in vitro antitubercular effect of inh-conjugates and in silico identified drug candidates szilvia...
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In vitro antitubercular effect of INH-conjugates and in silico identified drug candidates
Szilvia Bősze1, Kata Horváti1, Nóra Szabó2, Vince Grolmusz3, Éva Kiss4, Katalin Hill4, Gábor Mező1, Ferenc Hudecz1,5 and Beáta G. Vértessy6
1Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest, Hungary
2Korányi National Institute for TB and Pulmonology, Budapest, Hungary3Department of Computer Science, Eötvös Loránd University, Budapest, Hungary4Department of Physical Chemistry, Eötvös Loránd University, Budapest, Hungary5Department of Organic Chemistry, Eötvös Loránd University, Budapest, Hungary
6Institiute of Enzymology, BRC, Hungarian Academy of Sciences, Budapest, Hungary;
M. tuberculosis has evolved extremely efficiently todeal with the human condition:
(i) Stops the normal progression of the phagosomes(ii)Avoids the development of a localised and productive immune response
Site of actionSite of action
receptor
degradation
lysosome
degradation
fluidicendocytosis
receptor mediatedendocytosis
diffusion/activetransport
Uptake of bioactive entities
Takakura Y., Hashida M. Crit.Rev.Oncol.Hematol. 18: 207 (1994)
lysosome
(i) Receptor mediated drug targeting
(i) Receptor mediated drug targeting
(ii) New drug candidates (in silico identified)
Receptor families that can be used for delivery:
mannosyl-fucosyl receptors galactosyl receptors scavenger receptors tufsin receptor
Specific delivery of INH into macrophagesSpecific delivery of INH into macrophages
+
lysosome
endosome
phagocytosis
intracellularparasite
specific receptor
I NH targetingmoiety
spacer / linkercarrier
I NH targetingmoiety
spacer / linkercarrier
NH2
NHO
N
Taylor, P. R. et al, Annu. Rev. Immunol. (2005) 23: 901–44Becker, M.et al, Eur J Immunol. (2006) 36: 950-60Basu, Biochem. Pharmacol., (1995) 40:1941-1946H. Soyez et al. 1996, Adv. Drug Delivery Rew. 21: 81-86
frontline drug, min. 6 months therapybactericide prodrug inhibits the formation of cell wall
Application of carrier/targeting molecules
increasing the solubility
influence of biodistribution
decreasing of toxicity (continuous liberation of drug molecule)
improvement of selectivity
retarded effect application of multi copy of
the drug moiety
Polymers
polylysine branched chain polypeptide polytuftsin N-vinyl-pirrolidone - maleic acid copolymer stirene-maleic acid copolymer
Molecules with defined structure
lysine dendrimers sequential oligopeptides cell penetrating peptides GnRH-derivatives antimicrobial peptides (NK lysin, granulysin)
Tuftsin
Thr-Lys-Pro-Arg (human); Thr-Lys-Pro-Lys (dog)
(leukokinin) 289-292 position, liberation in two enzymatic cleavage steps
stimulation of phagocytosis immunmodulatory activity chemotactic activity on monocytes antitumour activity increase the production of TNF and ILs
Derivatives: Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg (dituftsin)
Thr-Lys-Pro-Arg-Gly
Fridkin, M., Najjar, V.: Crit. Rev. Biochem. Mol. Biol. 24 (1989) 1
Development of new tuftsin based carrier molecules
H-[Thr-Lys-Pro-Lys-Gly]n-NH2 (n=1,2,4,6,8)
defined carrier molecule well-characterized conjugates
tuftsin-like effects
40% of amino acids can be substituted
application of orthogonal protecting groups on Lys side chains:
selective coupling of drug molecules differently cleavable spacers by enzymes coupling of fatty acids
presence of OH-groups increase the solubility (purification, biology) glycopeptide derivatives G. Mező et al. Biopolymers
Fluorescent labelling5(6)-
carboxyfluoresceinAntitubercular drugs Antitubercular drugs or/andor/andaantintimicrobialmicrobial peptidespeptides
Receptor-specificitySR-A
OC
CH2
NH
COO-
CH2
Succ
-OOC
C O
CH3
NH
Ac
-OOC
Hudecz, et al, J. Controlled Release, 1992 Hudecz, et al, Bioconjugate Chem. 1999
Rajnavölgyi et al, Mol. Immunol., 1986 Rajnavölgyi et al, Chimica Oggi, 1990Clegg et al, Bioconjugate Chem., 1990 Hudecz et al. Bioconjugate Chem., 1999 Pimm et al, J. Controlled Release, 1995
poly[Lys(Succ-Glui-DL-Alam)]; Succ-EAK
Branched chain polypeptides
NH2NH2
NH2CF-NH
poly[Lys(DL-Alam)]; AK
Succ-NHSucc-NH
Succ-NHCF-NH
COOH
COOH
HOOC
HOOC
Ac-NHAc-NH
Ac-NHCF-NH
COOH
COOH
HOOC
HOOC
poly[Lys(Ac-Glui-DL-Alam)]; Ac-EAK
Mal-NHMal-NH
Mal-NHCF-NH
COOH
COOH
HOOC
HOOC
poly[Lys(Mal-Glui-DL-Alam)]; Mal-EAK
Fluidic endocytosis(polycationic polymer)
Receptor mediatedendocytosis
(scavanger receptors)(polyanionic polymer)
Szabo, R., et al. Bioconjugate 16, 1442-1450 (2005)
1. treatment of the cells
2. washing 1x SFM/HPMI
3. tripsinisation
4. Flow cytometry (BD LSR II) 10000 events
CF:CF: ex=488 nm; em=519 nm
*HPMI: glucose, NaHCO3, NaCl, HEPES, KCl, MgCl2, CaCl2, Na2HPO4 x 2H2O
Cellular uptake of the carrier/targeting molecules
MonoMac6
Cellular uptake of CF-GC-T20 and CF-GFLGC-T20 of MonoMac6
CF-GC-T20, c=3,7×10-5 M CF-GFLGC-T20, c=3,3×10-5 M
Kontroll 5 min 15 min 45 min 75 min105 min
0 20 40 60 80 100 120400
600
800
1000
1200
1400
1600
1800
2000
2200 CF-GFLGC-T20 CF-GC-T20
fluo
resz
cenc
ia
idõ [perc]time[min]
Internalisation of bioconjugate containing carboxy-fluoresceine into THP-1 monocytes
Control
CF-T20
1 min
15 min 60 min
[TKPKG]4-NH2
CH2CO
CF-GFLGC-NH2
CF: 5(6)-carboxy-fluoresceine
Images were recorded byconfocal laser scanning microscopy
1 min1 min
60 min60 min
fixed cellsfixed cells
Uptake polylysine based polypeptides J774 cellsUptake polylysine based polypeptides J774 cells
SAKcontrol AK
EAK Ac-EAK Succ-EAK
PiK
Synthesis of carrier peptide – INH conjugates -1
*Geoghean, K. F. and Stroh, J. G. Bioconjugate Chem. 1992, 3: 138-142.
INH-92EFAGAGFVRAGAL104 (hydrazone) (INH-oxAGA)INH-91SEFAYGSFVRTVSLPV106 (hydrazone) (INH-oxSer)
91SEFAGAGFVRAGAL104
S-91SEFAYGSFVRTVSLPV106
N-glyoxylil-92EFAGAGFVRAGAL104
N-glyoxylil- 91SEFAYGSFVRTVSLPV106
RNH2
OH
O
NH
R
O
O
NH
+ NH3 +O
CH2 + IO 3 -
IO 4 -
92EFAGAGFVRAGAL104
92EFAGAGFVRAGAL104
4 equiv. NaIO4 10 equiv. Met (scavenger)
1 % NH4HCO3 (pH=8.3)
+ 10 equiv. ethyleneglycol, RP-HPLC
+ 50 equiv. INH
0.1M NH4OAc (pH=4.6)
RP-HPLC
NH2
NHO
N
R
O
O
NH
+R
O
NH
NNHO
N
92EFAGAGFVRAGAL104
Glyoxylic acid, as a heterobifunctional linker ->
-> Coupling INH to peptides on solid phase
-> Reduction before coupling (-> hidrazide)
NH2
NHO
N
O
O
OH
+
O
OH
NNHO
N
H2O : AcN10 : 1
1h RT
yield: 98%
1 8 0 1 9 0 2 0 0 2 1 0
1 .0
1 .51 0 0 1 2 0 1 4 0 1 6 0 2 2 0 2 4 0 2 6 0 m /z
1 8 0 1 9 0 2 0 0 2 1 0
1 9 4 .0
1 9 5 .0
m /z1 8 0 1 9 0 2 0 0 2 1 0
1 .0
1 .51 0 0 1 2 0 1 4 0 1 6 0 2 2 0 2 4 0 2 6 0 m /z
1 8 0 1 9 0 2 0 0 2 1 0
1 9 4 .0
1 9 5 .0
1 8 0 1 9 0 2 0 0 2 1 0
1 .0
1 .51 0 0 1 2 0 1 4 0 1 6 0 2 2 0 2 4 0 2 6 0 m /z
1 8 0 1 9 0 2 0 0 2 1 0
1 8 0 1 9 0 2 0 0 2 1 01 8 0 1 9 0 2 0 0 2 1 0
1 .0
1 .51 0 0 1 2 0 1 4 0 1 6 0 2 2 0 2 4 0 2 6 0 m /z
1 8 0 1 9 0 2 0 0 2 1 0
1 9 4 .0
1 9 5 .0
m /z
Mp: 206.5 – 207.0 oC
Elemental Analysis: (calculated) foundN% (21.75) 22.15; 22.24C% (49.72) 49.76; 49.68H% (3.63) 3.53; 3.42
[M+H]+
Mmo (calculated) = 193.1
Synthesis of carrier peptide – INH conjugates -2
O
OH
NHNHO
N
1.0 equiv. NaCNBH3
methanol (suspension)
NH2
NHO
N
O
O
OH
+
O
OH
NNHO
N
H2O : AcN10 : 1
1h RT
NH2
NHO
N
O
O
OH
+
O
OH
NNHO
N
H2O : AcN10 : 1
1h RT
+ M S , 0 . 2 - 1 . 3 m i n ( # 1 2 - # 1 0 2 )
0 . 0
0 . 5
1 . 0
1 . 5
6x 1 0I n t e n s .
1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 m / z
+ M S , 0 . 2 - 1 . 3 m i n ( # 1 2 - # 1 0 2 )
0 . 0
0 . 5
1 . 0
1 . 5
6x 1 0I n t e n s .
1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 m / z
1 9 6 . 0
1 9 7 . 0
m / z
+ M S , 0 . 2 - 1 . 3 m i n ( # 1 2 - # 1 0 2 )
0 . 0
0 . 5
1 . 0
1 . 5
6x 1 0I n t e n s .
1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 m / z
+ M S , 0 . 2 - 1 . 3 m i n ( # 1 2 - # 1 0 2 )
0 . 0
0 . 5
1 . 0
1 . 5
6x 1 0I n t e n s .
1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 m / z
1 9 6 . 0
1 9 7 . 0
m / z
[M+H]+
Mmo (calculated) = 195.1
reduced form of glyoxylic acid derivatives(hydrazide)
Couple to the N-terminus of a peptide on solid phase
5 equiv. INH-gli(red) / NMP
5 equiv. DIC / HOBt
Synthesis of carrier peptide – INH conjugates -3
glyoxylic acid derivative of INH(hydrazone)
6 4 5 .9
9 6 8 .4
+ M S , 0 .3 -1 .5 m i n (# 2 1 -# 1 0 3 )
0
1
2
3
4
6x 1 0In te n s.
4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 m /z
[M + 2 H ] 2 +
[M + 3 H ] 3 +
6 4 5 .9
9 6 8 .4
+ M S , 0 .3 -1 .5 m i n (# 2 1 -# 1 0 3 )
0
1
2
3
4
6x 1 0In te n s.
4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 m /z
[M + 2 H ] 2 +
[M + 3 H ] 3 +
INH-91SEFAYGSFVRTVSLPV106
(INH-red-Ser, hydrazide)
Mav(calculated) = 1935.0
Mav(measured) = 1934.8
RP-HPLC, Knauer, Eurospher-100 C18, 5m, 250x4mm column, =214nm, gradient: 5-60B% 35min. A eluent: H2O+0,1 v/v% TFA, B eluent: AcN: H2O =80:20 (v/v) +0,1 v/v% TFA
5 10 15 20 25 30 35 400
50
100
150
200
250
300
350
A
t / min
O
OH
NHNHO
N
91SEFAYGSFVRTVSLPV106
Synthesis of carrier peptide – INH conjugates -4
Stability of INH(red)-SEFAYGSFVRTVSLPV (hydrazide) conjugate
RP-HPLC, Knauer, Eurospher-100 C18, 5m, 250x4mm column, =214nm, gradient: 5-60B% 35min. A eluent: H2O+0,1 v/v% TFA, B eluent: AcN: H2O =80:20 (v/v) +0,1 v/v% TFA
semisynthetic Sula media, pH = 6.8 37 oC, c0 = 0.5 mg/ml
Determination of minimum inhibitory concentration (MIC)Determination of minimum inhibitory concentration (MIC)
M. tuberculosis H37RV
Drug / conjugateMIC (g/ml)
CFU
INH 0.16 12
INH-gli(ox) (hydrazone, Figure 1) 0.40 60
INH-gli(red) (hydrazide, Figure 2) 0.40 6
INH-92EFAGAGFVRAGAL104
(hydrazone, Figure 3)0.24 40
INH-91SEFAYGSFVRTVSLPV106 (hydrazone) 0.18 30
INH-91SEFAYGSFVRTVSLPV106 (hydrazide) 0.16 2
GTKPK(INH)G (hydrazide, Figure 4) 0.18 2091SEFAGAGFVRAGAL104 - -91SEFAYGSFVRTVSLPV106 - -
GTKPKG - -
O
OH
NHNHO
N
NH2
NHO
N
O
O
OH
+
O
OH
NNHO
N
O
OH
NHNHO
N
GTKPKGNH2
NHO
N
R
O
O
NH
+R
O
NH
NNHO
N
92EFAGAGFVRAGAL104
Determination of MIC/CFU of INH, „INH-linker”, INH-conjugates and carriers
Figure 1
Figure 2
Figure 4Figure 3
Cytostatic effect of INH, INH-conjugates and carriers
INHIC50 > 3.6*10-2M
MIC=1.4 *10-6 M
1E-7 1E-6 1E-5 1E-4
0
20
40
60
80
100 Data: Data1_BModel: Logistic Chi^2 = 1.22039R^2 = 0.42933 A1 8.9567 ±0.72729A2 -2.20406 ±306804840.52571x0 0.00043 ±11063.22402p 18.96518 ±492771983.71424
cyto
sta
sis
%
lg (c/M)
T6 carrier
T6IC50 > 5.0*10-4 M
MIC=1.4 *10-6 M
1E-7 1E-6 1E-5 1E-4
0
20
40
60
80
100 Data: Data1_BModel: Logistic Chi^2 = 0.05595R^2 = 0.9706 A1 -2.042E-6 ±0.89422A2 5.8245 ±1.57824x0 9.2994E-8 ±1.9725E-7p 4.47112 ±46.33094
cyto
sta
sis
%lg (c/M)
T6(INH) conjugate
T6(INH) conjugateIC50 > 5.0*10-4 M
MIC=1.4 *10-6 M
Gerlier D, Thomasset N. Use of MTT colorimetric assay to measure cell activation. J Immunol Methods. 1986,20;94(1-2):57-63.
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/well PBMC 1.105 cell/well
DMSO, = 540 nm
Small molecules based therapies are the most important interventions for TB.
computer cluster RS-PDB database (highly structured and repaired version of PDB)
new molecular-dynamic docking algorithms
drug database (ZINC)
MTB proteins (known 3D structure)
(they are crucial for the maintance of cellular integrity and survival of the pathogen)
In silico identified drug candidates
Holton, S.J., King-Scott, S., Eddine, A.N., Kaufmann, S.H., Wilmanns, M. Structural Diversity in the Six-Fold Redundant Set of Acyl-Coa Carboxyltransferases in Mycobacterium Tuberculosis. FEBS Lett. (2006) 580 6898-6892
2bzr, ACYL-COA CARBOXYTRANSFERASE, EC 6.4.1.3
(Rv3280, AccD5)
Fleischmann, R.D., Alland, D., Eisen, J.A., Carpenter, L., White, O., Peterson, J., DeBoy, R., Dodson, R., Gwinn, M., Haft, D., Hickey, E., Kolonay, J.F., Nelson, W.C., Umayam, L.A., Ermolaeva, M., Salzberg, S.L., Delcher, A., Utterback, T., Weidman, J.,Khouri, H., Gill, J., Mikula, A., Bishai, W., Jacobs, W.R. Jr., Venter, J.C., and Fraser, C.M. "Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains." J. Bacteriol. (2002) 184:5479-5490.Camus, J.C., Pryor, M.J., Medigue, C., and Cole, S.T. "Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv." Microbiology (2002) 148:2967-2973.
CH3
CH3
N
N
N
N
Monoisotopic Mass = 382.309646 Da
Molecular Form ula = C 24 H 38 N 4
Lig 14, C24H38N4, Mw = 382.3
Lig 22, C23H34N4O2S, Mw = 430.2
CH3
CH3
OO S
N
N
N
N
Molecular Form ula = C 23 H 34 N 4 O 2 S
Monoisotopic M ass = 430.240247 Da
NN
CH3
N N
NN
CH3
Lig 35, C22H36N6, Mw = 384.3
birA, biotin-protein ligase [Mycobacterium tuberculosis H37Rv]EC 6.3.4.15
(Rv3279c)
Lig 4, C22H36N6, Mw = 384.3
NHN
N
N
F
Molecular Form ula = C 14 H 19 F N 4
Monoisotopic Mass = 262.159374 Da
NN
CH3
N N
NN
CH3
Lig 5, C14H19FN4, Mw = 262.2
dUTPase, nucleotidohydrolase [Mycobacterium tuberculosis H37Rv]EC 3.6.1.23
(Rv2697c)
DUT 32, C27H36N6O2, Mw = 376.3
DUT 1, C24H19N3O7S, Mw = 493.1
N NH
N
N
O
N
ON
M onoisotopic Mass = 476.289974 Da
M olecular Form ula = C 27 H 36 N 6 O 2
O
CH3
NH
O
O
NH
O
O
NH
S
O
O
Molecular Form ula = C 24 H 19 N 3 O 7 S
Monoisotopic Mass = 493.094373 Da
DUT 44, C25H28N2O5, Mw = 436.2
OH
N
N
O
O
CH3
O
OH
DUT 13, C25H31N5O3S, Mw = 384.3
DUT3, C25H38N4O, Mw = 410.3
N
OHCH3
N
N NH
CH3
O
CH3N
O
NH
O
N
NHN
S
Monoisotopic Mass = 481.214761 Da
Molecular Form ula = C 25 H 31 N 5 O 3 S
Determination of minimum inhibitory concentration (MIC)Determination of minimum inhibitory concentration (MIC)
M. tuberculosis H37RV
Docked moiety MIC (µg/ml) CFU
DUT I/4 25 n.d.
DUT 3 5 5
DUT 13 15 42
DUT 32 30 n.d.
DUT 44 25 n.d.
Rv3279c Lig 4 30 50
Rv3279c Lig 5 25 40
2Bzr Lig 14 25 6
2Bzr Lig 22 30 n.d.
2Bzr Lig 35 25 n.d.
+/- : no invisible growth (CFU)
Determination of MIC/CFU of in silico identified candidates
OH
N
N
O
O
CH3
O
OH
1. treatment of the cells 2. washing 1x SFM/HPMI3. tripsinisation4. Flow cytometry (BD LSR II)
10000 events
CF:CF: ex=488 nm; em=530 nm (FL2)
*HPMI: glucose, NaHCO3, NaCl, HEPES, KCl, MgCl2, CaCl2, Na2HPO4 x 2H2O
Emission spectra and cellular uptake of the DUT 44
10-5 M, 1%DMSO / HPMIex=488nm
control
1.10-5 M
5.10-5 M
1.10-4 M
1x10-7 1x10-6 1x10-5 1x10-4
0
20
40
60
80
100
IC50
=6.35*10-5
Data: Data1_BModel: ExpGro1 Chi^2 = 632.36626R^2 = 0.5879 y0 0 ±0A1 19.99146 ±8.14982t1 0.0003 ±0.00009
cyto
sta
sis
%
lg (c/M)
Lig 3HepG2, 3h kezelés, 3nap kultúrában
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
HepG2IC50 = 6.35.10-5 MMIC = 1.21. 10-5 M
1x10-6 1x10-5 1x10-4 1x10-3
0
20
40
60
80
100
IC50
=2.11*10-5
Data: Data1_BModel: Logistic Chi^2 = 9.79033R^2 = 0.99703 A1 -4.98413 ±1.51658A2 89.50155 ±1.66826x0 0.00002 ±7.584E-7p 4.23567 ±2.53917
cyto
sta
sis
%
lg (c/M)
Lig 3PBMC (EÜ2), 3,5h kezelés, 3 nap kultúrában, citosztázis
PBMCIC50 = 2.11.10-5 MMIC = 1.21. 10-5 M
Cytostatic effect of DUT 3 ligand on HepG2 and PBMC
1x10-6 1x10-5 1x10-4 1x10-3
0
20
40
60
80
100
IC50
=1.60*10-5
Data: Data1_BModel: Logistic Chi^2 = 14.65181R^2 = 0.96761 A1 4.87387 ±1.63647A2 85.65138 ±0.3894x0 0.00001 ±1.1867E-6p 2.95128 ±0.55818
cyto
stas
is %
lg (c/M)
Lig 3PBMC (EÜ1)3,5h kezelés, 2 nap inkubálás3,5h kezelés, 3 nap inkubálás
PBMC double treatmentIC50 = 1.60.10-5 MMIC = 1.21. 10-5 M
1x10-6 1x10-5 1x10-4 1x10-3
0
20
40
60
80
100
IC50
=3.45*10-5
Data: Data1_BModel: Logistic Chi^2 = 2.0648R^2 = 0.99759 A1 4.44142 ±1.10457A2 86.87049 ±0.46924x0 0.00003 ±3.2792E-6p 3.30751 ±0.73207
cyto
sta
sis
%
lg (c/M)
Lig 3PBMC (EÜ2), 3,5h kezelés, citotoxicitás
PBMC cytotoxicityIC50 = 3.45.10-5 MMIC = 1.21. 10-5 M
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nm
3 h 72 h
treatment wash, culture MTT
HepG2 5.103 cell/ wellPBMC 1.105 cell/ well
DMSO, = 540 nmwash
Cytotoxic and cytostatic effect of DUT 3 ligand on PBMC
Interaction with lipid monolayer of INH and INH(red)-SEFAYGSFVRTVSLPV
Langmuir balance
Wilhelmy-type surface tension sensors:surface pressure isotherms
Lipid = 85% of distearoyl phosphatidyl choline (DSPC) / 15 % of dipalmitoyl phosphatidyl choline (DPPC)
Wilhelmy plate
water
Wilhelmy plate
electrobalance
movablebarrier
Langmuir trough
compression/expansion: 24 cm2/min
electrobalance
Incorporation of INH and INH(red)-SEFAGSFVRTVSLPV into the lipid film is reflected in the shape of the isotherms.
Isotherms of lipid and mixed Langmuir films
(i) completely reproducible isotherms for the pure lipid film(ii) IHN low interaction with the monolayer(iii) dramatic change in the isotherms, higher stability
(i) completely reproducible isotherms for the pure lipid film(ii) IHN low interaction with the monolayer(iii) dramatic change in the isotherms, higher stability
2-3-5 consequtive compression/expansion cycles,3 μg lipid or lipid+drug(conjugate) with molar ratio of 5:1 in dichloromethane
Isotherms after compression cycles of pure and mixed monolayers
0 1000 2000 3000 4000
-2
0
2
4
6
8
10
12
14
16
18
20
22
24Stability of phospholipon on water
(m
N/m
)
t (sec)
INH 2M
0 1000 2000 3000 4000
-2
0
2
4
6
8
10
12
14
16
18
20
22
24Stability of phospholipon on water
(m
N/m
)t (sec)
INH, 2M
0 1000 2000 3000 4000
-2
0
2
4
6
8
10
12
14
16
18
20
22
24Stability of phospholipon on water
(m
N/m
)
t (sec)
DUT3, 2M
0 1000 2000 3000 4000
-2
0
2
4
6
8
10
12
14
16
18
20
22
24Stability of phospholipon on water
(m
N/m
)
t (sec)
DUT 3, 2M
Wilhelmy plate
surface pressuresensor
movablebarrier
water
Langmuir balance
compression/expansion: 24 cm2/min
Wilhelmy plate
drug
1
2
surface pressuresensor
Wilhelmy plate
surface pressuresensor
movablebarrier
water
Langmuir balance
compression/expansion: 24 cm2/min
Wilhelmy plate
drug
1
2
surface pressuresensor
Comparison of INH or DUT 3 penetration into lipid monolayer
(1)lipid monolayer
(2)injection of INH/DUT 3 into the subphase
(i) pink line was the reference (pure lipid film)
(ii) penetration of DUT 3 was indicated by the difference between the pink and black line
(iii) DUT 3 shows a significant affinity to lipid layer, this tendency for INH is lower
Jelölés 5(6)-karboxifluoreszceinnel
5(6)-karboxifluoreszcein-szukcinimid-észter(CF-SE)
OHO O
C
O
ON
O
O
C
O
OH
+ NH2R
OHO O
C
O
RNH
C
O
OHpH=9,2
1 óra
CF-polipeptid
Tisztítás: Sephadex G25Eluens: desztillált vízMosás: 1% ecetsav (v/v)
Karboxifluorszcein-tartalom meghatározása:Savas hidrolízis (6M HCl, 24 óra)Analitikai HPLC: CF kalibrációs görbe alapján
NH3+
CO
CH CH3
m
n]CONH[
CH2
CH2
CH2
CH2
NH
AK
NH
CO
CH CH3
m
NH3+
COO (CH2)2
CO
CH
n]CONH[
CH2
CH2
CH2
CH2
NH
EAK
Structure and charge of the side-chainsStructure and charge of the side-chains
NH3+
CO
CH
NH
CO
CH CH3
m
CHCH3
OH
n]CONH[
CH2
CH2
CH2
CH2
NHTAK
CH2 OH
NH3+
CO
CH
NH
CO
CH CH3
m
n]CONH[
CH2
CH2
CH2
CH2
NHSAK
salt bridge
NH
CO
CH CH3
m
NH
COO (CH2)2
CO
CH
n]CONH[
CH2
CH2
CH2
CH2
NH
AcEAK
C O
CH3
OC
CH2
COO
CH2
NH
CO
CH CH3
m
NH
COO (CH2)2
CO
CH
n]CONH[
CH2
CH2
CH2
CH2
NH
SuccEAK
Structure and charge of the side-chainsStructure and charge of the side-chains
Lys-(Aaa)n-Ala-OH
Lys
Lys
Lys
Lys
Lys
Lys
NH2
NH2
NH2NH2
NH2
NH2
NH2
NH2
oligopeptide based carrier molecules
„Lysine tree”:
SOC (Sequential Oligopeptide Carrier):
Ac-[Lys-Aib-Gly]n-OH (n=3-7) 310-hélix
Tam, J. P.: Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9084
Tsikaris, V., et al.: Biopolymers 38 (1996) 291
1. 1. Poly[L-Lys] backbone (polimerisation degree: 60-120)Poly[L-Lys] backbone (polimerisation degree: 60-120)
2. 2. Oligo[DL-Ala] sidechains 3 Ala/Lys) Oligo[DL-Ala] sidechains 3 Ala/Lys)
3. 3. Different amino acids at the N-terminus of the branchesDifferent amino acids at the N-terminus of the branches
Hudecz, et al, J. Controlled Release, 1992 Hudecz, et al, Bioconjugate Chem. 1999
Rajnavölgyi et al, Mol. Immunol., 1986 Rajnavölgyi et al, Chimica Oggi, 1990Clegg et al, Bioconjugate Chem., 1990 Hudecz et al. Bioconjugate Chem., 1999 Pimm et al, J. Controlled Release, 1995
poli[Lys(Xpoli[Lys(Xii)])] poli[Lys(DL-Alapoli[Lys(DL-Alamm)])] poli[Lys(Xpoli[Lys(Xii-DL-Ala-DL-Alamm)])]
Carrier molecules
A) Natural compounds
BSA, KLH, ovalbumine,tetanus toxoid, dextrane
B) Synthetic products
• biodegradable• biocompatible, but
non-degradable
Polymers polylysine branched chain polypeptide polytuftsin N-vinyl-pirrolidone -
- maleic acid copolymer stirene-maleic acid copolymer
Molecules with defined structure
lysine dendrimers sequential oligopeptides
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