1. Tuberculosis Sandra Ferreira

2. Agenda What is Tuberculosis History of Treatment Our Immune Response PA-824 Conclusions 2

3. The Global Burden ~2 billion people infected 9.2 million new cases in 2006 1.7 million deaths occurred in 2006 7 th leading cause of death 3 Global Tuberculosis Control, 2008 World Health Organization

4. What is tuberculosis Tuberculosis is an infectious lung disease caused by the bacteria Mycobacterium tuberculosis (Mtb) Spread from person to person There are two types of infections: ~90% Latent TB ~10% Active TB 4 Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino Sylvia Cardoso LeãoViviana Ritacco

5. Symptoms of Tuberculosis Sweating and fever Weight loss Cough, begins dry but becomes productive with mucous Thoracic pain 5 Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

6. History of Tuberculosis Found in mummies thousands of years old “Phthitis”- Hippocrates 450BC 1852 Robert Koch and Julius Richard Petri- isolated the bacteria Finally in 1943, the first antibiotic was used to treat tuberculosis 6 Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

7. Targets of Current Drug Treatment 7 Ruben, E. J., Nature Medicine 2003, 13, 279-280

8. Targets of Current Drug Treatment 8 Streptomycin 1943 Ruben, E. J., Nature Medicine 2003, 13, 279-280

9. Targets of Current Drug Treatment 9 Para amino salicylic acid (PAS) 1944 Ruben, E. J., Nature Medicine 2003, 13, 279-280

10. Targets of Current Drug Treatment 10 Isoniazid 1953 Ruben, E. J., Nature Medicine 2003, 13, 279-280

11. Targets of Current Drug Treatment 11 Ethambutol 1960 Ruben, E. J., Nature Medicine 2003, 13, 279-280

12. Targets of Current Drug Treatment 12 Rifampin 1970’s Ruben, E. J., Nature Medicine 2003, 13, 279-280

13. Targets of Current Drug Treatment 13 Pyrazinamide 1980’s Ruben, E. J., Nature Medicine 2003, 13, 279-280

14. Current Treatment of Tuberculosis First Line Drugs Isoniazid Rifampin Pyrazinamide Ethambutol Streptomycin 14 These are the five first Line drugs Three or more of them are Used in combinations For up to 6 months However a larger % Of patients are not finishing The drug treatments and Or being prescribed the Wrong treatment times Drug resistant strains Began to immerge Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

15. Definitions: Multidrug and Extremely Multidrug resistant Mtb In 2006, half a million people Had mdr-tb. Approximately 4.8% of Cases of TB were Reported to be Multidrug resistant. Very Important because isoziazid Is the most powerful drug Against active tuberculosis And rifampacin is important For curing latent forms of Tuberculosis??? Is that true Extreme multidrug resistance Is multidrug resistance but is also Resistant to three or more of MDR-TB Isoniazid and Rifampin. XDR-TB Isoniazid, Rifampin and three or more second line drugs. 15 Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

16. Current Treatment of Tuberculosis Second Line Drugs Rifampentine Capreomycin Rifambutin Cycloserine Ethionamide Levofloxacin Amikacin Moxifloxacin Kanamycin Gatifloxin Para-amino salicylic acid 16 Many second line Drugs are available But they are More toxic Up to a thousand Times more expensive And require drug Treatments of up to 2 years Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

17. Drug Treatment to Date 17 All of these antibitics Were found 30 years Ago. The latest of which Is the flouroquinolones Interfer with dna gyrase As can be seen from Long treatment Times and drug Resistance we need New drugs With new modes of Action to help when Out immune system Is not enough. 1970’s1980’s1944 1950-19621980’s1953 No new first line drugs since 1980’s Need new targets Need better understanding of the infection Ruben, E. J., Nature Medicine 2003, 13, 279-280

18. What Happens After Infection with Mtb Inhale the bacteria 1. Spontaneous healing! 3. Latent Tuberculosis - Granulomas 2. Active Tuberculosis 18

19. 19 Phagocyte Phagolysosome Bacteria How the Immune System Kills Mtb Lysosome

20. 20 Phagocyte Bacteria Bacteria has been able to stop this process Deretic, V., PNAS 2005, 12, 4033–4038 How the Immune System Kills Mtb Phagolysosome Lysosome

21. How the Immune System Kills Mtb 21 Phagocyte Bacteria Volker Brinkmann, Max Planck Institute, Press Release, March 24, 2004 Lysosome Phagolysosome Deretic, V., PNAS 2005, 12, 4033–4038

22. Phagocyte 22 Bacteria TNF-alpha and IFN-gamma Other immune responses How the Immune System Kills Mtb Lysosome Phagolysosome Deretic, V., PNAS 2005, 12, 4033–4038

23. Immune response O2O2 Respiratory Burst Reactive Oxygen Intermediates-ROI phox 23 TNF-alpha and IFN-gamma 3O23O2 O2-O2- Superoxide dismutase H2O2H2O2 melyoperoxidase ClO - H2OH2O 1O21O2 H2O2H2O2 Cl - Foote C.S.; Wexler S., J. Am. Chem. Soc. 1964, 86, 3879–3880, Winterbourn, C.C., Blood 1998, 92, 3007-3017

24. RNI- Reactive Nitrogen Intermediates NO ●  Cytotoxic to Mycobacterium tuberculosis  Highly reactive and diffusible free radical  Capable of reacting with ROI’s O 2 - + NO ONOO - Highly antibacterial 24 ● Winterbourn, C.C., Blood 1998, 92, 3007-3017

25. Immune Response Nitric oxide Synthase- Produces NO NOS discovered in 1989 Tens of thousands of papers have been published, ~ 50 papers/week Nobel prize awarded for discovery NO as biological mediator There are three kinds of NOS Type 1 nNOS- neuronal Type 2 iNOS- inducible Type 3 eNOS- endothelial This is my plug on why its Important to spend a little Time talking about how this Works. Should still Scifinder NOS to check the Number of papers published 25 ●

26. iNOS - inducible Nitric Oxide Synthase iNOS can be induced to produce nitric oxide for hours or even days iNOS dimer L-arginine L-citrulline 26

27. 27 iNOS Dimer reductase oxygenase NADPH NADP + FADH 2 FADH ● reductase e-e- FMNH 2 FMNH ● Fe(lll)Fe(ll) Knowles, R. G., Biochem. J. 2001, 357, 593-615 iNOS - inducible Nitric Oxide Synthase

28. iNOS Dimer reductase oxygenase reductase e-e- Fe(lll)Fe(ll) 28 Fe(ll)Fe(lll) e-e- iNOS - inducible Nitric Oxide Synthase Knowles, R. G., Biochem. J. 2001, 357, 593-615

29. ~ Fe L-arginine 29 iNOS - inducible Nitric Oxide Synthase Knowles, R. G., Biochem. J. 2001, 357, 593-615

30. iNOS - Oxygenase Domain Marletta, M. A., J. Am. Chem. Soc. 2009, 131, 297–305. [Fe(lll)] O 2 Arginine e-e- [Fe(ll)] O 2 Arginine [Fe(ll)] O 2 ●- Arginine e-e- 2H + H2OH2O [Fe(lll)] N-hydroxyl amine 30

31. [Fe(ll)] e-e- [Fe(ll)] O 2 O2O2 H+H+ H 2 O + NO ● [Fe(lll)] iNOS - Oxygenase Domain N-hydroxyl amine 31 [Fe(lll)] O 2 Arginine e-e- [Fe(ll)] O 2 Arginine [Fe(ll)] O 2 ●- Arginine e-e- 2H + H2OH2O [Fe(lll)] N-hydroxyl amine Citrulline Marletta, M. A., J. Am. Chem. Soc. 2009, 131, 297–305. N-hydroxyl amine

32. Tuberculosis Fights Back 1. Many strains of Tuberculosis have shown resistance to ROI’s 32 Shiloh, M. U., PNAS 2000, 97, 8841–8848

33. Tuberculosis Fights Back 32 1. Many strains of Tuberculosis have shown resistance to ROI’s 2. Live tuberculosis bacteria is able to resist the accumulation of iNOS surrounding the phagosome A B C 1 2 Deretic V., PNAS, 2007, 3, 1887- 1894 Shiloh, M. U., PNAS 2000, 97, 8841–8848

34. Containment of Tuberculosis Complex collection of cells: - phagocytes - T cells - necrotic tissue - giant cells Decrease oxygen availability Detrimental to both the bacteria and the host 34 Granuloma Imposes bacteriostatis Barry, E. C., Nature Reviews Microbiology 2005, 3, 70-80

35. Containment of Tuberculosis Depletion of oxygen 35 Forces bacteria into a latent state Decrease in ROIs and RNIs Bacteria can persist Not able to fully irradicate TB Barry, E. C., Nature Reviews Microbiology 2005, 3, 70-80

36. New Drugs 36 New Drugs ? Ruben E.J., Nature Medicine, 2003, 13, 279-280

37. 1989 - Hindustan Ciba-Geigy Research Centre - Bombay, India Kuppuswamy Nagarajan Nitroimidazoles as Antimicrobials Anaerobic bacteria Anti tuberculosis activity Found to be mutenigenic In murine model 37 Nagarajan K., J. Chem. Sci., 2006, 291-309 Nargarajan, K., Eur. J. Med. Chem. 1989, 24, 631-633

38. Kuppuswamy Nagarajan 38 Nagarajan K., J. Chem. Sci., 2006, 291-309 1989 - Hindustan Ciba-Geigy Research Centre - Bombay, India 2 6 Nargarajan, K., Eur. J. Med. Chem. 1989, 24, 631-633

39. Activity of PA-824 39 PA-824 Baker, W.R., Nature 2000, 405, 962-966

40. Activity of PA-824 40 Active Tuberculosis Isoniazid 0.03-0.06 (µg/mL) PA-824 0.02-0.25 (µg/mL) MDR-TB Activity PA-824 0.03-0.25 (µg/mL) PA-824 Minimum Inhibitory Concentration Baker, W.R., Nature 2000, 405, 962-966

41. Activity of PA-824 41 Active Tuberculosis Isoniazid 0.03-0.06 (µg/mL) PA-824 0.02-0.25 (µg/mL) MDR-TB Activity PA-824 0.03-0.25 (µg/mL) Minimum Anaerobic Concentration Latent Tuberculosis PA-824 0.25 (µg/mL) PA-824 Minimum Inhibitory Concentration Baker, W.R., Nature 2000, 405, 962-966

42. Synthesis of PA-824 42 PA-824 53% 79%73% 62% 86% Baker, W.R., US Patent # 5668127, 1997 99% 65% Sharpless, B.K., JACS 1987, 109, 5765-80 Marko, I.S., Tetrahedron 2006, 47, 5933-37

43. 43 Nitroimidazoles Mode of Action iNOS O2O2 Dying Bacteria host Active Aerobic Nathan, C., Science 2008, 322, 1337-1338 NO

44. Nitroimidazoles Mode of Action 44 host Anaerobic Limited iNOS Latent iNOS O2O2 Aerobic Dying Bacteria host Active NO Nathan, C., Science 2008, 322, 1337-1338

45. Nitroimidazoles Mode of Action 45 Dying Bacteria host Limited iNOS ENZYME Latent iNOS O2O2 NO Dying Bacteria host Active Anaerobic Aerobic NO Nathan, C., Science 2008, 322, 1337-1338

46. F 420 - dependant glucose- 6 phosphate dehydrogenase (Ddn) Glucose-6-phosphate F 420 - coenzyme Manjunatha, U. H., PNAS 2006, 103, 431–436 Baker W.R., Nature 2000, 13, 962-966. Mtb Enzyme Needed for PA-824 Activity Bashiri, J., J Biol Chem. 2008, 283, 17531-41

47. Hydride Transfer to Cofactor 47 Bashiri, J., J Biol Chem. 2008, 283, 17531-41

48. Mode of Action in Latent TB 48 Manjunatha, U.H., PNAS, 2006, 103, 431–436 Many Products and NO

49. Gave Rise to More Polar Metabolites A B C A- PA-824 B- Conversion of PA-824 by whole cells of Mtb C-Conversion of PA-824 using Ddn and F 420 49 Singh, R., Science, 2008, 322, 1392-1395 Des Nitro

50. Analysis of Hydride Transfer 50 Reduction Ddn NaBH 4 Des Nitro and metabolites PA-824 Singh, R., Science, 2008, 322, 1392-1395

51. Analysis of Hydride Transfer 51 Reduction Ddn NaBH 4 NaBH 4 / MeOH 32 Singh, R., Science, 2008, 322, 1392-1395

52. Analysis of Hydride Transfer 52 Reduction Ddn NaBH 4 NaBH 4 / MeOH NaBD 4 / MeOH 32 Singh, R., Science, 2008, 322, 1392-1395

53. Analysis of Hydride Transfer 53 Reduction Ddn NaBH 4 NaBH 4 / MeOH NaBH 4 / MeOD NaBD 4 / MeOH 32 Singh, R., Science, 2008, 322, 1392-1395

54. 54 Des Nitro Reduction of Nitroimidazole by Ddn Singh, R., Science, 2008, 322, 1392-1395 1

55. Reduction of Nitroimidazole by Ddn 55 2 Singh, R., Science, 2008, 322, 1392-1395

56. Reduction of Nitroimidazole by Ddn 56 3 Singh, R., Science, 2008, 322, 1392-1395

57. What is Killing Tuberculosis? 57 2HNO 2 2N 2 O 3 + H 2 O N2O3N2O3 NO + NO 2 HNO 2 + 123 Singh, R., Science, 2008, 322, 1392-1395

58. Detection of RNI’s 1. Griess Reagent 58 They needed to test The hypothesis that the Reduction of PA-824 Sulfanilic Acid reacts with nitrite to poduce a diazonium Salt. Then upon Addition of napthyl Amine is added if froms A azo dye which is a pink color H 3 PO 4

59. Detection of RNI’s 59 2. DAF-FM diaminofluorescein Nitrous acid breaks Down to produce Nitric oxide. And this Can be montitored Using diamino Flourescein Which reacts to form N 2 O 3 C-PTIO 2HNO 2 2N 2 O 3 + H 2 O N2O3N2O3 NO + NO 2

60. NO and Antimicrobial Activity Monitoring PA-824 reduction by Ddn and F 420 using griess reagent Using DAF-FM 60 Singh, R., Science, 2008, 322, 1392-1395

61. Aerobic and Anaerobic Activity of Analogues 61 MACZ R Singh, R., Science, 2008, 322, 1392-1395

62. Correlation Between Anaerobic Killing and NO 62 Singh, R., Science, 2008, 322, 1392-1395

63. Where is PA-824 now TB Alliance and Chiron have agreed to produce PA-824 for “not for profit” to developing countries for the purpose of tuberculosis chemotherapy Currently in phase II 63

64. Conclusions Our immune system produce RNI’s and ROI’s to kill bacteria Bacteria can adapt to our defence system to protect itself Prodrug PA-824 uses the bacteria’s enzyme, to kill the LATENT bacteria 64

65. The End - Thank you 65 Dr. Robert Ben Roger Tam Pawel Czechura Jennifer Chaytor John Trant Wendy Campbell Jackie Tokarew Liz Von Moos Gloria Gong Mike Souweha Mathieu Leclere Cole Stevens Taline Boghossian

66. Hydrogen Bonding 66

67. Co- enzyme 420 67