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