1. In vitro antitubercular effect of INH-conjugates and in silico identified drug candidates Szilvia Bősze 1, Kata Horváti 1, Nóra Szabó 2, Vince Grolmusz 3, Éva Kiss 4, Katalin Hill 4, Gábor Mező 1, Ferenc Hudecz 1,5 and Beáta G. Vértessy 6 1 Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest, Hungary 2 Korányi National Institute for TB and Pulmonology, Budapest, Hungary 3 Department of Computer Science, Eötvös Loránd University, Budapest, Hungary 4 Department of Physical Chemistry, Eötvös Loránd University, Budapest, Hungary 5 Department of Organic Chemistry, Eötvös Loránd University, Budapest, Hungary 6 Institiute of Enzymology, BRC, Hungarian Academy of Sciences, Budapest, Hungary;

2. 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

3. Site of action receptor degradation lysosome degradation fluidic endocytosis receptor mediated endocytosis diffusion/active transport Uptake of bioactive entities Takakura Y., Hashida M. Crit.Rev.Oncol.Hematol. 18: 207 (1994) lysosome

4. (i) Receptor mediated drug targeting

5. (ii) New drug candidates (in silico identified)

6. Receptor families that can be used for delivery: mannosyl-fucosyl receptors galactosyl receptors scavenger receptors tufsin receptor Specific delivery of INH into macrophages + lysosome endosome phagocytosis intracellular parasite specific 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 frontline drug, min. 6 months therapy bactericide prodrug inhibits the formation of cell wall

7. 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)

8. 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

9. Development of new tuftsin based carrier molecules H-[Thr-Lys-Pro-Lys-Gly] n -NH 2 (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

10. Fluorescent labelling 5(6)-carboxyfluorescein Antitubercular drugs or/and antimicrobial peptides Receptor-specificity SR-A OC CH 2 NH COO - CH 2 Succ - OOC C O CH 3 NH Ac - OOC Hudecz, et al, J. Controlled Release, 1992 Hudecz, et al, Bioconjugate Chem. 1999 Rajnavölgyi et al, Mol. Immunol., 1986Rajnavölgyi et al, Chimica Oggi, 1990 Clegg et al, Bioconjugate Chem., 1990 Hudecz et al. Bioconjugate Chem., 1999 Pimm et al, J. Controlled Release, 1995

11. poly[Lys(Succ-Glu i -DL-Ala m )]; Succ-EAK Branched chain polypeptides

12. 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, NaHCO 3, NaCl, HEPES, KCl, MgCl 2, CaCl 2, Na 2 HPO 4 x 2H 2 O Cellular uptake of the carrier/targeting molecules MonoMac6

13. 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 min 105 min time[min]

14. Internalisation of bioconjugate containing carboxy- fluoresceine into THP-1 monocytes Control CF-T20 1 min 15 min 60 min [TKPKG] 4 -NH 2 CH 2 CO CF-GFLGC-NH 2 CF: 5(6)-carboxy-fluoresceine Images were recorded by confocal laser scanning microscopy 1 min 60 min fixed cells

15. Uptake polylysine based polypeptides J774 cells SAK control AK EAK Ac-EAK Succ-EAK PiKPiK

16. Synthesis of carrier peptide – INH conjugates -1 *Geoghean, K. F. and Stroh, J. G. Bioconjugate Chem. 1992, 3: 138-142. INH- 92 EFAGAGFVRAGAL 104 (hydrazone) (INH-oxAGA) INH- 91 SEFAYGSFVRTVSLPV 106 (hydrazone) (INH-oxSer) 91 SEFAGAGFVRAGAL 104 S- 91 SEFAYGSFVRTVSLPV 106 N-glyoxylil- 92 EFAGAGFVRAGAL 104 N-glyoxylil- 91 SEFAYGSFVRTVSLPV 106 92 EFAGAGFVRAGAL 104 4 equiv. NaIO 4 10 equiv. Met (scavenger) 1 % NH 4 HCO 3 (pH=8.3) + 10 equiv. ethyleneglycol, RP-HPLC + 50 equiv. INH 0.1M NH 4 OAc (pH=4.6) RP-HPLC 92 EFAGAGFVRAGAL 104

17. Glyoxylic acid, as a heterobifunctional linker -> -> Coupling INH to peptides on solid phase -> Reduction before coupling (-> hidrazide) H 2 O : AcN 10 : 1 1h RT yield: 98% Mp: 206.5 – 207.0 o C 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 [M+H] + Mmo (calculated) = 193.1 Synthesis of carrier peptide – INH conjugates -2

18. 1.0 equiv. NaCNBH 3 methanol (suspension) [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)

19. INH- 91 SEFAYGSFVRTVSLPV 106 (INH-red-Ser, hydrazide) M av(calculated) = 1935.0 M av(measured) = 1934.8 RP-HPLC, Knauer, Eurospher-100 C18, 5  m, 250x4mm column, =214nm, gradient: 5-60B% 35min. A eluent: H 2 O+0,1 v/v% TFA, B eluent: AcN: H 2 O =80:20 (v/v) +0,1 v/v% TFA 91 SEFAYGSFVRTVSLPV 106 Synthesis of carrier peptide – INH conjugates -4

20. Stability of INH(red)-SEFAYGSFVRTVSLPV (hydrazide) conjugate RP-HPLC, Knauer, Eurospher-100 C18, 5  m, 250x4mm column, =214nm, gradient: 5-60B% 35min. A eluent: H 2 O+0,1 v/v% TFA, B eluent: AcN: H 2 O =80:20 (v/v) +0,1 v/v% TFA semisynthetic Sula media, pH = 6.8 37 o C, c 0 = 0.5 mg/ml

21. Determination of minimum inhibitory concentration (MIC) M. tuberculosis H 37 RV

22. Drug / conjugate MIC  (  g/ml) CFU INH0.1612 INH-gli(ox) (hydrazone, Figure 1)0.4060 INH-gli(red) (hydrazide, Figure 2)0.406 INH- 92 EFAGAGFVRAGAL 104 (hydrazone, Figure 3) 0.2440 INH- 91 SEFAYGSFVRTVSLPV 106 (hydrazone)0.1830 INH- 91 SEFAYGSFVRTVSLPV 106 (hydrazide)0.162 GTKPK(INH)G (hydrazide, Figure 4)0.1820 91 SEFAGAGFVRAGAL 104 -- 91 SEFAYGSFVRTVSLPV 106 -- GTKPKG-- 92 EFAGAGFVRAGAL 104 Determination of MIC/CFU of INH, „INH-linker”, INH-conjugates and carriers Figure 1 Figure 2 Figure 4Figure 3

23. Cytostatic effect of INH, INH-conjugates and carriers INH IC 50 > 3.6*10 -2 M MIC=1.4 *10 -6 M T6 IC 50 > 5.0*10 -4 M MIC=1.4 *10 -6 M T6(INH) conjugate IC 50 > 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 h72 h treatment wash, culture MTT HepG2 5. 10 3 cell/well PBMC 1. 10 5 cell/well DMSO, = 540 nm

24. 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

25. 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.

26. Lig 14, C 24 H 38 N 4, Mw = 382.3 Lig 22, C 23 H 34 N 4 O 2 S, Mw = 430.2 Lig 35, C 22 H 36 N 6, Mw = 384.3

27. birA, biotin-protein ligase [Mycobacterium tuberculosis H37Rv] EC 6.3.4.15 (Rv3279c) Lig 4, C 22 H 36 N 6, Mw = 384.3 Lig 5, C 14 H 19 FN 4, Mw = 262.2

28. dUTPase, nucleotidohydrolase [Mycobacterium tuberculosis H37Rv] EC 3.6.1.23 (Rv2697c) DUT 32, C 27 H 36 N 6 O 2, Mw = 376.3 DUT 1, C 24 H 19 N 3 O 7 S, Mw = 493.1

29. DUT 44, C 25 H 28 N 2 O 5, Mw = 436.2 DUT 13, C 25 H 31 N 5 O 3 S, Mw = 384.3 DUT3, C 25 H 38 N 4 O, Mw = 410.3

30. Determination of minimum inhibitory concentration (MIC) M. tuberculosis H 37 RV

31. Docked moietyMIC  (µg/ml)CFU DUT I/425n.d. DUT 355 DUT 131542 DUT 3230n.d. DUT 4425n.d. Rv3279c Lig 43050 Rv3279c Lig 52540 2Bzr Lig 14256 2Bzr Lig 2230n.d. 2Bzr Lig 3525n.d. +/- : no invisible growth (CFU) Determination of MIC/CFU of in silico identified candidates

32. 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 =5 30 nm (FL2) *HPMI: glucose, NaHCO 3, NaCl, HEPES, KCl, MgCl 2, CaCl 2, Na 2 HPO 4 x 2H 2 O Emission spectra and cellular uptake of the DUT 44 10 -5 M, 1%DMSO / HPMI ex =488nm control 1. 10 -5 M 5. 10 -5 M 1. 10 -4 M

33. HepG2 IC 50 = 6.35. 10 -5 M MIC = 1.21. 10 -5 M PBMC IC 50 = 2.11. 10 -5 M MIC = 1.21. 10 -5 M Cytostatic effect of DUT 3 ligand on HepG2 and PBMC

34. PBMC double treatment IC 50 = 1.60. 10 -5 M MIC = 1.21. 10 -5 M PBMC cytotoxicity IC 50 = 3.45. 10 -5 M MIC = 1.21. 10 -5 M wash Cytotoxic and cytostatic effect of DUT 3 ligand on PBMC

35. 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 movable barrier Langmuir trough compression/expansion: 24 cm 2 /min electrobalance

36. 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

37. (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

38. 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

41. Jelölés 5(6)-karboxifluoreszceinnel 5(6)-karboxifluoreszcein-szukcinimid-észter (CF-SE) O HO O C O ON O O C O OH + NH 2  R O HO O C O R  NH C O OH pH=9,2 1 óra CF-polipeptid Tisztítás: Sephadex G25 Eluens: desztillált víz Mosás: 1% ecetsav (v/v) Karboxifluorszcein-tartalom meghatározása: Savas hidrolízis (6M HCl, 24 óra) Analitikai HPLC: CF kalibrációs görbe alapján

42. NH 3 + CO CH CH 3 m n ] CONH [ CH 2 NH AK NH CO CH CH 3 m NH 3 + COO  (CH 2 ) 2 CO CH n ] CONH [ CH 2 NH EAK Structure and charge of the side-chains NH 3 + CO CH NH CO CH CH 3 m CH CH 3 OH n ] CONH [ CH 2 NH TAK CH 2 OH NH 3 + CO CH NH CO CH CH 3 m n ] CONH [ CH 2 NH SAK salt bridge

43. NH CO CH CH 3 m NH COO  (CH 2 ) 2 CO CH n ] CONH [ CH 2 NH AcEAK C O CH 3 OC CH 2 COO  CH 2 NH CO CH CH 3 m NH COO  (CH 2 ) 2 CO CH n ] CONH [ CH 2 NH SuccEAK Structure and charge of the side-chains

44. Lys-(Aaa) n -  Ala-OH Lys NH 2 oligopeptide based carrier molecules „Lysine tree”: SOC (Sequential Oligopeptide Carrier): Ac-[Lys-Aib-Gly] n -OH (n=3-7) 3 10 -hélix Tam, J. P.: Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9084 Tsikaris, V., et al.: Biopolymers 38 (1996) 291

45. 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 Hudecz, et al, J. Controlled Release, 1992 Hudecz, et al, Bioconjugate Chem. 1999 Rajnavölgyi et al, Mol. Immunol., 1986Rajnavölgyi et al, Chimica Oggi, 1990 Clegg et al, Bioconjugate Chem., 1990 Hudecz et al. Bioconjugate Chem., 1999 Pimm et al, J. Controlled Release, 1995 poli[Lys(X i )] poli[Lys(DL-Ala m )] poli[Lys(X i -DL-Ala m )]

46. 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