conditions - elteszerves.chem.elte.hu/oktatas/ea/gyogysz/1_eloadas_2017.pdf · 2018. 9. 20. ·...
TRANSCRIPT
Conditions
1 Two components two sets of lecturers
2 Lectures 1-5 Prof F Hudecz Lectures 6-10 Dr Gy Domaacuteny 3 Examination two parts determined by the lecturers and one mark - option A written test - option B presentation based on literature - option C oral examination 4 Participation at lectures gt 70 fhudeczeltehu
First generatioin Second generation New generation
Oxytocin (L) Carbetocin (S) Abarelix (GnRH) (L) ACTH (1-24) amp (1-39) (LS) Terlipressin (LS) Cetrorelix (GnRH) (L) Vasopressin (LS) Felypressin (LS) Ganirelix (GnRH) (L) Insulin (ESS R) Buserelin (LS) Eptifibatide Glucagon (ESR) Deslorelin (LS) Bivalirudin (L) Calcitonins (LSR) Goserelin (L) Copaxone (L) TRH (L) Histrelin (L) Techtide P-289(S) Gonadorelin (LS) Leuprolide (LS) Cubicin (F) Somatostatin (LS) Nafarelin (S) Fuzeon (antiHIV (H) GHRH (1-29) amp (1-44) (S) Tryptorelin (LS) Ziconotide (pain) (S) CRF (Human amp Ovine) (S) Lecirelin (S) Pramlintide (diabetes) (S) Cyclosporin (F) Lanreotide (S) Exenatide (diabetes) (S) Thymopentin (L) Octreotide (LS) Icatibant (brady-rec) Thymosin Alpha-1 (S) Atosiban (L) Romiplostim (hormon) Secretins (Human amp Porcine) (ES) Desmopressin (LS) Degarelix (GnRH) Parathyroid Hormone (1-34) amp (1-84)(S) Lypressin (L) Mifamurtide (raacutek adj) Vasoactive Intestinal Polypeptide (S) Ornipressin Ecallantide (oumldeacutema) Brain Natriuretic Peptide (R) Pitressin (L) Liraglutide (diabetes) Cholecystokinin (L) ACE Inhibitors (Enalapril Lisinopril) (L) Tesamorelin Tetragastrin (L) HIV Protease Inhibitors (L) Surfaxin Pentagastrin (L) Peginesatide Eledoisin (L) Carfilzomib Linaclotide (enzinh)
L = in solution S = on solid phase E = extraction F = fermentation H = hybrid synthesis R = recombinant SS = semi-synthesis
Some Approved Peptide Pharmaceuticals and their Methods of Manufacture
Oxytocin
Structure 1953 V du Vigneaud Synthesis 1954 V du Vigneaud
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2
V du Vigneaud (Nobel prize 1955)
Vasopressin
Hormones hypophysis
ACTH (Adrenocorticotropic hormone
corticotropin) (1-39 1-24)
H-Ser1-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- -Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys- -Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Leu-Ala- -Glu-Ala-Phe-Pro-Leu-Glu-Phe39-OH
Synthesis (1971) S Bajusz L Kisfaludy K Medzihradszky
First generation the first ones
Hormones hypothalamus
CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)
H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41
GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)
The first generation
TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)
(pyro)Glu-His-Pro-NH2
(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)
A long-acting derivative of somatostatin
Sandostatin (Peptidomimetic)
Hormones
Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix
Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a
polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood
whereas calcitonin acts to decrease calcium concentration
hPTH-(1-34) crystallizes as a slightly bent long helical dimer
The extended helical conformation of hPTH-(1-34) is
the likely bioactive conformation
The first generation
salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
Hormones pancreas The first generation
Glucagon (29 amino acids) Insulin
Vasoactive intestinal peptide (VIP) (28 amino acids)
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin
Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers
blood glucose levels
Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found
in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the
gastrointestinal tract
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
First generatioin Second generation New generation
Oxytocin (L) Carbetocin (S) Abarelix (GnRH) (L) ACTH (1-24) amp (1-39) (LS) Terlipressin (LS) Cetrorelix (GnRH) (L) Vasopressin (LS) Felypressin (LS) Ganirelix (GnRH) (L) Insulin (ESS R) Buserelin (LS) Eptifibatide Glucagon (ESR) Deslorelin (LS) Bivalirudin (L) Calcitonins (LSR) Goserelin (L) Copaxone (L) TRH (L) Histrelin (L) Techtide P-289(S) Gonadorelin (LS) Leuprolide (LS) Cubicin (F) Somatostatin (LS) Nafarelin (S) Fuzeon (antiHIV (H) GHRH (1-29) amp (1-44) (S) Tryptorelin (LS) Ziconotide (pain) (S) CRF (Human amp Ovine) (S) Lecirelin (S) Pramlintide (diabetes) (S) Cyclosporin (F) Lanreotide (S) Exenatide (diabetes) (S) Thymopentin (L) Octreotide (LS) Icatibant (brady-rec) Thymosin Alpha-1 (S) Atosiban (L) Romiplostim (hormon) Secretins (Human amp Porcine) (ES) Desmopressin (LS) Degarelix (GnRH) Parathyroid Hormone (1-34) amp (1-84)(S) Lypressin (L) Mifamurtide (raacutek adj) Vasoactive Intestinal Polypeptide (S) Ornipressin Ecallantide (oumldeacutema) Brain Natriuretic Peptide (R) Pitressin (L) Liraglutide (diabetes) Cholecystokinin (L) ACE Inhibitors (Enalapril Lisinopril) (L) Tesamorelin Tetragastrin (L) HIV Protease Inhibitors (L) Surfaxin Pentagastrin (L) Peginesatide Eledoisin (L) Carfilzomib Linaclotide (enzinh)
L = in solution S = on solid phase E = extraction F = fermentation H = hybrid synthesis R = recombinant SS = semi-synthesis
Some Approved Peptide Pharmaceuticals and their Methods of Manufacture
Oxytocin
Structure 1953 V du Vigneaud Synthesis 1954 V du Vigneaud
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2
V du Vigneaud (Nobel prize 1955)
Vasopressin
Hormones hypophysis
ACTH (Adrenocorticotropic hormone
corticotropin) (1-39 1-24)
H-Ser1-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- -Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys- -Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Leu-Ala- -Glu-Ala-Phe-Pro-Leu-Glu-Phe39-OH
Synthesis (1971) S Bajusz L Kisfaludy K Medzihradszky
First generation the first ones
Hormones hypothalamus
CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)
H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41
GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)
The first generation
TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)
(pyro)Glu-His-Pro-NH2
(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)
A long-acting derivative of somatostatin
Sandostatin (Peptidomimetic)
Hormones
Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix
Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a
polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood
whereas calcitonin acts to decrease calcium concentration
hPTH-(1-34) crystallizes as a slightly bent long helical dimer
The extended helical conformation of hPTH-(1-34) is
the likely bioactive conformation
The first generation
salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
Hormones pancreas The first generation
Glucagon (29 amino acids) Insulin
Vasoactive intestinal peptide (VIP) (28 amino acids)
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin
Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers
blood glucose levels
Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found
in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the
gastrointestinal tract
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Oxytocin
Structure 1953 V du Vigneaud Synthesis 1954 V du Vigneaud
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2
V du Vigneaud (Nobel prize 1955)
Vasopressin
Hormones hypophysis
ACTH (Adrenocorticotropic hormone
corticotropin) (1-39 1-24)
H-Ser1-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- -Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys- -Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Leu-Ala- -Glu-Ala-Phe-Pro-Leu-Glu-Phe39-OH
Synthesis (1971) S Bajusz L Kisfaludy K Medzihradszky
First generation the first ones
Hormones hypothalamus
CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)
H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41
GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)
The first generation
TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)
(pyro)Glu-His-Pro-NH2
(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)
A long-acting derivative of somatostatin
Sandostatin (Peptidomimetic)
Hormones
Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix
Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a
polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood
whereas calcitonin acts to decrease calcium concentration
hPTH-(1-34) crystallizes as a slightly bent long helical dimer
The extended helical conformation of hPTH-(1-34) is
the likely bioactive conformation
The first generation
salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
Hormones pancreas The first generation
Glucagon (29 amino acids) Insulin
Vasoactive intestinal peptide (VIP) (28 amino acids)
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin
Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers
blood glucose levels
Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found
in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the
gastrointestinal tract
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Hormones hypothalamus
CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)
H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41
GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)
The first generation
TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)
(pyro)Glu-His-Pro-NH2
(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)
A long-acting derivative of somatostatin
Sandostatin (Peptidomimetic)
Hormones
Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix
Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a
polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood
whereas calcitonin acts to decrease calcium concentration
hPTH-(1-34) crystallizes as a slightly bent long helical dimer
The extended helical conformation of hPTH-(1-34) is
the likely bioactive conformation
The first generation
salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
Hormones pancreas The first generation
Glucagon (29 amino acids) Insulin
Vasoactive intestinal peptide (VIP) (28 amino acids)
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin
Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers
blood glucose levels
Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found
in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the
gastrointestinal tract
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)
(pyro)Glu-His-Pro-NH2
(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)
A long-acting derivative of somatostatin
Sandostatin (Peptidomimetic)
Hormones
Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix
Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a
polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood
whereas calcitonin acts to decrease calcium concentration
hPTH-(1-34) crystallizes as a slightly bent long helical dimer
The extended helical conformation of hPTH-(1-34) is
the likely bioactive conformation
The first generation
salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
Hormones pancreas The first generation
Glucagon (29 amino acids) Insulin
Vasoactive intestinal peptide (VIP) (28 amino acids)
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin
Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers
blood glucose levels
Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found
in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the
gastrointestinal tract
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Hormones
Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix
Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a
polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood
whereas calcitonin acts to decrease calcium concentration
hPTH-(1-34) crystallizes as a slightly bent long helical dimer
The extended helical conformation of hPTH-(1-34) is
the likely bioactive conformation
The first generation
salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
Hormones pancreas The first generation
Glucagon (29 amino acids) Insulin
Vasoactive intestinal peptide (VIP) (28 amino acids)
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin
Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers
blood glucose levels
Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found
in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the
gastrointestinal tract
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Hormones pancreas The first generation
Glucagon (29 amino acids) Insulin
Vasoactive intestinal peptide (VIP) (28 amino acids)
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin
Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers
blood glucose levels
Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found
in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the
gastrointestinal tract
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Cholecystokinin (CCK-8) C-terminal octapeptide
Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of
gastrin
Hormones digestion
Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2
The first generation
Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)
From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating
the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product
eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Cyclosporin (cyclosporin A) immunesuppressantfungi natural product
11 amino acids cyclic D-amino acids
Peptides acting on the immune system
Arg-Lys-Asp-Val-Tyr
Thymosin α1 T-cell immune stimulant 28 amino acids fragment
Thymopentin (TP-5) T-cell immune stimulant
The first generation
Isolation 1971 fromTolypocladium inflatum Medical use 1983
2009 treatment of hepatitis BC
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Eledoisin 11 amino acids octopus (Eledone) origin
Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
Brain natriuretic peptide (BNP) 32 amino acids cyclic
Antihypertensive drugs
The first generation
Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The
physiologic actions include decrease in systemic vascular resistance and central venous pressure
as well as an increase in natriuresis The net effect a decrease in blood volume
which lowers systemic blood pressure
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
New generation
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Cetrorelix (synthetic)
Hormones GnRH antatogists - peptidomimetics
Abarelix (Plenaxis)
Ganirelix Degarelix
New generation
In oncology to reduce the amount of testosterone in patients with advanced
symptomatic prostate cancer
In assisted reproduction to control ovulation
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2
httpwwwusermedscom
Enfuvirtide 36 amino acids
HIV fusion inhibitor antiviral
(binding to gp41 protein)
Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)
13 amino acids D-amino acid non-natural amino acid
N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone
Antibacterial drugs inhibitors of interactions
New generation
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10
Ziconotide ω-conotoxin peptide bdquoConus magusrdquo
Ca channel blocking (non-opioid non-NSAID) pain killer
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2
J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85
Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis
random copolymer polymer
Mifamurtide (Mepact) muramyl tripeptide
phosphatidylethanolamine osteosarcoma
httpwwwmedkoocomAnticancer-trialsMifamurtidehtm
New generation
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Carfilzomib tetrapeptide multiple myeloma
proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012
httpwwwlookchemcomcas-868868540-17-4html
N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide
Enzyme inhibitors
Linaclotide (Linzess) 14 amino acids 3 cycles
guanlylate cyclase 2C inhibitor
irritable bowel syndrome FDA 30 August 2012
HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH
New generation
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Peptide
Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )
40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10
18 16 14 12 10 8 6 4 2 0
Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT
Product Indication Length Status
Degludec Type 1 and type 2 diabetes -- NDA Pending
Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending
Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)
Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)
MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections
Pasireotide Cushingrsquos disease 6 Phase III
Albiglutide Type 2 diabetes -- Phase III
E75 Breast cancer (therapeutic vaccine) -- Phase IIIII
Pexiganan Diabetic foot infections 22 Phase IIIII
Cilengitide Glioblastoma 5 Phase III
KAI-4169 Seconday hyperparathyroidism -- Phase II
TRV120027 Acute heart failure -- Phase II
MIM-D3 Dry eye -- Phase II
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
API Date Manufacturing Method Indication
Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]
Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes
PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]
Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome
Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome
RECENTLY APPROVED PEPTIDES
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines
Main fields of applications
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Number of Peptides Approved or in Active Development
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
generic
name
year of
approval
therapeutic
area
country of
approval
generic name year of
approval
therapeutic area country
of
approval
atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU
taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU
aviptadil 2000 urology EU tesamorelin 2010 antiinfective US
carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US
nesiritide 2001 cardiovascular US peginesatide 2012 hematology US
teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU
enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US
abarelix 2003 oncology US linaclotide 2012 gastroenterology US
ziconotide 2004 pain US teduglutide 2012 gastroenterology EU
pramlintide 2005 metabolic
disease
US lixisenatide 2013 metabolic disease EU
exenatide 2005 metabolic
disease
US albiglutide 2014 metabolic disease EU
icatibant 2008 hematology EU oritavancin 2014 antiinfective US
romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US
degarelix 2008 oncology US afamelanotide 2014 dermatology
Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414
Top Selling Non-Insulin Peptide Sales 2015
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions
Targets
Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Advantages - Disadvantages
Specificity Potency Low toxicity
Low stability Short half-life Enzymatic decomposition Lack of oral application
Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav
Sources
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
NUMBERS
Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)
1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)
1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION
1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection
bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection
(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)
bull nerve impulses bull growth bull differentiation
6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support
bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
RECOGNITION PHENOMENA
Interaction Kd[M]
1 Enzyme ndash substrate 10-3 ndash 10-5
2 Transporter ndash ligand 10-6 ndash 10-8
3 Hormone ndash receptor 10-9
4 Antibody ndash antigen 10-7 ndash 10-11
5 Storage protein ndash ligand
6 Toxin ndash receptor
7 Protein ndash protein (in a contractile super assembly)
8 Lectin ndash carbohydrate 10-4 ndash 10-7
9 Avidin ndash biotin 10-15
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Chemical modification bull side chain modification bull conjugation Fragmentation
bull enzymatic (eg trysin) bull chemical (eg BrCN)
Separation bull centrifugation bull chromatography bull electrophoresis
Identification bull amino acid analysis bull sequencing bull mass spectrometry
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
I Experimental methods
Genetic engineering bull deletion bull chimeric proteins bull mutagenesis
ndash site directed ndash random
bull phage display libraries
Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
An example Identification of epitope sequences in protein antigens
Whole virus
Viral envelope proteins (mixture)
1 Affinity chromatography 2 Gradient centrifugation
Amino acid sequence Immunodominant protein component komponens
Immuneprecipitation (Gelelectrophoresis)
Sequencing
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Identification of peptid epitopes
protein
prediction of 3D structure
epitope bdquomaprdquo
Prediction of hydrophilicity
modeling
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Identification of short sequences responsible for activity
bdquooverlappingrdquo strategy
bdquopredictedrdquo
Gaacutet
laacutes
TPTPTGTQ
PTGTQ
TGTQ
0
20
40
60
80
100
001 1 100 cpeptid (mmoll)
bdquoshorteningrdquo strategy
intuicioacute
bdquocombinatorialrdquo strategy
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
IITheoretical methods
Quantum chemistry bull molecular mechanics bull molecular dynamics
Predictions from the primary structure
bull Probabilistic (statistical) 1970 ndash
bull Physicochemical 1974 ndash
bull Information theory 1974 -
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Approaches for the localisation of functionally relevant domains in proteins
Protein primary structure
Know Unknown
3D Structure
Know Unknown
Isolation
Fragmentation separation
Functional assay with fragments
Selection
Structure elucidation
Prediction of
bull secondary structure
bull functionally relevant domain
Fragmentation separation
Functional assay with fragments
Selection
3D Structure elucidation
Chemical modification
Genetic engineering
Chemical synthesis
Smallest functional domain
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Strategies for determinations of 3D structures
Experimental methods Theoretical methods
X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)
bull crystal bull time-intensive
NMR
CD spectroscopy FT-IR spectroscopy
Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
Prediction of secondary structures (helix b-turn)
Prediction of hydrophobic ndash hydrophilic regions
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Techniques for the detection of interactionrecognition phenomena
1 Molecular level
Detection with Separation Detection without Separation
Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis
Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation
Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy
Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)
Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Techniques for the detection of interactionrecognition phenomena
2 Cellular level
Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
In vivo post-translational modifications
alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)
Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)
Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
Ubiquitination
bull Ubiquitin protein (76 amino acids 85 kDa)
bull Almost all tissues of eukaryotic organisms
bull It can signal the degradation of the attached protein by transporting to proteasome
bull Isopeptide linkage (4)
bull Enzymes involved
ndash E1 (ub activation)
ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)
ndash E3 ( ub ligation)
Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)
bull Function
activationregulation of ubiquitin
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)
SUMOylation (Small Ubiquitin-like Modifier)
bull SUMO proteins 100 aa 12 kDa 4 isoforms
bull Post-translational modification
bull Aktivation cleavage of 4 residues at the C-terminal
bull Attachment to target protein by using three enzymes
Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation
R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)