OD values were observed in
the systems 1, 6 and 15. However, the addition of QM31 in cell system 15 inhibited
cell growing when compared with the control (Figure 5.6). These results indicate that
QM31 does not inhibit Apaf-1 oligomerization but it impedes the interaction between
Apaf-1-CARD and procaspase 9-CARD domains as it was demonstrated in yeast cell
system 15.
94
Figure 5.5. Yeast two-hybrid system. (A) Bait and prey plasmids transformations made in yeast EGY048 strain. (B) Restrictive medium lacking glucose, his and trp was employed to select cells transformed with both bait and prey plasmids. (C) Restrictive medium lacking glucose, his, trp and leu was used to select the existence of two-hybrid interactions among bait and prey plasmids. (D) Restrictive medium lacking galactose, his, trp and leu to demonstrate the dependence of hybrid-protein expression on GAL1 promoter. In the presence of glucose, GAL1 promoter is inhibited and no expression of fusion proteins or his and trp aminoacids is obtained.
95
00,20,40,60,8
11,21,41,61,8
pGilda/pJG4-5 pGilda Apaf-1CARD/pJG4-5 C9
CARD
pGilda C9CARD/pJG4-5Apaf-1 CARD
pGilda Apaf-1 (95-530)/pJG4-5 Apaf-
1 (95-530)
OD
(600
nm
)1.8
1.41.6
1.21
0.80.60.40.2
000,20,40,60,8
11,21,41,61,8
pGilda/pJG4-5 pGilda Apaf-1CARD/pJG4-5 C9
CARD
pGilda C9CARD/pJG4-5Apaf-1 CARD
pGilda Apaf-1 (95-530)/pJG4-5 Apaf-
1 (95-530)
OD
(600
nm
)1.8
1.41.6
1.21
0.80.60.40.2
0
Figure 5.6 OD values at 600 nm observed 96 h after the treatment. Absorbance values for yeast cells treated with DMSO or QM31 at concentrations of 1.25, 2.5 and 5 μM (black, striped, grey and white bars, respectively) are depicted in the graphic. No significant differences in cell growing were observed at lower time-points (data not shown).
5.1.4 In silico modelization analysis
This study was performed by our collaborators Drs. Jordi Bujons and Ángel Messeguer
(Department of Chemical and Biomolecular Nanotechnology, IQAC, CSIC, Barcelona).
In a preliminary assay Apaf-1 protein surface was explored in order to determine the
QM31 potential binding points, the most probable point of interaction was located in a
cavity located in the interphase between Apaf-1 CARD and NOD domains. This cavity
is close to a probable nucleotide binding point, derived form the localization of an ADP
molecule in the crystal structure of Apaf-1 (CARD-NOD) [48]. In the next step, a
docking study focused on this cavity was done. The docking process consisted of: a)
generation of a great Lumber of QM31 conformers (max 50,000), b) study of the
possible dispositions of this ligand making use of an algorithm based on the alignment
of inertia moments, c) elimination of duplicates and evaluation of each conformer
bound to the protein employing a scoring function (London ΔG), d) selection of the 20
% more favorable conformers and optimization through a minimization of their energy,
e) elimination of duplicates and re-evaluation of the scoring function [230].
96
By this way, 1748 different dispositions were obtained representing the possible
conformations of the QM31 bound to the cavity aforementioned. From these
dispositions, Figure 5.7 represents the best result obtained for QM31 (the one with less
binding energy). The cavity is composed of the aminoacidic residues Tyr24, Asp27,
His28, Ser31, Asp32, Val69, Ser70, Asn73, Ala74, His77, Ser98, Lys100, Asp101,
Ser104, Ile106, Arg111, Val124, Val125, Arg122, Phe126, Val127, Thr128, Glu293,
Leu297, Gln416 and Asn420. As depicted in Figure 5.7, the complex is stabilized
through interactions cation−π, among the Lys100 and Arg 111 residues and two of the
aromatic substituents of QM31; hydrogen bridge interactions, among the residues His28
and Ser31 and two of the apolar residues from the carbonylic groups of the compound
and hydrophobic interactions with the apolar residues Ile106, Val124, Val125, Phe126,
Val127 and Leu297 (Figure 5.8).
The localization of QM31 in this cavity allows us to propose a possible mechanism that
explains the Apaf-1 proapoptotic activity inhibitory effect of QM31. First, this cavity is
connected through a channel with the ADP binding site, as a consequence it is possible
that QM31 blocks the access of ATP/dATP, necessary for Apaf-1 activation, to this
place. Secondly, Riedl et al. [48] propose that the CARD-CARD interaction between
Apaf-1 and procaspase 9 needs a previous conformational change of Apaf-1 in order to
show some residues less accessible in the “closed” structure of Apaf-1 (CARD-NOD).
The interaction with QM31 could stabilize the Apaf-1 “closed” structure avoiding the
interaction with the procaspase 9 CARD and inhibiting, thus, the formation of the
apoptosome. Finally, it cannot be forgotten that QM31 could be also able to bind to the
CARD-CARD complex, if this is assembled, perturbing its function.
97
Figure 5.7. Model of the complex CARD-NODApaf-1/QM31 obtained for docking. (A) The model represents the energetically most favorable disposition obtained for QM31 (yellow), while in green and blue the CARD and NOD Apaf-1 domains are represented, respectively [48]. (B) Representation of the most energetically favorable disposition of QM31 and the surface of the protein colored according to electrostatic potential (negative in red and positive in blue).
Figure 5.8. Diagram of QM31 and Apaf-1 interaction determined by docking.
98
5.2 Characterization of peptoid 1 binding site in whole cell systems
Previous results using cell extract-based experiments [158] (see chapter 4) together with
those showed in the present chapter suggest that the molecular mechanism of action of
peptoid 1, and their derivatives, will imply binding to the CARD domain of Apaf-1
inducing a conformational change that will preclude the interaction with the CARD
domain of procaspase 9. Then, the formation of the apoptosome is inhibited and the
mitochondria-dependent apoptosis signaling is interrupted. In order to further
demonstrate the proposed mechanism of action, a series of chemical biology
experiments were designed to address this question in the whole cell environment.
5.2.1 Cell extracts fractionation studies
HeLa extract cells were treated with control vehicle PBS, dox or dox in the presence of
QM56 (see Materials and Methods for further detail). Then, the different samples
where subjected to gel filtration and aliquots blotted against Apaf-1 and procaspase 9
[55]. In the control extracts, without apoptosis induction (Figure 5.9 A), there is no
evidence for the presence of procaspase 9 in those fractions that correspond to the Mw
of the apoptosome (approximately 750 kDa – circled in orange). When apoptosis is
induced after treating cells with dox (Figure 5.9 B), procaspase 9 is found at the high
Mw fractions that correspond to the apoptosome (with the antibody used for Apaf-1 we
could not detect Apaf-1 in these fractions and only the monomeric Apaf-1 in
equilibrium is detected). However, in cells treated with dox in the presence of QM56
the recruitment of procaspase 9 by the apoptosome is inhibited (Figure 5.9 C). In case
of Apaf-1 blot, it was not possible to find the protein in the blot conditions employed in
fractions higher than those expected for the monomer (MW= 135 kDa – circled in
green), so further experiments should be developed in order to improve Apaf-1
detection.
99
Figure 5.9. Gel filtration results for HeLa cells. Cells were treated with PBS A), dox 1.25 μM B) or dox in the presence of QM56 50 μM, drug-equivalents C), for 24 hours. Cells extracts were obtained and a gel filtration chromatography was developed. 750 μL fractions were collected and a Western-Blot against Apaf-1 (circled green) and procaspase 9 (circled orange) was performed.
5.2.2 Inhibition of apoptosome-dependent apoptosis induction
As previously shown in chapter 4, an “only intrinsic” apoptosis system was employed in
order to determine the specificity of peptoid 1 derivatives for its target, Apaf-1. This
system was based on the doxy-dependent overexpression of the MOMP inducing
protein Bax. In the attempt to better characterize peptoid 1 target and discard any
possible off-target effect of our compounds related to mitochondrial proteins, apoptosis
was induced in an apoptosome exclusive-dependent manner by the addition of the
apoptosome assembly activator described by Nguyen et al. [151], compound 2 (Figure
5.10 - see Introduction)
100
N
O O
Cl Cl
Figure 5.10. Chemical structure of compound 2.
For our purposes, U937 cell lines were plated and treated with 20 μM compound 2
alone of in combination with the polymeric conjugates QM56 and QM31-P4 at 50 μM
drug-equivalents (see materials and methods for further information) [200] and
incubated for 24 and 48 h. Caspase 3 activity was analyzed at different times and a
markedly caspase 3 activity inhibition was observed in the presence of both QM56 and
QM31-P4, which would indicate a specificity of action of peptoid 1 and QM31 for the
apoptosome (Table 5.1).
Table 5.2. Percentage of caspase 3 activity inhibition after apoptotic induction with compound 2. Data are expressed as the mean ± SE (n = 3).
Cell line U937 Saos-2
Compounds 24 h 48 h 24 h 48 h
QM56 82 ± 1 64 ± 1 ND ND
QM31-P2 27 ± 6 95 ± 2 22 ± 2 41 ± 14
The main objective in this chapter was to map the binding site of peptoid 1 and its
derivatives on Apaf-1 and to analyze the in cell molecular mechanism of action. The
combined results from the different experimental settings that included in vitro, in silico
or ex vivo approaches converge to a selective mechanism of action. Peptoid 1 binds to
Apaf-1 CARD domain and precludes procaspase 9 recruitment as demonstrated from
fluorescence spectroscopy and NMR experiments. Such a molecular mechanism of
action occurs in vitro and in vivo as demonstrated from the Y2H and cell extracts
fractioning experiments. The binding site of peptoid 1 on Apaf-1 is probably located at
the CARD-NOD interface (docking experiments) however the ability of Apaf-1 to
oligomerize is not interfered by peptoid 1 derivatives (from Y2H experiments). Finally,
a specific apoptosome assembly activator was employed and peptoid 1 derivatives were
able to inhibit the apoptosis induction, proving again the specificity of the compounds
for its target and their efficiency inhibiting apoptosis.
101
CHAPTER 6. CHARACTERIZATION OF THE
CYTOPROTECTIVE EFFECT OF QM31
As mentioned in the introduction a critical feature of apoptosis is MOMP that leads to
the egress of proteins that activate the proteases responsible for cell death. We were
interested in the study of the mitochondria events that precede apoptosome formation
under conditions of apoptosome inhibition. The experiments shown in this section were
in part performed in the laboratories of Prof. Guido Kroemer (Cancer, Apoptosis and
Immunology laboratory at the Institut Gustave Roussy in Paris, France) and Prof.
Douglas R. Green (Department of Immunology of St. Jude’s Children Research
Hospital in Memphis, USA).
The study was mainly based on flow cytometry (FC) analysis of: i) Caspase 3 cleavage;
ii) Annexin V/Propidium iodide (Ann V/PI) staining; iii) 3,3'- Dihexyloxacarbocyanine
iodide /PI (DiOC6(3)/PI) staining; iv) Cyt c release measurements. Also a cell
clonogenic assay was implemented.
i) As it was commented in chapter 1, caspases are present in the cells as inactive
zymogens that are proteolytically activated upon apoptosis induction. The caspase 3
zymogen is hydrolyzed into the two subunits p17 and p19 that will form the active
dimer. Specific antibodies have been developed against both subunits. In the present
study we used a fluorescent labeled anti-p17 to monitorize caspase 3 cleavage. [231].
ii) The Ann V labeling was first described by Fadok et al. [232, 233]. Ann V is a
protein belonging to the family of annexin proteins. Although there is no clear function
attributed to Ann V, this family of proteins acts as membrane scaffolds and they are
implicated in the inflammatory response and apoptosis among other functions. The Ann
V labeling or affinity assay is based on the ability of this protein to bind to
phosphatidylserine (PS) membrane containing surfaces. PS exposure on the surface of
cellular membranes is a universal phenomenon of all the cells initiating the process of
apoptosis [233] and Ann V is quickly bound to this phospholipid. The conjugation of
102
Ann V to a fluorescent marker, such as the fluorescein derivative FITC (fluorescein
isothiocyanate), and its subsequent addition to cells allows detecting by fluorescence
measurements specifically cells that have initiated apoptotic process (AnnV+) [234].
However, one of the “drawbacks” of this technique is the low binding of Ann V to cells
undergoing necrosis, so in order to determine “completely dead” cells, a different
staining has to be done [233, 235]. In order to determine the amount of dead cells
propidium iodide (PI) staining is employed. PI is a fluorescent molecule able to
intercalate in the DNA only in those cells in which the plasmatic membrane is broken or
seriously damaged (PI+) [236]. For this reason, it is has been broadly used as a
complementary staining of Ann V in order to differentiate between apoptotic
(AnnV+/PI-) and necrotic cells (AnnV+/PI+).
iii) 3,3’-Dihexyloxacarbocyanine iodide (DiOC6(3)) is a green fluorescent dye
employed for the staining of vesicles, mitochondria and reticulum in the cells. In the
study of apoptosis, this dye has been employed as a marker of mitochondria membrane
potential (ΔΨm) that is related to the degree of mitochondrial integrity [236, 237]. Only
cells with an intact mitochondrial membrane are able to be stained with DiOC6(3) and
as a consequence dead cells or cells undergoing apoptosis present low DiOC6(3)
staining profile. Like Ann V, DiOC6(3) only indicates cells that undergo apoptosis but
cannot distinguish between apoptotic and dead cells. For this reason, this dye is usually
employed in combination with PI.
iv) Furthermore, the analysis of cyt c release was also performed by FC. The assay is
based on the two different localizations that cyt c can have in the cell: mitochondrial
(healthy cells) and cytosolic (apoptotic cells after MOMP). A restrictive fixation and
permeabilization of cells allows the elimination of cytosolic components without
affecting organelles. As a consequence, cytoplasmic released cyt c is washed away.
The subsequent staining with a specific anti-cyt c antibody and revelation with a
fluorescent secondary antibody (e.g. FITC conjugated) allows selective staining of cell
containing mitochondrial cyt c [238]. In order to complement the FC study of cyt c
release, cyt c based immunofluorescence studies were also performed. Here we
followed the characteristic mitochondrial network provided when cyt c is inside the
mitochondria in healthy cells that turns out to a diffuse fluorescent signal in cells
103
undergoing MOMP with cyt c released into the cytosol [239, 240]. Cyt c release studies
were complemented with an kinetic analysis. To this end a HeLa cell line stably
transfected with pBabe cyt c-GFP (Heltog) was chosen as cellular model [36, 37].
Green fluorescent protein (GFP) has broadly used as fluorogenic marker of the fused
protein under study [241]. The GFP is composed of 238 amino acids (26.9 kDa) and it
was originally isolated from the jellyfish Aequorea victoria that fluoresces green when
exposed to blue light. When compared to normal HeLa, Heltog cell lines presented a
higher division rate and were more resistant to apoptotic insults. At the same time,
when fluorescent studies were performed a green cytoplasm background was observed.
Whether or not this background was consequence of a basal leakage of cyt c was not
determined, however a selection of lowered fluorescent background cells was
performed to diminish interferences with the actual apoptosis-dependent cyt c release.
The clonogenic assay is considered as an appropriate test to determine whether or not
cells can recover after apoptotic insults. This assay is based on the ability of a cell to
proliferate indefinitely, thereby retaining its reproductive ability to form a large colony
or a clone [242, 243]. Then, cells are seeded at extremely low density (see Materials
and Methods for further information) and treated with the apoptosis-inducing agent
alone or in combination with apoptosis inhibitors. After 2-3 weeks, the amount of
colonies formed (those containing more than 50 cells) is quantified.
6.1 Characterization of QM31 cytoprotective effect in different cellular models: 6.1
Characterization of QM31 cytoprotective effect in different cellular models”
As shown in Section 4.3, QM31 was able to inhibit apoptosis in the different
transformed human cell lines tested. Moreover, QM31 efficiently inhibited cell death
induced by doxy-inducible Bax over expression in human osteosarcoma cells [200].
These functional assays, performed on intact cells, suggested that QM31 may constitute
the first representative of a new class of cytoprotective agents that inhibit the
apoptosome yet have no direct effect on the proteolytic activity of caspases.
The objective here was a comparative evaluation in terms of efficiency and final
outcome of two different antiapoptotic strategies, caspase inhibition versus apoptosome
104
inhibition. The pan-caspase peptide-based inhibitor Z-Val-Ala-Asp-fluoromethylketone
modified with an ester group Z-Val-Ala-Asp(OMe)fluoromethylketone (Z-VAD-fmk)
(Figure 6.2) to increase its cellular permeabilization was used.
6.1.1 Cytoprotective effects of QM31 and Z-VAD-fmk in A549 cells
Non small cell lung cancer (NSCLC) A549 cells are sensitive to the chemotherapeutic
drug cisplatin (cis-diamminedichloridoplatinum (II) - CDDP - Figure 6.1) and apoptosis
cell death is activated with this treatment. CDDP is a platinum-based drug employed in
the treatment of different types of cancer including NSCLC. It acts by forming
platinum complexes inside the cells. These complexes bind to DNA causing
crosslinkings that in the last term trigger apoptosis. The mechanism of apoptosis
induction is p53-dependent.
Pt
Cl NH3
Cl NH3
Figure 6.1. CDDP chemical structure.
Z-VAD-fmk is a cell-permeant pan caspase inhibitor that irreversibly binds to the
catalytic site of caspases and can inhibit induction of apoptosis. Its effect as caspase
inhibitor was first described over a decade ago [244-247], nowadays it is broadly used
as apoptosis inhibitor control. However, Z-VAD-fmk also inhibits other proteolytic
enzymes like cathepsin B and calpain I [248].
O
O
HN
O
NH
O
HN
O
O
O
F
Figure 6.2. Z-Val-Ala-Asp-fmk chemical structure.
To proceed with the comparative study, cells were pretreated with Z-VAD-fmk before
the addition of CDDP. In the case of QM31, the apoptotic insult was added before the
105
addition of QM31. Cells were then incubated for 48 h till the different stainings and
flow cytometry analysis were performed.
The proteolytic maturation of caspase 3 was monitored 48 after the addition of the
treatments. The results obtained are depicted in Figure 6.3 A,B. As expected, both Z-
VAD-fmk and QM31 relevantly reduced the percentage of cells displaying caspase 3
activation upon CDDP treatment (Figure 6.3 B). These data are in agreement with
previous data showed in chapter 5 in which caspase 3 enzymatic activity was measured
in cell extracts. When the fate of cells was monitored by the combined staining with the
vital dye PI and Ann V (Figure 6.3 C,D), we found that Z-VAD-fmk and QM31 were
equally efficient in reducing the frequency of dead (PI+) and dying cells (PI
-/AnnV
+)
(Figure 6.3 D). The fact that QM31 is slightly less potent than Z-VAD-fmk in
inhibiting caspase activation (Figure 6.3 B), yet displays an analogous efficacy in
preventing cell death (Figure 6.3 D), suggests that the cytoprotective effects of QM31
may not be exclusively mediated by caspase inhibition.
Intrigued by the cellular events underlying the aforementioned discrepancy, the effect of
QM31 and Z-VAD-fmk on mitochondrial alterations that precede cell death and in
particular on MOMP and ΔΨm dissipation was compared. MOMP was monitored by
assessing the mitochondrial release of cyt c, either by in situ immunofluorescence
microscopy, or by cytofluorometry (Figure 6.4 C,D). Using either of these two
methods, it was found that CDDP-induced MOMP was only partially inhibited by Z-
VAD-fmk, yet strongly reduced by QM31 (Figure 6.4 B,D). The loss of ΔΨm and
subsequent cell death were also determined by simultaneous staining with the ΔΨm-
sensitive cationic DiOC6(3) and PI (Figure 6.4 E,F). In accord with published
observations, Z-VAD-fmk was able to stabilize the ΔΨm even in cells that had
undergone MOMP. Z-VAD-fmk and QM31 were equally potent in maintaining high
ΔΨm levels (Figure 6.4 F). This Apaf-1-dependent inhibition of cyt c release is in
agreement with previous studies in which a MOMP amplification loop was described
involving apoptosome formation and subsequent caspases 9 and 3 activation [249]. In
this model, caspase 3 would be able to process Bid into its truncated form tBid that
subsequently would activate Bax/Bak MOMP induction. The inhibition of Apaf-1 or
caspase 9 markedly reduced the amount of cyt c released into the cytosol [42, 250].
106
However, there is still certain controversy about the existence of this loop and further
experiments should be developed in order to ensure the observations made.
Figure 6.3. A549 cells were left untreated or incubated with 50 μM Z-VAD-fmk for 1h, followed or not by the administration of 50 μM cisplatin (CDDP). When required, 1 h later, QM31 was added at the final concentration of 5 μM and cells were maintained in culture for additional 48 h prior to the cytofluorometric assessment of apoptosis-related parameters. Panels A and C depict representative caspase 3 activation profiles and AnnV/ PI dot plots, respectively. Quantitative data are reported in panels B and D (mean ± SEM, n = 4). In panel B, columns indicate the percentage of cells which stained positive for active caspase 3 (Casp-3a+ cells). In panel D, black and white columns illustrate the percentage of dead (PI+) and dying (PI-/AnnV+) cells, respectively. Asterisks indicate statistically significant data (Student’s t test, p < 0.05), as compared to CDDP-treated cells. FITC, fluorescein isothiocyanate.
107
Figure 6.4. Non-small cell lung cancer (NSCLC) A549 cells treated as in figure 7.7 were fixed, permeabilized and stained for the immunofluorescence microscopy-assisted detection of cyt c (Cyt c) release (A,B), or subjected to the cytofluorometric determination of parameters that reflect the mitochondrial status during cell death (C-F). In panel (A), representative immunofluorescence microscopy pictures are shown to exemplify mitochondrial (Cyt c mito) vs. released (Cyt c rel) Cyt c (green signal). Nuclei appear in blue due to Hoechst 33342 chromatin counterstaining. White bars indicate picture scale. Columns in panel B report the percentage of cells (mean ± SEM, n = 4) which exhibited diffuse Cyt c staining (Cyt c rel cells). Panels C and E illustrate representative Cyt c release profiles and DiOC6(3)/propidium iodide (DiOC6(3)/ PI) dot plots, respectively. Quantitative data are reported in panels D and F (mean ± SEM, n = 4). In panel D, columns indicate the percentage of cells (mean ± SEM, n = 4) exhibiting Cyt c release (Cyt c rel cells). In panel F, white and black columns report the percentage of cells characterized by mitochondrial transmembrane dissipation alone (DiOC6(3)low/PI-) or in combination with plasma membrane breakdown (PI+). Asterisks mark statistically significant results (Student’s t test, p < 0.05), as compared to cells treated with cisplatin (CDDP) alone. FITC, fluorescein isothiocyanate.
108
6.1.2 Cytoprotective effect of QM31 and q-VD-OPH in Heltog cell line
With the objective to better understand the effect of a chemically inhibited Apaf-1 in
apoptosis-induced mitochondrial cyt c release and the possible implication of Apaf-1 in
this phenotype, a more specific cellular model for the study of mitochondrial proteins
was employed. In these experiments, staurosporine (STS) was used as apoptosis
inducer. Due to the HeLa p53 low protein levels, cells were resistant to CDDP
treatment. STS is a natural high affinity but low specificity ATP-competitive kinase
inhibitor isolated from Streptomyces staurosporeus (S. staurosporeus) [251]. STS is
broadly used as an apoptosis inductor although the molecular mechanism is poorly
understood [252] (Figure 6.5).
Figure 6.5. Chemical structure of STS.
As a caspase inhibitor control (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-
methyl ketone (q-VD-OPH) was employed [248, 253, 254]. This compound is a pan
caspase inhibitor less effective in vitro than Z-VAD-fmk but having better cell
internalization properties (Figure 6.6) [254].
NNH
HN
O O
O
O
O
O
F
F
Figure 6.6. Chemical structure of q-VD-OPH.
In this study cells were treated with STS alone or in combination with q-VD-OPH or
QM31 according to the protocol described in the Materials and Methods section. Then,
two different assays were performed: Ann V labeling and cyt c release kinetic studies.
109
The former was employed as a control of the appropriate activity of the inhibitors in the
cellular model. Treatments were added and 24 h later the cytoprotective effect of both
compounds was evaluated through FC employing the Ann V (Figure 6.7). None of the
compounds were toxic at the concentrations tested. However, STS-induced apoptosis
could not be inhibited by QM31 in this particular model cell line. The results obtained
differ from the ones observed in A549 cell line in which CDDP plus QM31 treated cells
showed a markedly decrease in cyt c release when compare to the CDDP control. A
possible explanation of this unexpected inability of QM31 to inhibit apoptotic cell death
could be related to the extended kinase inhibition state of the cell or could be related to
the use of Heltog cell line. As stated before, a green background was observed in the
cytoplasm of the cell, but we could not determine it this background was caused by a
basal cyt c-GFP release in the cytosol or by a GFP unexpected overexpression. Also,
cyt c-GFP can force apoptosome rearrangements and difficult QM31 binding to Apaf-1.
0102030405060708090
100
Control - QM31 - QM31
STS 0.25uM STS 0.25uM + qVD 20 uM
% A
nnex
in V
+ ce
lls (%
Tot
al c
ells
)
Figure 6.7. Heltog cell lines were left untreated or incubated with 0.25 μM STS for 6 h, when required QM31, q-VD-OPH or both were added at the final concentration of 5 μM and 20 μM, respectively. Then, medium was changed and fresh medium with the required compounds was added. Cells were maintained in culture for additional 24 and 48 h (black and grey bars, respectively) prior to the cytofluorometric assessment of Ann V staining.
Despite the lack of apoptotic inhibitory effect of QM31, some experimental evidences
suggested differences on cyt c release on q-VD-OPH- or QM31-treated cells. Then, cyt
c release kinetics were performed according to the protocol described in Materials and
Methods section. Briefly, a confocal microscope was employed and FITC and dsRed
fluorescence corresponding to cyt c-GFP and PI dye were measured by taking pictures
110
of the different samples in study for a period of 37 h. The counting of the time-point in
which individual cells released cyt c and stained for PI was done and then a comparison
among the different samples was carried out. Results obtained are depicted in Figure
6.8. According to previous data, QM31 was not able to inhibit cell death when
compared to STS control. However, a markedly delay in the cyt c release was observed
in the samples treated with q-VD-OPH plus QM31 when compared to the q-VD-OPH
treated cells. Then, QM31 exerted a mitoprotective effect that included the delay of cyt
c mitochondrial release.
0
5
10
15
20
25
30
35
DMSO QM31 q-VD-OPH q-VD-OPH +QM31
STS
Tim
e (h
)
B
0
5
10
15
20
25
30
35
DMSO QM31 q-VD-OPH q-VD-OPH +QM31
STS
Tim
e (h
)
B
Figure 6.8. Heltog cell line treated with STS 0.25 μM for 6 h alone or in combination with QM31 5 μM and/or q-VD-OPH 20 μM. After 6 h, STS was removed and freshly medium was with QM31 and/or q-VD-OPH. Pictures from the different samples were taken employing a confocal microscope (FITC and dsRed filters) for an interval of 37 h every 15 min added. (A) A representative example of the pictures taken is shown. (B) Quantification of the cyt c release and PI+ kinetics.
A more detailed experiment was then set up. The kinetic experiments started with
apoptosis induction on Heltog cells (see Material and Methods for further information)
111
and were followed by continuous fluorescence microscopy (pictures of the cells were
taken at different time points for a period of 37 h). As a result, samples treated with
QM31 and q-VD-OPH presented a delay in cyt c release in comparison with those
solely treated with q-VD-OPH. A short delay was also observed in cell death when
QM31 plus q-VD-OPH treatment was compared (Figure 6.8).
6.2 Clonogenic cell survival effect of QM31 and q-VD-OPH in Heltog cell line
Intrigued by the results obtained when studying cyt c release kinetics, a clonogenic cell
survival assay was performed. Our objective was to determine if the treatment with the
Apaf-1 inhibitor QM31 could have a long-time effect. In A549 cell lines we observed a
clear QM31-induced inhibition of cyt c release and in Heltog cell line a delayed release.
The question was how these and previous observations can influence cell fate? Can
QM31 rescued apoptotic cell and modify cell fate? In order to find answer to these
questions a clonogenic assay was performed. Cells were seeded and appropriately
treated (see Materials and Methods section) and 2-3 weeks later the number of colonies
formed was quantified. It was demonstrated that cells treated with QM31 in the
presence of q-VD-OPH presented a significative increase in the number of colonies
when compared to the control samples. Although, further experiments should be done,
these results could suggest the existence of an Apaf-1-dependent amplification loop in
MOMP induction. The reduction of this amplification loop due to an inhibited Apaf-1
may offer a recovery-window to cells and have time to design a cell strategy directed to
restore vital functions (Figure 6.9).
112
DMSO QM31QM31 +q-VD-OPHq-VD-OPH
CONTROL
STS
DMSO QM31QM31 +q-VD-OPHq-VD-OPH
CONTROL
STS
Figure 6.9. Clonogenic assays in Heltog. 2500 cells/well were seeded in a 12-well plate and after 48 hours cells were treated with DMSO, QM31 5 μM, q-VD-OPH 20 μM or QM31 5 μM plus q-VD-OPH 20 μM alone or in combination with STS 2 μM for six hours. Then, STS was removed and fresh medium containing the compounds was added. 24 h later, medium plus the different compounds was added and cells were let grow for 10-15 days. After that time, medium was removed and colonies were stained with methylene blue (0.5% in MeOH:H2O 1:1).
113
CHAPTER 7. APAF-1 NON-APOPTOTIC FUNCTIONS IN
CELL
Multiprotein complexes have become interesting points of chemical intervention for
therapeutic gain due to their key role in the regulation for cellular signalling pathways
[255-258]. This also applies to apoptosis-related proteins and protein complexes [259-
261]. On the other hand, it is now well recognized that protein and/or protein
complexes originally described as members of the ‘death machinery’ are implicated in
non-apoptotic functions [262-266]. Interestingly, Apaf-1, the central component of the
apoptosome, has been also described to contribute to a specific intra-S-phase DNA
damage checkpoint [94]. However, the molecular mechanism by which Apaf-1 exerts
its role in cell cycle it is not fully understood.
In previous chapters, the ability of peptoid 1 derivatives to inhibit apoptosis by
inhibiting Apaf-1 has been demonstrated. Now, the question that arises is whether or
not chemical inhibition of Apaf-1 applies exclusively to the apoptotic role of Apaf-1 or
is also extensive to the postulated role of Apaf-1 in the control of the cell cycle.
Furthermore, unexpected Apaf-1-related cell-signalling events were also evaluated from
experimental observations derived from the mechanism of action of peptoid 1
derivatives in the presence of the amino acid glutamine.
7.1 Inhibition of the intra-S-phase DNA damage checkpoint by QM31
Knockdown of Apaf-1 in human cells as well as knockout of apaf-1 in mice implicated
Apaf-1 in DNA damage-induced cell cycle arrest (reviewed in [262]). Thus Apaf-1 loss
compromises the DNA damage checkpoints elicited by ionizing irradiation or
chemotherapy. Apaf-1 depletion also reduces the activating phosphorylation of the
checkpoint kinase 1 (Chk1) provoked by DNA damage[94] and knockdown of Chk1
abrogates Apaf-1-mediated cell cycle arrest.[93]
The ability of our compounds to inhibit the apoptotic role of Apaf-1 was demonstrated
in chapter 5, but now the question to answer is the potential of these compounds to also
inhibit the Apaf-1 capability to induce cell cycle arrest after a sub-lethal damage to the
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cell. With this objective, the NSCLC A549 cell line was chosen as cellular model and
CDDP at low-doses as sub-apoptotic damage inductor. As stated in chapter 5, CDDP
induces DNA damage and apoptosis at high-doses (50-100 μM, 24-48 h treatment) in a
p53 dependent way. However, when CDDP is added to non-synchronized cells at sub-
apoptotic dosis (10-30 μM approximately, 24 h treatment) a markedly increased in the
percentage of cells in S-phase is found, which is characterized by a gradual increase
from a 2N DNA content (at the G1-S transition) to 4N one (in cells that are entering
mitosis), but no apoptotic induction is observed during the first 24 h [94].
A549 cells were treated with QM31 5 μM one hour prior the addition of CDDP at low
dosis (20 μM, 24 h). As assessed by cytofluorometric analysis of cell cycle distribution,
this manifestation of the intra-S-phase DNA damage checkpoint was gradually
subverted by increasing concentrations of QM31 (Figure 7.1 A,B). An Apaf-1-specific
siRNA (Figure 7.1 C) also reduced the number of cells retained in the S phase of the
cell cycle following CDDP sub-apoptotic stimulation (Figure 7.1 B). The degree of this
reduction (~10-15%) could have been negatively influenced by the incomplete siRNA-
mediated downregulation of Apaf-1 (Figure 7.1 C), yet was in line with previously
published results [93, 94]. In this setting, siRNA-mediated partial depletion of Apaf-1
and QM31 administration failed to exhibit additive or synergistic effects (Figure 7.1 C),
suggesting that the Apaf-1-specific siRNA and QM31 affect the same molecular
pathway of CDDP-induced cell cycle blockade.
One of the principal mediators of the Apaf-1-dependent intra-S-phase DNA damage
checkpoint is the checkpoint kinase Chk1 [94]. Chk1 activation in S phase cells delays
DNA replication, stabilizes stalled replication forks and blocks mitotic entry [267]. As
detectable by both immunoblotting (Figure 7.2 A) and immunofluorescence staining
(Figure 7.2 B), QM31 partially reduced the activating phosphorylation of Chk1
following CDDP-triggered DNA damage (Figure 7.2 C). Together this data indicate
that chemical inhibition of Apaf-1 seem to exert and inhibitory effect of the apoptotic
role of Apaf-1 but also of its cell-cycle arrest function in a similar way than siRNA for
Apaf-1.
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Figure 7.1. Non-small cell lung cancer (NSCLC) A549 cells were left untreated or transfected with a control (siUNR) or an Apaf-1-specific (siApaf-1) siRNA for 48h, and then incubated with the indicated concentration of QM31 for 1h. Thereafter, cells were cultured in the absence or in the presence of 20 μM cisplatin (CDDP) for additional 24 h, followed by cytofluorometric analysis of the cell cycle (A,B) or immunoblotting to check for Apaf-1 downregulation (C). Panel A reports representative cell cycle profiles, as obtained in a single experiment upon propidium iodide (PI) staining. 2n and 4n indicate the peaks corresponding to the G0/G1 and G2/M phases, respectively. In panel B, black, white, and grey columns illustrate the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle, respectively (mean ± SEM, n = 3). Please note how the percentage of S phase cells recorded upon CDDP administration is gradually reduced by increasing concentrations of QM31, down to a minimum that matches the value of siApaf-1-transfected cells. Panel C illustrates the partial downregulation of Apaf-1 observed after the transfection of A549 cells with siApaf-1 as compared to siUNR. An antibody recognizing β-actin was employed to control equal loading.
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Figure 7.2. Non-small cell lung cancer (NSCLC) A549 cells were transfected for 48 h with a control (siUNR) or an Apaf-1-specific (siApaf-1) siRNA, and then left untreated or incubated with 5 μM QM31 for 1 h. Finally, cells were treated or not with 20 μM cisplatin (CDDP) for 24 h, prior to immunoblotting (A) or immunofluorescence microscopy (B,C) for the assessment of the phosphorylation status of Chk1. Panel A illustrates the protein levels of phosphorylated (pChk1) and total (tChk1) Chk1, as well as those of Apaf-1 and β-actin (which was monitored to check equal loading). Panel B reports representative immunofluorescence microphotographs of untreated and CDDP-treated cells, which exhibit negative (pChk-) and positive (pChk+, nuclear bright dots) staining for phosphorylated Chk1, respectively. White bars indicate scale. In panel C, columns depict the percentage (mean ± SEM, n = 3) of pChk+ cells recorded upon the administration of CDDP to siUNR- and siApaf-1-transfected cells in the absence or presence of QM31. Asterisks indicate values that are significantly different (Student’s t test, p < 0.05) from those observed in siUNR-transfected, CDDP-treated cells.
7.2 Influence of high doses of L-glutamine in cell survival after peptoid 1
derivatives treatment
The implication of Apaf-1 in cell cycle could not be the only “extra role” exerted by the
protein. An unexpected cytotoxicity of the Apaf-1 inhibitors was observed when cells
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were grown in a medium containing permanent high concentrations of L-glutamine (2
mM). This L-glutamine high concentration environment was achieved by adding
GlutaMAX 2 mM (L-alanyl-L-glutamine dipeptide employed to elongate L-glutamine
lifetime in the medium) to the cell culture medium. In line with these observations,
recent studies have suggested a possible cytotoxic effect of L-glutamine in tumour cells
[268, 269].
In chapter 5, the lack of toxicity of peptoid 1 derivatives was demonstrated in a wide
variety of tumour cell lines. However, when such compounds were tested in
combination with high concentrations of L-glutamine (GlutaMAX addition) in the
absence of any apoptotic insult a markedly reduction in cell survival was observed.
Saos-2 and HeLa cells cultured with DMEM containing L-glutamine or GlutaMAX in
equivalent concentration were seeded in a 96-well plate and QM56 and QM31 were
added at concentrations of 100, 50 and 20 μM drug-equivalents and 10, 5 and 1 μM,
respectively. Then, MTT cell viability assays were developed following the protocol
described in Materials and Methods (Figure 7.3). A dose-dependent decrease in cell
viability was observed in both cell lines in the presence of GlutaMAX, more
pronounced in HeLa cells. Consequently, no cell recovery was observed when cells
challenged with dox or doxy were treated with QM56 or QM31 in the presence of
GlutaMAX in the cell culture media (Figures 7.3 A,B and 7.4 A,B).
In order to exclude any unspecific problem in the cells growing with high doses of L-
glutamine, GlutaMAX was removed from the culture medium of these cells and
substituted by L-glutamine. Cells were purged for 7 days and the MTT assays were
developed. Then, the cytotoxicity of both QM56 and QM31 went back to normal levels
and both compounds were able to inhibit caspase 3 activity (Figures 7.3 C and 7.4 C).
However, cell recovery assays suggested that treated cells continue dying despite the
Apaf-1-mediated QM56- and/or QM31-induced caspase 3 activity inhibition (Figures
7.3 B and 7.4 B). One month after the removal of GlutaMAX, QM56 toxic effect was
lost and the compound was able to induce cell viability recovery and caspase 3
inhibition after dox/doxy-induced apoptosis both in HeLa and Saos-2 cell lines (data not
shown).
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Figure 7.3. Influence of GlutaMAX and L-glutamine on QM56 cell toxicity in HeLa and Saos-2 cell lines. In panels A and B, cells were treated with QM56 100, 50 and 20 μM drug-equivalent (black, grey and white bars, respectively) plus 2 mM GlutaMAX, 2 mM L-glutamine after growing cells in 2 mM GlutaMAX or 2 mM L-glutamine. (A) MTT cell viability studies were developed. (B) Cell-viability recovery studies were done after adding 1 μM dox (HeLa) or doxy 2 μg/μL (Saos-2 ) to the QM56 treated cells. (C) Caspase 3 activity inhibition studies after treating cells with QM56 50 μM drug-equivalents in the presence of 2 mM GlutaMAX, 2 mM L-glutamine after growing cells in 2 mM GlutaMAX or 2 mM L-glutamine. Data depicted are mean ± SE (n > 3).
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Figure 7.4. Influence of GlutaMAX and L-glutamine on QM31 cell toxicity in HeLa and Saos-2 cell lines. In panels A and B, cells were treated with QM31 10, 5 and 1 μM drug-equivalent (black, grey and white bars, respectively) plus 2 mM GlutaMAX, 2 mM L-glutamince after growing cells in 2 mM GlutaMAX or 2 mM L-glutamine. (A) MTT cell viability studies were developed. (B) Cell-viability recovery studies were done after adding 1 μM dox (HeLa) or doxy 2 μg/μL (Saos-2 ) to the QM31 treated cells. (C) Caspase 3 activity inhibition studies after treating cells with QM31 5 μM drug-equivalents in the presence of 2 mM GlutaMAX, 2 mM L-glutamince after growing cells in 2 mM GlutaMAX or 2 mM L-glutamine. Data depicted are mean ± SE (n > 3).
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The initial conclusion from these experimental observations is that in the presence of
high glutamine concentration the chemical inhibition of Apaf-1 is deleterious to the cell.
Which can be the molecular mechanism of action? At this point and in the absence of
more detailed studies, I can only speculate. In order to address the problem studies of
autophagy induction were performed. Autophagy, a catabolic process in which cell
degrades part of the components of the cell in order to maintain homeostasis between
synthesis and degradation processes and to reallocate nutrients from unnecessary
processes to more-essential ones. Under certain cell death stimuli a massive autophagy
induction (macroautophagy) has been observed. This macroautophagy (that it will be
referred from this point as autophagy) has made consider autophagy as a type of cell
death although there are no conclusive evidences of this fact [270]. Nowadays, there is
still controversy in this point, but new evidences seem to indicate that autophagy is
more a side effect of the induction of cell death than a cell death process per se [4, 271-
273].
The molecular basis of autophagy was first described in yeast and homolog proteins
developing similar functions have been found in mammals [264, 274]. The key protein
in the initiation of autophagy is a serine threonine protein kinase, the mammalian target
of rapamicin (mTOR) that acts as an inhibitor of the process. In the absence of
nutrients, energy or with high redox levels, mTOR is inhibited and the autophagy is
initiated. The inhibition of mTOR allows activation of the proteins implicated in the
vesicle nucleation. Then, the double membrane vesicle is elongated surrounding the
cytoplasm portion to be degradated. Finally, the formed vesicle, autophagosome, fuses
to a lysosome to constitute autolysosome and the different organelles and protein
contained inside the vesicle will be degradated (see reviews [274, 275] for further
information). The techniques available to study autophagy are diverse and they are well
revised in Klionsky et al. [276]. One common way to study autophagy is the study of
the microtubule-associated protein 1 light chain 3 alpha (LC-3) distribution in the cell
[277]. This protein has a cytosolic distribution, but when autophagy is induced the LC-
3 cytoplasmic form (LC-3-I) is processed and recruited to the autophagosomes where
LC-3-II is generated by site specific proteolysis and C-terminal lipidation. The fussion
of a fluorescent marker to LC-3 makes possible to follow the fate of this protein. The
fussion of GFP to LC-3 protein allows the identification of its cytoplasmic diffused
localization (green diffused fluorescence all over the cell). When autophagy is induced,
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LC-3 is processed and it translocates to the autophagosomes. As a consequence of
autophagy-induction, LC-3 protein relocalizes to the autophagosomes and fluorescent
signal concentrates in small dots corresponding to the autophagosomes organelles
(Figure 7.5 A).
MCF-7 cells transfected with pEGFP vector containing LC-3 protein (MCF-7-eGFP-
LC3) [278] were used in an attempt to further analyze the effects of high-glutamine in
Apaf-1-inhibited cells. MCF-7-eGFP-LC3 cells were grown in DMEM containing
GlutaMAX and treated with QM31 and QM56 (see Materials and Methods section).
The eGFP-LC3 translocation to the autophagosomes was quantified 24 h after the
addition of the treatments. As a result, over 60 % of cells treated with QM31 5 μM
presented green fluorescent dots indicating the formation of autophagosomes (Figure
7.5 B). However, the counting for QM56 treated samples could not be made as a
significative percentage of the treated cells died and were detached from the cover slip.
To determine the origins of QM56-induced cell death in the current experimental
conditions, an MTT assay was performed seeding cells in a 96-well plate and treating
them with QM56 50, 25 and 10 μM drug-equivalents. The possible effect of the
polymeric carrier PGA on the cell toxicity was also studied (Figure 7.5 C). As a result,
no cell toxicity associated to the PGA carrier was found, but higher cell growing was
observed due to introduction of L-glutamic acid moieties that can be used as a source of
�"�rgy.
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Figure 7.5. MCF-7-eGFP-LC3 cells were culture in the presence of 2 mM GlutaMAX plus QM31 5, 1 μM or QM56 or PGA 50, 25 and 10 μM drug-equivalents and LC-3 translocation to the autophagosomes was studied. In panel A, representative immunofluorescence microscopy pictures are shown to exemplify cytosolic (eGFP-LC3 diffused) vs. autophagosome LC-3 localization (eGFP-LC3 dots) (green signal). Panel B shows LC-3 translocation to autophagosomes after QM31 24 h treatment. Data are expressed as mean ± SE (n =3). Finally, panel C depicts an MTT cell viability assay after the addition of QM56. Measurements were done after incubating cells for 24, 72, 96 and 120 h (black, dark grey, light grey and white bars, respectively).
The results suggest that autophagy could be implicated in a high-L-glutamine Apaf-1-
inhibited cell death. Then we would like to riskily postulated as working hypothesis the
existence of a possible connection among Apaf-1, L-glutamine metabolism and ATP
production. To the best of our knowledge only a reference on that hypothesis was
reported by Pascal Meier’s group in a D. melanogaster-based study [279]. In such a
report, glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2) was described as
an Apaf-1 interacting protein. This protein discovered in 1999 [280] is an isoform of
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GFAT1, the first rate-limiting enzyme of the hexosamine pathway that it is implicated
in the synthesis of glycoproteins. GFAT1 and 2 control the flux of glucose into the
hexosamine pathway (HP) and catalyzes the formation of glucosamine-6-phosphate.
Activation of the hexosamine pathway due to high concentrations of glucose in the
medium has been implicated in the progress of different pathologies associated to
hyperglycaemia [281-284] and alterations of AMP-activated protein kinases (AMPK)
and Ca+2 levels [281], that in turn could be correlated with both, autophagy and a state
of metabolic pseudo-hypoxia due to decreased ATP levels [285]. Interestingly,
overexpression of GFAT or treatment with glucosamine (GFAT substrate) reproduce
some of the cellular phenotypes described [283].
With these precedents we wanted to explore in an in vitro reconstituted apoptosome
assay a possible interaction between GFAT2 and Apaf-1. Recombinant GFAT2
(rGFAT2) was incubated with rApaf-1 previous to the addition of other components of
the apoptosome (see Materials and Methods for further information). As shown in
Figure 7.6, the activity of the apoptosome (measured as caspase 9 activity) was sensitive
to the presence rGFAT2. As control of the output of the assay we used recombinant
Bcl-xL (rBcl-xL) as an early described Apaf-1-interacting protein [286] that also
induced a decrease in the activity of the apoptosome and recombinant cyclin dependent
kinase-2 (rCDK-2) as unrelated protein that had no effect on the output of the assay.
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According to the data obtained in our laboratory, GFAT2 and Apaf-1 could directly
interact in normal conditions in the cell, although we are also exploring the contribution
of other proteins in a putative multiprotein comples. Further experiments should be
carried out in order to better determine the effects of this interaction in the activity of
the selected proteins. According to the preliminary data obtained, a possible
explanation for our results could be the existence of GFAT2 and Apaf-1 mutual
regulation of their activities in normal conditions. This complex (or a more populated
protein complex) would be sensitive to the inhibition of one of their main components,
Apaf-1. In the presence of high concentrations of L-glutamine, this would also lead to
the activation of hexosamine pathway by GFAT2 and the appearance of the cytotoxic
phenotypes observed, consequence of the HP activation.
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CHAPTER 8. PRIMARY CULTURE AND IN VIVO
HYPOXIA EXPERIMENTS
Different studies carried out in animals and humans have demonstrated that apoptosis is
implicated in both acute and chronic phases of ischemia related pathologies as heart
failure, stroke and those derived from organ transplantation [95, 96]. Specifically,
evidence suggests that prolonged ischemia is associated with an increased rate of
necrosis, but that reperfusion leads to an enhancement in apoptosis. Hypoxia-inducible
factor (HIF) is the principal transcription factor involved in the regulation of
transcriptional responses to hypoxia. HIF-alpha increments and induces expression of
genes involved in glycolysis, glucose metabolism, mitochondrial function, cell survival,
apoptosis, and resistance to oxidative stress. There are some proteins which expression
could be regulated by HIF and reported to bind to Apaf-1 [98, 99], so the inhibition of
Apaf-1 could have direct effect in the inhibition of hypoxia-induced apoptotic damage.
Preliminary experiments employing primary cultured cardiomyocytes were developed
and QM31 and QM56 evaluated as hypoxia-induced apoptosis inhibitors. Furthermore,
the beneficial, in vitro and ex vivo properties of the compounds as inhibitors of
apoptosis through Apaf-1 interaction make them good candidates for validation in
preclinical assays. In particular, an animal-based model for a possible application of
Apaf-1 inhibitors in organ transplantation has been also evaluated. In collaboration
with the research group of Dr. Antonio Alcaraz (Hospital Clinic, Barcelona) hot
ischemia experiments were set up in a rat model. It is known that one of the main
problems associated to both, hot and cold ischemia is the induction of apoptosis. The
time of hot and cold ischemia is determinant in the kidney viability. Hot ischemia time
is defined as the time elapsed since the heart stops beating till the start of the renal
perfusion with the preservation fluid.
The results presented in this section should be considered as preliminary in that only a
moderate number of replica have been processed due to an incomplete pharmacological
development (currently in progress, including appropriate drug-vehiculization).
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8.1 Analysis of apoptosis inhibition in primary culture cells: cardiomyocytes
under hypoxia conditions
Myocardial damage induced by ischemia-reperfusion has been shown to be directly
associated to apoptosis. Furthermore, after myocardial infarction, the presence of
apoptotic cells has been demonstrated both in the infarct area and in the border zone of
the infarction [287]. Although the signal transduction pathways are not completely
defined, it is known that mitochondrial dysfunction is one of the most critical events
associated with myocardial ischemia-reperfusion injury [288]. In this sense, previous
studies showed that very low levels of cardiac myocyte apoptosis are sufficient to
produce heart failure [289]. In addition, reduction of myocyte apoptosis by the potent
polycaspase inhibitor IDN-1965, improved left ventricular function and survival of mice
with an induced peripartum cardiomyopathy [290].
In collaboration with Drs. J. A. Montero and Pilar Sepúlveda (Cardioregeneration Unit,
CIPF), the ability of QM31 and QM56 as potential inhibitors of hypoxia-induced
apoptosis was analyzed in cardiomyocytes as an ex vivo model of myocardial infarction.
Then, primary cultures of neonatal rat cardiomyocytes were established and subjected to
hypoxic conditions. A double immunohistochemistry-based procedure clearly showed
the presence of apoptotic cells and active caspase 3 demonstrating the active role of
apoptosis in hypoxia-induced cardiomyocytes cell death (Figure 8.1) [200]. The
presence of Apaf-1 protein in neonatal cardiomyocytes was also determined by western
blot (data not shown).
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Nx HxNx Hx
Figure 8.1. Double immunohistochemistry of cardiomyocytes with anti-Caspase 3 (green) and anti-α-Actinine (red) antibodies analyzed by confocal microscopy. (40x magnification). Nucleous are stained with DAPI (blue). Nx: normoxia; Hx: hypoxia.
These results provide a validation of the cellular model selected for our ex vivo studies.
In order to complete the validation study, live-cell confocal QM56-OG internalization
studies were done incubating cardiomyocites primary culture cells in the presence of
QM56-OG at 4 ºC and 37 ºC (Figure 8.2). The quantification of the polymer cellular
internalization showed a higher internalization of QM56-OG at 37 ºC. This fact is in
agreement with the internalization mechanism proposed for polymers: endocytosis.
This mechanism requires energy consumption from the cell and the decrease of the
temperature inhibits or partially reduces the cellular uptake of the compound.
Once confirmed the cellular model for the study of apoptosis inhibition by our Apaf-1
inhibitors, neonatal cardiomyocites were subjected to hypoxia conditions (see Materials
and Methods for further information). Importantly, when the neonatal cardiomyocytes
were subjected to the hypoxia conditions in the presence of QM31 or QM56, the
percentage of cell death was clearly diminished when compared to the control cell
population (Table 8.1). Furthermore, to determine whether or not the compounds could
decrease caspase 3 activity in this cell system, cells from a 2-days old established
culture were subjected to the hypoxia conditions in the presence of different
concentrations of QM31 or QM56 and caspase 3 activity was assessed (Table 8.1).
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40
50
60
0 30 60 90 120 150 180
Time (min)
Cel
l-ass
ocia
ted
Fluo
resc
ence
(FU
)
37 ºC4 ºCB
Figure 8.2. Cellular uptake of QM56-OG in cardiomyocite primary culture cells. (A) Live-cell fluorescence confocal images at different incubation times. Images correspond to the polymer internalization (green) in the presence of Texas red-dextran (red) as lysosomal marker. (B) Graphical view of the quantification of QM56-OG internalization analyzed by flow cytometry at 4 ºC (○) and 37 ºC (♦). Data are expressed as the mean ± SD (n > 3).
129
Table 8.1. Percentage of cell recovery and inhibiton of caspase 3 activity obtained with QM31 and QM56 in hypoxia-induced apoptosis in cardiomyocytes.
QM31 QM56
% Cell-viability recovery
% Caspase 3 activity inhibition
% Cell-viability recovery
% Caspase 3 activity inhibition
Concentration (μM) Concentration (μM, drug-equivalents)
5 100 50 100 50
84 ± 2 51 ± 20 91 ± 4 49 ± 5 94 ± 3 91 ± 7
Data expressed as mean ± SE (n > 3)
Then, we have obtained initial evidences for an application of QM31 and QM56 as
inhibitors of hypoxia-induced cell death in cardiomyocytes. If the compounds here
analyzed further demonstrate to be able to reduce hypoxia-induced cardiomyocyte
apoptosis in vivo, it would result in a prevention of ventricular remodelling and
improvement of damaged cardiac function. That, in turn, would open new strategies to
improve the therapeutic resources for the treatment of this, unfortunately, very common
pathology.
8.2 Analysis in animal models
Although the results shown in the previous section are derived from ex vivo studies, we
moved ahead to the next level of complexity that represents an appropriate in vivo
model. As stated before, attention was centered to a different and very interesting
application of apoptosis inhibitors as compounds that will increase the time window of
organs from donors for transplants. However, previous to the initiation of these
experiments a safety and toxicology evaluation of a selected group of peptoid 1
derivatives in mice was performed.
130
8.2.1 Safety and toxicology evaluation of peptoid 1 derivatives in mice
The compounds selected for this detailed toxicology analysis were those that presented
improved biological activity in previous in vitro experiments and were judged to have
an appropriate therapeutic window: QM31, QM57, QM56 and QM31-P2.
CD1 mice were divided into groups of at least 3 animals, and each group was
intravenously inoculated with different dose of the compound under analysis. Mortality
was scored over a period of 7 days. Then, three animals of each group were further
subjected to blood analysis in order to evaluate from a biochemical point of view any
alteration in critical organs such as kidney (creatinin and urea), liver (creatin
phophokinase, CPK), heart (creatinine), muscle (glutamate pyruvate transaminase,
GPT). Results are depicted in Tables 8.1 and 8.2.
Table 8.2. Evaluation of toxicity in mice. It was not possible to determine the exact LD50 values due to solubility limitations.
Compound LD50
QM57
> 275 μM (DMSO, 15 mg/Kg single injection)
> 550 μM (DMSO, 30 mg/Kg double injection)
QM56 > 1840 μM (PBS, 114 mg/Kg)
QM31-P2 > 298 μM (PBS, 24 mg/Kg)
When compared to the corresponding control samples, QM57, QM56 and QM31-P2 did
not alter urea or creatinine blood levels indicating a lack of alteration in kidneys or heart
due to the presence of the compound. No alteration was observed in case of CPK levels
of the animals so no hepatotoxicity was caused. Finally, the study of GPT showed no
alteration of muscle biochemical parameters. Notwithstanding, an increase in GPT
levels were observed when PBS was employed in the drug-vehiculization. Due to the
low number of animals employed for the blood analysis (three animals per group), in
order to discard any possible damage in muscle tissues caused by PBS further
experiments employing a higher number of animals should be done.
131
Table 8.3. Blood analysis parameter results.3 Mice blood analysis parameter results after peptoid 1 derivatives treatment”
Samples Sm-UREA
(8 – 33) mg/dl
Sm-CREATININE (0.2 – 0.9) mg/dl
Sm-CPK (350 – 1800)
U/L
Sm-GPT (17 – 77) U/L
Control 1 52 0.03 6194 68 Control 2 50 0.06 11850 93 Control 3 47 0.07 9932 70
Control Average 50 ± 3 0.05 ± 0.02 9325± 2876 77 ± 14
DMSO 1 38 0.18 4291 13 DMSO 2 49 0.10 - 144 DMSO 3 31 0.53 2437 -
DMSO Average 39 ± 9 0.27 ± 0.23 3364±1311 79 ± 65 QM 57 1 47 - 2570 62 QM57 2 43 0.01 2368 35 QM57 3 31 0.53 2437 -
QM57 Average 40 ± 8 0.27 ± 0.26 2458 ± 103 49 ± 13
PBS 1 40 0.02 - 64 PBS 2 48 0.08 23807 180 PBS 3 57 - 28326 -
PBS Average 48 ± 9 0.05 ± 0.04 26067 ± 3195 122 ± 58 QM56 1 25 0.05 5965 82 QM56 2 27 0.08 12940 140 QM56 3 26 0.06 9120 93
QM56 Average 26 ± 1 0.06 ± 0.02 9342 ± 3493 105 ± 31 QM31-P2 1 32 - 10239 105 QM31-P2 2 53 0.17 - 78 QM31-P2 3 35 0.15 10260 -
QM31-P2 Average 40 ± 11 0.16 ± 0.01 10250 ± 10 92 ± 14
8.2.2 Analysis of apoptosis inhibition in an animal model of kidney hot ischemia
The shortage of donor kidneys is a critical limiting factor for renal transplantation in
treatment of end-stage renal diseases. Organs from non-heart-beating donors (NHBD)
seem to be an option to alleviate this problem effectively. However, the main obstacle
to the use of kidneys from NHBD is hot ischemia-induced kidney damage. Moreover,
in kidney transplantation, the allograft sustains inevitable cold ischemia in addition to
re-warming injury during liver reperfusion.
132
Therefore, hot ischemia-reperfusion injury of kidney grafts has become a hot topic with
theoretical and clinical significance. In particular, an application in organ
transplantation of Apaf-1 inhibitors was pursued due to the beneficial aspects that
inhibition of apoptosis can offer in order to increase the allowed time window for
functional organs from donors in organ transplantation procedures. In this study, an
observation of the effect of Apaf-1 inhibitors in Wistar rats under a different hot
ischemia time by pathological analysis, terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) analysis and evaluation of caspase 3 activity was
performed. With this purpose, seven working groups were designed with 8
animals/group (Table 8.4). At the time of the experimental design and taking into
account data from previous experiments using cultured cells, compounds QM57 and
QM31-P2 at two different concentrations were selected. In addition, QM56 was also
evaluated. Animal were intravenously injected with the compounds in the appropriate
vehicle 6 hours before application of the hot ischemia protocol. Then, a 90 min post-
mortem ischemia was applied to the kidneys that were extracted and analyze (Figure
8.2)
Table 8.4. Description of the groups of animals and pre-treatment applied at each group.
Group Pre-treatment
I (1-8) PBS control (vehicle for PGA-based derivatives)
II (9-16) DMSO control
QM57 solubilized in PBS containing a 30% DMSO (v/v) (vehicle for QM57)
III (17-24) QM57 (5 mg/Kg)
IV (25-32) QM57 (10 mg/Kg)
V (33-40) QM56 (70 mg/Kg)
VI (41-48) QM31-P2 (100 mg/Kg)
VII (49-56) QM31-P2 (45 mg/Kg)
Pathological analysis. The pathological analysis of the different kidney tissues was
based on the observation of three main parameters: nuclear loss, tubular simplification
and the existence of mitosis. It has been observed that the increase of these parameters
can be considered as an indication of tissue damage [291]. In the experiments here
133
performed a score from 0-4 was stablished. For our purposes, 90 min hypoxia tissue
damage was scored as 4 and 0 was considered as healthy tissue.
TUNEL analysis. As stated above, the TUNEL assay is a method employed in the
detection of DNA fragmentation due to the existence of apoptosis by labeling the
terminal end of nucleic acids. The assay is based on the ability of the terminal
deoxynucleotidyl transferase to detect the presence of nicks in the DNA and,
subsequently, catalyze the addition of dUTPs in the 3’ ends of DNA that are secondarily
labeled with a marker [292]. The subsequent identification of the markers by
microscopy allows to quantify the apoptosis induction in the different samples studies.
Caspase 3 assay. The assay employed was the same that the one described in chapter 4.
From the compounds selected, the heterocyclic compound QM57 in a dose-dependent
manner was the more effective on decreasing the score associated to the overall kidney
damage in pathological analysis (Figure 8.3). Differences in pharmacokinetic profile of
the macromolecular QM31-P2 and QM56 could be the reason of the differences
observed with QM57. Different kidney preparations from the animal groups above
mentioned subjected to 90 min hot ischemia, were stained for TUNEL and the results
analyzed. The analysis of variance (ANOVA) test revealed statistical differences
among the groups (p = 0.07). Interestingly, kidney preparations from animals pre-
treated with QM57, QM56 and QM31-P2 showed a reduced number of apoptotic cells
(Figure 8.4)
134
A
PBSDMSO
QM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
QM57 10 mg/Kg
QM57 5 mg/Kg
B C
AA
PBSDMSO
QM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
QM57 10 mg/Kg
QM57 5 mg/Kg
B C
PBSDMSO
QM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
QM57 10 mg/Kg
QM57 5 mg/Kg
B C
-ns0.234Kruskal Wallis
-ns0.241Anova
Pairing results
Summaryp
valueStatistical test
-ns0.234Kruskal Wallis
-ns0.241Anova
Pairing results
Summaryp
valueStatistical test
-ns0.096Kruskal Wallis
DMSO vs QM57 10 mg/Kg *0.044Anova
Pairing results
Summaryp
valueStatistical test
-ns0.096Kruskal Wallis
DMSO vs QM57 10 mg/Kg *0.044Anova
Pairing results
Summaryp
valueStatistical test
Figure 8.3. Pathological analysis of histological preparations of mice subjected to hot ischemia. (A) Score used ranged from 0 to 4; 0 is healthy kidney while 5 refers to seriously damaged kidneys. (B,C) Animals were treated with the compound subjected to analysis 6 h before the procedure of hot ischemia (90 min). Results were evaluated by Anova and the Kruskal-Wallis test. PBS and DMSO are vehicle controls. DMSO means that the compounds were administered solubilised in PBS containing a 30% DMSO (v/v).
135
A B
C D
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
A
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
A
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
AA
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Pairing results
Summary
p valueStatistical test
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Pairing results
Summary
p valueStatistical test
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Pairing results
Summary
p valueStatistical test
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Pairing results
Summary
p valueStatistical test
E F
A B
C D
A B
C D
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
A
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
A
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
AA
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Pairing results
Summary
p valueStatistical test
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Pairing results
Summary
p valueStatistical test
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Pairing results
Summary
p valueStatistical test
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Pairing results
Summary
p valueStatistical test
E FB
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
A
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
A
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
B
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
AA
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Pairing results
Summary
p valueStatistical test
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Pairing results
Summary
p valueStatistical test
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Pairing results
Summary
p valueStatistical test
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Pairing results
Summary
p valueStatistical test
E F
Figure 8.4. TUNEL assay applied to selected preparations of the hot ischemia animal model. Panels A-D. Non-normalized fields of TUNEL analysis. Bright spots label for fragmented DNA indicative of apoptotic cells. Preparations from: A. Mice 9 group DMSO control. B. Mice 18 treated with QM57 at 5 mg/kg. C. Mice 1 group PBS control. D. Mice 47 treated with QM31-GFRK(ε)-PGA at 100 mg/kg. Results were evaluated by the Kruskal-Wallis test. PBS and DMSO are vehicle controls (DMSO, refers to PBS with a 30 % DMSO content).
Finally, cellular extracts prepared from kidneys of treated and control animals were
evaluated for caspase 3 activity. As shown in Table 4 and Figure 8.5 the levels of
136
caspase 3 activity in QM56 and QM31-P2 pre-treated animals were lower that those
from control animals achieving more than 70% caspase 3 activity inhibition.
Table 8.5. Analysis of caspase 3 activity in kidney extracts. Data depicted are mean ± (SE < 10%).
Compound % Caspase 3 activity inhibiton
PBS (control) 0
DMSO (control) 0
QM57** 5 mg/Kg 0
QM57** 10 mg/Kg 0
QM56 70 mg/Kg 79
QM31-P2 45 mg/Kg 75
QM31-P2 100 mg/Kg 60 ** Visual inspection of the frozen samples suggested a possible interference of the DMSO vehicle in these samples
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
DE
VD
ase
activ
ity(Δ
F/m
in)
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
DE
VD
ase
activ
ity(Δ
F/m
in)
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
DE
VD
ase
activ
ity(Δ
F/m
in)
-**0.0025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b***0.0002Anova
Pairing resultsSummaryp valueStatistical test
-**0.0025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b***0.0002Anova
Pairing resultsSummaryp valueStatistical test
Figure 8.5. Analysis of caspase 3 activity in kidney extracts.
Collectively, the data from this section suggest that inhibition of apoptosis by Apaf-1
inhibitiors might be effective in acute indications such as renal hot-ischemia. The
present section needs from more experiments before the selection of a unique drug-
candidate. Thus, while the heterocyclic compound QM57 could be the choice
combining results from previous sections, pathological analysis and from TUNEL
137
results, QM56 and/or QM31-P2 offered better results in caspase 3 inhibition in treated
kidney extracts. The differences observed could be due on one side to the use of 30% of
DMSO as vehicle for QM57 due to its low solulity in buffered solution, and in the case
of the nanoconjugates the different pharmacokinetics could yield to a non-improvement
macroscopically. These facts could affect the output of the experiment. Almost
certainly, there is still a wide range of improvement for these first-in-class apoptosis
inhibitors by improving vehiculization for QM57 and timing schedule for the
nanoconjugates that will be probably done in a near future.
138
139
CHAPTER 9. GENERAL DISCUSSION
The main objective of the present work was the development of a second and improved
generation of chemical (pharmacological) Apaf-1 inhibitors, the characterization of the
molecular mechanism of action and the analysis of their convenience as drug
candidates. The proposal for the development of Apaf-1 inhibitors came from the
urgent need of efficient apoptotic inhibitors that could be advanced to drugs for the
treatment of the unwanted cell death associated to some diseases. Initial strategies to
inhibit apoptosis were focused on the development of caspase inhibitors although
twenty years later, the caspase-inhibitors proposed, failed to change cell’s fate due to
the existence of cell death compensatory mechanisms and other problems associated to
the required chemical properties for efficient in vitro inhibition [293]. The strategic
situation of Apaf-1, a key protein of the apoptosome, upstream of caspase 3 and
downstream of mitochondria makes this protein an interesting target in the development
of new chemical modulators. Previous to our work, only a family of urea-based
derivatives was proposed as efficient Apaf-1 inhibitors however they were identified
from a cell extract-based screening and a direct prove of Apaf-1 binding was not shown
[155]. Furthermore, neither the molecular mechanism of action nor follow up
optimization studies on these compounds have been reported. It was then proposed in
our lab that the more convenient way to find inhibitors of a protein-based
macromolecular complex should rely in the in vitro reconstitution of such a complex
using recombinant (or highly purified) proteins. We developed a medium-throughput
assay with purified recombinant Apaf-1, cytochrome c, dATP and [35S]-Met
procaspase-9. Such an assay was useful for the identification of the initial peptoid-
based hit compounds that inhibited the activity of the apoptosome.
Drug discovery is a long and tedious process involving huge amount of scientific
labour. It involves several phases from target or hit identification to preclinical
development. The process of transforming a hit into high-content lead series (hit-to-
lead) includes the following steps: hit confirmation and lead generation and
optimization phases. The hit confirmation step includes a crucial sub-step; the
compound has to exhibit cell membrane permeability. The initial peptoid 1 hit,
however, did not accomplish such a requirement and different strategies were designed
to overcome such shortage mainly defining two lead series: conformationally restricted-
140
and polymer conjugate-derivatives of the initial hit peptoid 1. The accuracy of our
proposal was early confirmed when these series, more suitable for ex vivo assays, were
tested in different cellular models and demonstrated their ability to reduce cell death.
The cell death inhibition not only was achieved when inductors of the intrinsic
apoptosis pathway were used (dox, CDDP, STS - [196, 200, 240]) but also with more
specific inductors of mitochondrial MOMP (doxy-dependent Bax overexpression -
[196, 200]) or apoptosome-formation inducers (Wells’ compound 2 - [151, 200]). .
Subsequently, we demonstrated the specificity of peptoid 1 and derivatives for targeting
Apaf-1 in different peptoid 1/Apaf-1 binding site determination assays. These assays
revealed the molecular mechanism by which peptoid 1 exerts its Apaf-1 inhibitory
effect. The Apaf-1 inhibitors bind to the interface of the CARD-NOD domains of Apaf-
1 in a way that precludes procaspase 9 recruitment without affecting Apaf-1
oligomerization.
A crucial question still to be solved in apoptosis is whether or not a cell that has
initiated the death pathway can recovery and several points of no return have been
postulated [4]. In my work I have tried to approach the problem. The results here
presented point to a positive answer: the chemical (pharmacological) inhibition of Apaf-
1 after apoptotic cell death induction not only markedly reduced apoptotic phenotype,
but also diminished the amount of cyt c release facilitating cell recovery. Encouraged
with such results and recent reports on the existence of a caspase 9-, caspase 3- and Bid-
depending MOMP amplification loop different clonogenic cell survival assays were
performed [42, 249, 250]. It was demonstrated that cells treated with QM31 showed a
significative increase in the number of colonies when compared to the control samples.
For a long time it has been assumed that caspases (as proteases responsible of the
execution of cell death), proapoptotic proteins from the Bcl-2 family as well as Apaf-1
would play a part only in cell death signaling pathways. However, a shift in thought is
now occurring and an increasing number of reports on how proteins involved in the
induction and execution of apoptosis also exhibit cell death-unrelated functions [93, 94,
294, 295]. In fact, the chemical (pharmacological) inhibition of Apaf-1 was useful to
confirm the participation of Apaf-1 in cell cycle-related events [240]. Furthermore, I
would like to hypothesize here a new role, outside from apoptosis, for Apaf-1. Cells
with Apaf-1 inhibited are extremely sensitive to high dosage of L-glutamine. A protein-
141
protein interaction between Apaf-1 and GFAT2 [280] was early reported [279] and we
have found evidences of a partial inhibitory effect of GFAT2 on the apoptosome
activity. My hypothesis, that needs now further experimentation, proposes that GFAT-2
and Apaf-1 could be mutually regulated, then having a role on the regulation of the
GFAT2 dependent hexosamine pathway activation. If confirmed, the assigned Apaf-1
cellular role move ahead as we will be in front of a key protein in the determining
cellular fate when apoptosis is induced and the metabolic machinery has to be adapted
to the new cell conditions.
Once demonstrated the efficiency of inhibiting Apaf-1 as a strategy to recover cell
viability after apoptosis induction, we moved a step forward in order to find an
appropriate therapeutic application. A kidney hot ischemia animal model was
developed and upon the treatment with peptoid 1 derivatives we obtained a markedly
reduction in kidney tissue damage. A reduction in apoptotic phenotype was obtained
but also pathology studies demonstrated the improvement in the conditions of the cell.
This fact opens the door of a possible application of Apaf-1 selective inhibitors in the
treatment of ischemia damage or in organ transplantation in order to extend the time-
lapse in which an organ can be transplanted. Besides, this is not the only possible
application. The crucial role of Apaf-1 in neuronal cell death together with the
evidences that implicate Apaf-1 in neurodegenerative diseases such as Huntington
disease make us to further explore exciting new pharmacological applications [141].
142
143
CHAPTER 10. CONCLUSIONS
1. A second generation of peptoid 1 derivatives has been synthesized (PEN-1, TAT-1,
QM31 and derivatives and QM56 and derivatives) and compounds have been proved to
be active inhibiting apoptosis in different cellular models
2. The molecular mechanism of peptoid 1 derivatives Apaf-1 inhibitory activity has
been elucidated employing different in vitro and in silico assays and different cellular
models
3. Inhibition of Apaf-1 in different cellular models in the presence of the
conformationally constrained peptoid 1 derivative, QM31, alone or in combination with
q-VD-OPH results in an inhibition/delay of cyt c release and clonogenic cell survival
after an apoptotic insult
4. The ability of QM31 to inhibit other Apaf-1 roles in the cell such as S-phase cell
cycle arrest has been demonstrated and the possible implication of Apaf-1 in the control
of metabolism has been proposed
5. A validation of Apaf-1 inhibition as a therapeutic strategy in the treatment of
ischemia damage and organ transplantation has been done through the development of
an ex vivo hypoxia cardiomyocyte primary culture and an in vivo model of kidney hot
ischemia
144
145
CHAPTER 11. MATERIALS AND METHODS
11.1 Equipment and material
11.1.1 Equipment and material employed in the synthesis of peptoid 1 and peptoid 1
derivatives
a) Equipment
Penetratin, TAT, PEN-GG-peptoid and TAT-GG-peptoid sequences were synthesized
employing an automatic peptide synthesizer 433A from Applied Biosystems.
Purifications and analysis of PEN-1, TAT-1, PEN-1-CF, TAT-1-CF and low Mw
peptoid 1 derivatives (i. e. peptoid 1-GlyGly) by preparative HPLC were carried out by
means of a Merck Hitachi L-2130 HPLC pump and a sample carrier L-2200 with a
Lichrospher® 100 C18 (250 x 10 mm) column. The eluent was monitored at 220 nm
employing a Merck Hitachi UV Detector L-2400 spectrophotometer. NMR spectra
were recorded on a Bruker Ultrashield plus 600 MHz NMR. GPC analysis was
performed using a Merck Hitachi L-2130 HPLC pump and L-2200 autosampler, with
two TSK-gel columns in series (G3000 PWXL and G2500 PWXL) and a guard column
(PWXL Guardcol). The eluent was monitored by an UV-visible spectrophotometer at
220 nm (Merck Hitachi UV Detector L-2400). A flow rate of 1 mL/min, and a mobile
phase 0.1 M PBS buffer was used. EzChrom Elite Instrument software was employed
for data analysis. Analytical RP-HPLC was performed using the same system as the
one described for GPC but with a Lichrospher® 100 C18 (150 x 3.9 mm) column, and
as mobile phase different acetonitrile gradients in aqueous 0.1 % TFA. The UV spectra
were recorded on a Jasco V-530 UV/Vis spectrophotometer. Analytical
ulracentrifugation (AUC) experiments were conducted using a Beckman Optima XL-A
equipped with a UV detection system and fitted with an An-Ti50 rotor and 6-channel,
12 mm thick charcoal-epon centrepieces. Characterization of QM56-OG was done in a
Jasco 810 Spectrofluorimeter.
146
b) Materials
PGA salt (17000-40000 Mw), DIC, 1-hydroxybenzotriazol (HOBt), di-
isopropylethylamine (DIEA), anhydrous methylformamide (DMF), HPLC grade
aminoacids and bovine cathepsin B were purchased from Sigma-Aldrich Química S. A.
(Madrid, Spain) and used without further purification. All solvents including HPLC
grade were obtained from Merck (Barcelona, Spain). All other reagents were of general
laboratory grade and were purchased from Merck unless otherwise stated. OG was
purchased from Molecular probes-Invitrogen (Barcelona, Spain). A PD-10 desalting
column from GE Healthcare España (Barcelona, Spain) was employed for QM56-OG
purification.
11.1.2 Materials and equipment employed in the biological assays
a) Equipment
Bacteria and yeast were grown separately in Binder stoves with or without the help of a
shaker IKA KS 130 Basic. Sterility was achieved by means of a Bunsen burner. Optical
density (OD) determinations and subsequent protein quantification assays were
performed in UV-Vis spectrophotometer Thermo Spectronic. Insect cells were grown
in a Raypa stove with the help of a shaker and mammal cells in a Steri-cycle CO2
incubator from Termo Electron Corporation. The manipulation of insect and mammal
cells was done in a culture hood Telstar Bio-II-A and cells were counted employing an
Olympus CKX-41 microscope, employing a Neubauer counting chamber (0.1 mm deep
and 0.0025 mm2). Centrifugations were done in an Eppendorf Centrifuge 5810R
Olympus CKX-41 microscope and ultracentrifugations in a Beckman Coulter Optima
MAX Ultracentrifuge. Protein purifications and cell extract fractionations were
performed in a FPLC® System with a LKB-UV-M-II detector from Amersham
Pharmacia Systems. Fluorescence polarization, MTT and caspase 3 activity
measurements were done in a Wallac Victor2V 1420 Multilabel HTS. FC studies were
performed employing a Cytomics FC 500 (Beckman Coulter Inc.) equipped with an
argon laser (488 nm) and emission filter for 530 nm (Vector Lab. Inc., Burlingame), a
FACSCalibur equipped with Cell Quest Pro software (Becton Dickinson) equipped with
an argon laser (488 nm) and a Violet laser (405 nm) and emission filters for 455 and
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530 nm, respectively. PEN-1, TAT-1 and QM56-OG cellular internalization studies
were performed with a Leica microscope equipped with a λ-blue 63X oil immersion
objective and handled with a TCS SP2 system, equipped with an acoustic optical beam
splitter (AOBS). Excitation was done with an argon laser (548, 476, 488, 496 and 514
nm) and blue diode (405 nm). In case of immunofluorescence studies with A549 cell
line a LSM 510 confocal fluorescence microscope (Carl Zeiss AG, Oberkochen,
Germany) was employed and the quantitative immunofluorescence assessments were
carried out on a DMIR2 inverted fluorescence microscope equipped with a DC300F
camera (Leica Microsystems GmbH, Wetzlar, Germany). NMR spectra were measured
employing a Bruker Avance Ultrashield Plus 600 MHz.
b) Materials
The necessary reagents for the preparation of bacteria and yeast medium and the
different buffers employed for the assays described were purchased from Sigma-Aldrich
Química S. A. (Madrid, Spain) unless otherwise stated. Protease inhibitors aprotinin,
leupeptine and pepstatin and Complete or Complete Mini EDTA-free Protease Inhibitor
Cocktail Tablets were purchased from Roche-Applied Science España (Barcelona,
Spain). For the isolation of plasmids the kit QIAprep Spin MIDIprep kit from Quiagen
Iberia S. L. (Madrid, Spain) was employed. Protein content was determined by means
of the Bicinchoninic Acid Protein Assay from Pierce Technology Corporation (New
Jersey, USA). His Trap HP 5 mL and Hi-Trap Q 5 mL columns from GE Healthcare
España (Barcelona, Spain) were employed for the purification of histidine tagged
proteins and anion exchange chromatography, respectively and BD TALON resin was
also employed BD Pharmingen (San Diego, USA). Resin Ni-NTA Agarose from
Qiagen Iberia S. L. (Madrid, Spain) was employed in the purification of rApaf-1 and Y-
M10 columns Millipore Ibérica (Madrid, Spain) were employed for the subsequent
concentration of the protein. A superpose 6 10/300 column GE Healthcare España
(Barcelona, Spain) was used for molecular exclusion chromatography. Media and the
different reagents necessary to grow insect, mammal and primary culture
cardiomyocytes cells were purchased from Gibco-Invitrogen (Barcelona, Spain) unless
a different supplier is indicated. Tissue culture grade DMSO, MTT, trypan blue
solution (0.4%) (cell culture grade), Epidermal Growth Factor from murine
submaxillary gland (EGF), dox, doxy, CDDP, STS, horse cyt c, dATP and
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paraformaldehyde were from Sigma-Aldrich Química S. A. (Madrid, Spain). Caspase 3
and caspase 9 fluorogenic substrates Ac-DEVD-afc and Ac-LEDH-afc, respectively,
were purchased from Biomol International (Grupo Taper, Madrid, Spain). Pan-caspase
inhibitors Z-VAD-fmk and q-VD-OPH were purchased from Promega Biotech Ibérica
S. L. (Madrid, Spain) and Biovision , Inc. (Mountain View, USA), respectively. The
antibody for Apaf-1 (mouse monoclonal, #Mab868) was purchased from R&D Systems
(Minneapolis, USA) and for procaspase 9 (rabbit polyclonal, sc-81663) was provided
for Santa Cruz Biotechnology, Inc. (California, USA). Cyt c antibody (mouse
monoclonal, #556432) was purchased from BD Pharmingen (San Diego, USA). In case
of anti-R-actinine (sarcomeric), cleaved caspase 3 (rabbit polyclonal, #9661), phospho-
Chk1 (Ser317, rabbit polyclonal, #2344) and Chk1 (rabbit polyclonal, #2345) Cell
Signaling Technology (Beverly, USA) was the supplier. β-actin antibody (mouse
monoclonal, #MAB1501) was purchase from Chemicon International (Temecula,
USA). Secondary antibodies for western-blot revelations were purchased from GE
Healthcare España (Barcelona, España). Alexa Fluor® 488 (emitting in green)
fluorochrome from Molecular Probes-Invitrogen (Barcelona, Spain) was used for
immunofluorescence revelation assays and Hoechst 33342 for nuclei staining from
Molecular Probes-Invitrogen (Barcelona, Spain). For cytometry assays BD
Pharmingen™ FITC Active Caspase 3 Apoptosis Kit from BD Pharmingen (San Diego,
USA) was employed. DiOC6(3) was obtained from Molecular Probes-Invitrogen
(Barcelona, Spain) and the PI was purchased from Sigma-Aldrich Química S. A.
(Madrid, Spain). The Annexin V-FITC and Pacific blue kits were provided by Miltenyi
Biotec GmbH (Bergisch Galdbach, Germany) and GE Healthcare, Inc. (California,
USA), respectively. Cyt c release cytofluorometry marcage was done employing the
InnocyteTM Flow Cytometric Cytochrome c Release kit Calbiochem (San Diego,
USA). The different siRNA employed were acquired from Sigma-Aldrich (Paris,
France) and the Oligofectamine® was procured by Invitrogen (Barcelona, Spain). For
TUNEL analysis the FragEL™ DNA Fragmentation Detection Kit, Fluorescent - TdT
Enzyme was employed Calbiochem (Madrid, Spain).
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11.2 Peptoid 1 and peptoid 1 derivatives synthesis
11.2.1 Synthesis of peptoid 1
Peptoid 1 was synthesized in the department of Chemical and Biomolecular
Nanotechnology, IQAC-CSIC in Barcelona supervised by Ángel Messeguer. For the
synthesis of the different peptoid 1 derivatives a peptoidyl resin (a polystyrene resin
functionalized with peptoid 1 (f = 0.75 mmol/g)) was used and Fmoc-solid phase
synthesis was employed to incorporate the different aminoacidic sequences and linkers.
11.2.2 Cell-penetrating peptides and cell-penetrating peptide/peptoid hybrids synthesis
a) Synthesis of PEN, TAT, PEN-1 and TAT-1
For the synthesis of hybrids peptide-peptoid, peptoid 1 was fused to the peptide carriers
PEN (RQIKIWFQNRRMKWKK-NH2) and TAT (YGRKKRRQRRRG-NH2) peptides.
PEN and TAT control peptides were synthesized by Fmoc-based solid phase synthesis
[196]. The hybrids PEN-1 and TAT-1 molecules were also synthesized using the same
procedure but loading the synthesizer with the pre-assembled peptoidyl 1-resin.
Peptides and conjugates were cleaved from the resin by treatment with trifluoroacetic
acid (TFA, 70%) and purified by preparative RP-HPLC using different acetonitrile
gradients in aqueous 0.1% TFA. The identity and purity was confirmed by HPLC and
MALDI-TOF mass spectrometry (Figure 4.2). Stock solutions of control peptides and
peptide/peptoid hybrids were prepared in PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3
mM Na2HPO4, 1.4 mM KH2PO4 pH 7.3) and the concentrations were determined by
UV spectroscopy and quantitative amino acid analysis.
b) Synthesis of CF-PEN-1 and CF-TAT-1
Synthesis of PEN-1-CF and TAT-1-CF was similar to PEN-1 and TAT-1, but
employing a petoidyl resin containing CF attached to the peptoid 1.
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11.2.3 Synthesis of QM56 and QM56-OG
11.2.3.1 Synthesis of QM56
QM56 was synthesized using a DIC-mediated coupling through a diglycyl spacer (using
the peptoid 1-GG as a reagent) to contain peptoid 1 up to a 12 mol % loading (free drug
content <1 %wt of total drug), as determined by UV-Vis spectrophotometry and HPLC
analysis [158, 192]. The synthesis can be divided in two parts: the coupling of the
linker to peptoid 1 molecule, and the conjugation of the GG-peptoid 1 to the polymeric
carrier.
The two Gly residues that act as a linker were incorporated to the pre-assembled
peptoidyl 1-resin through Fmoc-solid phase chemistry. Peptoid-GG derivative was
cleaved from the resin by treatment with TFA and purified by a preparative HPLC. The
identity and purity (95 %) was confirmed by HPLC and matrix -assisted laser
desorption ionization-time-of-flight mass spectrometry.
For the second part of the synthesis, to a solution of PGA sodium salt (0.1 g, 0.5 mmol)
with a Mw range 17000-43000 Da in water, 0.2 M HCl was added until the pH of the
solution was adjusted to 2.0. The precipitate was collected, dialyzed against water, and
lyophilized. PGA in its protonic form was dissolved in dry DMF (7.5 mL) and DIC (3
eq) and HOBt (3 eq) were added and left stirring for 15 min. Then peptoid 1-GGNH2
(0.052 mmol (12 mol %) 1 eq) was added and the pH adjusted to 8 with DIEA (1.5 mL).
The reaction was allowed to proceed at room temperature for 36 h. Thin layer
chromatography (TLC, silica) showed complete conversion of peptoid 1 (Rf = 0.5) to
polymer conjugate (Rf=0; 85:10:5/MeOH:H2O:AcOH). To stop the reaction, the
mixture was poured into CHCl3. The resulting precipitate was collected and dried in
vacuum. The sodium salt of PGA-peptoid conjugate was obtained by dissolving the
product in 1.0 M NaHCO3. This aqueous solution was dialyzed against distilled water
(MWCO 6,000-8000 Da). Lyophilization of the dialyzate yielded the product as a white
powder. A second purification step was performed by SEC using a G-25 sephadex
column (PD-10 column) in order to remove any remaining unreacted free peptoid 1.
The total peptoid content in the polymer as determined by UV (λ= 282 nm) was in the
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range of 25-30 % (w/w) (10-12 mol % functionalization), free drug content < 1wt %
total drug.
a) Determination of total drug content by UV spectroscopy
A stock solution of peptoid 1-GG-PGA in MeOH was prepared (1 mg/mL). To obtain a
calibration curve, samples were diluted using MeOH to give a concentration range of
0 –1 mg/mL. The total drug loading of the conjugates was determined by measuring the
OD at 272 nm and 281 nm in H2O. PGA in the same concentration range as the
analyzed conjugate (0–5 mg/mL) in H2O was used as blank. The extinction coefficient
found for peptoid 1-GG-PGA at 281 nm in MeOH at RT was ε = 713 L mol-1 cm.
b) Determination of free drug content by HPLC
Aqueous solutions of QM56 (1 mg/mL) were prepared, and an aliquot (100 μL) was
added to a polypropylene tube and made up to 1 mL with water. The pH of the samples
was adjusted to 8 with ammonium formiate buffer (100 μL, 1 M, pH 8.5), and then
CHCl3 (5 mL) was added. Samples were then thoroughly extracted by vortexing (3 x 10
sec). The upper aqueous layer was carefully removed and the solvent was evaporated
under N2. The dry residue was dissolved in 200 μL of HPLC grade acetonitrile. In
parallel the same procedure was carried out for the parent compound (using 200 μL of a
1 mg/mL stock aqueous solution). Addition of 1 mL of acetonitrile to redissolve the
product gave a 200 μg/mL stock from which a range of concentrations was prepared (2
to 100 μg/mL). The free amount of drug in the conjugates was determined by HPLC
and a flow rate 1 mL/min using an acetonitrile gradient in aqueous 0.1 % TFA (from 20
to 100 % of acetonitrile in 20 min). The retention time was tr = 14.3 min for peptoid 1-
GG (λ = 220 nm).
c) Determination of average molecular weight, polydispersity and behaviour in solution
of QM56
The average molecular weight (Mw), polydispersity (Mw/Mn) and the behavior of both
QM56 and control PGA in solution were analyzed by SEC, AUC and sedimentation
equilibrium.
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Analytical Ultracentrifugation. The knowledge of the partial specific volume (vbar)
(mL/gr) of PGA and PGA-peptoid was required for the evaluation of the AUC data. In
principle vbar for a protein can be calculated from its amino acid sequence, so vbar =
0.6580 or 0.6215 for PGA and PGA-peptoid, respectively. The density of the buffer
solution (Phosphate Buffer Saline (PBS)) was determined as 1.0048 g/L.
Sedimentation equilibrium studies. The six-channel charcoal-filled epon centrepieces
were filled with 85 μl of PGA or PGA-peptoid at two loading concentrations, 0.3 and 1
mg/mL. Sedimentation equilibrium was attained at a rotor temperature of 20°C at rotor
speeds of 12.5, 14 and 16 krpm, respectively, and absorbance profiles were acquired at
230 nm. Once the sedimentation equilibrium was reached at each of the defined
velocities, the apparent average molecular weight (Mw,a) was determined. The Mw,a
can be determined in a sedimentation equilibrium experiment independently of the
compound shape. It is calculated by adjusting the experimental data to the equation that
describes the radial distribution of the gradient of concentration in equilibrium, using
EQASSOC program (Beckman software packages) for a macromolecular solute.
Sedimentation velocity experiments. The sedimentation coefficient S of PGA and PGA-
peptoid was measured at 20 °C by AUC sedimentation velocity experiment using the
apparatus as described above. S (in Svedberg unit [10-13 s]) represents the speed at
which solute molecules are sedimenting to the bottom of the centrifuge cell under the
influence of a strong force field during the experiment. Epon double-sector
centerpieces were filled with 400 μLf sample solution and PBS, respectively, and
centrifuged at a rotor speed of 50 krpm at rotor temperatures of 20°C. Absorbance data
were acquired at 230 nm, and in time intervals of 2 min, with the radial increment set to
0.002 cm and taking two averages per scan; interference scans were taken in time
intervals of 1 min. The differential sedimentation coefficient distributions, c(s), were
calculated by modelling of the sedimentation velocity data using the least-squares
method. The program SEDFIT was used for this analysis.
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d) Nuclear Magnetic Resonance analysis of QM56
QM56 was dissolved in H2O+D2O at a concentration of 1mM drug-equiv. NMR
spectra were recorded on a Bruker Avance Ultrashield Plus 600 spectrometer equipped
with a 5-mm single-axis gradient TCI cryoprobe. NMR data were processed using the
program Topspin (Bruker GmbH, Karlsruhe, Germany). Homonuclear experiments
included 2D 1H, 1H NOESY and TOCSY with spectral widths of 8 kHz in both
dimensions, using a WATERGATE scheme to suppress the water signal for the H2O.
Mixing times were 400 and 80 ms for NOESY and TOCSY experiments respectively.
e) Degradation study of PGA and QM56 on incubation with Cathepsin B. Evaluation of
Mw loss and peptoid 1 release profile from QM56
Cathepsin B (5 U) was added last to a solution of 3 mg PGA or QM56 in 1 mL of a pH
6 buffer composed by 20 mM sodium acetate, 2 mM ethylenediaminetetraacetic acid
(EDTA) and 5 mM DTT. Incubation was carried out at 37°C. Aliquots (150 μL) were
taken at times up to 72 h, immediately frozen in liquid nitrogen, and stored frozen in the
dark until assayed by HPLC (analysis of 100 μL aliquots after extraction procedure)
and/or GPC (direct analysis of 50 μL aliquots). In control experiments polymers were
incubated in buffer alone (without addition of cathepsin B) to assess non-enzymatic
hydrolytic cleavage. In addition, free drug (0.75 mg/mL) was also incubated under
same conditions and later used as the reference control.
Then, released peptoid 1 was evaluated through HPLC employing the extraction method
described in section b). Mass analysis of the released compounds was carried out by
MALDI TOF TOF, being G-peptoid the major metabolite (Mw = 786 Da).
GPC Evaluation of Polymer Mw loss. The 50μL aliquots was diluted up to 200μL with
buffer (PBS) and the Mw determined by GPC using PEG standards.
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f) Stability in plasma studies
To assess degradation in plasma, the same procedure above described was followed
using as buffer PBS at pH 7.4 with a 10% of FBS.
11.2.3.2 QM56-OG (peptoid 1-GG-PGA-oregon green)
For the synthesis of QM56-OG, 30 mg of PGA (1 eq) in its protonic form were
dissolved in dry DMF (7.5 mL) and DIC (3 eq) and HOBt (3 eq) were added and left
stirring for 15 min. Then 1 mg of OG was added (1 mg, 0.01 eq, 1 % mol) was added
and the pH adjusted to 8 with DIEA (2 mL). The reaction was allowed to proceed at
room temperature for 36 h protected from light. Thin layer chromatography (TLC,
silica) showed the appearance of a fluorogenic stain in the point of application of the
sample (Rf = 0; 100% MeOH). DMF was evaporated to dryness using a high vacuum
purp. The residue was rediossolved in the minimum volume of a 1 M NaHCO3 solution
in order to obtain the insoluble salt. Then, this solution was purificed by SEC and at the
same time to determine conjugation efficiency. PGA-OG was eluted with water and 1
mL samples were collected. Fluorescence (λexc = 488 nm and λem = 530 nm) was
measured in the different fractions in order to determine the fluorescence associated to
PGA-OG samples containing the PGA-OG (first fractions) and the OG alone (final
fractions). For this purpose 5 μL of the different fractions were solved in MeOH.
Finally, fractions containing the PGA-OG were mixed and lyophilized.
Once the PGA-OG was obtained, the fluorescent QM56 analogue (QM56-OG) was
synthesized following the same protocol described for the conjugation of PGA to the
peptoid 1-GG in Section 11.2.3.1. The total drug content was determined by SEC
(PD10 columns) and fluorescence spectroscopy (yield 70-80%, OG loading 0.7-0.8 mol
%).
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11.2.4 Synthesis of conformationally constrained peptoid 1 mimetics: 1-(3’,3’-
diphenylpropyl)-4-[2’-(2’’,4’’-dichlorophenyl)ethyl]-7-[N-aminocarbonylmethyl-N-[2’-
(2’’,4’’-dichlorophenyl)ethyl]aminocarbonyl]perhydro-1,4-diazepine-2,5-dione
(QM31)
11.2.4.1 Synthesis of QM31
The synthesis of this heterocyclic derivative was carried out following a solid-phase
general procedure with microwave activation developed in at Messeguer’s lab.
(unpublished results).
11.2.4.2 Synthesis of QM31 derivatives
a) R1 moiety derivatives
Optimization of QM31 chemical structure was done by modifying R1 moiety. A series
of eleven derivatives QM31a-QM31m were initially designed. These derivatives where
synthesized employing the same strategy than for the QM31, but introducing different
substituents at R1.
b) Synthesis of QM57
Compound QM57 was synthesized employing a chemical strategy similar to than
reported for QM31. Due to patent pending issues, no chemical structure or synthesis
strategy of QM57 or its derivatives will be displayed in this section.
c) Synthesis of QM57 and analogues
The compounds were synthesized following the procedure for QM57 but by coupling
the corresponding N-alkylglycine fragment to the common intermediate. A total of
twelve QM57 derivatives were synthesized (QM57a, QM57b, QM57c, QM57d,
QM57e, QM57f, QM57g, QM57h, QM57i, QM57j, QM57k, QM57l and QM57m).
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11.2.5 Synthesis of QM56 and heterocycles QM31 and QM57 polymeric derivatives
A series of QM56 analogues and QM31 and QM57 polymeric derivatives, all of them
with different peptide linkers were synthesized. Similar synthetic protocols were
followed in order to obtain this family of nanoconjugates. However, due to patent
pending issues no structural characteristics or synthetic procedures will be shown in
detail.
11.3 E. coli manipulation
11.3.1 E. coli strains and plasmid constructs
Max Efficiency DH5α, BL21(DE3) and BL21(DE3)pLysS Codon + competent bacteria
cells (Invitrogen, Barcelona, Spain) were employed for the assays. Plasmid pET15b
was purchased from Merck Chemicals Ltd. (Nottingham, UK) and CARDApaf-1 cDNA
was subcloned into the plasmid (pET15b-CARDApaf-1, NdeI and XhoI restriction sites).
The plasmid pET23b constaining human procaspase 9 cDNA (pET23b-pc9) was kindly
donated by James A. Wells (Sunesis Pharmaceuticals, San Francisco, USA). The strain
DH5α was employed for the isolation and manipulation of pET15b-CARDApaf-1 and
pET23b-pc9 and its subsequent storage in glycerol 15 % at – 80 ºC. For the
manipulation of pET15-pc9 and pET15b-CARDApaf-1 BL21(DE3) and
BL21(DE3)pLysS Codon + competent cells were employed, respectively.
11. 3. 2 Culture conditions and manipulation of E. coli
a) Transformation and storage of the plasmids in DH5α
The strain DH5α was grown in LB (Luria Bertani) liquid (1 % (w/v) bacteriological
triptone, 0.5 % (w/v) yeast extract, 1 % (w/v) NaCl) or solid (1.5 % (w/v)
bacteriological grade agar is added to the previous recipe) medium. When transformed
with the different plasmids, LB medium was supplemented with 100 μg/ml of sterile
ampicilin (Amp).
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Transformed bacteria where then grown in 100 mL of LB containing 100 μg/ml of
sterile ampicilin (Amp) at 37 ºC overnight and the following day plasmidic DNA
extraction was done (Qiagen kit). The plasmidic DNA was sequenced and possible
mutations discarded.
b) Transformation of BL21(DE3)pLysS Codon +
BL21(DE3)pLysS Codon + were transformed with pET23b-pc9 and transformed
colonies were selected by growing bacteria in 2XTY medium (1 % (w/v) bacteriological
triptone, 1.6 % (w/v) yeast extract, 0.5 % (w/v) NaCl and pH = 7 adjusted with NaOH)
containing 100 μg/ml of sterile Amp.
BL21 plysS DE3 codon + were transformed with pET15b-CARDApaf-1. Selection of
transformed bacteria was made by adding Amp 100 μg/mL and chloramphenicol (Chlo)
25 μg/mL to the bacteria culture medium LB.
11.3.3 Obtention of recombinant proteins
a) Human procaspase 9 expression and purification
BL21(DE3)pLysS Codon + transformed with pET23b-pc9 were grown in a 1.5 mL pre-
culture at 37 ºC overnight at 255 rpm in 2XTY medium containing 100 μg/ml of sterile
Amp. The following day, the pre-culture was added to 50 mL of 2XTY containing 50
μg/mL Amp and grown for 1.5 h at 37 ºC and 200 rpm. Then, 25 mL of this culture
was added to 500 mL of the same medium and was grown at 37 ºC and 200 rpm till an
ODλ=600nm around 0.6-0.7. Then, the culture was cold till 30 ºC and expression of
induced by the addition of 0.2 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) for
3 h at 30 ºC and shaking rate of 170 rpm. Finally, culture was cold in ice and
centrifuged at 3000 g, 4 ºC for 15 min. Supernatant was discarded and pellet frozen at -
80 ºC.
The following day, pellets were thaw and resuspended in 50 mL of buffer A (100 mM
Tris-HCl pH = 8, 0.1 M NaCl) with the help of a pipette. Then, sample was sonicated
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on ice for 3 min with pauses every min to avoid sample heating and an amplitude (A) of
35 %. Once sonicated, sample was centrifuged at 17000 g at 4 ºC for 45 min and
supernatant was recovered and 0.45 μM filtered.
Then, protein was purified employing a Fast Protein Liquid Chromatography (FPLC)
apparatus and a His Trap HP 5 mL column. Column was equilibrated in buffer A and
sample was added at 4 mL/min rate and then washed with 15 mL of buffer A at 3.5
mL/min. In the next step, 225 mL of 90 % buffer A and 10 % buffer B (100 mM Tris-
HCl pH = 8, 0.5 M NaCl, 200 mM imidazole) were added. Finally, protein was eluted
with 100 mL and a gradient from 10 % of buffer B in buffer A till 100 % of buffer B.
Elution rate was 5 mL/min. The procaspase 9 subunits p35 and p10 are eluted, but
although the protein is processed there is no activation of this protein.
The eluent was purified again employing a His Trap Q HP 5 mL column. For this
purpose, column was equilibrated in buffer Ai (20 mM Tris-HCl pH = 8, 5 % (v/v)
glycerol, 1 mM dithiotreitol (DTT)). Sample was loaded at 3.5 mL and column washed
with 225 mL of buffer Ai. Then Protein was eluted with 100 mL of a gradient from
buffer Ai 100 % to buffer B 100 % (20 mM Tris-HCl pH = 8, 5 % (v/v) glycerol , 1 M
NaCl, 1 mM DTT) and a rate of 3.5 mL/min. Eluation peak was frozen at -80 ºC and
quantified employing Bradford assay and taking into account the presence of DTT in
the sample.
b) Apaf-1 XL CARD domain expression and purification
Bl21 plysS DE3 codon + transformed with pET15b-CARDApaf-1 were grown in 5 mL of
LB containing Amp and Chlo at 37 ºC, 255 rpm overnight. This preculture was
inoculated in 500 mL of LB medium supplemented with Amp 100 μg/mL and Chlo 25
μg/mL. Bacteria culture was grown at 37 ºC, 300 rpm till an ODλ = 600 nm of 0.6. Then
protein expression induction was done by adding 0.5 mM of IPTG and letting the cells
grow overnight at 20 ºC, 300 rpm.
Bacteria culture was centrifuged at 3500 rpm, 4 ºC for 20 min. Pellet was resuspended
in 25 mL of lysis buffer (5mM Tris-HCl pH 8, 300 mM NaCl and 10% glycerol)
containing 25 mg of lysozime and Complete EDTA-free Protease Inhibitor Cocktail and
159
it was incubated on ice for 30 minutes. Then, pellet was sonicated (35 % amplitud for 1
min three times).
Once the bacteria were lysated, sample was centrifuged at 12000 rpm for 20 min at 4
ºC. Supernatant was taken and centrifuged again at 12000 rpm for 1 h at 4 ºC. Finally,
supernatant was purified employing 2 mL BD TALON resin at room temperature in a
econopack 10mL from BIORAD. Column was equilibrated in 20 mL of lysis buffer
and sample was loaded by gravity. Then, the resin was washed with 20 mL of washing
buffer (25mM Tris-HCl pH 8, 300mM NaCl, 10% glycerol and 3mM imidazole).
Protein was eluted with 10 mL of elution buffer (25mM Tris-HCl pH 8, 300mM NaCl
and 250 mM imidazole) in 1 mL fraction that were kept in individual eppendorf for
subsequent characterization of the protein content. During this time, column was at
room temperature but samples were kept on ice. Finally, protein content was quantified
employing Bradford assay and the most concentrated fractions of protein were dialyzed
in a 6000-8000 MW dialysis membrane in 1 liter of dialysis buffer (20mM NaH2PO4
pH = 6) for 16 h at 4 ºC. Then, protein was kept at 4 ºC or for long storage terms at -80
ºC.
c) Apaf-1 15N-CARD domain production
Bl21 plysS DE3 codon + transformed with pET15b-CARDApaf-1 were grown in 5 mL of
LB containing Amp and Chlo at 37 ºC overnight. This preculture was inoculated in 500
mL of M9 minimal medium (12.8 g/L Na2HPO4·7H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1
g/L MgSO4·7H2O, 20 mg/L CaCl2·2H2O, 10 mg/L FeCl3. 6H2O, 1mg/L ZnCl2, 0.5mg/L
Na2MoO4. 2H2O, 0.2 mg/L MnCl2. 4H2O, 0.2 mg/L CoCl2. 6H2O, 0.1 mg/L CuCl2.
2H2O, 2 mg/L thiamine·HCl, 2 g/L glucose, 1 g/L 15NH4Cl and final pH 7.4)
supplemented with Amp 100 μg/mL and Chlo 25 μg/mL. Bacteria culture was grown at
37 ºC, 300 rpm till an ODλ = 600 nm of 0.6. Then protein expression induction was done
by adding 0.5 mM of IPTG and letting the cells grow overnight at 20 ºC, 300 rpm.
160
The purification protocol followed was the same than the one described above for the
CARD domain production with no marcage. Protein was stored at 4 ºC.
11.4 S. cerevisiae manipulation
11.4.1 S. cerevisiae strain
Genetically modified EGY048 yeast strain (MATα trp1, ura3, his3 leu2::p6LexAop-
LEU2) was employed for the development of the different yeast two-hybrid
experiments and was kindly donated by Patrick Fitzgerald (St. Jude Children’s Research
Hospital, Memphis, USA).
11.4.2 Culture conditions and manipulation of EGY048 strain
EGY048 yeast cells were grown at 30 ºC, 250 rpm in liquid Synthetic-Complete drop
out medium (SC-drop out). This medium is composed of 2 % β-galactose, 0.67 %
bacto-yeast nitrogen-base w/o amino acids and 0.2 % of drop out mix (2 g of the
different amino acids except for leucine (10 g), 0.5 g adenine, 2 g uracil, 2 g inositol and
2 g p-amino benzoic acid with a total weight of 54 g).
Yeast cells were cotransformed with the different pGilda and pJG4-5 construct plasmids
kindly provided by Patrick Fitzgerald (St. Jude Children’s Research Hospital, Memphis,
USA) (Figure 6.5). Selection was made by growing cells in SC-drop out lacking
histidine and tryptophan (SC-His-Trp/galactose). The selected cells were grown in
different media in order to test the proper working of the system chosen (Figure 6.5). In
all cases, medium employed was SC-drop out with different variations in their
composition: taking away His and Trp (SC–His-Trp/galactose); removing His, Trp and
Leu from the final mixture (SC-His-Trp-Leu/galactose) and finally eliminating His, Trp
and Leu and substituting galactose for glucose (SC-His-Trp-Leu/glucose). The
objective was to select the cells transformed with both plasmids (SC-His-Trp/galactose),
to select the cells in which there was a two-hybrid protein interaction and to test the
dependence on β-galactose of the promoters of the pGilda and pJG4-5 plasmids
introduced in the cells (SC-His-Trp-Leu/glucose).
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11.4.3 Yeast two-hybrid system assay
Colonies transformed with the systems 1, 6, 12 and 15 (see Figure 6.5 for further
information) were inoculated in 4 10 mL polypropylene tubes with 1 mL of SC-His-
Trp-Leu/galactose. Then, the 4 tubes of the different systems were treated with 5 μL
DMSO:SC-His-Trp-Leu/galactose (1:2) alone or containing 5, 2.5 and 1.25 μM of
QM31. Cells were then incubated at 30 ºC and shaked at 300 rpm up to 96 h.
Measurements of OD 600 nm were carried out daily till significant differences in cell
growing were observed.
11.5 Insect cell manipulation
11.5.1 Insect cell lines
SF9 and SF21 insect cell lines derived from pupal ovarian tissue of the fall armyworm
(Spodoptera frugiperda) were purchased from the German Collection of
Microorganisms and Cell Cultures (Braunschweig, Germany) and Invitrogen (Paisley,
UK), respectively.
11.5.2 Culture conditions and manipulation of insect cells
Insect cells were grown at 27 ºC in the absence of humity in a Raypa stove. Grace’s
medium complemented with 10% FBS, yeastsolate 4 g/L, gentamycin 50 ug/mL,
amphotericin B 0.25 ug/mL was employed and Pluronic F-68 0.1% was added for insect
cells growing in suspension.
Insect cells were grown in glass bottles with screw top for allowing the oxygenation of
the culture at 27 ºC in the absence of humidity. Bottles were filled one third from its
total capability and shaked at 130 rpm in an orbital shaking platform. Cell density and
culture viability was followed daily. Culture density oscillated between 0.3·106 to 2·106
cells/mL. Cells presented 98-100% cell viability and 2·106 cells/mL in the moment of
the virus infection.
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11.5.3 Baculovirus manipulation and recombinant Apaf-1 production
a) Baculovirus obtention
Recombinant Apaf-1-XL (rApaf-1) was obtained as previously reported [38]. Briefly,
Apaf-1 cDNA (kindly provided by G. Núñez, Michigan University) was subcloned into
pFastBac I vector with a C-terminal 9-His-tag. The expression plasmid was
transformed into DH10Bac Escherichia coli bacteria. The recombinant bacmids were
purified as recommended by the manufacturer (Invitrogen) and used to transfect SF9
insect cells.
b) Amplification and titration of viral stock
SF9 insect cells were infected in order to amplify the viral stock. For this purpose SF9
cells growing at 2·106 cells/mL were inoculated with an average of 0.1 particles of virus
per cell (multiplicity of infection, MOI). The the following formula to calculate how
much viral stock to add to obtain a specific MOI:
(pfu/ml)stock viralof Titelcells ofnumber Total x (pfu/cell) MOI(mL) required Inoculum = ,
pfu is the number of plaque forming colonies.
Cells were grown till infection phenotype appeared approximately 72-108 h after the
virus inoculation. Then, the culture was centrifuged at 800g for 5 minand supernatant
containing viruses was stored at 4 ºC protected from light.
Virus stocks were titrated employing the BacPAK Baculovirus Rapid Titer Kit
(Clontech) according to manufacturer’s instructions. Briefly, SF21 cells were seeded in
a 96 well-plate and infected with serial dilutions of the virus sample. One hour later,
virus was eliminated and methyl cellulose was added as a protective cover. The plate
was incubated for 45 h at 27 ºC. Then, methyl cellulose layer was eliminated by adding
freshly prepared ice-cold formyl buffered acetone and an immunoassay employing gf64
protein antibody, a virus envelope protein, was done and revealed employing a
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secondary antibody conjugated to peroxidase. Chromogenic peroxidase showed the
blue nucleus corresponding to the infection foci. The number of foci was quantified
employing and the viral titter was estimated following formula:
40factor x dilution x foci/well ofnumber (pfu/mL) titer Virus = ,
being 40 an empiric correction factor inherent in this assay [296].
c) Apaf-1 protein production
For the obtaining of recombinant Apaf-1, 300 mL of SF9 cells growing at 2·106
cells/mL were infected with 3 MOI, approximately 4-9 mL from virus stock with a
2·108 pfu/mL titer. The infected cells were harvested after 40 h, washed in cold
phosphate buffered saline (PBS), and resuspended in 5 volumes of lysis buffer (20 mM
Hepes-KOH pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 1 mM NaEDTA, 1 mM EGTA
(glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid), 1 mM DTT (dithiothreitol))
supplemented with Complete Protease Inhibitor Cocktail. Cells were lysated in a
dounce homogeniser with 10-14 strokes with a loose pestle. The cell lysated was
centrifuged at 10000 g for 1 h and the supernatant was injected into a Ni-NTA (Ni2þ-
nitrilotriacetate)-agarose column and it was washed with 40 mL of washing buffer
(20mM Hepes–KOH pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM NaEDTA, 1 mM
EGTA, 1 M NaCl, 25 mM imidazol, 1mM DTT). The protein was eluted in 600 ul of
elution buffer (20mM Hepes–KOH pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM
NaEDTA, 1 mM EGTA, 250 mM imidazol, 1mM DTT). Protein was concentrated
employing Y-M10 (Millipore) and buffer A to eliminate imidazol or was desalted with a
PD-10 column and then eluted with buffer A and concentrated with a Y-M10 column.
Finally, the purified protein was stored at –80 ºC in aliquots in buffer A (20 mM Hepes–
KOH pH 7. 5, 10 mM KCl, 1.5 mM MgCl2, 1 mM NaEDTA, 1 mM EGTA, 0.1 mM
PMSF, 1 mM DTT) plus 20% glycerol after being snap frozen in liquid nitrogen. The
quantification of the protein was done employing the bicinchoninic acid kit and its
ability to activate caspases was tested by mixing the protein with FT, a fraction from
HEK 293 cell extracts that lacks Apaf-1 but contains caspase 9, 3 and cyt c [87, 202]
(see section 11.4.3 for further information).
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11.6 Mammal cell lines manipulation
11.6.1 Mammal cell lines
U937 human histiocytic lymphoma and the U-2 OS human osteosarcoma cell lines were
obtained from the American Type Culture Collection (Rockville, MD). Human
embrional kidney 293 (HEK 293), A549 human lung carcinoma and HeLa human
cervix adenocarcinoma were purchased from the German Collection of Microorganisms
and Cell Cultures (Braunschweig, Germany). The Saos-2 human osteosarcoma cell line
was kindly provided by Karin Vousden (Cancer Research U. K., Glasgow). HeLa
expressing cyt c-GFP (Heltog) generated by retroviral transduction employing pBABE
mouse cyt c-GFP virus [36, 37] were kindly provided by Prof. Douglas Green (St. Jude
Children’s Research Hospital, Memphis, USA). MCF-7 peGFP-LC3 transformed cells
were kindly provided by Dr. Marja Jäättellä (Kraefte Bekaempelse, Copenhagen,
Denmark).
11.6.2 Culture conditions
U937 and MCF-7 peGFP-LC3 cells were grown in suspension in RPMI 1640 medium
supplemented with 10% FBS and 2 mM L-glutamine. The HEK 293, U-2 OS, Saos-2
and HeLa, cells were grown in Dulbecco’s modified Eagle’s medium supplemented
with 15% FBS, 2 mM L-glutamine was also added except for Saos-2. A549 were
routinely maintained in F-12 medium supplemented with 10% fetal bovine serum
(FBS), 10 mM Hepes buffer, 100 units/mL penicillin G sodium, and 100 μg/mL
streptomycin sulfate. Cells were maintained at 37 ºC in an atmosphere of 5% carbon
dioxide and 95% air and underwent passage twice weekly.
11.6.3 HEK 293 cell extracts preparation and cell-free caspase activation assays
(DEVDase activity)
Cells were seeded in 15 cm diameter plates at 1–2·106 cells per plate and harvested
when confluent (approximately 107 cells per plate). S-100 extracts from HEK 293 cells
were prepared from 2·108 cells and fractionated essentially as described by Fearnhead et
al. [147, 202]. Briefly, fractionation of extracts was carried out by ion exchange
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chromatography. An S-100 extract was separated into three fractions (F1, F2 and FT)
and its composition was confirmed by immunoblot. F1 (5 mg of protein/mL) contained
Apaf-1 and FT (30 mg of protein/mL) contained caspases-3 and -9, and cyt c. None of
these fractions had caspase activity but a combination of active recombinant Apaf-1 and
FT reconstituted caspase activation.
In order to perform the cell-free caspase activation assay, rApaf-1 (5–10 ng) was added
to FT fraction (5 μL) and incubated with 1 mM ATP or dATP at 37 ºC, for 30 min.
Then, a 3 μL aliquot was mixed with 200 μL of caspase assay buffer (PBS, 10%
glycerol, 0.1 mM EDTA, 2 mM DTT) containing 20 μM Ac-DEVD-afc. This assay is
based on the caspase 3 cleavage sequence of PARP (poli(ADP-ribose)polimerase)
protein. Caspase activity was continuously monitored following the release of
fluorescent afc (λexc = 400 nm; λem = 508 nm) in a multimarcage Wallac Victor2V 1420
Multilabel HTS. DEVDase activity was expressed as either as increase of relative
fluorescence units per min (ΔFU/min) or as percentage of the initial fluorescence signal
value obtained in the absence of inhibitor when the inhibitory activity of compounds
was evaluated [158, 200].
11.6.4 In vitro reconstitution assay of the apoptosome
For the in vitro apoptosome reconstitution assay, 5 μL of 600 μM compounds were
tested at 10 μM in 100% DMSO and 181.7 μL of master mix containing 136.7 μL
water, 40 μL 5X assay buffer (100 mM Hepes-KOH pH = 7.5, 500 mM KCl and 5 mM
DTT) and 5 μL for a final concentration of 40 nM. rApaf-1 and the compounds were
incubated for 15 min at room temperature before adding 5 μL ATP/Mg from a stock 80
mM in water and 2 uL of cyt c at concentration of 10 μM After 10 min, 5 μL of
procaspase 9 from 16 μM stock was added. The mixture was incubated at room
temperature for 5 more minutes and 5 μL of 4 mM caspase 9 fluorogenic substrate stock
was added, Ac-LEDH-afc. Caspase activity was continuously monitored following the
release of fluorescent afc at 37 ºC using a Wallac 1420 Workstation (λexc = 400 nm; λem
= 535 nm). DEVDase activity was expressed as the increase of relative fluorescence
units per minute (ΔFU/min). All the assays were validated employing different internal
controls (Table 3. 1).
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Table 11.1. Internal controls of the yeast two-hybrid assay. SAMPLE rApaf-1 Procaspase 9 Z-VAD-fmk QM31
Procaspase 9 control + - - - Apaf-1 control - + - -
Inhibition control + + + - QM31 control + + - +
GFAT2/Apaf-1 interaction studies. For study of Apaf-1/GFAT2 possible interaction,
recombinant GFAT2-GST (OriGene, USA), Bcl-xL-GST [223] and CDK2-GST [297]
proteins were employed. 40 μM rApaf-1 was incubated with GFAT2, Bcl-xL and
CDK2 proteins at a ratio concentration 1:1, 1:2 and 1:3 for 30 min at 30 ºC. Then, the
remaining components of the apoptosome were added as previously described and
caspase 9 activity was measured and results were expressed as caspase 9 activity
inhibition.
11.6.5 MTT cell viability assays
Cells were cultured in sterile 96-well microtiter plates at a seeding density of 104, 3·103,
2.5·103, and 2·103 cells/well for Saos-2, U-2 OS, HeLa, and A549, respectively, and
2.5·103 and 4·103 cells/well in the case of U937, and they were allowed to set for 24 h
[200]. Doxy (2 μg/mL in PBS) for Saos-2 cells and dox in the cases of A549, HeLa, U-
2 OS, and U937 at concentrations of 1, 1.5, 2, and 0.25 μg/mL in PBS, respectively,
were then added. After 30 min cells were treated with compounds (0.2 μm filter
sterilized) at final concentrations of 1, 5, and 10 μM in the cases of PEN-1, TAT-1, and
QM31 and its derivatives, while polymer conjugates (QM56, QM31-P and QM57-P)
were tested at concentrations of 20, 50, and 100 μM drug-equivalents. Compounds
were incubated for 19, 43 or 67 h, and then MTT (20 μL of a 5 mg/mL solution) was
added to each well. Cells were further incubated for 5 h (a total of 24 h of incubation
with the inhibitors was therefore studied). In the next step, medium was removed, the
precipitated formazan crystals were dissolved in optical grade DMSO (100 μL), and
plates were read at 570 nm.
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11.6.6 Caspase activation assays in cellular models (DEVDase activity)
Cell extracts were prepared from cells seeded in 3.5 cm diameter plates at seeding
densities of 2·105 cells per plate in the case of Saos-2, 3·105 and 3.5·105 cells per plate
for HeLa and U-2 OS, and 4·105 and 4. 5·105 cells per plate for U937, and they were
allowed to set for 24 h. Then cells were treated with doxy (2 μg/mL in PBS) for Saos-2
cells and dox in the cases of HeLa, U-2 OS, and U937 at concentrations of 2, 2.5, and
0.25 μg/mL in PBS, respectively.
After 30 min, PEN-1 and compound QM31 derivatives at 5 μM and polymer conjugates
(QM56, QM31-P and QM57-P) at 50 and 100 μM drug-equivalents were added. Cells
were harvested after 24, 48, and 72 h of incubation and pellets resuspended in 30 μL of
extraction buffer (50 mM PIPES, 50 mM KCl, 5 mM EDTA, 2 mM MgCl2, 2 mM DTT
supplemented with protease inhibitor cocktail from Sigma) and kept on ice for 5 min.
Once pellets were frozen and thawed three times, cell lysates were centrifuged at 14,000
rpm for 5 min and supernatants were collected. Quantification of total protein
concentration from these cell extracts was performed using the bicinchoninic acid
method.
Aliquots were prepared at the same concentration, and afterwards mixed with 200 μL of
caspases assay buffer (PBS, 10% glycerol, 0.1 mM EDTA, 2 mM DTT) containing 20
μM Ac-DEVD-afc. Caspase activity was continuously monitored following the release
of fluorescent afc at 37 ºC using a Wallac 1420 Workstation (λexc = 400 nm; λem = 535
nm). DEVDase activity was expressed as the increase of relative fluorescence units per
minute (ΔFU/min).
11.6.7 Cellular internalization studies by flow cytometry and confocal fluorescence
microscopy
a) Uptake of PEN-1 and TAT-1 by U937 human histiocytic lymphoma cells
U937 cells were seeded in 6-well plates at a density of 106 cells/mL and allowed to
settle for 24 h. CF labelled conjugates (CF-PEN-1 and CF-TAT-1, both 1 mM) were
added and the cells incubated for 0–60 min at 37 ºC. At each sample time, cells were
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placed on ice, the cell suspension removed and then centrifuged at 4 ºC (600 g for 10
min). Pellet was resuspended in ice-chilled PBS (5 mL), this washing procedure was
repeated three times and finally pellet was resuspended in ice-chilled PBS (200 μL) and
collected in tubes. Cell-associated fluorescence was then analysed. Data collection
involved 25,000 counts per sample and the data were analyzed with Beckman Coulter
CXP software. Data are expressed by plotting the shift in geometric mean of the entire
population. Cells incubated without the compounds were used to account for the
background fluorescence [196].
b) Cellular internalization of CF-PEN-1, CF-TAT-1 and QM56-OG by Saos-2
osteosarcoma cells
For confocal microscopy internalisation studies, 105 Saos-2 cells were seeded in glass
bottom culture dishes and left to adhere to the cover slips for 24 h. Cells were
incubated for either 0, 15, 30 min or 1 h at 37 ºC with the CF-peptides (1 μM). After
this time cells were maintained on ice, washed twice with chilled PBS and fixed with
paraformaldehyde (PFA) for 30 min at 37 ºC. Then cells were washed three times with
PBS and the samples prepared using mounting medium for fluorescence with DAPI.
Images were captured with a confocal microscope. Excitation was done with an argon
laser (548, 476, 488, 496 and 514 nm) and blue diode (405 nm).
For live-cell imaging, Saos-2 cells were seeded in glass bottom culture dishes and left to
adhere to the coverslips for 24 h. To assess the subcellular localization of the hybrids
peptide/peptoid 1, cells were incubated for either 15, 30 min or 1 h at 37 ºC in complete
medium containing CF-PEN-1, CF-TAT-1 (1 μM) or QM56-OG (0. 125 mg/mL).
Before visualization, medium containing the conjugates was removed from the cells by
aspiration, and the cells were washed three times with fresh medium (37 ºC). Finally,
clear complete media was added and cells were subsequently viewed for a maximum of
30 min. Images were captured with a confocal Leica microscope equipped with a l-blue
20X objective and handled with the same system described above [196].
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11.6.8 Circular Dichroism spectroscopy measurements
CD spectra were acquired at 5 ºC on a Jasco-810 spectropolarimeter equipped with a
Peltier temperature control in quartz cells of 0.1 cm path length. Peptides and hybrids
were analyzed at 15 μM in all cases. CD spectra were the average of 20 scans made at
1 nm intervals, and always the same buffer without peptides, used as baseline, was
subtracted. In the model membrane interaction studies, stock solutions of sodium
dodecyl sulfate (SDS) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocoline (LPC)
were prepared in PBS buffer. Results are expressed as mean molar residue ellipticities
[u]MR (deg cm2/dmol) [196].
11.6.9 Fluorescence polarization assay
Apaf-1 CARD domain protein was solved in 20mM NaH2PO4 pH = 6 buffer at
increasing concentrations (0-120 nM). Then, CF-peptoid 1 was solved in the same
phosphate buffer and added at a final concentration of 240 nM. Then, sample was
incubated for 15 min and fluorescence polarization measurements were carried out at 30
ºC at wavelengths λexc = 485 and λem = 535 nm. Data were recorded in
millipolarization units (mP) versus Apaf-1 CARD protein concentration and adjusted to
a two-state binding model.
11.6.10 NMR 15N-CARD peptoid 1 interaction assays
15N enriched CARD domain from Apaf-1 (15N-CARD) 15N-HSQC spectra was
measured. For this purpose, CARD protein was diluted in 20mM NaH2PO4 pH 6 buffer
at concentration of 75 μM. Then, to this sample PEN-1 solved in the same buffer was
added at a final concentration of 100 μM and 15N-HSQC spectra was measured again.
All the spectra were carried out in a Bruker Avance Ultrashield Plus 600 MHz.
11.6.11 In silico modelization analysis
Docking Studies were done employing the module Dock included in the program MOE
(Chemical Computing Group, Montreal, Canada), in which protein is considered as a
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rigid structure and the ligands as flexible molecules. For the energy calculations, the
strength fieldMMFF94x (modification of the MMFF94s strength field, [298, 299]) was
employed and conditions of continuous solvent (GB/VI, [230]) were chosen.
11.6.12 Gel filtration studies in whole cells
7·106 HeLa cells were seeded in 15 cm diameter plates. 24 h later, cells were treated
with dox 1 μM or dox plus QM56 at 50 μM drug-equivalents. After 24 h, cells were
harvested and centrifuged at 800 g for 5 min. Then, buffer A was added and cells were
incubated on ice for 30 min. Then, cells were centrifuged at 100000 g for 45 min.
Pellet was removed and S-100 fraction protein content quantified employing the
bicinchoninic acid kit.
Equal amounts of protein content from the different S-100 fractions were fractionated in
a molecular fractionation column previously equilibrated with gel fractionation buffer
(Hepes-NaOH pH 7 20 mM, NaCl 50 mM, Chaps 0.5 % (w/v) and sucrose 5 % (w/v)).
750 μL fractions were collected and protein content precipitated by adding TFA (1
g/mL) at a final concentration of 20 % (v/v). Then samples are incubated on ice for 30
min. Once the proteins precipitated, samples were centrifuged at 12000 rpm for 20 min
and supernatant carefully removed. Pellet was washed with cold acetone and
centrifuged again at 12000 for 15 min. Finally, acetone was removed and pellet air-
dried. 25 μL of loading buffer 1X were added to each sample and after boiling samples
for 5 min an acrylamide 10 % gel was run and samples blotted against Apaf-1 and
procaspase 9 according to manufacturer’s instructions.
11.6.13 Apoptosis induction experiments in A549 cell line
For apoptosis induction experiments, cells were either left untreated or incubated with
50 μM Z-VAD-fmk for 1 h, followed or not by the administration of 50 μM CDDP.
When required, 5 μM QM31 was administered 1h after CDDP addition, and cells were
maintained in culture for 48 h.
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a) Cytofluorometry assays in A549
Caspase 3 activation was monitored by staining the cells with a FITC-conjugated
monoclonal antibody specific for active caspase 3. To quantify apoptosis-associated
parameters, live cells were co-stained with 40 nM DiOC6(3) which measures
mitochondrial transmembrane potential (ΔΨm), and 1 μg/mL PI, which identifies cells
with ruptured plasma membrane [300]. To assess phosphatidylserine exposure and to
quantify the release of cyt c from mitochondria, the Annexin V-FITC kit and the
InnocyteTM Flow Cytometric Cytochrome c Release kit were employed, respectively,
according to the manufacturer’s recommendations. For cell cycle analysis, cells were
fixed in 80% ice-cold ethanol and labelled with propidium iodide (PI, 50 μg/mL) in the
presence of 500 μg/mL RNAse. Finally, cytofluorometric determinations were
performed.
b) Immunofluorescence microscopy in A549
Immunofluorescence assessments were carried out as previously reported [94, 301].
Briefly, cells were fixed in 4% PFA, permeabilized with 0. 1% sodium dodecylsulfate
and labelled with primary antibodies specific for cyt c according to the manufacturers’
instructions. Then, goat anti-mouse antibody coupled to Alexa Fluor® 488 (emitting in
green) fluorochrome was used for revelation. Nuclei were counterstained with 10 μM
Hoechst 33342 and slides were observed on a LSM 510 confocal fluorescence
microscope. Quantitative immunofluorescence assessments were carried out on a
DMIR2 inverted fluorescence microscope equipped with a DC300F camera.
11.6.14 Apoptosis induction experiments in Heltog cell line
HeLa expressing cyt c-GFP (Heltog) were generated by retroviral transduction
employing pBabe mouse cyt c-GFP virus [36, 37]. In this cellular model, STS was used
in order to induce cell death. CDDP could not be employed due to the low levels of p53
protein in HeLa cell line that make impossible the induction of cell death by CDDP.
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a) Heltog cell line cytometry assays
50·103 cell/well were seeded in a 12-well plate. After 24 h, cells were treated with STS
at 0.25 uM alone or in combination with QM31 5 μM, q-VD-OPH 20 μM or QM31 5
μM plus q-VD-OPH 20 μM for 6 h. Then medium was removed and fresh medium
containing QM31 5 μM, q-VD-OPH 20 μM or QM31 5 μM plus q-VD-OPH 20 μM
was added. 24 and 48 h after the treatment, phosphatidylserine exposure was quantified
by Annexin V-Pacific blue staining according to manufacturer’s recommendations.
Then cells were analyzed.
b) Clonogenic assays with Heltog cell line
2. 5·103 cells per well were seeded in a 12-well plate. After 48 h, cells were treated
DMSO, QM31 5 μM, q-VD-OPH 20 μM or QM31 5 μM plus q-VD-OPH 20 μM were
added to these cells for cytotoxicity studies. In case of clonogenic survival assays cells
were also treated with STS at different concentrations 0. 5, 1, 2 and 4 μM for 6 h. Then
medium was removed and freshly medium with DMSO, QM31 5 μM, q-VD-OPH 20
μM or QM31 5 μM plus q-VD-OPH 20 μM were added. 24 h later, medium was
changed and q-VD-OPH and QM31 compounds were added to the corresponding
samples. Cell growing was observed during the following days and after 10-15 days till
colonies were visible at sight. Then, medium was removed and wells were washed with
PBS before adding 100-200 μL of methylene blue 0.5% in methanol/water 1:1. Cells
were washed twice with PBS and plates were scanned.
c) Cytochrome c release studies in Heltog cell line
In a 12-glass bottom well plate a solution of epithelial growth factor 0. 1 mg/mL was
added for 5 min at room temperature. Then the solution was removed and after washing
the wells with PBS 50·104 cells/well Heltog cells were seeded. 24 h later, cells were
treated with STS 0.1 μM for 6 h. During this incubation time DMSO, QM31 5 μM, q-
VD-OPH 20 μM or QM31 5 μM plus q-VD-OPH 20 μM were also added to the
corresponding samples. Afterwards, medium was removed and freshly medium with
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the compounds but no STS was added. 24 h later cyt c release was quantified in the
microscope. For this purpose, 100-200 cells were counted per triplicate per well.
d) Microscopy studies with STS and QM31 and q-VD-OPH
In order to study the kinetics of cyt c release, 12. 5·104 cells were seeded in each of an 8
well-glass chamber. This chamber has been previously covered with epithelial growth
factor in order to help cells to adhere to the glass surface of the chamber. 24 h after
seeding cells, they were treated with DMSO, QM31 5 μM, q-VD-OPH 20 μM or QM31
5 μM plus q-VD-OPH 20 μM with or without STS 0.25 μM for 6 h. Then, medium was
changed and freshly medium containing Hepes 20 mM, 55 μM 2-mercaptoethanol and
PI 1:200 (1 mg/mL solution in water) and the different compounds were added to the
corresponding samples. Cyt c release kinetic was followed by taking pictures with a
confocal microscope of the cFITC and cRFP channels from 4-8 different points in the
same well of the chamber every 15 minutes for a period of 37 h.
11.6.15 Cell cycle experiments
a) siRNA transfections
A549 cells were transfected with an Apaf-1-specific siRNA (siApaf-1, sense 5’-
GAGCAGCUAUGCUGAUUAAdTdT-3’) or with an irrelevant control siRNA (siUNR,
sense 5’- GCCGUAUGCCGGUUAAGUdTdT-3’), by means of oligofectamineTM
transfection reagent, according to the manufacturer’s recommendations. Briefly, 2.
5·105 cells/well were seeded in a six-well plate and after 24 h, medium was removed
and replaced with 800 μL of F-12 medium without antibiotics or FBS. 2 μL of siRNA
100 μM were mixed with 178 μL of serum-free F-12 medium, while 4 μL of tempered
oligofectamine were added to 16 μL of serum-free F-12 medium. After five min,
oligofectamine solution was mixed with the siRNA and incubated at room temperature
for 25 min. Then, 200 μL of the mixture was added drop by drop to each well and the
plate was put back to the incubator. 4 h later, 500 μL of F-12 with 30% FBS was added
to each well. The following day, cells were reseeded and after 24 h the correspondent
treatments were added.
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b) Cell cycle arrest experiments
For cell cycle arrest assays, cells that had previously been transfected with the indicated
siRNAs (or left untreated) for 48 h were incubated with 2.5, 5 or 7.5 μM QM31 for 1 h,
followed or not by the administration of 20 μM CDDP for a total time of 24 h. For
chromosomal instability determinations, cells were pre-treated or not with 5 μM QM31
for 1h and then subjected to 5 J/m2 UVC irradiation (upon removal of the culture
medium), followed by culture in normal conditions for 24 h. During the irradiation
period, control cultures were left untreated in the absence of the culture medium.
c) Immunofluorescence cell cycle arrest experiments
Immunofluorescence assessment was carried out as described in 3. 4. 13 b section.
Briefly, cells were fixed in 4% PFA, permeabilized with 0. 1% sodium dodecylsulfate
and labelled with the primary antibody phospho-Chk1, according to the manufacturers’
instructions. Then, goat anti-rabbit antibodys coupled to Alexa Fluor® 488 (emitting in
green) fluorochrome was used for revelation. Nuclei were counterstained with 10 μM
Hoechst 33342 and slides were observed on a confocal fluorescence microscope.
Quantitative immunofluorescence assessments were carried out on an inverted
fluorescence microscope.
d) Immunoblotting cell cycle arrest experiments
For immunoblotting determinations, cells were lysed on ice in a buffer containing 1%
NP40, 20 mM Hepes pH 7.9, 10 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM
orthovanadate, 1 mM PMSF, 1 mM DTT, and 10 μg/mL aprotinin, leupeptin, pepstatin
followed by centrifugation (20 min, 14,000 rpm) and collection of supernatants. Total
protein extracts were then separated on precast 4-12% polyacrylamide gradient gels and
analyzed following standard immunoblotting procedures [29, 30], by using primary
antibodies specific for Apaf-1, Chk1, phospho-Chk1 or β-actin, which was employed to
ensure equal loading of lanes.
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11.7 Cardiomyocyte primary culture
11.7.1 Culture of neonatal rat cardiomyocytes in hypoxic conditions
Rat neonatal cardiomyocytes were prepared from ventricles of 1–2 days Wistar rats by a
previously described method [290, 302] with some modifications. Briefly, hearts were
isolated, auricles were discarded, and ventricles were minced and digested with 2
mg/mL of collagenase type II for 1 h and, subsequently, with 1.2 U/mL dispase for 30
min with gently shaking. After two washes primary cultures were pre-plated for 90 min
to eliminate contaminating fibroblasts. Hypoxia was induced by incubating the
cardiomyocytes cultures in an atmosphere of 1% O2. Experiment was performed three
times with different breedings. Each procedure was repeated twice for two identically
treated culture flasks.
11.7.2 Immunohistochemistry studies in cardiomyocytes
Cardiomyocytes were fixed in 2% PFA. Immunohistochemistry was performed with
antibodies against caspase 3 (1:100) and anti-R-actinine (sarcomeric) (1:800) followed
by detection with secondary antibodies coupled to either cyanine or FITC.
11.7.3 Cellular internalization of QM56-OG in cardiomyocites
For confocal microscopy internalization studies, cardiomyocites were seeded in glass
bottom culture dishes and left to adhere to the cover slips. Cells were incubated for
either 0, 15, 60, 120 and 240 min at 4 ºC and 37 ºC with complete medium containing
the QM56-OG (50 μM drug-equivalents). Before visualization, medium containing the
conjugates was removed from the cells by aspiration, and the cells were washed three
times with fresh medium (37 ºC and 4 ºC). Finally, clear complete media with Texas
Red dye was added (20 μg/mL)was added and cells were subsequently viewed for a
maximum of 240 min.
176
11.8 In vivo kidney hot ischemia models
11.8.1 Kidney hot ischemia induction
For the development of this in vivo model, Wistar rats were chosen and 7 working
groups with 8 animals/group were designed (Table 11.2) according to the number of
compounds selected for the in vivo experiments and the corresponding vehicles used as
controls.
Hot ischemia was induced on Wistar rats at different times and kidney samples analysed
by pathological studies, TUNEL and caspase 3 activity quantification. Compounds
were previously dissolved according to Table 8.4. Then, the compounds were
intravenously injected through the tail vein 6 hours before induction of the hot ischemia
protocol. After 90 min post-mortem kidneys were extracted and divided in 3 parts and
treated accordingly to the analysis experiment to carry out: pathological studies,
TUNEL analysis or caspase 3 activity measurements.
After this time, kidneys were removed from the animal and kidneys were divided in
three parts in order to develop pathological studies, TUNEL analysis and caspase 3
activity measurements.
11.8.2 Pathological studies
Kidney tissue were fixed in 10% buffered formaldehyde, and then washed in 0.1 M
phosphate buffer pH = 6. Then, samples were embedded in paraffin and sectioned (5
μm) [303]. These sections were stained with Masson Trichrome, and/or hematoxylin
and eosin, and viewed by light microscopy. A pathologist then performed morphologic
evaluation in blinded, randomized sections of the kidney and liver tissue. Ischemia
damaged was scored semiquantitatively from normal tissue (0) till severely damaged
tissue (5). The parameters taken into consideration were: tubular simplification,
existence of mitosis and nuclear loss.
177
11. 8. 3 TUNEL analysis
Kidney tissues were stored in formol plus 20 % sucrose overnight. The following day,
samples were washed in 0. 1 M phosphate buffer pH = 6, embedded in paraffin and
sectioned (5 μM) and stored at – 20 ºC. In order to perform the TUNEL assay paraffin
was eliminated by immersin the samples in xylene for 5 min twice and ethanol solutions
(100 %, 90 %, 80 % and 70 %) for 3 min each at room termperature. Then, slides were
briefly washed in Tris Buffer Solution (TBS) (20 mM Tris pH 7.6, 140 mM NaCl) and
carefully dry the glass slide around the sample.
Sample were permeabilized by diluting 2 mg/ml proteinase K 1:100 in 10 mM Tris pH
8, covering them completely with this solution and incubating them at room temperature
for 20 min. Then, samples were washed with TBS. Before the staining samples were
equilibrated in the antibody buffer terminal deoxynucleotidyl transferase (TdT) for 30
min. Equilibrating solution was removed and the TdT Labeling Reaction Mixture
(LRM) composed of the TdT enzyme, labelled-deoxynucleotidyl triphosphate (dNTP)
and unlabelled-dNTP according to manufacturer’s indications was added to the sample.
The slides were incubated for 30 min at room temperature and LRM was removed and
samples washed in TBS before mounting the slides employing mounting medium.
11.8.4 Caspase 3 activity in kidney extracts
Liquid nitrogen frozen kidneys were smashed while keeping them completely frozen. A
kidney powder was obtained and 500 μL extraction buffer (50 mM Hepes-KOH pH = 7,
10% sucrose, 0.1% CHAPS, 5 mM DTT, 5 mM GSSG) was added to all the samples.
Then, suspension was sonicated to break kidney cells for intervals of 15 s a total time of
60 s. After the sonication, samples were kept on ice for 15 min, centrifuged at 12000
rpm, 15 min and protein content determined employing the bicinchoninic acid assay.
Once samples were quantified, 100 μL of 800 μg protein aliquots were prepared and
mixed with 100 μL of caspase assay buffer (PBS, 10% glycerol, 0.1 mM EDTA, 2 mM
DTT) containing 20 μM Ac-DEVD-afc. Caspase activity was continuously monitored
following the release of fluorescent afc at 37 ºC using a plate reader (λexc = 400 nm; λem
= 535 nm). DEVDase activity was expressed as the increase of relative fluorescence
units per minute (ΔFU/min).
178
179
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DISCOVERY AND DEVELOPMENT OF APAF-1 INHIBITORS: ADVANCING TOWARDS UNWANTED
CELL DEATH TREATMENTS
Resumen en castellano del proyecto de tesis presentado por
Laura Mondragón Martínez
con el fin de obtener el Doctorado Europeo en Bioquímica
Directores de tesis
Dr. Enrique Pérez Payá y
Dra. María J. Vicent Docón
1
CAPÍTULO 1: INTRODUCCIÓN
1. Introducción
La muerte celular programada (programmed cell death, PCD) es un proceso de vital
importancia en el desarrollo, mantenimiento de la homeostasis de los tejidos y la
eliminación de las células dañadas [4]. Recientes estudios han dividido a la PCD en tres
categorías apoptosis, autofagia, necrosis y cornificación basándose en criterios
morfológicos, señales iniciadoras de muerte o la implicación de diferentes familias de
proteínas (Tabla 1. 1). Pese a ser clasificados de forma independiente estos procesos se
encuentran interconectados siendo en ocasiones difícil distinguir qué proceso es
iniciador de la cascada de muerte celular.
Tabla 1. 1. Diferentes tipos de muerte (modificado de [4]) Tipo de muerte celular Características morfológicas Apoptosis Células redondeadas
Retracción de los pseudópodos Reducción del volumen nuclear y citoplasmático Fragmentación nuclear Fragmentación del citoplasma en vesículas Fagocitación de los fragmentos de la célula por fagotitos presentes, in vivo
Muerte relacionada con autofagia
Ausencia de cromatina condensada Masiva formación de vesículas en el citoplasma Acumulación de vesículas de autofagia (doble membrana) Pequeña fagocitación de dichas células por fagotitos presentes, in vivo
Necrosis Rotura de la membrana celular Aumento de tamaño del citoplasma Aumento de tamaño de los orgánulos citoplasmáticos Pequeño porcentaje de cromatina condensada
Cornificación Eliminación de orgánulos citosólicos Modificaciones de la membrana plasmáticas Acumulación de lípidos en gránulos F y L Extrusión de lípidos en el espacio extracelular Descamación (pérdida de corneocitos) por activación de proteasas
2
1. 1. Apoptosis La apoptosis se define como un proceso de desmantelamiento celular inducido por la
propia célula de una forma activa y programada que lleva a su autodestrucción de una
forma silenciosa (Figura 1. 2). En mamíferos, existen dos rutas generales de activación
de la apoptosis denominadas vía extrínseca (ruta receptores de muerte) y vía intrínseca
(ruta mitocondrial). La vía extrínseca es activada por estímulos que comprenden
señales externas como la unión de ligandos de muerte a los receptores de la superficie
celular. En otros casos, la apoptosis es iniciada por señales internas que incluyen daño
en el DNA inducido por radiación o factores químicos, deprivación de factores de
crecimiento o estrés oxidativo. Estos estímulos internos suelen iniciar por lo general la
apoptosis vía mitocondrial [276].
La vía extrínseca es mediada por receptores de muerte de superficie tales como es el
caso de Fas, el receptor del factor de necrosis tumoral, o los receptores TRAIL. La
estimulación de estos receptores por parte de los ligandos de muerte da lugar a su
oligomerización y al reclutamiento de una proteína adaptadora, FADD (Fas-associated
death domain) junto con la caspasa 3 (proteasa perteneciente a la familia de proteínas
caspasas que constituyen la maquinaría ejecutora de la apoptosis), formando el
complejo de inducción de muerte DISC (death-inducing signalling complex). La
autoactivación de la caspasa 3 en el DISC induce la activación de caspasas efectoras
como la caspasas 3, 6 y 7, que llevan a cabo el programa de muerte celular.
Por su parte, la vía intrínseca es activada por diversos estímulos apoptóticos que
convergen en la mitocondria. La liberación de citocromo c (cit c) de la mitocondria al
citoplasma constituye el inicio de la cascada de activación de las caspasas. El
citocromo-c citosólico se une a Apaf-1 (protease activating factor 1) y procaspasa 9,
generando un complejo intracelular semejante al DISC conocido como apoptosoma. En
el apoptosoma, la caspasa 9 es activada, la cual encargándose de la activación de la
caspasa 3 y como consecuencia del resto de proteasas y nucleasas que conducen a los
eventos finales de la muerte celular programada. (Figura 1. 2)
3
Pese a sus diferencias, ambas rutas se encuentran interconectadas en diferentes pasos de
este proceso, siendo la permeabilización de la membrana mitocondrial externa y la
activación de las caspasas los procesos que marcan el destino final de la célula.
Figura 1. 2. Modelo simplificado de las principales proteínas implicadas en el proceso de apoptosis
1. 2. Caspasas
Las caspasas son las proteínas ejecutoras dentro del proceso de apoptosis. Son
sintetizadas como zimógenos inactivos (procaspasas, 30-50 kDa) y se componen de tres
dominios: un prodominio N-terminal, una subunidad grande y otra pequeña, al tiempo
que pueden incluir dominios de reclutamiento de caspasas (caspase-recruitment
domains, CARDs) y dominios efectores de muerte (death effector domains, DEDs)
(Figure 1. 3) [19]. La familia de las caspasas puede dividirse en dos grupos: las
caspasas iniciadoras (p. ej. , caspasas 8, 9 y 10) y las caspasas efectoras (p. ej. ,
caspasas 3 y 7). Las caspasas iniciadoras son las primeras en ser activadas tras la
activación de la apoptosis y, a continuación, se encargan de activar a las caspasas
efectoras. Dichas caspasas efectoras serán las que finalmente ejecutarán el proceso de
muerte mediante el procesamiento de un selecto número de proteínas dianas implicadas
4
en procesos esenciales para la células como son el mantenimiento del citoesqueleto,
cese del metabolismo, destrucción del ADN,. . . [19]
Subunidad grande Subunidad pequeñaCARD
DED
Caspasa 1
Caspasa 2
Caspasa 3
Caspasa 4
Caspasa 5
Caspasa 6
Caspasa 8
Caspasa 9
Caspasa 10
Caspasa 14
Caspasa 7
Subunidad grande Subunidad pequeñaCARD
DED
Subunidad grande Subunidad pequeñaCARD
DED
Caspasa 1
Caspasa 2
Caspasa 3
Caspasa 4
Caspasa 5
Caspasa 6
Caspasa 8
Caspasa 9
Caspasa 10
Caspasa 14
Caspasa 7
152 316
29 175
270
311
23 179
23 198
374
316
219 415
331 316
175
270
311
194 179
385
198
374
316
219 415
290
331
242
Figura 1. 3. Estructura de las diferentes caspasas (modificado de [20])
1. 3. Familia de proteínas Bcl-2
La mitocondria es el elemento clave de la vía intrínseca. La familia de proteínas de Bcl-
2 la que regula la permeabilización de la membrana mitocondrial por las acciones
opuestas de sus miembros proapoptóticos y antiapoptóticos en un proceso intrincado y
no del todo conocido [24]. Las proteínas Bcl-2 pueden contener de uno a cuatro
dominios de homología a Bcl-2 (BH, Bcl-2 homology). Entre ellos, el dominio BH3 es
esencial y suficiente para llevar a cabo la actividad proapoptótica y supresora tumoral.
Las interacciones opuestas entre miembros de la familia Bcl-2 establecen un equilibrio
entre los estímulos de señalización proapoptóticos y antiapoptóticos. Esta familia de
proteínas se subdivide en tres grupos según los dominios BH que las integran (figura 1.
5): antiapoptóticas y oncogénicas, las que comparten homología de secuencia en los
dominios BH1, BH2, BH3 y BH4, como son Bcl-2, Bcl-XL, Mcl-1, A1; proapoptóticas,
que comparten la homología de los dominios BH1, BH2 y BH3, como son Bax y Bak; y
proapoptóticas, las que comparten la secuencia de homología sólo en el dominio BH3,
como son Bid, Bik, Noxa, Puma, Bad, Bim [74, 153]
5
Tras un estímulo apoptótico, el balance de proteínas proapoptóticas aumenta y como
consecuencia se produce la permeabilización mitocondrial a través de Bax y Bak, con lo
que se produce un descenso en el potencial de membrana (ΔΨm) y la salida de factores
apoptóticos entre los que se incluyen cit c, Smac, OmiHtra2 y AIFs entre otros [117].
AIF es una proteína que estimula la apoptosis a nivel nuclear independientemente de las
caspasas [71]. La función de la proteína Smac/DIABLO es bloquear la acción de las
proteínas inhibidoras de la apoptosis (IAP, Inhibitor of Apoptosis Protein).
1. 4. El apoptosoma
En el citoplasma, cit c ejerce su papel proapoptótico, estimulando la activación de
caspasas a través de la formación de un complejo proteico con la proteína Apaf-1
(Apoptotic Protease-activating factor-1) y una caspasa iniciadora, procaspasa 9 dando
lugar a la formación de una estructura macromolecular denominada apoptosoma. La
formación de esta estructura induce la activación de la caspasa 9 que activará a la
caspasa efectora caspasa 3 desencadenándose así el proceso apoptótico.
Apaf-1 es una proteína citoplasmática de unos 130 kDa que se compone de tres
dominios funcionales: el dominio de reclutamiento de caspasas N-terminal (CARD), el
dominio de unión a nucleótidos y oligomerización (nucleotide-binding and
oligomerization domain, NOD) y un dominio C-terminal que contiene de 12 a 13
motivos WD40 y a través del que se une al cit c [38].
La importancia del apoptosoma ha sido demostrada en experimentos desarrollados con
ratones deficientes de Apaf-1. Estos ratones mostraron letalidad embrionaria con
marcados daños en el sistema nervioso y deformaciones craneofaciales [56, 57]. Por
estas razones, hay diversos mecanismos en la célula dedicados a la regulación del buen
funcionamiento de este complejo proteico. En el caso de Apaf-1, la proteína que va a
ocupar la temática principal de la presente tesis, los diversos moduladores encontrados
se muestran en la Figura 1. 4. Se trata de proteínas pertenecientes a la familia de Bcl-2,
diversas fosfoquinasas, proteínas chaperonas, proteínas implicadas en el ciclo celular,. .
.[304]
6
H1.2NuclingPPI
124898
NBD WD40CARDCARD4121
Apo cyt cDiva/BooHsp 70Hsp 90
Hsp70/CAS/PHAPIJNK
ATP/dATPHCA66PARCS
ADPBcl-XL
Diva/BooHCA66S
APIPAIP Aven
Bcr-Abl
Procaspasa 9NAC
Cytochromo c
H1.2NuclingPPI
H1.2Nucling
H1.2NuclingPPIPPI
124898
NBD WD40CARDCARD4121
Apo cyt cDiva/BooHsp 70Hsp 90
Hsp70/CAS/PHAPIJNK
ATP/dATPHCA66PARCS
ADPBcl-XL
Diva/BooHCA66S
APIPAIP Aven
Bcr-Abl
Procaspasa 9NAC
Cytochromo c
124898
NBD WD40CARDCARD4121 124898
NBD WD40CARDCARD4121
Apo cyt cDiva/BooHsp 70Hsp 90
Hsp70/CAS/PHAPIJNK
ATP/dATPHCA66PARCS
ADPBcl-XL
Diva/BooHCA66S
APIPAIP Aven
Bcr-Abl
Procaspasa 9NAC
Cytochromo c
Figure 1. 4. Diferentes proteínas que interaccionan con Apaf-1 y que regulan su función. En negro se muestran las proteínas inhibidoras de la función, mientras que en blanco se muestran las proteínas activadoras En gris se muestran las proteínas cuya función sobre Apaf-1 todavía no ha sido descrita.
1. 5. Implicación del apoptosoma y la apoptosis en el desarrollo de enfermedades
Las alteraciones en la formación del apoptosoma también se han asociado con
patologías humanas. Estudios acerca de la expresión de Apaf-1 en tumores, y estudios
mecanísticos en modelos celulares indican que la vía intrínseca y el apoptosoma son
importantes para el desarrollo y la progresión del cáncer. Se ha demostrado una
expresión de Apaf-1 deficiente por mutaciones en su locus génico en melanomas
malignos, sobre todo en melanomas metastáticos; cáncer gastrointestinal,
adenocarcinoma pancreático ductal y en glioblastoma. Asimismo las alteraciones en la
regulación transcripcional y post-transcripcional de Apaf-1 se han asociado con otros
tipos de cáncer. En células de cáncer de ovario se ha correlacionado la
quimiorresistencia de los tumores con una actividad deficiente del apoptosoma.
Estudios dirigidos a restaurar los niveles de Apaf-1 en diversas líneas tumorales se han
correlacionado con una pérdida en la quimiorresistencia característica de estas líneas.
Del mismo modo, se han relacionado con procesos tumorales las alteraciones en la
expresión de proteínas que regulan la formación y actividad del apoptosoma, como las
Hsp, ProT, XIAP, ML-IAP y PKB.
7
La inhibición de la apoptosis cobra especial importancia en el caso de las enfermedades
neurodegenerativas ya que, aunque no la única, es una de las formas principales de
muerte neuronal. La apoptosis es una característica tanto de las enfermedades
neurológicas agudas como crónicas. La necesidad de inhibir la muerte neuronal y sus
causas se agrava por la escasa o nula capacidad regenerativa que tiene el sistema
nervioso central. En un proceso agudo, como una lesión traumática, tras la isquemia, la
necrosis y la apoptosis coexisten. La necrosis se encuentra en el centro de la lesión,
donde la hipoxia es más severa, pero en las zonas de penumbra aparece flujo sanguíneo
lateral y se crea un gradiente de tejido perfundido donde se establece un umbral de
muerte por apoptosis.
En las enfermedades neurodegenerativas crónicas la apoptosis es la forma principal de
muerte neuronal. Hay evidencias de que las caspasas tienen un papel importante en la
esclerosis lateral amiotrófica (ELA), Parkinson, Huntington, Alzheimer y demencia
asociada a la infección por HIV. En fases tempranas se crea una activación de caspasas
subletal que provoca la disfunción celular. Aunque la causa que origina estas
enfermedades no se conoce con exactitud, se ha encontrado activación de caspasas. Por
otro lado, en un tejido donde las células mueren por apoptosis, existe un fenómeno de
“contagio” en el que una célula que sufre apoptosis, produce moléculas de señalización
de muerte celular que difunden y que inducen apoptosis en células vecinas, creando un
efecto dominó de expansión de muerte celular. Este fenómeno de apoptosis contagiosa
ocurre tanto en procesos agudos como crónicos y es importante desde el punto de vista
terapéutico antiapoptótico, porque un inhibidor de apoptosis no solo retrasará la muerte
de una neurona particular sino que también puede inhibir los factores difusibles tóxicos
de apoptosis en neuronas vecinas.
Según estudios preclínicos en modelos animales, la inhibición de caspasas resulta una
terapia prometedora en ELA, enfermedad de Parkinson, lesiones cerebrales traumáticas
y epilepsia, meningitis bacteriana, sepsis y en modelos de lesión de isquemia-
reperfusión. Muchas compañías farmacéuticas están desarrollando nuevas generaciones
de inhibidores de caspasas suficientemente potentes y capaces de atravesar la membrana
de las células. Las aplicaciones clínicas de compuestos inhibidores de Apaf-1 estarían
sujetas a las mismas consideraciones que los inhibidores de caspasas con la ventaja de
encontrarnos un paso por encima de las caspasas efectoras.
8
La comprensión de los mecanismos moleculares de las asociaciones entre apoptosis y
enfermedad descritas ha permitido el desarrollo de terapias de las que enfermedades
neurodegenerativas, cáncer y procesos autoinmunes ya se están beneficiando, pero
todavía existen grandes posibilidades de mejora en el tratamiento de estas
enfermedades.
Investigaciones recientes han identificado tres clases de moléculas que directa o
indirectamente modulan la actividad del apoptosoma y que podrían tener una
proyección terapéutica. Los tres tienen en común el haberse identificado mediante la
utilización de colecciones de compuestos y ensayos en extractos celulares. El
compuesto PETCM se identificó a partir del cribado de quimiotecas de compuestos
orgánicos como activador de caspasa 3 [139]. PETCM estimula la activación del
apoptosoma inhibiendo la proteína ProT (protimosina), una oncoproteína que actúa
como regulador negativo de la formación del apoptosoma. Otros compuestos también
identificados mediante el cribado de quimiotecas utilizando ensayos en extractos
celulares, activan la formación del apoptosoma, aparentemente disminuyendo los
umbrales de cit c requeridos para la formación del apoptosoma [151]. Estos
compuestos han demostrado ser efectivos en la inducción química de la apoptosis en un
amplio espectro de líneas celulares. Finalmente, también se han encontrado moléculas
que actúan como inhibidores de la formación del apoptosoma. Una familia de
diarilureas han demostrado ser inhibidores efectivos de la activación de caspasas en
extractos celulares y aunque su mecanismo de acción no se conoce, provocan la
inhibición de la formación del apoptosoma [155].
1. 7. Identificación de un inhibidor de Apaf-1, el peptoide 1, a través del
cribado de una quimioteca de N-alquilglicinas
Uno de los principales proyectos en desarrollo en nuestro laboratorio está basado
en la búsqueda de moduladores del apoptosoma. En ausencia de una información
estructural detallada, en la actualidad no existe información en la literatura que
describan la identificación de moduladores artificiales del apoptosoma que
interaccionen físicamente con el complejo e interfieran con alguno de los pasos
necesarios para su formación y actividad. Por esta razón, la reconstitución in vitro del
apoptosoma a través de sus constituyentes proteicos recombinantes se planteó como una
9
interesante estrategia a seguir en el desarrollo de moduladores apoptóticos basados en
las interacciones proteína-proteína [158]. La reconstitución del apoptosoma se efectuó a
partir de Apaf-1 recombinante obtenido mediante el sistema de baculovirus Bac-to-Bac
en células de insecto, cit c, dATP y procaspasa 9 transcrita y traducida in vitro. El
procesamiento de procaspasa 9 se monitoriza mediante marcaje radiactivo de
procaspasa 9 con [35S]Met, resolución de las muestras en SDS-PAGE y autorradiografía
(Figura 1. 5).
Figura 1. 5. Estructura del apoptosoma y seguimiento del procesamiento de la [35S]Met procaspasa 9 en SDS-PAGE y autorradiografía [158]
La quimioteca elegida para este cribado fue una quimioteca de trímeros de N-
alquilglicinas en formato de rastreo posicional e incorporando una amplia variedad de
grupos químicos. La quimioteca se ensayó con el objetivo de encontrar moléculas que
inhibiesen el procesamiento de procaspasa 9 in vitro utilizando el ensayo de
reconstitución de apoptosoma y se cuantificó el porcentaje de activación de la
procaspasa 9. El cribado de esta quimioteca llevó a la identificación de una serie de
pseudopéptidos de entre los cuales el peptoide 1 (figura 1. 6) mostraba la mayor
actividad inhibitoria [158].
10
Cl
Cl
HN
N
ON
ON
O
Cl
Cl
O
N
ONH2
N
Cl
Cl
NN
ON
ONH2
O
Cl
ClO
N O
NH
N
Cl
Cl
HN
N
ON
ONH2
O
Cl
Cl
Peptoide 1bPeptoide 1a
Peptoide 1
Figura 1. 6. Estructura química del peptoide 1 y de sus derivados peptoide 1a y peptoide 1b
Sin embargo, esta molécula presentaba problemas de reproducibilidad en los
ensayos in vitro debido a su baja solubilidad y tendencia a la agregación. Por esta
razón, resultó necesario mejorar sus propiedades. Experiencia previa de nuestro
laboratorio en el diseño de péptidos bioactivos [157] indicaba que la presencia de
cadenas laterales con carga positiva con una glicina espaciadora en el extremo N-
terminal o C-terminal podía incrementar la solubilidad general del compuesto sin
afectar a la conformación estructural o las propiedades biológicas. Con estos
antecedentes se sintetizaron los peptoides 1a y 1b (figura 1. 9), los cuales
presentaban dos residuos adicionales de N-alquilglicina situados en el extremo C-
terminal o N-terminal del trímero original respectivamente. Ambos compuestos
mejoraron la reproducibilidad en ensayos in vitro sin afectar a la actividad. No
obstante, cuando estos compuestos se testaron en ensayos ex vivo resultaron
tóxicos por lo que nuevas estrategias con el fin de mejorar dicha internalización
fueron seguidas y se mostrarán a lo largo de esta tesis.
11
CAPÍTULO 2: OBJETIVOS
Mi tesis se basa en el descubrimiento y desarrollo de una serie de inhibidores del
apoptosoma a través de la proteína Apaf-1. El trabajo aquí presentado es una
continuación de la línea investigadora seguida en nuestro laboratorio con el fin de
mejorar las propiedades químicas del peptoide 1, un trímero de N-alquilglicinas
descubierto a través del cribado de una librería química empleando un ensayo de
reconstitución in Vitro del apoptosoma. Sin embargo, debido a su baja capacidad para
atravesar la membrana celular nuevos derivados fueron sintetizados a través del uso de
diferentes portadores o modificaciones en su estructura que disminuyeran la flexibilidad
de la molécula.
Los objetivos que se han definido son:
1. Síntesis de derivados del peptoide 1
2. Estudio sistemático de las propiedades biológicas de estos derivados así como su
toxicidad en células
3. Mejora de la estructura química de los derivados con mayor actividad biológica
encontrados
4. Caracterización del sitio de unión de los derivados del peptoide 1 a Apaf-1
5. Caracterización del mecanismo molecular de acción del peptoide 1 y sus
derivados en célula (consecuencias para la célula incluyendo análisis del papel
no apoptótico de Apaf-1)
6. Desarrollo de un modelo in vivo de rata de isquemia en caliente en riñón y,
posteriores estudios del efecto terapéutico de los compuestos sintetizados en
dicho modelo en la mejora del daño tisular causado por la deprivación de
oxígeno.
12
13
CAPÍTULO 3: SÍNTESIS DEL PEPTOIDE 1 Y SUS DERIVADOS
Con el fin de mejorar la capacidad de internalización del peptoide 1 se llevaron a cabo
modificaciones estructurales de este compuesto con el fin de mejorar su baja solubilidad
y baja capacidad de atravesar la membrana celular. Las estrategias seguidas fueron:
a) Fusión a péptidos con capacidad de permear
b) Conjugación a un portador polimérico
c) Síntesis de un derivado de conformación restringida
a) Péptidos con capacidad de permear
Durante la pasada década, fueron descubiertas diversas proteínas con capacidad de
atravesar la membrana celular. Estas proteínas eran capaces de llegar incluso al núcleo
y retener su actividad biológica. Los responsables de la internalización celular de estas
proteínas son determinados dominios proteicos de pequeño tamaño capaces por sí solos
de atravesar las membranas biológicas de manera eficiente e independiente de proteínas
transportadoras o receptores específicos. Estos dominios proteicos o péptidos con
capacidad de permear (cell-penetrating peptides, CPPs) pueden internalizar también
distintas moléculas unidas covalentemente al interior de las células [162]. Un amplio
abanico de biomoléculas como péptidos antigénicos, híbridos péptido-ácidos nucleico,
oligonucleótidos antisentido, proteínas completas o incluso nanopartículas y liposomas
han sido transportados de este modo convirtiéndose en una excelente estrategia para la
mejora de las propiedades farmacológicas.
Entre los dominios más utilizados y mejor caracterizados en el transporte de péptidos a
través de la membrana plasmática se encuentran la proteína TAT (YGRKKRRQRRR)
del virus de inmunodeficiencia humana VIH-1, la tercera hélice α del homeodominio de
Antennapedia de Drosophila o sus derivados como la penetratina
(RQIKIWFQNRRMKWKK), y la proteína V22 del virus herpes simple. Además de
péptidos naturales, se han diseñado una amplia variedad de secuencias transportadoras
artificiales basadas en los residuos de lisina y arginina de la secuencia del péptido TAT.
En concreto, se ha encontrado homología estructural entre el dominio TAT y
homopolímeros de aminoácidos básicos como poliarginina o polilisina, entre otros, que
14
han resultado ser muy útiles en el transporte de moléculas a través e la membrana
plasmática.
El mecanismo de internalización de los CPPS y sus moléculas cargo no se ha definido y
existe cierta controversia en torno al mismo. En un principio, se postuló se trataba de
un transporte directo a través de la bicapa lipídica de las membranas e independiente de
endocitosis o receptores de membrana [173]. Sin embargo, estudios recientes han
demostrado que la endocitosis sí podría jugar un papel importante en la internalización
de los compuestos[174].
Con el fin de aumentar las propiedades de permeabilidad de membrana del peptoide 1,
se sintetizaron análogos del peptoide 1 fusionados a secuencias de transducción al
interior celular, penetratina (PEN-1) y TAT (TAT-1). Ambas secuencias polibásicas
fueron seleccionadas por ser las mejor descritas y empleadas con mayor éxito en la
internalización de moléculas de diferente naturaleza.
b) Conjugación del peptoide 1 a un transportador polimérico
La expresión polímero terapéutico es el término empleado para describir una
familia que incluye fármacos poliméricos, conjugados polímero-fármaco, conjugados
polímero-proteína, micelas poliméricas en las que el fármaco está unido de forma
covalente, y poliplejos multicomponentes que son usados en el desarrollo de vectores no
virales. Todas estas subclases emplean polímeros solubles en agua, bioactivos por ellos
mismos o como una parte funcional inerte de una macromolécula cuyo propósito es el
de mejorar la liberación del fármaco, proteína o gen. [180]
Los polímeros terapéuticos pueden considerarse como las primeras
nanomedicinas poliméricas. Estas nuevas entidades químicas son nanoconstrucciones
híbridos que unen covalentemente una agente bioactivo con un polímero con el fin de
asegurar no solo una eficiente liberación del fármaco en un determinado compartimento
celular, sino que aseguran su disponibilidad en un periodo de tiempo específico. Las
excelentes cualidades de estos compuestos se han puesto de manifiesto a través del
XYOTAX®, un conjugado polímero-fármaco (ácido poli-(L-glutámico)-paclitaxel,
PGA-paclitaxel), que en el próximo año se convertirá en el primer fármaco polimérico
15
anticancerígeno en salir al mercado dados los espectaculares resultados obtenidos en
ensayos clínicos en fase III. Este éxito no ha sido fruto de la casualidad, en la pasada
década, más de 10 conjugados polímero-fármaco entraron en fases clínicas I/II como
agentes anticancerígenos. Esta lista incluye seis conjugados basados en copolímeros de
N-(2-hidroxipropil)metacrilamida (HPMA) y, más recientemente, series de conjugados
de polietilenglicol (PEG) y PGA [180].
Debido a esto, en el presente trabajo, se hipotetizó que la conjugación del
peptoide 1 a un transportador polimérico podría ofrecer una internalización celular más
específica. Se escogió como polímero transportador al PGA, por ser un polímero
biodegradable por catepsina B (una enzima lisosomal), estable en plasma lo que permite
su inoculación a través de la vía sistémica, y disponer de una gran cantidad de grupos
funcionales a los que unir el fármaco. Además, estudios en fase clínica ya han sido
llevados a cabo con este polímero a través del OPAXIO®, por lo que su farmacología
está ampliamente descrita.
En los conjugados poliméricos, el fármaco permanece en el interior del polímero
de modo que el fármaco es fácilmente solubilizado. Debido a su elevado tamaño, estos
conjugados poliméricos tienen restringida su entrada en la célula por la vía endocítica.
Una vez internalizado el conjugado pasa por el endosoma hasta llegar al lisosoma. Una
vez en el lisosoma debido a la disminución del pH y la presencia de enzimas tales como
las catepsinas, el conjugado es degradado liberando de este modo el agente bioactivo
que pasa al citosol por difusión, consiguiendo alcanzar así la diana molecular
seleccionada [180, 192]
c) Análogos de conformación restringida
Otro de los mecanismos de internalización celular es la difusión pasiva. Se trata de un
proceso sin consumo de energía dado con moléculas de pequeño tamaño no cargadas,
las cuales pueden atravesar la membrana libremente en función del gradiente. Se
postula que este mecanismo sería por el cual fármacos de bajo peso molecular como el
peptoide 1 serían internalizados por la célula. Por tanto, dependerá fuertemente de las
propiedades intrínsecas del fármaco tales como solubilidad, lipofilicidad y capacidad de
interacción con los componentes de la membrana.
16
Dado que las membranas controlan los procesos de distribución de los fármacos, en el
diseño y mejora de fármacos evitar cualquier posible interacción del fármaco con la
membrana celular que pudiera afectar a las propiedades de la misma constituye un
objetivo prioritario. En el caso de moléculas con estructuras flexibles resulta
especialmente difícil la no interacción con el entorno, dada su capacidad de adoptar gran
variedad de conformaciones. Por esta razón, una de las estrategias en la optimización
de fármacos cabeza de serie se basa en la restricción conformacional de su estructura
En este sentido y con el objetivo de evitar posibles interacciones del peptoide 1 con la
membrana plasmática y mejorar sus propiedades de solubilidad, se efectuó el diseño y
síntesis de un derivado de conformación restringida, el ciclopeptoide QM31 y una
colección de moléculas derivadas de este. La rigidez de su estructura debería facilitar
su internalización celular al reducir la posibilidad de interacción del compuesto con los
componentes de la membrana plasmática.
3. 1. Síntesis del peptoide 1
Para llevar a cabo la síntesis de los derivados peptídicos, el peptoide 1 necesario fue
producido en el laboratorio del Dr. Ángel Messeguer del grupo de Química Orgánica
Biológica del IIQAB (CSIC) de Barcelona. El esquema de dicha síntesis se muestra en
la Figura 3. 1.
NH2
1. BrCH2COOH, DIPCDI, DMF
NH2
HN
ONH
2. TEA, DMF,
1. BrCH2COOH, DIPCDI, DMF
2. TEA, DMF,
NH2
Cl
1. TFA:DCM:H2O (60:40:2)
Cl
ClCl
Cl
1. BrCH2COOH, DIPCDI, DMF
2. TEA, DMF,
NH2
HN
ON
Cl
Cl
NHO
Cl
HN
ON
Cl
Cl
NO
NHO
Cl Cl
H2N
ON
Cl
Cl
NO
NHO
Cl Cl
Figura 3. 1. Esquema de la síntesis química del peptoide 1
17
3. 2. Péptidos con capacidad de permear: híbridos péptido-peptoide Para la síntesis de los híbridos péptido-peptoide el peptoide 1 se fusionó a secuencias
ampliamente utilizadas en el transporte de moléculas al interior celular, como son
penetratina y TAT. La síntesis se efectuó utilizando química F-moc en fase sólida,
posterior purificación por HPLC y caracterización por espectroscopia de masas
MALDI-TOF (Figura 3. 2) [196].
Cl
Cl
N
NO
NO
NH2O
Cl
Cl
RQIKIWFQNRRMKWKKGG
A
Cl
Cl
N
NO
NO
NH2O
Cl
Cl
YGRKKRRQRRRGGG
B
Figura 3. 2. Estructura del PEN-1 (A) y del TAT-1 (B)
3. 2. 1. CF-TAT-1 y CF-PEN-1
Asimismo, se llevó a cabo la síntesis de una serie de conjugados PEN-1 y TAT-1
empleando carboxifluoresceína (CF) como marcador fluorescente. Para ello se
empleó el mismo tipo de síntesis pero una resina conteniendo al peptoide 1 ya
marcada con la CF.
3. 3. Conjugación a un transportador polimérico: PGA-peptoide
La conjugación del peptoide 1 a un transportador polimérico se efectuó en dos pasos.
En primer lugar, mediante química F-moc en fase sólida se incorporaron dos glicinas al
peptoide 1. Y, a continuación, el peptoide 1-GG se fusionó al poli-(L-ácido glutámico)
transformado en su forma protonada (Figura 3. 3). El contenido total de peptoide en
este conjugado polimérico fue determinado por UV (λ = 270 y 282 nm) se situó entre el
25-30 % (m/m) (10-12 mol % funcionalización) (Figura 3. 4) [192].
18
Figura 3. 3. Esquema de la síntesis química del PGA-peptoide
Longitud de onda (nm)Longitud de onda (nm) Figura 3. 4. Espectro UV del PGA, del PGA-peptoide y del GG-peptoide 1 en la determinación del grado de funcionalización del PGA-peptoide
3. 3. 2 QM56-OG (peptoide 1-GG-PGA-oregon green)
El análogo fluorescente del QM56 se sintetizó siguiendo el mismo protocolo
anteriormente descrito seguido de la conjugación del PGA al peptoide 1-GG (Figure 3.
5). El contenido total de fármaco se determine por UV (peptoide 1) y espectroscopia de
fluorescencia (contenido de Oregon Green, OG).
19
+ DIC/HOBt
DIEA/DMFanh
Cl
Cl
NN
ON
ONH2
O
Cl
Cl
O
NH
HN
O
1
HN
O
20
O
HN O
NH
O
O
79
+Na-O O
NH
O
HN
O
HOF
OF
OHO
Cl
Cl
NN
ON
ONH2
O
Cl
Cl
O
NH
HN
O
O
80
+Na-O
HN
O
20
O
HN ONHO
H2N
OHO
F
O
F
OH
O
Figura 3. 5. Esquema de la síntesis del QM56-OG
3. 4. Derivado peptídico de conformación restringida: ciclopeptoide
Tanto la síntesis del ciclopeptoide como la de los diferentes derivados cíclicos se
efectuó en el laboratorio del Dr. Ángel Messeguer. En la figura 3. 6 se muestra el
esquema de síntesis del ciclopeptoide.
NH2
1. BrCH2COOH, DIPCDI, DMF
NH2
HN
ONH
OO
HOO
2. TEA, DMF,
HN
ON
O
HN
HN
ON
OO O
1. TEA, DMF
1. BrCH2COOH, DIPCDI, DMF
2. TEA, DMF,
O O
1. PhSiH3, Pd(PPh3)4 DCM 1. PyBOP, HOBt, DIEA,
DMF
2. TFA:DCM:H2O (60:40:2) N
N
O
O
Cl
N
OH2N
O
Cl
Cl
Cl
Cl
Cl
Cl
NH2
Cl
Cl
NH2Cl
Cl
HN
ON
O
N
O O
NHO
Cl
Cl
Cl
Cl
HN
ON
O
N
O OH
NHO
Cl
Cl
Cl
Cl
ClCl
Cl 2 Figura 3. 6. Esquema de la síntesis química del ciclopeptoide
Diversos derivados del QM31 fueron sintetizados con el fin de llevar a cabo un estudio
comparativo actividad-estructura. Para ello, se modificó el grupo funcional R1 (Figure
3. 7). Los diversos QM31 derivados se muestran en la tabla 3. 1.
20
R1 NOO
O
NN
O
Cl
Cl
NH2
GROUP I
Figura 3. 7. Estructura química del QM31 y sus derivados
Tabla 3. 1. Estructura química de los sustituyentes del grupo R1 utilizados
Análogos QM31 R1
QM31 Cl
Cl
QM31a F
QM31b
QM31c
QM31d O
QM31e F
QM31f Cl
QM31g y QM31h
(mezcla) y O
QM31 i NH
O
QM31j O
QM31k Cl
QM31l
21
3. 4. 1. Síntesis del QM57 y derivados
Asimismo, otra estrategia seguida fue la modificación del anillo central de la molécula.
De este modo fueron sintetizados el QM57 y una serie de derivados de dicha molécula.
Sin embargo, debido a procesos de patentabilidad ninguna de sus estructuras o métodos
de síntesis será mostrada.
3. 5. Síntesis del QM56 y derivados poliméricos de los heterociclos QM31 y QM57
Una serie de análogos del QM56 y del QM31 y QM57 fueron sintetizados. El objetivo
fue el de conjugar los diferentes compuestos al transportador polimérico con un número
diferente de aminoácidos de distinta naturaleza y así conseguir diferentes velocidades de
liberación del fármaco gracias a la diferente afinidad presentada por la catepsina B
(encargada de romper el enlace peptídico que une polímero-fármaco) por dichos
aminoácidos. Debido a procesos de patente ninguna estructura o síntesis de dichos
compuestos será mostrada (Tabla 3. 2).
Tabla 3. 2. Estructura esquemática del QM56 y polímeros derivados del QM31 y el QM57
PGA derivados Estructura esquemática
QM56 Peptoide 1-GG-PGA
QM56-P1 Peptoide 1-R1R2-PGA
QM56-P2 Peptoide 1-R1R3-PGA
QM31-P1 QM31-R4-PGA
QM31-P2 QM31-R5R3R6R4-PGA
QM31-P3 QM31-R1R3R6R4-PGA
QM31-P4 QM31-R5R5R4-PGA
QM57-P1 QM57-R5R3R6R4-PGA
QM57-P2 QM57-R5R5R4-PGA
22
23
CAPÍTULO 4: ANÁLISIS SISTEMÁTICO DE LA INHIBICIÓN DE
APOPTOSIS EJERCIDA POR LOS DERIVADOS DEL PEPTOIDE 1
Sintetizados los compuestos, nuestro siguiente objetivo fue el de determinar la validez
de nuestras estrategias para conseguir la internalización de dichos fármacos. Para ello,
se llevaron a cabo tres tipos de estudios: a) estudio de la actividad biológica de los
compuestos in vitro; b) estudios de viabilidad celular a través de ensayos de MTT y
estudios de actividad biológica tras inducción de apoptosis a través de ensayos de MTT
y de medida de activación de la caspasas 3; c) ensayos de internalización celular en
célula viva. Los detalles de cada una de las estrategias seguidas se describen en el
capítulo 9 de Materiales y Métodos.
4. 1. Capacidad de inhibición de los híbridos péptido-peptoide: PEN-1 y TAT-1
4. 1. 1 Actividad antiapoptótica del PEN-1 y el TAT-1 en extractos de células
La capacidad inhibitoria del PEN-peptoide y el TAT-peptoide se evaluó mediante el
ensayo anteriormente descrito de reconstitución del apoptosoma en extractos celulares
(Capítulo 9) (Figura 4. 1).
Figura 4. 1. Cuantificación del porcentaje de inhibición de formación del apoptosoma. Los híbridos péptido-peptoide y sus respectivos controles fueron analizados a concentraciones de 1 μM, 5 μM, 10 μM y 20 μM (barras negras, rayadas, grises y blancas, respectivamente)
Tanto el PEN-peptoide como el TAT-peptoide mostraron capacidad inhibitoria
dependiente de la concentración.
0
2
4
6
8
10
Control positivo
Peptoide 1a PEN-1 PEN TAT-1 TAT
% A
ctiv
idad
Cas
pasa
3
24
4. 1. 2 Evaluación de la toxicidad celular y actividad biológica del PEN-1 y del TAT-1
a) Ensayos de MTT: ensayos de toxicidad y de recuperación de la viabilidad
celular
Las gráficas muestran en los ensayos de viabilidad como el TAT-1 induce una elevada
toxicidad a concentraciones de 10 y 5 μM desde las 24 horas de incubación del
compuesto (Figura 4. 2). Por el contrario, en el caso del PEN-1, molécula de igual
naturaleza no se observaba ningún efecto citotóxico. Este hecho justifica la ausencia de
recuperación de la viabilidad celular en el caso del TAT-1 tras la inducción de muerte
en las células (Figura 4. 3).
24H48H
72HU 2 - OS
HeLaA 54 9
U 9 3 7Saos- 2
0
20
40
60
80
100B
24H48H
72HA 54 9
HeLaU 2 - OS
U 9 3 7Saos- 2
0
20
40
60
80
100
A
24H48H
72HU 2 - OS
HeLaA 54 9
U 9 3 7Saos- 2
0
20
40
60
80
100B
24H48H
72HA 54 9
HeLaU 2 - OS
U 9 3 7Saos- 2
0
20
40
60
80
100
A
Figura 4. 2. Porcentaje de viabilidad de los derivados del peptoide 1: TAT-1 (A), PEN-1 (B), Los derivados del peptoide 1 se probaron a concentraciones de 10, 5 y 1 μM (barras negras, grises y blancas, respectivamente).
25
24H48H
72HSaos-2A549HeLaU-2 OSU937
0
10
20
30
40
50
60
% R
ecup
erac
ión
celu
lar
(% C
élul
asto
tale
s)
Figura 4. 3. Porcentaje de recuperación de la viabilidad celular del PEN-1 en diferentes líneas celulares. El PEN-1 se testó a concentraciones de concentraciones de 10, 5 y 1 μM (barras negras, grises y blancas, respectivamente).
b) Ensayos de medida de la actividad caspasa 3
La medida de caspase 3 se empleó como un método de medida indirecto de la
formación del apoptosoma y más específico que el MTT, que no nos permite distinguir
entre el tipo de muerte existente. PEN-1 inhibió la actividad caspase 3 en diferentes
modelos celulares, no fue así en el caso del TAT-1 que no mostró capacidad inhibidora
(datos no mostrados). Los péptidos control fueron también analizados no mostrando
actividad inhibidora de la apoptosis (datos no mostrados) [196, 200] (Figure 4. 4).
010
2030
4050
6070
80
Saos-2 HeLa U-2 OS U937
% C
aspa
se 3
act
ivity
inhi
bitio
n%
Inhi
bici
ónac
tivid
adca
spas
a3
Figure 4. 4. Medida de la actividad caspasa 3 en diversas líneas celulares. PEN-1 se testó a 5 μM en todas las líneas excepto en Saos-2 (2 μM). Las medidas se llevaron a cabo tras 24, 48, y 72 h de incubación (barras negras, grises y blancas, respectivamente). Los datos se expresan como media ± ES (n> 3).
26
4. 1. 3 Internalización celular y caracterización estructural del PEN-1 y el TAT-1
Con el fin de determinar el aumento de internalización celular del peptoide 1 tras su
conjugación al PEN y al TAT. PEN-1 y TAT-1 fueron marcados con CF. Los estudios
mediante citometría indicaron una rápida internalización de los compuestos tras su
adición a las células (menos de 1 hora). Sin embargo, cuando se efectuaron estudios de
internalización en célula viva se observó cómo el CF-TAT-1 dañaba la membrana de la
célula, lo que causaría su toxicidad. Estudios posteriores empleando técnicas de
dicroísmo circular indicaron que el peptoide 1 influía en la estructura del TAT de modo
significativo. Este hecho podría causar variaciones en su mecanismo de internalización
que explicaría la no eficacia de dicho híbrido para atravesar la membrana celular . En
caso del PEN-1 este era internalizado correctamente y la estructura del PEN no se veía
significativamente afectada por la adición del peptoide 1 [196].
4. 2. Capacidad de inhibición del conjugado polimérico: QM56
Debido a su naturaleza química la evaluación en ensayos in Vitro de la actividad
biológica del QM56 no pudo efectuarse, ya que sería necesaria la presencia de catepsina
B y un entorno ligeramente ácido para poder conseguir la liberación del fármaco al
medio. No obstante se efectuaron estudios de internalización celular del QM56
marcado con Oregon Green (OG) con el fin de comprobar la correcta internalización del
compuesto y así asegurar de su futura actividad biológica. Los resultados de
internalización celular mostraron como el QM56 era internalizado por las células a
través de mecanismos de endocitosis a tiempos bajos, lo que auguraba buenos
resultados en el caso de los estudios de citotoxicidad y actividad biológica que se
muestran a continuación.
4. 2. 2 Evaluación de la toxicidad celular y actividad biológica del QM56
a) Ensayos de MTT: ensayos de toxicidad y de recuperación de la viabilidad celular
El QM56 no mostró ningún tipo de toxicidad celular en las células tratadas. Asimismo,
cuando se testó su capacidad biológica tras la inducción de muerte en la célula se
observó una clara capacidad de recuperación de la viabilidad celular (Figure 4. 5).
27
24H48H
72HHeLaSaos-2A549U-2 OSU937
0
20
40
60
80
100
24H48H
72HSaos- 2
A 54 9HeLaU 2 - OS
U 9 3 70
20
40
60
80
100%
Via
bilid
adce
lula
r(%
Cél
ulas
tota
les)
% R
ecup
erac
ión
celu
lar
(% C
élul
asto
tale
s)
A B
24H48H
72HHeLaSaos-2A549U-2 OSU937
0
20
40
60
80
100
24H48H
72HSaos- 2
A 54 9HeLaU 2 - OS
U 9 3 70
20
40
60
80
100%
Via
bilid
adce
lula
r(%
Cél
ulas
tota
les)
% R
ecup
erac
ión
celu
lar
(% C
élul
asto
tale
s)
A B
Figura 4. 5. Porcentaje de viabilidad y de recuperación celular (A y B, respectivamente) del QM56 a concentraciones de 100, 50 y 20 μM equivalentes de fármaco (barras negras, grises y blancas, respectivamente).
b) Ensayos de medida de la actividad caspasa 3
El QM56 se testó junto con sus dos derivados QM56-P1 y QM56-P2 en ensayos de
inhibición de la actividad caspasa 3. En todos los casos, los compuestos fueron capaces
de inhibir la activación de la caspasa 3, aunque ninguno de los derivados del QM56
sintetizados presentó una mayor actividad biológica de modo significativo con respecto
al QM56. Se muestran como ejemplo las medidas efectuadas en la línea Saos-2 (Tabla
4. 1).
Tabla 4. 1. Porcentaje de inhibición de la apoptosis por medida de la actividad caspase 3 en los diferentes derivados del QM56
Saos-2
(%) Compuesto
24 h 48 h
QM56 54±3 (n=20) 93±5 (n=10)
QM56-P1 75±12 (n=3) 93±23 (n=3)
QM56-P2 67±4 (n=3) 34±5 (n=3)
28
4. 3. Capacidad de inhibición del derivado de conformación restringida QM31 y
sus derivados
4. 3. 1. Actividad biológica de los diversos derivados del QM31 en los ensayos de
reconstitución in vitro del apoptosoma
Los compuestos fueron testados en el ensayo de reconstitución in vitro del apoptosoma,
la subsiguiente medida de la activación de la procaspasa 9 nos indicaba el grado de
inhibición de los compuestos ejercido sobre Apaf-1. Los valores de inhibición
obtenidos abarcan un rango del 10-20 % de inhibición de la actividad caspasa 9. La
explicación de este valor un tanto bajo, podría deberse a la dificultad de solubilizar los
compuestos testados en las condiciones óptimas del ensayo. No obstante, posteriores
ensayos de MTT y medidas de caspasa 3 nos permitieron caracterizar la actividad
biológica de dichos compuestos.
4. 3. 2. Evaluación de la toxicidad celular y la actividad biológica del QM31 y
derivados
a) Ensayos de MTT: toxicidad y recuperación de la viabilidad celular del QM31
Los ensayos de viabilidad celular mostraron como el QM31 y los diferentes derivados
sintetizados del QM31 y el QM57 no resultan tóxicos para las células en las condiciones
de ensayo estudiadas. Asimismo, su capacidad de recuperación de la viabilidad celular
tras la inducción de muerte en las células mostró como las moléculas sintetizadas eran
capaces de aumentar, en diferente medida, el porcentaje de células viables. No obstante
ninguno de los compuestos testados mostró una actividad biológica significativamente
mayor a la del compuesto líder, el QM31. A modo de ejemplo se muestran los valores
obtenidos en los diferentes ensayos para esta molécula (Figuras 4. 6 y 4. 7).
29
24H48H
72HA 54 9
HeLaU 2 - OS
U 9 3 7Sao s- 2
0
20
40
60
80
100
24H48H
72HA549U-2 OSHeLaSaos-2U937
0
10
20
30
40
50%
Via
bilid
adce
lula
r(%
Cél
ulas
tota
les)
% R
ecup
erac
ión
celu
lar
(% C
élul
asto
tale
s)
A B
24H48H
72HA 54 9
HeLaU 2 - OS
U 9 3 7Sao s- 2
0
20
40
60
80
100
24H48H
72HA549U-2 OSHeLaSaos-2U937
0
10
20
30
40
50%
Via
bilid
adce
lula
r(%
Cél
ulas
tota
les)
% R
ecup
erac
ión
celu
lar
(% C
élul
asto
tale
s)
A B
Figuras 4. 6. Porcentaje de viabilidad y de recuperación celular (A y B, respectivamente) del QM31 a concentraciones de 10, 5 y 1 μM (barras negras, grises y blancas, respectivamente).
-10
0
10
20
30
40
50
60
70
Saos-2 HeLa U2-OS U937
% C
aspa
se 3
act
ivity
inhi
bitio
n%
Inhi
bici
ónac
tivid
adca
spas
a3
-10
0
10
20
30
40
50
60
70
Saos-2 HeLa U2-OS U937
% C
aspa
se 3
act
ivity
inhi
bitio
n%
Inhi
bici
ónac
tivid
adca
spas
a3
Figura 4. 7. Medida de la actividad caspasa 3 en diversas líneas celulares. QM31 se testó a 5 μM en todas las líneas. Las medidas se llevaron a cabo tras 24, 48, y 72 h de incubación (barras negras, grises y blancas, respectivamente). Los datos se expresan como media ± ES (n> 3).
4. 4. Capacidad de inhibición de los derivados poliméricos del QM31 y el QM57
Nuestra estrategia sintetizando estos derivados era el de sacar provecho de la eficiente
internalización del peptoide 1 obtenida tras su conjugación al QM56 y, al mismo
tiempo, sacar provecho de la significativa actividad biológica obtenida por los derivados
cíclicos. Con el fin de testar estos nuevos derivados poliméricos, se efectuaron medidas
de su capacidad de inhibir la caspasa 3 (Tabla 4. 2).
30
Tabla 4. 2. Inhibición de la apoptosis por inhibición de la actividad caspase 3 por los diferentes conjugados ciclopeptoide-PGA
U937 Saos-2 Compuesto
24 h 48 h 24 h 48 h
QM31-P2 74±3 (n=3) 85±1 (n=3) 67±4 (n=3) ND
QM31-P3 ND ND 61±7 (n=9) 84±9 (n=6)
QM31-P4 ND ND 53±3 (n=9) 100±3 (n=6)
QM57-P1 ND ND 58±5 (n=6) 100±10 (n=6)
QM57-P2 ND ND 27±9 (n=6) 95±20 (n=6)
Los datos obtenidos muestran como en todos los casos los derivados poliméricos
mostraron una marcada capacidad de inhibir la actividad caspase 3.
31
CAPÍTULO 5: DERIVADOS DEL PEPTOIDE 1 COMO
INHIBIDORES DEL APOPTOSMA: CARACTERIZACIÓN DEL
MECANISMO MOLECULAR DE ACCIÓN
En previos ensayos, se ha postulado la posible interacción del peptoide 1 con Apaf-1
[158]. Con el fin de caracterizar de un modo más preciso esta interacción diversos
ensayos in Vitro y ex vivo se llevaron a cabo y se muestran en el presente capítulo.
5. 1. Ensayos in vitro de caracterización del sitio de unión peptoide 1/Apaf-1
a) Medidas de polarización de fluorescencia
Tal y como se explica en el capítulo 9 de materiales y métodos, se sintetizó un peptoide
1 marcado con CF y su interacción con el dominio CARD de Apaf-1 fue testada. Como
resultado se vio una marcada variación en la fluorescencia de polarización que indicaría
la existencia de una unión entre ambas entidades (Figura 5. 1). Específicamente el
peptoide 1 sería capaz de interaccionar con Apaf-1 a través de su dominio CARD.
Subsiguientes ensayos empleando resonancia magnética nuclear y el CARD de Apaf-1
marcado con 15N demostraron esta misma interacción en presencia de uno de los
derivados del peptoide 1 el PEN-1.
32
405060708090
100110
10 30 50 70 90 110[CARD] nM
mP
O COOH
FO
FHO
O
HN
O
NH
NN
NN
NNH2
O
N
O
O
Cl
Cl
O
O
O
Cl
Cl
A
B
405060708090
100110
10 30 50 70 90 110[CARD] nM
mP
O COOH
FO
FHO
O
HN
O
NH
NN
NN
NNH2
O
N
O
O
Cl
Cl
O
O
O
Cl
Cl
405060708090
100110
10 30 50 70 90 110[CARD] nM
mP
O COOH
FO
FHO
O
HN
O
NH
NN
NN
NNH2
O
N
O
O
Cl
Cl
O
O
O
Cl
Cl
A
B
Figure 5. 1. El CF-peptoide 1 se une al rCARDApaf-1. (A) Estructura química del CF-peptoide 1 (B) Se empleó una solución a 60 nM del CF-peptoide 1 en tampón A y fue titulada con crecientes concentraciones de rCARDApaf-1 (■). La fluorescencia de polarización fue medida a 30 ºC. Los datos se muestran como (media ± s. d. ; n=3) y fueron tomados en unidades de milipolarización (mP) ajustando los datos a un modelo de dos estados.
b) Ensayos de doble híbrido Los ensayos de doble híbrido han sido ampliamente utilizados en la determinación de
interacciones proteína-proteína. Para un mayor conocimiento de la base de dichos
ensayos son interesantes los trabajos presentados por S. Fields [219, 224, 225]. Un
pequeño esquema de la base de este ensayo se muestra en la Figura 5. 2.
Se emplearon las construcciones mostrados en la Tabla 5. 1 con el objetivo de
determinar el efecto de la interacción de nuestros compuestos con Apaf-1. El
objetivo era determinar si esta interacción inhibe la oligomerización de Apaf-1 o
el reclutamiento de procaspasa 9.
33
promotor Gen indicador
X Y
DADU
Maquinaria detranscripción
activo
I
promotor Gen indicador
X
Y
DA
DU inactivo
XAD
DU
Z
inactivo
promotor Gen indicador
A
B
C
promotor Gen indicador
X Y
DADU
Maquinaria detranscripción
activo
promotor Gen indicador
X Y
DADU
X Y
DADADU
Maquinaria detranscripción
activo
I
promotor Gen indicador
X
Y
DA
DU inactivo
I
promotor Gen indicador
X
Y
DA
Y
DADA
DU inactivo
XAD
DU
Z
inactivo
promotor Gen indicador
XADAD
DU
Z
inactivo
promotor Gen indicador
A
B
C
Figure 5. 2. Ensayo de doble híbrido (A) el dominio de unión (DU) se fusiona a la proteína Y. a menos que dicha proteína Y se una a X, el dominio de activación (DA) no se encontrará próximo al DU y la maquinaria de transcripción no será reclutada. Si las células crecen en un medio restrictivo el y el gen marcador de esta interacción es autotrófico no se producirá el crecimiento de las células. (B) Interacción entre las proteínas X e Y resulta en el reclutamiento de la maquinaria de transcripción, la expresión del gen autotrófico (en nuestro modelo de ensayo) y el consiguiente crecimiento de las células en el medio restrictivo. (C) La introducción de una molécula inhibidora (I) de la interacción entre X e Y lleva a la inhibición de la expresión en el gen autotrófico y la ausencia de crecimiento de las células en medio mínimo. Tabla 5. 1. Construcciones empleadas en los experimentos
Plásmido Proteínas de fusión
pGilda
(contiene DU)
(1) Vacío
(2) Apaf-1 (95-530)
(3) Apaf-1 CARD
(4) Procaspasa 9 CARD
pGJ4-5
(contiene DA)
(1) Vacío
(2) Apaf-1 (95-530)
(3) Apaf-1 CARD
(4) Procaspasa 9 CARD *Expresión inducible por galactosa. No expresión en presencia de glucosa.
La cepa de levadura empleada fue transfectada con los diferentes plásmidos pGilda y
pGJ4-5 del ensayo dando lugar a los sistemas 1, 2, 3 y 4. Una vez seleccionadas las
cepas con los diferentes plásmidos, se trataron con el QM31 a diferentes
concentraciones y se siguió su crecimiento con el fin de determinar la capacidad de
34
romper las diferentes interacciones proteicas del QM31. Los resultados se muestran en
la Figura 5. 3. La disminución del crecimiento celular en el sistema 2 tras tratar a las
células con QM31 indicaría que nuestros compuestos son capaces de inhibir la
interacción entre los CARD de Apaf-1 y de procaspasa 9.
00,20,40,60,8
11,21,41,61,8
pGilda/pJG4-5 pGilda Apaf-1CARD/pJG4-5 C9
CARD
pGilda C9CARD/pJG4-5Apaf-1 CARD
pGilda Apaf-1 (95-530)/pJG4-5 Apaf-
1 (95-530)
DO
(600
nm
)
Figura 5. 3. Se efectuaron medidas de la densidad óptica a 600 nm tras 96 h de incubación. Las células se trataron con DMSO o QM31 a concentraciones de 1.25, 2.5 and 5 μM (barras negras, rayadas, grises o blancas, respectivamente).
c) Ensayos in silico
Los ensayos in silico efectuados permitieron confirmar la existencia de una interacción
entre el QM31 y Apaf-1 a través de su dominio CARD (Figuras 5. 4 y 5. 5). La
localización de QM31 en esta cavidad permite formular hipótesis sobre la forma en que
inhibe la actividad proapoptotica de Apaf-1. Por un lado, esta cavidad se encuentra
conectada mediante un canal al lugar de unión de ADP, por tanto es plausible que la
presencia de QM31 bloquee el acceso a dicho lugar por el ATP/dATP necesario para la
activación del apoptosoma. Por otra parte, Riedl y colaboradores. postulan que la
interacción CARD-CARD entre Apaf-1 y procaspasa 9 requiere un cambio
conformacional previo en Apaf-1 que expone residuos que son menos accesibles en la
estructura “cerrada” de Apaf-1(CARD-NOD). La interacción con QM31 podría
estabilizar la estructura de Apaf-1 en dicha conformación “cerrada” impidiendo así la
interacción con el CARD de procaspasa e inhibiendo la formación del apoptosoma.
35
Finalmente, no puede descartarse que QM31 sea capaz de unirse también al complejo
CARD-CARD, si este llega a formarse, perturbando su función.
Figura 5. 4. Modelo del complejo Apaf-1(CARD-NOD)/QM31 obtenido por docking. A. El modelo representa la pose más favorable obtenida en amarillo, mientras que en verde y azul se representan, respectivamente, los dominios CARD y NOD de Apaf-1 [48]. B. Representación de la misma pose y de la superficie de la proteína, coloreada en función del potencial electrostático (negativo en rojo, positivo en azul).
Figura 5. 5. Diagrama de interacciones determinadas por docking entre QM31 y Apaf-1
36
5. 2. Caracterización del sitio de unión en célula entera
Para ello se llevaron a cabo dos tipos de ensayos: el fraccionamiento de extractos
celulares tras tratar a las células con un inductor de muerte y uno de los inhibidores de
Apaf-1; o bien, tratando a las células con el activador del apoptosoma descrito por
Nguyen et al. [151].
5. 2. 1. Fraccionamiento de extractos
Tras tratar a las células tal y como se describe en el capítulo 9 y su subsiguiente
fraccionamiento tras cromatografía de exclusión molecular se analizó la presencia de
procaspasa 9 y Apaf-1 por western-blot. Los resultados indican, en acuerdo con los
resultados de doble híbrido, como en caso de inducir la apoptosis y tratar con el QM56
la procaspasa 9 no es reclutada al apoptosoma (no se observa en las fracciones
correspondientes a los pesos moleculares altos del apoptosoma), algo que sí se observa
en caso de inducir la muerte celular (Figura 5. 6)
5. 2. 2. Inhibición de la apoptosis inducida a través del apoptosoma
Las células fueron tratadas con el inductor de la apoptosis descrito por Nguyen et al. y
nuestros inhibidores. A continuación, se llevaron a cabo ensayos de medida de la
actividad caspasa 3 (Figura 5. 7). Estas medidas indicaron alta capacidad por parte de
nuestros compuestos de inhibir la apoptosis inducida a través del apoptosoma, lo que
corroboraría la especificidad de acción de nuestros compuestos por Apaf-1.
37
Fracción nº
a47 KDa Procaspasa 9
47 KDa Procaspasa 9
47 KDa Procaspasa 9
130 KDa Apaf-1
130 KDa Apaf-1
130 KDa Apaf-1
Fracción nº
a47 KDa Procaspasa 9
47 KDa Procaspasa 9
47 KDa Procaspasa 9
130 KDa Apaf-1
130 KDa Apaf-1
130 KDa Apaf-1
Figura 5. 6. Estudios de cromatografía de exclusión molecular en HeLa. Las células se trataron con PBS (A), doxorrubicina 1. 25 μM (B) o doxorrubicina en presencia de QM56 50 μM, equivalentes de fármaco (C), por 24 h. Los extractos celulares se obtuvieron y se llevó a cabo su fraccionamiento por cromatografía de exclusión molecular. Se recogieron fracciones de 750 μL y se analizó la presencia de Apaf-1 (círculos verdes) y procaspasa 9 (círculos naranjas) a través de Western-blot. Tabla 5. 2. Porcentaje de inhibición de la actividad caspase 3 tras la inducción de apoptosis con el compuesto 2. Los datos se muestran como media ± ES (n = 3).
Línea celular U937 Saos-2
Compuesto 24 h 48 h 24 h 48 h
QM56 82 ± 1 64 ± 1 ND ND
QM31-P2 27 ± 6 95 ± 2 22 ± 2 41 ± 14
38
39
CAPÍTULO 6: CARACTERIZACIÓN DEL EFECTO
CITOPROTECTOR DEL QM31
Demostrada la actividad biológica de los compuestos, el QM31 fue elegido para llevar
a cabo una serie de ensayos con el fin de mejorar la caracterización de su mecanismo de
acción dentro de la célula. Estas medidas fueron llevadas a cabo en los laboratorios del
Dr. Guido Kroemer en el Instituto Gustave Roussy (París, Francia) y el Prof. Douglas
Green en el Hospital de investigación San Judas para niños (Memphis, EE. UU. ).
Estas medidas se dividen en dos partes, una caracterización por citometría e
inmunofluorescencia de diferentes parámetros implicados en la muerte por apoptosis.
A continuación, un estudio más exhaustivo de la influencia de nuestros compuestos en
la cinética de liberación del cit c. Finalmente, un último ensayo efectuado fue un
ensayo de clonogenicidad con el fin de determinar la continuidad del efecto de nuestros
compuestos a largo plazo.
6. 1 Caracterización del efecto citoprotector del QM31 en célula
Para llevar a cabo esta caracterización se realizaron medidas de exposición de
fosfatidilserina por tinción con Anexina V (AnnV) que marcan específicamente el inicio
de la apoptosis; marcaje con yoduro de propidio (IP), como marcaje de la muerte de la
célula; tinción con DiOC6(3) que marca a las células con alto ΔΨm (potencial de
membrana mitocondrial) y, por último, marcaje del cit c en sus diferente localizaciones
mitocondrial (células sanas) y citoplasmática (células apoptóticas).
Los resultados obtenidos se muestran en las Figuras 6. 1 y 6. 2. Asimismo se introdujo
el inhibidor de caspasas Z-VAD-fmk como control para determinar el grado de
citoprotección ejercido por nuestros compuestos. En el caso de medidas de Ann V, PI y
DiOC6(3) QM31 y Z-VAD-fmk mostraron una capacidad de inhibición de la muerte
celular semejante. Sin embargo, cuando fueron comparadas su capacidad de inhibir la
liberación de cit c, el QM31 mostró una clara habilidad para parar este proceso, hecho
que no fue igualado por el Z-VAD-fmk.
40
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Con
trol
CD
DPEv
ento
s
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Con
trol
CD
DP
IP
CDDPZ-VAD-fmk
QM31
CDDPZ-VAD-fmk
QM31
Caspasa 3a-FITC
AnnV-FITC
% C
élul
as%
Cél
ulas
Cas
p-3a
-FIT
C
AnnV+
IP+
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Con
trol
CD
DPEv
ento
s
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Con
trol
CD
DP
IP
CDDPZ-VAD-fmk
QM31
CDDPZ-VAD-fmk
QM31
Caspasa 3a-FITC
AnnV-FITC
% C
élul
as%
Cél
ulas
Cas
p-3a
-FIT
C
AnnV+
IP+
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Con
trol
CD
DPEv
ento
s
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Con
trol
CD
DP
IP
CDDPZ-VAD-fmk
QM31
CDDPZ-VAD-fmk
QM31
Caspasa 3a-FITC
AnnV-FITC
% C
élul
as%
Cél
ulas
Cas
p-3a
-FIT
C
AnnV+
IP+
Figure 6. 3. A549 fueron tratada con cis-platino (CDDP) solo o en combinación con Z-VAD-fmk (pretratamiento de 1 h) 50 μM o QM31 5 μM (post-tratamiento, tras 30 min de la adición del CDDP). Tras 48 h de incubación se efectuó la medida de los diferentes parámetros de apoptosis tal y como se indica en el capítulo 9. Los paneles A y C muestran los datos crudos y la cuantificación de la caspasa 3 activa presente en las muestras (Casp-3a cells). En el caso de los paneles C y D se muestran los datos crudos y las cuantificaciones efectuadas tras la tinción con IP y Ann V (IP-/AnnV+). Los asteriscos indican los valores estadísticamente significativos (analizados con la t de Student, p < 0. 05).
41
Control CDDP
Cyt cmito Cyt ccitop
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
CDDPZ-VAD-fmk
QM31
CDDPZ-VAD-fmk
QM31
CDDPZ-VAD-fmk
QM31
% C
élul
asC
ytcci
top
% C
élul
asC
ytcci
top
% C
élul
as
Cyt c-FITC
DiOC6(3)
Con
trol
CD
DPEv
ento
s
Con
trol
CD
DP
IP
Cyt cmito Cyt ccitop
DiOC6(3) lowIP+
Control CDDP
Cyt cmito Cyt ccitop
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
Control Z-VAD-fmk QM31Z-VAD-fmk
+ QM31
CDDPZ-VAD-fmk
QM31
CDDPZ-VAD-fmk
QM31
CDDPZ-VAD-fmk
QM31
% C
élul
asC
ytcci
top
% C
élul
asC
ytcci
top
% C
élul
as
Cyt c-FITC
DiOC6(3)
Con
trol
CD
DPEv
ento
s
Con
trol
CD
DP
IP
Cyt cmito Cyt ccitop
DiOC6(3) lowIP+
Figura 6. 4. Las células A549 se trataron tal y como se indica en la figura 6. 3 y en el capítulo 9 de la tesis y se efectuó la posterior medida de la liberación de cit c. En el panel A, se muestra una inmunofluorescencia representativa de la localización del cit c mitocondrial (Cit c mito) vs. citosólico (Cit c rel) Cit c (señal verde). Los núcleos se han teñido en azul con Hoechst 33342. las barras blancas indican la escala de la fotografía. Columnas en el panel B indican el porcentaje de células con el cit c en el citosol. En el panel C y E se muestran los datos crudos de las tinciones de cit c y de DiOC6(3)/ PI) efectuadas. El análisis cuantitativo de las mismas se muestra en los paneles D y F. En todos los casos los datos se muestran como media ± ES, n = 4. Los asteriscos indican los resultados significativos estadísticamente (Test de student, p < 0. 05).
42
6. 2 Comparación de la supervivencia celular en Heltog tratadas con QM31 y q-
VD-OPH
Con el objetivo de estudiar de un modo más específico la influencia del QM31 sobre la
liberación del cit c y con la idea de una posible existencia de un bucle de amplificación
de la permeabilización de la membrana externa mitocondrial (MOMP, tomaremos la
abreviatura en inglés a partir de este momento). Se efectuaron estudios empleando la
línea celular HeLa expresando el cit c-GFP (Heltog) con el fin de efectuar el
seguimiento de esta proteína por microscopia confocal de fluorescencia. Se efectuó una
puesta a punto de las condiciones de ensayo empleando estaurosporina (STS), un
inhibidor inespecífico de fosfoquinasas, y se trataron las células con el QM31 y el q-
VD-OPH. Se observó que el QM31 necesitaba estar acompañado de q-VD-OPH en este
modelo para ejercer un efecto sobre la liberación del cit c. Este hecho podría deberse a
que la línea celular Heltog presenta una mayor resistencia a los estímulos apoptóticos y
una mayor velocidad de división. Asimismo, se observó un ruido de fondo verde en el
citosol que podría corresponder al cit c-GFP que estuviera presente en el citosol, aunque
este hecho no pudo ser corroborado. Este hecho podría afectar a su comportamiento
tras la inducción de la apoptosis y haría que nuestro compuesto únicamente no fuera
suficiente a la hora de frenar el proceso de muerte.
Por tanto, tratando a las células con STS y QM31 y q-VD-OPH, estos últimos solos o en
combinación se llevaron a cabo los diferentes estudios de la cinética de liberación del cit
c. Los resultados se muestran en la Figura 6. 3. Se observó, como la presencia de
QM31 produce un retraso en la liberación del cit c cuando se compara con el q-VD-
OPH solo. Este hecho confirma las observaciones hechas con la línea celular A549,
aunque en este caso el QM31 ejercía su función inhibidora de apoptosis sin ayuda del Z-
VAD-fmk.
43
B
0
5
10
15
20
25
30
35
DMSO QM31 q-VD-OPH q-VD-OPH +QM31
STS
Tie
mpo
(h)
BB
0
5
10
15
20
25
30
35
DMSO QM31 q-VD-OPH q-VD-OPH +QM31
STS
Tie
mpo
(h)
0
5
10
15
20
25
30
35
DMSO QM31 q-VD-OPH q-VD-OPH +QM31
STS
Tie
mpo
(h)
Figura 6. 3. Las Heltog fueron tratadas con STS 0. 25 μM por 6 h sola o en combinación con QM31 5 μM y/o q-VD-OPH 20 μM. Tras 6 h, la STS fue eliminada y se añadió medio fresco a las muestras junto con QM31 y/o q-VD-OPH en las muestras correspondientes. Tras añadir IP al medio, fotos de las diferentes muestras se tomaron durante 37 h (FITC, verde, y dsRed, rojo) a intervalos de 15 min. (A) Ejemplo representativo de las diferentes fotografías tomadas y los fenotipos obtenidos. (B) Cuantificación de la liberación de cit c y la tinción por IP.
6. 2. 2 Ensayos de clonogenicidad con Heltog
Intrigados por los resultados obtenidos y con el fin de determinar si la inhibición de
Apaf-1 puede ayudar verdaderamente a las células a sobrevivir tras la inducción de
apoptosis, se llevó a cabo un ensayo de clonogenicidad tras inducir la muerte en las
células con STS y tratarlas con QM31 y/o q-VD-OPH. Los resultados se muestran en la
Figura 6. 4. Estos datos indicarían que la inhibición de Apaf-1 puede ayudar a las
células a sobrevivir tras un estímulo de muerte al observarse un marcado incremento en
el número de colonias cuando QM31 en combinación con q-VD-OPH se añade a las
células (q-VD-OPH muestra una modesta capacidad de aumentar la formación de
44
colonias). Aunque quedan todavía experimentos por realizar, estos datos podrían
indicar que la inhibición de Apaf-1 aumenta el tiempo en el que las células pueden
recuperarse de un estímulo de muerte. Asimismo, los datos obtenidos indicarían
también la posible existencia de un bucle de amplificación del MOMP y que es
dependiente de Apaf-1 [42, 249, 250].
Figura 6. 9. Ensayo clonogénico en Heltog. 2500 células/pocillo fueron sembradas en una placa de 12 pocillos y tras 48 h las células fueron tratadas sin (A) o con STS 2 uM (B) por 6 h. Pasado este tiempo, la STS fue eliminada y medio fresco conteniendo los diferentes compuestos en concentraciones QM31 5 μM y q-VD-OPH 20 μM fueron añadidos. 24 h después se cambió el medio y los compuestos fueron añadidos de nuevo. Después de 2 semanas se efectuó la cuantificación de las colonias formadas.
45
CAPÍTULO 7: FUNCIONES DE APAF-1 NO RELACIONADAS
CON LA APOPTOSIS
Tras la publicación del artículo de Zermati et al. [94], se le ha atribuido un nuevo rol a
Apaf-1 independiente de la apoptosis como es el de efectuar un arresto en la fase S del
ciclo celular tras inducir un daño subletal a las células. Con el fin de comprobar que
dicho efecto era también inhibido por nuestros compuestos y que dichos compuestos no
eran simplemente inhibidores de la capacidad apoptótica de Apaf-1. Algunos de los
ensayos mostrados en dicho artículo fueron reproducidos en presencia de nuestros
compuestos.
7. 1. Inhibición del arresto celular en fase S por el QM31
De nuevo se tomó al QM31 como molécula modelo y tras tratar a las células A549 tal y
como se indica en el capítulo 9 con dosis subletales de CDDP y el QM31 se analizó el
porcentaje de células en cada una de las fases del ciclo celular. El resultado obtenido
demuestra cómo el QM31 es capaz de ejercer un efecto semejante al del siRNA de
Apaf-1 y reducir en un 10 % aproximadamente el porcentaje de células en fase S tras el
arresto producido en dicha fase por la adición del CDDP (Figura 7. 1). Se comprueba
así que nuestros compuestos son capaces de frenar otras funciones de Apaf-1 en la
célula diferentes a la apoptosis.
46
Eve
ntos
% C
élul
asto
tale
s
siUNR siApaf-1
siUNRsiApaf-1
Control QM31 5 μM QM31 7.5 μM
β-actina
Apaf-1
Con
trol
CD
DP
A
B
C
IP
130 kDa
47 kDa
G2/M
S
G0/G1
CDDPQM31 5 μM
QM31 7.5 μM
Eve
ntos
% C
élul
asto
tale
s
Eve
ntos
% C
élul
asto
tale
s
siUNR siApaf-1
siUNRsiApaf-1
Control QM31 5 μM QM31 7.5 μM
β-actina
Apaf-1
Con
trol
CD
DP
A
B
C
IP
130 kDa
47 kDa
G2/M
S
G0/G1
CDDPQM31 5 μM
QM31 7.5 μM
Figura 7. 1. A549 fueron transfectadas con siRNA control (siUNR)o específico para Apaf-1 (siApaf-1) por 48h. Tras este tiempo se trataron con el QM31 por una hora. Pasado este tiempo CDDP 20 μM fue añadido y 24 h después se efectuó la cuantificación de la reducción de Apaf-1 en el medio por el siApaf-1 (C) y el consiguiente análisis y cuantificación del porcentaje de células en los distintos estadíos del ciclo celular (A, B). Barras negras, blancas y grises marcan el porcentaje de células en las fases G0/G1, S y G2/M, respectivamente. Los resultados se muestran como media ES (n = 3).
7. 2. Influencia de dosis altas de L-glutamina en la supervivencia celular tras el
tratamiento con los derivados del peptoide 1
De modo accidental se observó cómo la presencia de dosis altas de L-glutamina
(GlutaMAX®) en el medio en presencia de los inhibidores de Apaf-1 causaban una
importante disminución de la viabilidad celular (Figure 7. 3). La toxicidad de la L-
47
glutamina en altas dosis se ha descrito anteriormente [268, 269], pero en este caso
solamente se apreciaba en combinación con nuestros compuestos. Con el fin de
caracterizar el tipo de muerte que podría ser inducida se efectuaron estudios de
inducción de autofagia mediante el estudio de la proteína LC-3 marcada
fluorescentemente en la línea celular MCF-7 (Figure 7. 4). Como resultado se observó
una inducción de la autofagia tras la adición del QM31 y del QM56 en presencia de
dosis altas de L-glutamina. En el caso del QM56 se observó incluso una disminución
de la viabilidad celular a concentraciones de 50 μM equivalentes de fármaco.
Los resultados obtenidos nos hicieron pensar en una hipótesis “arriesgada” como es la
existencia de una conexión entre Apaf-1, el metabolismo de la L-glutamina y la
producción de ATP. Esta hipótesis surgió a raíz del trabajo presentado por el grupo de
Pascal Meier en el que describían una posible interacción entre DARK (homólogo de
Apaf-1 en Drosophila) y GFAT2, una proteína implicada en la ruta de las hexosaminas
(RH) [279]. La glutamina-6-fosfoaminotransferasa 2 es una isoforma de la GFAT2.
Ambas proteínas controlan el flujo de glucosa en la RH y catalizan la reacción de
formación de glucosamina-6-fosfato. La activación de estas rutas debido a un exceso de
glucosa se ha relacionado con la progresión de diversas patologías asociadas a
hiperglicemia [281-284], alteraciones de las proteína quinasas activadas por AMP
(AMPK) y niveles de Ca+2 [281], este hecho podría relacionarse también con el aumento
de autofagia observado y el estado de pseudo-hipoxia metabólica debido a un descenso
en los niveles de ATP. Interesantemente, la sobreexpresión de GFAT o el tratamiento
con glucosamina (sustrato de GFAT) reproducen algunos de los fenotipos observados
[283]. Por esta razón, se llevó a cabo el ensayo de reconstitución in vitro del
apoptosoma con el fin de determinar la posible interacción entre GFAT2 y Apaf-1 en
humanos (Figura 7. 2). Como control positivo del experimento se utilizó Bcl-XL cuya
interacción con Apaf-1 [43] in vitro ha sido descrita y la adición de la proteína CDK2
sirvió como control negativo de interacción.
48
0
0,5
1
1,5
2
2,5
3
Control PGA QM56 PGA QM56 PGA QM56
50 25 10
OD
(570
nm
)
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM
A
C
B
eGFP-LC3 diffused
eGFP-LC3 dots
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM
A
C
B
50 μM50 μM 25 μM25 μM 10 μM10 μM
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM0
102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
ado
(% T
otal
cel
ls)
ControlQM31
1 μM 5 μM
A
C
B
eGFP-LC3 difuso
eGFP-LC3 vesicular
0
0,5
1
1,5
2
2,5
3
Control PGA QM56 PGA QM56 PGA QM56
50 25 10
OD
(570
nm
)
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM0
102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM
A
C
B
eGFP-LC3 diffused
eGFP-LC3 dots
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM0
102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM
A
C
B
50 μM50 μM50 μM50 μM 25 μM25 μM25 μM25 μM 10 μM10 μM10 μM10 μM
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
atio
n (%
Tot
al c
ells
)
ControlQM31
1 μM 5 μM0
102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
ado
(% T
otal
cel
ls)
0102030405060708090
Control 1 mM 5 mM
QM31% L
C-3
tran
sloc
ado
(% T
otal
cel
ls)
ControlQM31
1 μM 5 μM
A
C
B
eGFP-LC3 difuso
eGFP-LC3 vesicular
Figura 7. 2. MCF-7 sobreexpresando el LC3-GFP se cultivaron en presencia de 2 mM GlutaMAX con el QM31 5, 1 μM o QM56 o PGA 50, 25 and 10 μM equivalentes de fármaco. En el panel A, se muestra una fotografía ejemplo de la localización de LC-3-GFP en células sanas (eGFP-LC3 difuso) y en células en la que la autofagia ha sido inducida y se produce su translocación a los autofagosomas (eGFP-LC3 vesicular). En el panel B se muestra la cuantificación efectuada de las células en las que LC3 se transloca a los autofagosomas. Los datos se expresan en media ± ES (n =3). Finalmente, el panel C muestra un ensayo de viabilidad celular en presencia de QM56 y GlutaMAX® tras incubar las células por 24, 72, 96 y 120 h (barras negras, grises, grises con círculos y blancas, respectivamente).
49
0
10
20
30
40
50
1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1
GFAT2/Apaf-1 Bcl-XL/Apaf-1 CDK2/Apaf-1
% C
aspa
se 9
act
ivity
inhi
bitio
n%
Inhi
bici
ónac
tivid
adca
spas
a9
Figura 7. 3. Reconstitución in vitro del apoptosoma. Se incubó rApaf-1 40 nM con GFAT2, Bcl-XL y CDK2 en proporciones 1:1, 1:2 y 1:3 durante 30 min a 30 ºC. El resto de componentes del apoptosoma se añadió tal y como se describe en el capítulo 9. Finalmente, la actividad caspasa 9 fue medida y se expresó como inhibición de la actividad caspase 9.
De acuerdo a los datos obtenidos, podría existir una interacción entre GFAT2 y Apaf-1.
La proteína GFAT2 fue capaz de inhibir la actividad caspasa 9 al igual que Bcl-XL, tal
y como se esperaba de este control. En el caso de CDK2 no se observó su influencia en
la formación del apoptosoma. De acuerdo a estos datos preliminares, una posible
explicación a nuestros resultados podría encontrarse en la existencia de una interacción
GFAT2/Apaf-1 en la que ambas proteínas inhiben la función de la otra. Este complejo
(u otro conteniendo un mayor número de proteínas) podría verse afectado por la adición
de nuestros compuestos y debido a la existencia de altas concentraciones de L-
glutamina se produciría la activación de la RH con todas las consecuencias negativas
para la célula que hemos observado.
50
51
CAPÍTULO 8: EXPERIMENTOS DE HIPOXIA IN VIVO
Diversos estudios efectuados en humanos y animales han demostrado la importancia de
la apoptosis en procesos relacionados con isquemia (ataques, fallos cardíacos, procesos
derivados del transplante de órganos,. . . ) [95, 96]. Se ha descrito también que el
principal factor transcripcional implicado en la regulación de la respuesta a hipoxia
controla la expresión de diversas proteínas que se unen a Apaf-1. Por tanto la
inhibición de Apaf-1 puede ser una Buena estrategia con el fin de disminuir el daño
causado por la hipoxia. [98, 99]. Experimentos preliminares realizados en nuestro
laboratorio han demostrado la eficacia de los inhibidores de Apaf-1 en la disminución
del daño causado por la hipoxia en cultivos primarios de cardiomiocitos [200]. Con esta
premisa y con el objetivo de encontrar una aplicación terapéutica de nuestros
compuestos se desarrolló en colaboración con el Dr. Antonio Alcaraz (Hospital Clínic,
Barcelona) un modelo animal de isquemia en caliente, ya que este el tiempo de
isquemia es crucial en la determinación de la viabilidad de un órgano para ser
transplantado. En nuestro caso el estudio se llevó a cabo con riñones de ratón
sometidos a hipoxia. Se trata de experimentos preliminares que podrán mejorarse en
rondas sucesivas.
8. 2. Estudio de la inhibición de apoptosis en un modelo de hipoxia en rata
En el presente estudio, se seleccionaron una serie de inhibidores de Apaf-1 que
presentaron una mayor actividad biológica en ensayos anteriores y se testaron frene a
ratas Wistar bajo diferentes condiciones de isquemia en caliente. Los parámetros
estudios realizados fueron análisis patológicos, TUNEL y medida de la actividad
caspasa 3. Se escogieron 7 grupos de 8 animales cada uno (Tabla 8. 1). Tras pretratar a
las ratas durante 6 h con cada uno de los compuestos se sacrificaron y se mantuvieron a
37 ºC por 90 min. Pasado este tiempo los riñones fueron extirpados y divididos en tres
partes para llevar a cabo los análisis antes mencionados. Los resultados obtenidos se
muestran en las Figuras 8. 1, 8. 2 y 8. 3.
52
Tabla 8. 1. Descripción de los grupos de animales y pretratamientos aplicados a cada grupo.
Grupo Pretratamiento
I (1-8) Control PBS (vehículo para terapias con PGA)
II (9-16) Control DMSO
QM57 se solubilizó en PBS:DMSO (70:30) (v/v)
III (17-24) QM57 (5 mg/Kg)
IV (25-32) QM57 (10 mg/Kg)
V (33-40) QM56 (70 mg/Kg)
VI (41-48) QM31-P2 (100 mg/Kg)
VII (49-56) QM31-P2 (45 mg/Kg)
De los compuestos seleccionados el QM57 fue el que mostró una mayor capacidad de
disminuir el daño tisular en los ensayos patológicos. Asimismo, mostró capacidad de
reducir el fenotipo apoptótico en los ensayos por TUNEL. Sin embargo, mostró nula
capacidad para disminuir la actividad caspase 3. Este ultimo hecho podría deberse a
problemas en los ensayos con el DMSO utilizado para disolver el compuesto. En el
caso de los polímeros estos mostraron poca capacidad de disminuir el daño tisular en los
ensayos patológicos, pero disminuyeron el fenotipo apoptótico en los ensayos de
TUNEL e inhibieron la actividad caspasa 3. la causa del bajo efecto de los polímeros en
los ensayos patológicos podría deberse a problemas en el tiempo de tratamiento y de la
inducción del estímulo de muerte ya que deben ser procesados por la catepsina B para
ser liberado el fármaco. No obstante, estos problemas podrán se solucionados en un
futuro próximo.
En conjunto, podemos decir que la inhibición de apoptosis por Apaf-1 es una estrategia
efectiva a la hora de disminuir el daño renal por isquemia en caliente. De este modo nos
encontramos con una más que posible aplicación terapéutica de nuestros compuestos,
aunque para ello necesitaremos realizar un mayor número de experimentos con el fin de
seleccionar un único candidato.
53
A
PBSDMSO
QM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
QM57 10 mg/Kg
QM57 5 mg/Kg
B C
AA
PBSDMSO
QM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
QM57 10 mg/Kg
QM57 5 mg/Kg
B C
PBSDMSO
QM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
QM57 10 mg/Kg
QM57 5 mg/Kg
B C
-ns0.234Kruskal Wallis
-ns0.241Anova
ResultadosemparejadosResumenpTest estadístico
-ns0.234Kruskal Wallis
-ns0.241Anova
ResultadosemparejadosResumenpTest estadístico
-ns0.096Kruskal Wallis
DMSO vs QM57 10 mg/Kg *0.044Anova
ResultadosemparejadosResumenpTest estadístico
-ns0.096Kruskal Wallis
DMSO vs QM57 10 mg/Kg *0.044Anova
ResultadosemparejadosResumenpTest estadístico
Valo
raci
ón
Valo
raci
ón
ControlValoración 0
90 min isquemiaValoración 4
Figura 8. 1. Ensayos patológicos e histológicos de las preparaciones de los riñones sometidos a isquemia. (A) Rango de puntuaciones dado en función del estado del tejido: tejido sano (score 0) y tejido dañado (score 4). (B,C) Los animales fueron pretratados durante 6 h y se sometieron a hipoxia en caliente por 90 min. Los resultados se evaluaron por Anova y el test de Kruskal-Wallis. PBS y DMSO (PBS:DMSO, 70:30) fueron los vehículos control.
54
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
DMSOQM57 10 mg/Kg
QM57 5 mg/Kg
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
A B
C D
A B
C D
E FE F
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Resultadosemparejados
ResumenpTest estadístico
-*0.025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b**0.007Anova
Resultadosemparejados
ResumenpTest estadístico
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Resultadosemparejados
ResumenpTest estadístico
DMSO vs QM57 10 mg/Kg**0.002Kruskal Wallis
DMSO vs QM57 10 mg/Kg**0.007Anova
Resultadosemparejados
ResumenpTest estadístico
Pos
itivo
s/ca
mpo
Pos
itivo
s/ca
mpo
Figura 8. 2. Ensayo de TUNEL aplicado a un número determinado de preparaciones. Los resultados se evaluaron por el test de Kruskal-Wallis.
55
Tabla 8. 2. Análisis de la actividad caspase 3 en riñón
Compuesto % inhibición actividad caspasa 3
PBS (control) 0 DMSO (control) 0
QM57** 5 mg/Kg 0 QM57** 10 mg/Kg 0 QM56 70 mg/Kg 79
QM31-P2 45 mg/Kg 75 QM31-P2 100 mg/Kg 60
** Inspección visual de las muestras sugirió una posible interferencia del DMSO empleado como vehículo en la medida de esta actividad enzimática.
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
Act
ivid
ad D
EV
Das
a(Δ
F/m
in)
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
Act
ivid
ad D
EV
Das
a(Δ
F/m
in)
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
PBSQM56 70 mg/Kg
QM31-P2 100 mg/Kg
QM31-P2 45 mg/Kg
Act
ivid
ad D
EV
Das
a(Δ
F/m
in)
-**0.0025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b***0.0002Anova
Resultados emparejadosResumenpTest estadístico
-**0.0025Kruskal Wallis
PBS vs QM56PBS vs QM31-P2a
PBS vs QM31-P2b***0.0002Anova
Resultados emparejadosResumenpTest estadístico
Figura 8. 3. Análisis estadístico de los valores obtenidos de inhibición de la actividad caspasa 3
56
57
CAPÍTULO 9: CONCLUSIONES
El objetivo principal del trabajo presentado era el de desarrollar una segunda generación
mejorada de inhibidores de Apaf-1 así como la caracterización de los mecanismos
moleculares de acción de los mismos. El desarrollo de dichos inhibidores se llevó a
cabo ante la necesidad de inhibidores de apoptosis que fueran efectivos y pudieran
avanzar hasta convertirse en fármacos para el posible tratamiento de enfermedades
relacionadas con una muerte por apoptosis excesiva. Las estrategias iniciales se
dirigieron a la búsqueda de inhibidores de caspasas. Sin embargo, estos inhibidores no
han cumplido las expectativas debido a la existencia de problemas de internalización
celular y de mecanismos de compensación de muerte independiente de caspasas. La
situación estratégica del apoptosoma antes de la activación de la caspasa 3 y por debajo
de la inducción de MOMP ha llevado a considerar a este complejo proteico como una
más que interesante diana terapéutica. Hasta ahora, solamente se ha descrito un
inhibidor de Apaf-1 a través de un cribado de una quimioteca realizado con extractos
celulares [155]. No se ha descrito su mecanismo de acción o el tipo de interacción
existente entre dicha molécula y Apaf-1. Por esta razón, en nuestro laboratorio se
propuso un ensayo basado en la reconstitución del apoptosoma in vitro a través de sus
componentes recombinantes con el fin de conseguir compuestos que verdaderamente
interaccionaran con alguno de los componentes del apoptosoma y modularan su
actividad. El cribado de una quimioteca de N-alquilglicinas permitió la identificación
del peptoide 1, una molécula capaz de inhibir la formación del apoptosoma.
Sin embargo, problemas de internalización celular llevaron a la síntesis de derivados de
dicha molécula con el fin de conseguir una mayor capacidad de penetrar la membrana
celular. Se sintetizaron diversos derivados siendo los derivados de conformación
restringida (QM31 y familia) y la conjugación a un transportador polimérico las
estrategias que mejor funcionaron en diferentes ensayos ex vivo. En ambos casos,
QM31 y QM56 mostraron baja toxicidad y una actividad biológica significativa
reduciendo la muerte por apoptosis en las células. Posteriormente, se caracterizó el
modo de acción de los compuestos y el tipo de interacción con Apaf-1. Se determine
cómo los compuestos impiden el reclutamiento de procaspasa 9 por Apaf-1, ya que
estos se unen al dominio CARD de Apaf-1.
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Una cuestión crucial a ser contestada en apoptosis es si una célula es capaz tras iniciar el
proceso de apoptosis de recuperarse y ser viable. En la presente tesis se ha intentado
abordar este problema. Los resultados aquí presentados indican que la respuesta es sí
cuando se inhibe la apoptosis mediante inhibición de Apaf-1. Mediante ensayos de
clonogenicidad se demostró que la inhibición de Apaf-1 no sólo reduce de manera
marcada el fenotipo apoptótico sino que disminuye la cantidad de cit c liberado al
citosol. Con estos resultados en mente y diversos trabajos que apuntan a la existencia
de un bucle de amplificación de MOMP dependiente de las caspasas 9 y 3 y de Bid [42,
249, 250]se llevó a cabo un ensayo de clonogenicidad. En este ensayo se demostró
cómo las células tratadas con el QM31 mostraban un aumento significativo del número
de colonias cuando se comparaba con el control de la muestra.
Durante un largo tiempo se asumió que las caspasa (proteasas responsables de la
ejecución de la muerte celular), las proteínas proapoptóticas de la familia Bcl-2 y Apaf-
1 podrían jugar un papel importante en la señalización de la muerte celular. Sin
embargo, se produjo un cambio en el pensamiento con el aumento de publicaciones
indicando la realización de funciones no apoptóticas por parte de estas proteínas [93,
94]. De hecho, nuestros inhibidores sirvieron para confirmar la participación de Apaf-1
en procesos de control del ciclo celular. Lo que es más, en esta tesis quisiera también
hipotetizar una posible función extra de Apaf-1 en la célula diferente de la apoptosis. El
tratamiento de las células con los inhibidores de Apaf-1 en presencia de concentraciones
altas de L-glutamina induce una marcada toxicidad celular. La causa podría estar en
una interacción GFAT2-Apaf-1 indicada con anterioridad en Drosophila [279] y hemos
encontrado evidencias de una inhibición parcial de la formación del apoptosoma por la
GFAT2. Mi hipótesis, necesita todavía un largo proceso de experimentación, pero sería
la de proponer que GFAT2 y Apaf-1 interaccionan y ejercen un efecto inhibidor la una
sobre la otra y se regularía así la activación de la ruta de las hexosaminas. Si se
confirmara, el papel de Apaf-1 en célula cambiaría totalmente pasando a considerarse
una proteína clave en el control del metabolismo celular tras la inducción de muerte.
Demostrada la eficiencia de los inhibidores de Apaf-1 como una estrategia para
recuperar la viabilidad celular, pasamos a buscar una posible aplicación terapéutica a
nuestras moléculas. Se desarrolló un modelo animal de isquemia en caliente y tras
tratar a los animales con nuestros compuestos se obtuvo una marcada reducción del
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daño tisular en riñón. Asimismo, se obtuvo una reducción de los parámetros de
apoptosis. Este hecho abre las puertas a una posible aplicación de nuestros compuestos
en el tratamiento de enfermedades relacionadas con isquemia y el transplante de
órganos. Asimismo, esta no es la única posible aplicación de Apaf-1, ya que el papel
crucial de Apaf-1 en la muerte neuronal, abre las puertas a una posible aplicación en el
tratamiento de enfermedades neurodegenerativas como puede ser el Huntington [141].
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CAPÍTULO 10: MATERIALES Y MÉTODOS
10. 1. Síntesis del peptoide 1 y sus derivados
Las síntesis del peptoide 1, TAT-1, PEN-1, QM56, QM31 y derivados del QM31 se
hallan descritas en los artículos aquí referenciados [192, 196, 200]. En el caso del
QM57 y derivados y de los derivados del QM56 por razones de patentabilidad no se
efectuará la descripción de dichas síntesis.
10. 2. Manipulación de Escherichia coli: producción de procaspasa 9 y CARDApaf-1
Se emplearon células BL21(DE3) y BL21(DE3)pLysS Codon + de bacteria para la
expression de las proteínas procaspasa 9 y el dominio CARD de Apaf-1 cuyo ADN se
encontraba dentro de los plásmidos pET23b y pET15b, respectivamente. En el caso de
la procaspasa 9 se siguió el protocolo publicado por Denault et al [203]. En el caso del
dominio CARD de Apaf-1 se siguieron las indicaciones del artículo de Riedl et al. [48]
para la expresión de Apaf-1 1-591. En el caso de la expresión del 15N-CARD las
células se crecieron en medio mínimo buscando las condiciones de densidad óptica del
artículo de Riedls en presencia de 15NH4Cl.
10. 3. Manipulación de Saccharomyces cerevisiae
Se empleó la cepa EGY048 modificada genéticamente (MATα trp1, ura3, his3
leu2::p6LexAop-LEU2). Esta cepa se creció en medio SC-drop out eliminando los
aminoácidos trp e his para seleccionar las células transfectadas con los plásmidos
correspondientes. Por último, se eliminó la leu para determinar qué proteínas
expresadas por los plásmidos y fusionadas al DU y al DA eran capaces de interaccionar
y activar el gen que nos reporta la existencia de interacción entre las proteínas. .
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10. 4. Manipulación de células de insecto
Se efectuó de acuerdo a los protocolos descritos en el artículo referenciado [158].
10. 5. Manipulación de células de mamífero
Los protocolos correspondientes a los ensayos de MTT, de medidas de actividad
caspasa 3, de internalización celular, de medida de los diferentes parámetros de
apoptosis, de medida de diferentes parámetros del ciclo celular se encuentran descritos
en los artículos indicados [196, 200, 240]. El resto de experimentos se detallan a
continuación.
a) Medidas de polarización de fluorescencia
rCARDApaf-1 se disolvió en 20mM NaH2PO4 pH = 6 y se añadió a una solución de CF-
peptoide 240 nM. La polarización de fluorescencia se midió tras cada adición de
proteínas a 30 ºC. (λexc = 485 and λem = 535 nm).
b) Interacción 15N-CARD/peptoide por RMN
Se incubó el 15N-CARD junto con el PEN-1 en tampón 20 mM NaH2PO4 pH 6 a
concentraciones de 75 y 100 μM, respectivamente. Se efectuaron medidas de los
espectros de 15N-HSQC del CARD de Apaf-1 en presencia y en ausencia de PEN-1.
c) Estudios de interacción in silico
Los experimentos de docking se realizaron empleando el modulo Dock incluido en el
programa MOE (Chemical Computing Group, Montreal), considerando la proteína
rígida y los ligandos flexibles. Para el cálculo de energías se empleó el campo de
fuerzas MMFF94x y condiciones de solvente continuo[230, 298, 299].
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d) Fraccionamiento de extractos por cromatografía de exclusión molecular
Se trataron 7·106 células Hela con doxorrubicina sola o en combinación con QM56 a 50
μM equivalentes de fármaco por 24 h. Como control se añadió el volumen equivalente
de PBS. Pasadas 24 h las muestras se procesaron de acuerdo al artículo [110] con la
diferencia de que la columna empleada para el fraccionamiento fue una columna
Superosa 6 10/300 de Amersham.
e)Experimentos de inducción de apoptosis en Heltog
Las células Heltog fueron donadas por el Prof. Douglas Green (Hospital de
investigación San Judas, Memphis, EE. UU. ). Se trataron con STS 0. 25 μM por 6 h
sola o en combinación con QM31 5 μM y/o q-VD-OPH 20 μM. Pasado este tiempo se
cambió el medio eliminándose así la STS y el QM31 y q-VD-OPH se volvieron a
añadir. Así reefectuaron los estudios de cinética de liberación del cit c y los ensayos de
clonogenicidad. En el caso de los ensayos de clonogenicidad, se volvió a cambiar el
medio 24 h después. La tinción de las colonias se efectuó con azul de metileno 0.5 %
en una mezcla metanol:agua al 50 %.
10. 6. Experimentos in vivo de isquemia en caliente
Los compuestos fueron inyectados a las ratas mediante vía intravenosa. Pasadas 6 h los
animales fueron sacrificados y mantenidos a 37 ºC durante 90 min. A continuación, se
extirparon los riñones y se dividieron en tres partes para llevar a cabo los diferentes
ensayos: patológicos, TUNEL y medida de la actividad caspasa 3.
a) Estudios patológicos
Los riñones se fijaron en formaldehído y seguidamente se embebieron en parafina para
su posterior corte en láminas de 5 μM. Las tinciones utilizadas fueron Masson tricromo
y hematoxilina. Se evaluó la simplificación tubular, la existencia de mitosis y la
pérdida de núcleos en el tejido de riñón.
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b) Ensayo de TUNEL
Los riñones se conservaron en formol más 20 % sacarosa y se procesaron de acuerdo a
las indicaciones del artículo [305] indicado.
c) Medida de la actividad caspase 3
Los riñones fueron machacados hasta polvo tras ser congelados en nitrógeno líquido.
Seguidamente, se les añadió un tampón de extracción (50 mM Hepes-KOH pH = 7,
10% sacarosa, 0.1% CHAPS, 5 mM DTT, 5 mM GSSG). Seguidamente las muestras se
sonicaron y se procedió a medir la caspase 3 de acuerdo al protocolo descrito en los
artículos [196, 200] mencionados.