TuberculosisTuberculosis

Sandra Ferreira

AgendaAgenda

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

2

The Global BurdenThe 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

3Global Tuberculosis Control, 2008 World Health Organization

What is tuberculosisWhat 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

4Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino Sylvia Cardoso LeãoViviana Ritacco

Symptoms of TuberculosisSymptoms of Tuberculosis

Sweating and fever

Weight loss

Cough, begins dry but becomes productive with mucous

Thoracic pain

5Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

History of TuberculosisHistory 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

6Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

Targets of Current Drug TreatmentTargets of Current Drug Treatment

7Ruben, E. J., Nature Medicine 2003, 13, 279-280

Targets of Current Drug Treatment Targets of Current Drug Treatment

8

Streptomycin

1943

Ruben, E. J., Nature Medicine 2003, 13, 279-280

Targets of Current Drug TreatmentTargets of Current Drug Treatment

9

Para amino salicylic acid

(PAS)

1944Ruben, E. J., Nature Medicine 2003, 13, 279-280

Targets of Current Drug TreatmentTargets of Current Drug Treatment

1010

Isoniazid

1953

Ruben, E. J., Nature Medicine 2003, 13, 279-280

Targets of Current Drug TreatmentTargets of Current Drug Treatment

11

Ethambutol

1960

Ruben, E. J., Nature Medicine 2003, 13, 279-280

Targets of Current Drug TreatmentTargets of Current Drug Treatment

12

Rifampin

1970’sRuben, E. J., Nature Medicine 2003, 13, 279-280

Targets of Current Drug TreatmentTargets of Current Drug Treatment

13

Pyrazinamide

1980’s

Ruben, E. J., Nature Medicine 2003, 13, 279-280

Current Treatment of TuberculosisCurrent Treatment of Tuberculosis

First Line Drugs

Isoniazid

Rifampin

Pyrazinamide

Ethambutol

Streptomycin

14

These are the five firstLine drugs

Three or more of them are Used in combinations For up to 6 months

However a larger %Of patients are not finishingThe drug treatments andOr being prescribed the Wrong treatment timesDrug resistant strains Began to immerge

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

Definitions: Multidrug and Extremely Multidrug resistant Mtb

Definitions: Multidrug and Extremely Multidrug resistant Mtb

MDR-TB Isoniazid and Rifampin.

XDR-TBIsoniazid, Rifampin and

three or more second line drugs.

15Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

Current Treatment of TuberculosisCurrent Treatment of Tuberculosis

Second Line Drugs

Rifampentine Capreomycin

Rifambutin Cycloserine

Ethionamide Levofloxacin

Amikacin Moxifloxacin

Kanamycin Gatifloxin

Para-amino salicylic acid

16Tuberculosis 2007: From basic Science to Patient care. Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

Drug Treatment to DateDrug Treatment to Date

17

1970’s 1980’s 1944

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

What Happens After Infection with MtbWhat Happens After Infection with Mtb

Inhale the bacteria

1. Spontaneous healing!

3. Latent Tuberculosis - Granulomas

2. Active Tuberculosis

18

19

Phagocyte Phagolysosome

Bacteria

How the Immune System Kills MtbHow the Immune System Kills Mtb

Lysosome

20

Phagocyte Bacteria

Bacteria has beenable to stop this

process

Deretic, V., PNAS 2005, 12, 4033–4038

How the Immune System Kills MtbHow the Immune System Kills Mtb

Phagolysosome

Lysosome

How the Immune System Kills MtbHow 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

Phagocyte

22

Bacteria TNF-alpha and IFN-gamma

Other immune responses

How the Immune System Kills MtbHow the Immune System Kills Mtb

Lysosome

Phagolysosome

Deretic, V., PNAS 2005, 12, 4033–4038

Immune responseImmune response

O2

Respiratory Burst

Reactive Oxygen Intermediates-ROI

phox

23

TNF-alpha and IFN-gamma

3O2 O2-

Superoxidedismutase

H2O2

melyoperoxidaseClO-

H2O

1O2

H2O2

Cl-

Foote C.S.; Wexler S., J. Am. Chem. Soc. 1964, 86, 3879–3880, Winterbourn , C.C., Blood 1998, 92, 3007-3017

RNI- Reactive Nitrogen IntermediatesRNI- 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

24

●

Winterbourn , C.C., Blood 1998, 92, 3007-3017

Immune ResponseImmune 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- neuronalType 2 iNOS- inducible Type 3 eNOS- endothelial

25

●

NH2

NHHN

OOCNH3

+ O2

iNOS - inducible Nitric Oxide SynthaseiNOS - inducible Nitric Oxide Synthase

iNOS can be induced to produce nitric oxide for hours or even days

NH2

NHO

OOCNH3

+ NO

iNOS dimer

L-arginine

L-citrulline26

27

iNOS Dimer

reductase

oxygenase

oxygenase

NADPH

NADP+ FADH2

FADH●

reductase

e-

FMNH2

FMNH●

Fe(lll) Fe(ll)

Knowles, R. G., Biochem. J. 2001, 357, 593-615

iNOS - inducible Nitric Oxide SynthaseiNOS - inducible Nitric Oxide Synthase

iNOS Dimer

reductase

oxygenase

oxygenase

reductase

e-

Fe(lll) Fe(ll)

28

Fe(ll) Fe(lll)

e-

iNOS - inducible Nitric Oxide SynthaseiNOS - inducible Nitric Oxide Synthase

Knowles, R. G., Biochem. J. 2001, 357, 593-615

~Fe

L-arginine

29

iNOS - inducible Nitric Oxide SynthaseiNOS - inducible Nitric Oxide Synthase

Knowles, R. G., Biochem. J. 2001, 357, 593-615

iNOS - Oxygenase DomainiNOS - Oxygenase Domain

Marletta, M. A., J. Am. Chem. Soc. 2009, 131, 297–305.

[Fe(lll)] O2 NH2

NHHN

R

Arginine

e-[Fe(ll)] O2

NH2

NHHN

R

Arginine

[Fe(ll)] O2●-

NH2

NHHN

R

Arginine

e-2H+ H2O

[Fe(lll)]

NH2

NHN

R

HO

N-hydroxylamine

30

[Fe(ll)]

e-

[Fe(ll)] O2

O2

H+

H2O +

NO●[Fe(lll)]

iNOS - Oxygenase DomainiNOS - Oxygenase Domain

NH2

NHO

R

NH2

NHN

R

HO

N-hydroxylamine 31

[Fe(lll)] O2 NH2

NHHN

R

Arginine

e-[Fe(ll)] O2

NH2

NHHN

R

Arginine

[Fe(ll)] O2●-

NH2

NHHN

R

Arginine

e-2H+ H2O

[Fe(lll)]

NH2

NHN

R

HO

N-hydroxylamine

CitrullineMarletta, M. A., J. Am. Chem. Soc. 2009, 131, 297–305.

NH2

NHN

R

HO

N-hydroxylamine

Tuberculosis Fights BackTuberculosis Fights Back

1. Many strains of Tuberculosis have shown resistance to ROI’s

32Shiloh, M. U., PNAS 2000, 97, 8841–8848

Tuberculosis Fights BackTuberculosis 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- 1894Shiloh, M. U., PNAS 2000, 97, 8841–8848

Containment of Tuberculosis 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

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

Containment of TuberculosisContainment of Tuberculosis

Depletion of oxygen

35

Forces bacteria into a latent state

Decrease in ROIsand RNIs

Bacteria can persistNot able to fully

irradicate TBBarry, E. C., Nature Reviews Microbiology 2005, 3, 70-80

New DrugsNew Drugs

36

New Drugs

?

Ruben E.J., Nature Medicine, 2003, 13, 279-280

1989 - Hindustan Ciba-Geigy Research Centre - Bombay, India

1989 - Hindustan Ciba-Geigy Research Centre - Bombay, India

Kuppuswamy Nagarajan

Nitroimidazoles as Antimicrobials

Anaerobic bacteria

Anti tuberculosis activity

37Nagarajan K., J. Chem. Sci., 2006, 291-309

Nargarajan, K., Eur. J. Med. Chem. 1989, 24, 631-633

Kuppuswamy Nagarajan

38

N

NO2N

O

R

Nagarajan K., J. Chem. Sci., 2006, 291-309

1989 - Hindustan Ciba-Geigy Research Centre - Bombay, India

1989 - Hindustan Ciba-Geigy Research Centre - Bombay, India

2

6

Nargarajan, K., Eur. J. Med. Chem. 1989, 24, 631-633

Activity of PA-824Activity of PA-824

39

PA-824

Baker, W.R., Nature 2000, 405, 962-966

N

NO

O2N

O

OCF3

Activity of PA-824Activity 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

N

NO

O2N

O

OCF3

Baker, W.R., Nature 2000, 405, 962-966

Activity of PA-824Activity 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)

N

NO

O2N

O

OCF3PA-824

Minimum Inhibitory Concentration

Baker, W.R., Nature 2000, 405, 962-966

Synthesis of PA-824Synthesis of PA-824OH

OHO

N

N

NO2

OH

OTBSO

TBSO

N

N

NO2

OTHP

TBSO

N

N

O

OH

N

N

O

O

F3CO

D-(-) DIPT

TBHP,Ti(OiPr)4

TBS-Cl

imidazole

O2N O2N

O2NO2N

2, 4

dinitroimidazoleEtOH,70oC, 18h

DHP

PPTS

TBAF

THF

AcOH

THF

NaH

4-triflouromethoxybenzyl bromide

N

N

O

OTHP

O2N

42

PA-824

53%

79% 73%

62%86%

Baker, W.R., US Patent # 5668127, 1997

99%65%

Sharpless, B.K., JACS 1987, 109, 5765-80Marko, I.S., Tetrahedron 2006, 47, 5933-37

43

Nitroimidazoles Mode of ActionNitroimidazoles Mode of Action

iNOSO2

Dying Bacteria

host

Active

Aerobic

Nathan, C., Science 2008, 322, 1337-1338

NO•

Nitroimidazoles Mode of ActionNitroimidazoles Mode of Action

44

hostAnaerobic LimitediNOS

Latent

iNOSO2Aerobic

Dying Bacteria

host

Active

NO•

Nathan, C., Science 2008, 322, 1337-1338

Nitroimidazoles Mode of ActionNitroimidazoles Mode of Action

45

N

N

O

R

O2N

Dying Bacteria

hostLimitediNOS

ENZYMELatent

iNOSO2 NO•

Dying Bacteria

host

Active

Anaerobic

Aerobic

NO•

Nathan, C., Science 2008, 322, 1337-1338

F420 - dependant glucose- 6 phosphate dehydrogenase (Ddn)

OHO

HOOH

OH

OPO32-

Glucose-6-phosphate

N

NH

N O

OH

RHO

F420- coenzyme

Manjunatha, U. H., PNAS 2006, 103, 431–436 Baker W.R., Nature 2000, 13, 962-966.

Mtb Enzyme Needed for PA-824 ActivityMtb Enzyme Needed for PA-824 Activity

Bashiri, J., J Biol Chem. 2008, 283, 17531-41

Hydride Transfer to CofactorHydride Transfer to Cofactor

47Bashiri, J., J Biol Chem. 2008, 283, 17531-41

N

NH

N O

OH

RHO N

NH

HN O

O

RHO

H H

OHOHO

HHO

OPO32-

OHOHO

OHO

OPO32-

N

N

O2N O

O

OCF3

OH

Mode of Action in Latent TBMode of Action in Latent TB

48Manjunatha, U.H., PNAS, 2006, 103, 431–436

Many Products

and NO•

Gave Rise to More Polar MetabolitesGave Rise to More Polar Metabolites

A B C

A- PA-824

B- Conversion of PA-824 by wholecells of Mtb

C-Conversion of PA-824 using Ddnand F420

N

NO

R

N

NO

O

R

HH

N

H2NO

R

49Singh, R., Science, 2008, 322, 1392-1395

Des Nitro

Analysis of Hydride TransferAnalysis of Hydride Transfer

50

N

NO

R

O2N

H N

NO

R

H

H

ReductionDdn

NaBH4N

NO

R

O2N

H

Des Nitroand metabolitesPA-824 PA-824

Singh, R., Science, 2008, 322, 1392-1395

Analysis of Hydride TransferAnalysis of Hydride Transfer

51

N

NO

R

O2N

H N

NO

R

H

H

ReductionDdn

NaBH4

N

NO

R

O2N

H

NaBH4/MeOH

N

NO

R

H

H 3

2 3 2

Singh, R., Science, 2008, 322, 1392-1395

Analysis of Hydride TransferAnalysis of Hydride Transfer

52

N

NO

R

O2N

H N

NO

R

H

H

ReductionDdn

NaBH4

N

NO

R

O2N

H

N

NO

R

H

D

NaBH4/MeOH

NaBD4/MeOH

N

NO

R

H

H 3

2 3 2

Singh, R., Science, 2008, 322, 1392-1395

Analysis of Hydride TransferAnalysis of Hydride Transfer

53

N

NO

R

H

H 3

2

N

NO

R

O2N

H N

NO

R

H

H

ReductionDdn

NaBH4

N

NO

R

O2N

H

N

NO

R

H

D

N

NO

R

D

H

NaBH4/MeOH

NaBH4/MeOD

NaBD4/MeOH

3 2

Singh, R., Science, 2008, 322, 1392-1395

N

NO

NO

O

RH-

N

NO

NO

OH

R

HH

N

NO

NO

OH

R

HH

reduction

Protonation

N

NO

NO

O

R

H

H

H

N

NO

R

+HNO2

54

Des Nitro

Reduction of Nitroimidazole by DdnReduction of Nitroimidazole by Ddn

Singh, R., Science, 2008, 322, 1392-1395

1

Reduction of Nitroimidazole by DdnReduction of Nitroimidazole by Ddn

55

N

NO

N-O

O

RH-

N

NO

NO

OH

R

HH

N

NO

NO

OH

R

HH

reduction

Protonation

N

NO

N-O

O

R

H

H

H

N

NO

R

+HNO2

N

NO

N

R

H

H

OH

N

NO

O

R

H

H

+HNO

O

2

Singh, R., Science, 2008, 322, 1392-1395

Reduction of Nitroimidazole by DdnReduction of Nitroimidazole by Ddn

N

NO

N-O

O

RH-

N

NO

NO

OH

R

HH

N

NO

NO

OH

R

HH

reduction

Protonation Reduction

N

NO

N-O

O

R

H

H

H

N

NO

R

+HNO2

N

NO

N

R

H

H

OH

N

NO

O

R

H

H

2

+HNO

ON

NO

NHO

R

H

H

N

H2NO

R56

3

Singh, R., Science, 2008, 322, 1392-1395

What is Killing Tuberculosis?What is Killing Tuberculosis?

57

N

NO

O

R

HH

N

H2NO

R

2HNO2 2N2O3 + H2O

N2O3 NO• + NO2•

N

NO

R

HNO2

+1 2 3

Singh, R., Science, 2008, 322, 1392-1395

Detection of RNI’s Detection of RNI’s 1. Griess Reagent

58

NH2H2NO2SHNO2

N2+H2NO2S

NH

NH2

NH2NO2S NH

NH2

N

They needed to testThe hypothesis that the Reduction of PA-824

Sulfanilic Acid reacts with nitrite to poduce a diazoniumSalt. Then upon Addition of napthyl Amine is added if fromsA azo dye which is a pinkcolor

H3PO4

O-O O

FF

CO2-

NH2

NHCH3

O-O O

FF

CO2-

NN

NH3C

Detection of RNI’sDetection of RNI’s

59

2. DAF-FM diaminofluorescein

N2O3

C-PTIO

2HNO2 2N2O3 + H2O

N2O3 NO• + NO2•

NO and Antimicrobial ActivityNO and Antimicrobial Activity

Monitoring PA-824 reductionby Ddn and F420

using griess reagent

Using DAF-FM

60Singh, R., Science, 2008, 322, 1392-1395

Aerobic and Anaerobic Activity of Analogues

Aerobic and Anaerobic Activity of Analogues

61

MACZR

Singh, R., Science, 2008, 322, 1392-1395

Correlation Between Anaerobic

Killing and NO

Correlation Between Anaerobic

Killing and NO

62Singh, R., Science, 2008, 322, 1392-1395

Where is PA-824 nowWhere 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

Conclusions 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

The End - Thank youThe End - Thank you

65

Dr. Robert BenRoger TamPawel CzechuraJennifer ChaytorJohn TrantWendy CampbellJackie TokarewLiz Von MoosGloria GongMike SouwehaMathieu LeclereCole StevensTaline Boghossian

Hydrogen BondingHydrogen Bonding

66

Co- enzyme 420Co- enzyme 420

67