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, Hungary
3Department of Computer Science, Eötvös Loránd University, Budapest, Hungary
4Department of Physical Chemistry, Eötvös Loránd University, Budapest, Hungary
5Department of Organic Chemistry, Eötvös Loránd University, Budapest, Hungary
6Institiute of Enzymology, BRC, Hungarian Academy of Sciences, Budapest, Hungary;
M. tuberculosis has evolved extremely efficiently to
deal with the human condition:
(i) Stops the normal progression of the phagosomes
(ii) Avoids the development of a localised and productive immune response
Uptake of bioactive entities
fluidic
endocytosis
lysosome
diffusion/active
transport
receptor mediated
endocytosis
lysosome
Site of action
degradation
Takakura Y., Hashida M. Crit.Rev.Oncol.Hematol. 18: 207 (1994)
degradation
(i) Receptor mediated drug targeting
(i) Receptor mediated drug targeting
(ii) New drug candidates (in silico identified)
Specific delivery of INH into macrophages
endosome
+
O
NH
lysosome
NH2
frontline drug, min. 6 months therapy
bactericide prodrug
inhibits the formation of cell wall
N
phagocytosis
intracellular
parasite
specific
receptor
Receptor families that
can be used for delivery:
mannosyl-fucosyl receptors
galactosyl receptors
scavenger receptors
tufsin receptor
Taylor, P. R. et al, Annu. Rev. Immunol. (2005) 23: 901–44
Becker, M.et al, Eur J Immunol. (2006) 36: 950-60
Basu, Biochem. Pharmacol., (1995) 40:1941-1946
H. Soyez et al. 1996, Adv. Drug Delivery Rew. 21: 81-86
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
 defined carrier molecule
(n=1,2,4,6,8)
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 labelling
5(6)-carboxyfluorescein
Antitubercular drugs or/and
antimicrobial peptides
--OOC
OOC
NH
Ac
Succ
C
O
CH32
CH2
COO-
Hudecz, et al, J. Controlled Release, 1992
Rajnavölgyi et al, Mol. Immunol., 1986
Clegg et al, Bioconjugate Chem., 1990
Pimm et al, J. Controlled Release, 1995
Hudecz, et al, Bioconjugate Chem. 1999
Rajnavölgyi et al, Chimica Oggi, 1990
Hudecz et al. Bioconjugate Chem., 1999
Receptor-specificity
SR-A
Branched chain polypeptides
Szabo, R., et al. Bioconjugate 16, 1442-1450 (2005)
NH2
Ac-NH
HOOC
NH2
Fluidic endocytosis
(polycationic polymer)
Ac-NH
COOH
COOH
HOOC
Ac-NH
Receptor mediated
CF-NH
CF-NH
endocytosis
poly[Lys(DL-Alam)]; AK
(scavanger receptors) poly[Lys(Ac-Glui-DL-Alam)]; Ac-EAK
(polyanionic polymer)
Mal-NH
Succ-NH
Mal-NH
Succ-NH
HOOC
HOOC
COOH
COOH
NH2
HOOC
CF-NH
COOH
Succ-NH
poly[Lys(Succ-Glui-DL-Alam)]; Succ-EAK
HOOC
CF-NH
COOH
Mal-NH
poly[Lys(Mal-Glui-DL-Alam)]; Mal-EAK
Cellular uptake of the carrier/targeting molecules
1.
treatment of the cells
2.
washing 1x SFM/HPMI
3.
tripsinisation
4.
Flow cytometry (BD LSR II)
10000 events
CF: lex=488 nm; lem=519 nm
*HPMI: glucose, NaHCO3, NaCl, HEPES, KCl, MgCl2, CaCl2, Na2HPO4 x 2H2O
MonoMac6
Cellular uptake of CF-GC-T20 and CF-GFLGC-T20 of MonoMac6
CF-GFLGC-T20,
c=3,7×10-5 M
Kontroll
5 min
15 min
45 min
75 min
105 min
CF-GFLGC-T20
CF-GC-T20
2200
2000
1800
fluoreszcencia
CF-GC-T20,
1600
1400
1200
1000
800
600
400
0
20
40
60
idõ [perc]
time[min]
80
100
120
c=3,3×10-5 M
Internalisation of bioconjugate containing carboxyfluoresceine into THP-1 monocytes
[TKPKG]4-NH2
CH2CO
CF: 5(6)-carboxy-fluoresceine
CF-GFLGC-NH2
1 min
15 min
60 min
Control
CF-T20
1 min
60 min
fixed cells
Images were recorded by
confocal laser scanning microscopy
Uptake polylysine based polypeptides J774 cells
control
PiK
AK
SAK
EAK
Ac-EAK
Succ-EAK
Synthesis of carrier peptide – INH conjugates -1
91SEFAGAGFVRAGAL104
S-91SEFAYGSFVRTVSLPV106
O
H2N
EFAGAGFVRAGAL104
R92
NH
OH
4 equiv. NaIO4
10 equiv. Met (scavenger)
IO 4 -
1 % NH4HCO3 (pH=8.3)
O
N-glyoxylil-92EFAGAGFVRAGAL104
N-glyoxylil- 91SEFAYGSFVRTVSLPV106
NH
92EFAGAGFVRAGAL104
R
+ 10 equiv. ethyleneglycol, RP-HPLC
O
+ 50 equiv. INH
O
NH3
+
O
NH
O
104
NH2
INH-92EFAGAGFVRAGAL
(hydrazone)
R
(INH-oxAGA)
NH
106
INH-91SEFAYGSFVRTVSLPV
(hydrazone)
+
(INH-oxSer) O
N
*Geoghean, K. F. and Stroh, J. G. Bioconjugate Chem. 1992, 3: 138-142.
O
+
NH
0.1M NH4OAc (pH=4.6)
O
N
HN
N
IO 3 -
+
CH2
RP-HPLC
R
92
EFAGAGFVRAGAL104
Synthesis of carrier peptide – INH conjugates -2
Glyoxylic acid, as a heterobifunctional linker ->
-> Coupling INH to peptides on solid phase
-> Reduction before coupling (-> hidrazide)
O
NH
140
O
NH
O
NH2
160
220
H2O : AcN
10 : 1
OH
+
O
1h RT
N
240
O
N
260
OH
m/z
N
Mp: 206.5 – 207.0 oC
Elemental Analysis:
(calculated) found
N% (21.75) 22.15; 22.24
C% (49.72) 49.76; 49.68
H% (3.63) 3.53; 3.42
194.0
[M+H]+
Mmo (calculated) = 193.1
195.0
180
190
200
210
m/z
yield: 98%
Synthesis of carrier peptide – INH conjugates -3
O
NH
O
N
O
1.0 equiv. NaCNBH3
OH
N
NH
O
NH
OH
methanol (suspension)
N
N
glyoxylic acid derivative of INH
(hydrazone)
reduced form of glyoxylic acid derivatives
(hydrazide)
+MS, 0.2-1.3min (#12-#102)
196.0
Couple to the N-terminus of a
peptide on solid phase
[M+H]+
5 equiv. INH-gli(red) / NMP
Mmo (calculated) = 195.1
5 equiv. DIC / HOBt
197.0
180
190
200
210
m/z
220
230
m/z
Synthesis of carrier peptide – INH conjugates -4
INH-91SEFAYGSFVRTVSLPV106
350
300
(INH-red-Ser, hydrazide)
250
A
200
Mav(calculated) = 1935.0
150
100
50
0
5
10
15
20
25
30
35
40
t / min
RP-HPLC, Knauer, Eurospher-100 C18, 5mm, 250x4mm column, l=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
O
NH
[M+3H]
NH
OH
91SEFAYGSFVRTVSLPV
106
645.9
3+
O
+MS, 0.3-1.5min (#21-#103)
Mav(measured) = 1934.8
N
968.4
[M+2H]2+
600
700
800
900
1000
1100
1200
m/z
Stability of INH(red)-SEFAYGSFVRTVSLPV (hydrazide) conjugate
% of intact INH(red)-SEFAYGSFVRTVSLPV (hydrazide)
% of released SEFAYGSFVRTVSLPV peptide aldehyde
Data: Data2_C
Model: Boltzmann
100
90
Chi^2
R^2
= 6.62967
= 0.9964
80
A1
A2
x0
dx
-4.12959
±2.78891
84.00685
±1.84237
10.579 ±0.42459
3.20366
±0.342
semisynthetic Sula media, pH = 6.8
37 oC, c0 = 0.5 mg/ml
70
percentage
60
50
40
30
20
10
0
0
5
10
15
20
25
30
time / hours
RP-HPLC, Knauer, Eurospher-100 C18, 5mm, 250x4mm column, l=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
Determination of minimum inhibitory concentration (MIC)
M. tuberculosis H37RV
Determination of MIC/CFU of INH, „INH-linker”, INH-conjugates and carriers
Drug / conjugate
INH
O
NH
0.16
12
0.40
60
0.40
6
0.24
40
OH
+
INH-gli(red) (hydrazide, Figure 2)
N
INH-92EFAGAGFVRAGAL104
(hydrazone, Figure 3)
INH-91SEFAYGSFVRTVSLPV106 (hydrazone)
INH-91SEFAYGSFVRTVSLPV106
CFU
O
NH2
INH-gli(ox) (hydrazone, Figure 1)
MIC
g (mg/ml)
0.18
2
GTKPK(INH)G (hydrazide, Figure 4)
0.18
20
91SEFAGAGFVRAGAL104
-
-
91SEFAYGSFVRTVSLPV106
-
-
GTKPKG
-
-
NH
O
N
HN
N
O
NH
EFAGAGFVRAGAL104
Figure 3
N
Figure 1
O
N
NH
O
NH
OH
N
Figure 2
O
NH
O
N
OH
OH
GTKPKG
R
92
NH
30
0.16
O
(hydrazide)
O
O
Figure 4
Cytostatic effect of INH, INH-conjugates and carriers
treatment
wash, culture
MTT
3h
5.103 cell/well
1.105 cell/well
DMSO, l = 540 nm
T6 carrier
INH
INH
IC50 > 3.6*10-2M
MIC=1.4 *10-6 M
100
Data: Data1_B
Model:
80 Logistic
cytostasis %
cytostasis %
80
100
60
40
Chi^2
R^2
= 10.23021
= 0.92419
A1 60
A2
x0
p
8.37998
28.34909
0.00149
1.64607
T6
IC50 > 5.0*10-4 M
MIC=1.4 *10-6 M
100
±1.71451
±2.74491
±0.00062
±1.03003
40
60
20
0
0
0
1E-3
lg (c/M)
0.01
1E-7
1E-6
1E-5
Chi^2
R^2
= 1.22039
= 0.42933
A1
A2
x0
p
8.9567 ±0.72729
-2.20406
±306804840.52571
0.00043
±11063.22402
18.96518
±492771983.71424
40
20
1E-4
Data: Data1_B
Model: Logistic
80
20
1E-5
T6(INH) conjugate
IC50 > 5.0*10-4 M
MIC=1.4 *10-6 M
T6(INH) conjugate
cytostasis %
HepG2
PBMC
72 h
1E-4
lg (c/M)
Gerlier D, Thomasset N. Use of MTT colorimetric assay to measure cell activation. J Immunol Methods. 1986,20;94(1-2):57-63.
1E-7
1E-6
1E-5
lg (c/M)
1E-4
In silico identified drug candidates
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)
2bzr, ACYL-COA CARBOXYTRANSFERASE,
EC 6.4.1.3
(Rv3280, AccD5)
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
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.
Lig 14, C24H38N4, Mw = 382.3
Monoisotopic Mass = 382.309646 Da
CH3
Molecular Formula = C24 H38 N4
N
N
Lig 22, C23H34N4O2S, Mw = 430.2
N
N
Molecular Formu
H3C
CH3
N
Monoisotopic Ma
N
Lig 35, C22H36N6, Mw = 384.3
N
O
H3C
H3C
N
N
N
N
N
N
S
O
N
CH3
birA, biotin-protein ligase [Mycobacterium tuberculosis H37Rv]
EC 6.3.4.15
(Rv3279c)
Lig 4, C22H36N6, Mw = 384.3
H3C
H3C
N
N
N
N
N
N
Lig 5, C14H19FN4, Mw = 262.2
N
N
N
Molecular Formula = C14 H19 F N4
NH
Monoisotopic Mass = 262.159374 Da
F
dUTPase, nucleotidohydrolase [Mycobacterium tuberculosis H37Rv]
EC 3.6.1.23
(Rv2697c)
DUT 1, C24H19N3O7S, Mw = 493.1
O
O
S
NH
O
O
O
H3C
NH
O
NH
O
Molecular Formula = C24 H19 N3 O7 S
Monoisotopic Mass = 493.094373 Da
DUT 32, C27H36N6O2, Mw = 376.3
Monoisotopic Mass = 476.289974 Da
Molecular Formula = C27 H36 N6 O2
N
N
N
NH
N
N
O
O
DUT 44, C25H28N2O5, Mw = 436.2
H3C
DUT3, C25H38N4O, Mw = 410.3
O
O
O
N
N
H3C
N
NH
N
OH
OH
H3C
Monoisotopic M
N
DUT 13, C25H31N5O3S, Mw = 384.3
Molecular Form
OH
DUT 44
emissziós spektrum
lex=416nm
number of scans=1
Slit=2
6
2.0x10
lemmax=530nm
6
1.8x10
S
6
1.6x10
O
N
6
1.4x10
6
cps
1.2x10
NH
6
1.0x10
N
5
8.0x10
5
6.0x10
O
N
5
4.0x10
5
2.0x10
O
0.0
400
450
500
550
600
l / nm
650
700
750
800
850
CH3
NH
Determination of minimum inhibitory concentration (MIC)
M. tuberculosis H37RV
Determination of MIC/CFU of in silico identified candidates
Docked moiety
MIC g (µg/ml)
DUT I/4
CFU
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)
Emission spectra and cellular uptake of the DUT 44
H3C
1.
2.
3.
4.
O
O
treatment of the cells
washing 1x SFM/HPMI
tripsinisation
Flow cytometry (BD LSR II)
10000 events
CF: lex=488 nm; lem=530 nm (FL2)
O
*HPMI: glucose, NaHCO3, NaCl, HEPES, KCl, MgCl2, CaCl2, Na2HPO4 x 2H2O
N
OH
N
DUT 44
emissziós spektrum
lex=416nm
number of scans=1
Slit=2
OH
6
2.0x10
lemmax=530nm
6
1.8x10
6
1.6x10
6
1.10-5 M
5.10-5 M
1.4x10
6
1.2x10
cps
control
1.10-4 M
6
1.0x10
5
8.0x10
5
6.0x10
5
4.0x10
5
2.0x10
0.0
400
450
500
550
600
l / nm
10-5 M, 1%DMSO / HPMI
lex=488nm
650
700
750
800
850
Cytostatic effect of DUT 3 ligand on HepG2 and PBMC
treatment
wash, culture
MTT
3h
72 h
HepG2 5.103 cell/well
PBMC
Lig 31.105 cell/well
HepG2, 3h kezelés, 3nap kultúrában
100
DMSO, l = 540 nm
Lig 3
PBMC (EÜ2), 3,5h kezelés, 3 nap kultúrában, citosztázis
100
HepG2
IC50 = 6.35.10-5 M
MIC = 1.21. 10-5 M
80
80
Model: ExpGro1
60
60
cytostasis %
cytostasis %
PBMC
IC50 = 2.11.10-5 M
MIC
= 1.21. 10-5 M
Data: Data1_B
40
Chi^2 = 632.36626
R^2
= 0.5879
y0
A1
t1
0
±0
19.99146
±8.14982
0.0003 ±0.00009
40
IC50=6.35*10
-5
20
20
0
0
-7
1x10
-6
1x10
-5
1x10
lg (c/M)
-4
1x10
-6
1x10
-5
-4
1x10
1x10
lg (c/M)
-3
1x10
Cytotoxic and cytostatic effect of DUT 3 ligand on PBMC
treatment
treatment
33 hh
72 h
treatment
treatment
MTT
MTT
wash, culture
culture
wash,
MTT
wash,culture
culture
wash,
33hh
72 hh
72
.1033 cell/well
HepG2 55.10
HepG2
cell/well DMSO, l = 540 nm
.1055 cell/well
PBMC 11.10
PBMC
cell/well
wash
MTT
MTT
72hh
72
.10
3 3cell/well
HepG255.10
cell/well
HepG2
DMSO, ll == 540
540 nm
nm
DMSO,
.10
5 5cell/well
PBMC 11.10
cell/well
PBMC
DMSO,ll==55
DMSO,
MTT
72 h
Lig 3
PBMC (EÜ2), 3,5h kezelés, citotoxicitás
80
DMSO, l = 540 nm
100
PBMC cytotoxicity
IC50 = 3.45.10-5 M
MIC = 1.21. 10-5 M
80
Chi^2
R^2
cytostasis %
PBMC double treatment
IC50 = 1.60.10-5 M
MIC = 1.21. 10-5 M
Data: Data1_B
Model: Logistic
60
A1
A2
x0
p
40
= 2.0648
= 0.99759
cytostasis %
100
60
4.44142
86.87049
0.00003
3.30751
40
IC50=3.45*10
20
±1.10457
±0.46924
±3.2792E-6
±0.73207
-5
20
0
0
-6
1x10
-5
-4
1x10
1x10
lg (c/M)
1x10
-3
-6
1x10
-5
-4
1x10
1x10
lg (c/M)
-3
1x10
Interaction with lipid monolayer of INH and INH(red)-SEFAYGSFVRTVSLPV
electrobalance
electrobalance
Wilhelmy plate
movable
barrier
Langmuir balance
Wilhelmy-type surface
tension sensors:
surface pressure
isotherms
Langmuir trough
compression/expansion: 24 cm2/min
Lipid = 85% of distearoyl phosphatidyl choline (DSPC) / 15 % of dipalmitoyl phosphatidyl choline (DPPC)
Wilhelmy plate
water
Isotherms of lipid and mixed Langmuir films
60
lipid
lipid+INH
lipid+INH-red-Ser
50
 mN/m
40
30
20
10
0
50
100
150
200
250
300
350
400
450
2
A /molecule
Incorporation of INH and INH(red)-SEFAGSFVRTVSLPV into
the lipid film is reflected in the shape of the isotherms.
Isotherms after compression cycles of pure and mixed monolayers
40
2nd isotherm
3rd isotherm
5th isotherm
30
 mN/m
2-3-5 consequtive compression/expansion cycles,
3 μg lipid or lipid+drug(conjugate) with molar ratio of 5:1 in
dichloromethane
lipid: 85% DSPC, 15% DPPC
20
(i) completely reproducible isotherms for the pure lipid film
(ii) IHN low interaction with the monolayer
(iii) dramatic change in the isotherms, higher stability
10
0
50
100
150
200
250
300
350
400
450
2
A /molecule
60
60
lipid + INH
50
30
30
20
20
10
10
0
0
50
100
150
200
2
250
300
A /molecule
350
400
2nd isotherm
3rd isotherm
5th isotherm
40
 mN/m
 mN/m
50
2nd isotherm
3rd isotherm
5th isotherm
40
lipid+INH-red-Ser
450
50
100
150
200
2
250
300
A / molecule
350
400
450
Comparison of INH or DUT 3 penetration into lipid monolayer
(1) lipid monolayer
surface pressure
sensor
1
Wilhelmy plate
(2) injection of INH/DUT 3 into the subphase
surface pressure
sensor
Wilhelmy plate
movable
24
barrier
Stability of phospholipon on water
Stability of phospholipon on water
2
24
22
22
20
20
18
water
18
drug
16
14
 (mN/m)
 (mN/m)
16
DUT3, 2mM
14
12
Langmuir balance 10
compression/expansion: 24 8cm2/min
12
10
6
4
4
2
2
0
DUT 3, 2mM
8
6
0
(i) pink line was the reference (pure lipid film)
-2
0
1000
2000
3000
-2
4000
0
1000
t (sec)
(ii) penetration of DUT 3 was indicated by the difference
Stability of phospholipon on water
between the pink and black line
3000
4000
Stability of phospholipon on water
24
24
22
22
20
(iii) DUT 3 shows a significant affinity to lipid layer, this
tendency for INH is lower
20
18
18
16
16
14
12
10
8
6
INH 2mM
4
2
14
 (mN/m)
 (mN/m)
2000
t (sec)
12
10
8
INH, 2mM
6
4
2
0
0
-2
-2
0
1000
2000
3000
4000
0
1000
2000
t (sec)
3000
4000
Jelölés 5(6)-karboxifluoreszceinnel
O
HO
O
C
O
O
N
O
OH
+
O
pH=9,2
C
1 óra
O
NH2-R
C
R-NH
O
OH
C
O
O
5(6)-karboxifluoreszcein-szukcinimid-észter
(CF-SE)
Tisztítás:
Sephadex G25
Eluens: desztillált víz
Mosás: 1% ecetsav (v/v)
O
HO
CF-polipeptid
Karboxifluorszcein-tartalom
meghatározása:
Savas hidrolízis (6M HCl, 24 óra)
Analitikai HPLC: CF kalibrációs görbe
alapján
Structure and charge of the side-chains
[
NH
CO
]n
[
NH
CO
]n
[
NH
CO
]n
[
NH
CO
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
AK
TAK
NH
NH
NH
CO
CO
CO
CH
CH3
CH
NH3+
CH3
CH
NH
SAK
CO
CH
CH3
m
CO
CH3
CH
NH3+
CH
CH
OH
NH3+
CH3
NH
m
CO
EAK
NH
NH
m
]n
CH2
OH
m
CO
CH
NH3+
(CH2)2
COO -
salt bridge
Structure and charge of the side-chains
[
[
NH
CO
]n
NH
CO
]n
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
AcEAK
NH
SuccEAK
NH
CO
CO
CH3
CH
CH
CH3
NH
m
NH
CO
m
CO
CH
(CH2)2
COO -
(CH2)2
CH
NH
NH
C
CH3
C
O
O
CH2
CH2
COO -
COO -
oligopeptide based carrier molecules
„Lysine tree”:
Tam, J. P.: Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9084
NH2
NH2
NH2
Lys
Lys
NH2
NH2
Lys
NH2
NH2
Lys
Lys-(Aaa)n-Ala-OH
Lys
Lys
NH2
SOC (Sequential Oligopeptide Carrier):
Ac-[Lys-Aib-Gly]n-OH
(n=3-7)
310-hélix
Tsikaris, V., et al.: Biopolymers 38 (1996) 291
1. Poly[L-Lys] backbone (polimerisation degree: 60-120)
2. Oligo[DL-Ala] sidechains 3 Ala/Lys)
3. Different amino acids at the N-terminus of the branches
poli[Lys(Xi)]
poli[Lys(DL-Alam)]
Hudecz, et al, J. Controlled Release, 1992
Rajnavölgyi et al, Mol. Immunol., 1986
Clegg et al, Bioconjugate Chem., 1990
Pimm et al, J. Controlled Release, 1995
poli[Lys(Xi-DL-Alam)]
Hudecz, et al, Bioconjugate Chem. 1999
Rajnavölgyi et al, Chimica Oggi, 1990
Hudecz et al. Bioconjugate Chem., 1999
Carrier molecules
A) Natural compounds
BSA, KLH, ovalbumine,
tetanus toxoid, dextrane
Polymers
 polylysine
 branched chain polypeptide
 polytuftsin
 N-vinyl-pirrolidone - maleic acid copolymer
 stirene-maleic acid copolymer
B) Synthetic products
•
•
biodegradable
biocompatible, but
non-degradable
Molecules with defined
structure
 lysine dendrimers
 sequential oligopeptides