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Tuberculosis

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

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Tuberculosis

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  1. Tuberculosis Sandra Ferreira

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

  3. The Global Burden • ~2 billion people infected • 9.2 million new cases in 2006 • 1.7 million deaths occurred in 2006 • 7th leading cause of death 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 Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino Sylvia Cardoso LeãoVivianaRitacco

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

  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 Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, VivianaRitacco

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

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

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

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

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

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

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

  14. Current Treatment of Tuberculosis First Line Drugs Isoniazid Rifampin Pyrazinamide Ethambutol Streptomycin 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, VivianaRitacco

  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 Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, VivianaRitacco

  16. Current Treatment of Tuberculosis Second Line Drugs RifampentineCapreomycin RifambutinCycloserine EthionamideLevofloxacin AmikacinMoxifloxacin KanamycinGatifloxin Para-amino salicylic acid 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, VivianaRitacco

  17. Drug Treatment to Date 1953 1980’s 1950-1962 • No new first line drugs since 1980’s • Need new targets • Need better understanding of the infection 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’s 1980’s 1944 Ruben, E. J., Nature Medicine 2003, 13, 279-280

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

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

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

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

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

  23. Immune response Cl- Superoxide dismutase melyoperoxidase phox 3O2 O2- H2O2 ClO- H2O H2O2 1O2 O2 Reactive Oxygen Intermediates-ROI TNF-alpha and Respiratory Burst IFN-gamma Winterbourn , C.C., Blood 1998, 92, 3007-3017 Foote C.S.; Wexler S., J. Am. Chem. Soc.1964, 86, 3879–3880,

  24. RNI- Reactive Nitrogen Intermediates • NO● • Cytotoxic to Mycobacterium tuberculosis • Highly reactive and diffusible free radical • Capable of reacting with ROI’s O2-+ NO ONOO- ● Highly antibacterial 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

  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

  27. iNOS - inducible Nitric Oxide Synthase iNOS Dimer oxygenase Fe(lll) Fe(ll) reductase e- FMNH2 FADH● NADPH FMNH● FADH2 NADP+ reductase oxygenase Knowles, R. G.,Biochem. J.2001, 357, 593-615

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

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

  30. iNOS - Oxygenase Domain [Fe(lll)] [Fe(ll)] O2●- [Fe(lll)]O2 [Fe(ll)] O2 e- e- 2H+ H2O Arginine Arginine Arginine N-hydroxyl amine Marletta, M. A., J. Am. Chem. Soc.2009, 131, 297–305.

  31. iNOS - Oxygenase Domain [Fe(lll)] [Fe(ll)] O2●- [Fe(lll)]O2 [Fe(ll)] O2 e- e- 2H+ H2O e- N-hydroxyl amine Arginine Arginine Arginine N-hydroxyl amine N-hydroxyl amine [Fe(lll)] NO● [Fe(ll)] O2 [Fe(ll)] H+ O2 H2O + Citrulline Marletta, M. A., J. Am. Chem. Soc.2009, 131, 297–305.

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

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

  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 Granuloma Imposes bacteriostatis Barry, E. C., Nature Reviews Microbiology 2005, 3, 70-80

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

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

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

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

  39. Activity of PA-824 PA-824 Baker, W.R., Nature2000, 405, 962-966

  40. Activity of PA-824 Minimum Inhibitory Concentration • 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 Baker, W.R., Nature2000, 405, 962-966

  41. Activity of PA-824 Minimum Inhibitory Concentration • 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 Baker, W.R., Nature2000, 405, 962-966

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

  43. Nitroimidazoles Mode of Action host iNOS Aerobic O2 NO• Dying Bacteria Active Nathan, C., Science2008, 322, 1337-1338

  44. Nitroimidazoles Mode of Action host iNOS Aerobic O2 NO• Dying Bacteria Active host Anaerobic Limited iNOS Latent Nathan, C., Science2008, 322, 1337-1338

  45. Nitroimidazoles Mode of Action host iNOS Aerobic O2 NO• Dying Bacteria Active host Anaerobic Limited iNOS Dying Bacteria NO• Latent ENZYME Nathan, C., Science2008, 322, 1337-1338

  46. Mtb Enzyme Needed for PA-824 Activity F420 - dependant glucose- 6 phosphate dehydrogenase (Ddn) Glucose-6-phosphate F420- coenzyme Bashiri, J., J Biol Chem. 2008, 283, 17531-41 Manjunatha, U. H., PNAS2006, 103, 431–436 Baker W.R., Nature2000, 13, 962-966.

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

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

  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 F420 Des Nitro Singh, R., Science, 2008, 322, 1392-1395

  50. Analysis of Hydride Transfer Reduction Ddn NaBH4 Des Nitro and metabolites PA-824 PA-824 Singh, R., Science, 2008, 322, 1392-1395

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