tuberculosis sandra ferreira. agenda what is tuberculosis history of treatment our immune response...
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TuberculosisTuberculosis
Sandra Ferreira
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AgendaAgenda
What is Tuberculosis History of Treatment Our Immune Response PA-824 Conclusions
2
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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
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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
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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
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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
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Targets of Current Drug TreatmentTargets of Current Drug Treatment
7Ruben, E. J., Nature Medicine 2003, 13, 279-280
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Targets of Current Drug Treatment Targets of Current Drug Treatment
8
Streptomycin
1943
Ruben, E. J., Nature Medicine 2003, 13, 279-280
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Targets of Current Drug TreatmentTargets of Current Drug Treatment
9
Para amino salicylic acid
(PAS)
1944Ruben, E. J., Nature Medicine 2003, 13, 279-280
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Targets of Current Drug TreatmentTargets of Current Drug Treatment
1010
Isoniazid
1953
Ruben, E. J., Nature Medicine 2003, 13, 279-280
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Targets of Current Drug TreatmentTargets of Current Drug Treatment
11
Ethambutol
1960
Ruben, E. J., Nature Medicine 2003, 13, 279-280
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Targets of Current Drug TreatmentTargets of Current Drug Treatment
12
Rifampin
1970’sRuben, E. J., Nature Medicine 2003, 13, 279-280
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Targets of Current Drug TreatmentTargets of Current Drug Treatment
13
Pyrazinamide
1980’s
Ruben, E. J., Nature Medicine 2003, 13, 279-280
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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
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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
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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
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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
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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
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19
Phagocyte Phagolysosome
Bacteria
How the Immune System Kills MtbHow the Immune System Kills Mtb
Lysosome
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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
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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
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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
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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
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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
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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
●
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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
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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
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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
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~Fe
L-arginine
29
iNOS - inducible Nitric Oxide SynthaseiNOS - inducible Nitric Oxide Synthase
Knowles, R. G., Biochem. J. 2001, 357, 593-615
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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
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[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
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Tuberculosis Fights BackTuberculosis Fights Back
1. Many strains of Tuberculosis have shown resistance to ROI’s
32Shiloh, M. U., PNAS 2000, 97, 8841–8848
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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
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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
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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
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New DrugsNew Drugs
36
New Drugs
?
Ruben E.J., Nature Medicine, 2003, 13, 279-280
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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
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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
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Activity of PA-824Activity of PA-824
39
PA-824
Baker, W.R., Nature 2000, 405, 962-966
N
NO
O2N
O
OCF3
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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
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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
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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
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43
Nitroimidazoles Mode of ActionNitroimidazoles Mode of Action
iNOSO2
Dying Bacteria
host
Active
Aerobic
Nathan, C., Science 2008, 322, 1337-1338
NO•
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Nitroimidazoles Mode of ActionNitroimidazoles Mode of Action
44
hostAnaerobic LimitediNOS
Latent
iNOSO2Aerobic
Dying Bacteria
host
Active
NO•
Nathan, C., Science 2008, 322, 1337-1338
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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
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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
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Hydride Transfer to CofactorHydride Transfer to Cofactor
47Bashiri, J., J Biol Chem. 2008, 283, 17531-41
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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•
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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•
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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
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Aerobic and Anaerobic Activity of Analogues
Aerobic and Anaerobic Activity of Analogues
61
MACZR
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Correlation Between Anaerobic
Killing and NO
Correlation Between Anaerobic
Killing and NO
62Singh, R., Science, 2008, 322, 1392-1395
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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
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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
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The End - Thank youThe End - Thank you
65
Dr. Robert BenRoger TamPawel CzechuraJennifer ChaytorJohn TrantWendy CampbellJackie TokarewLiz Von MoosGloria GongMike SouwehaMathieu LeclereCole StevensTaline Boghossian
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Hydrogen BondingHydrogen Bonding
66
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Co- enzyme 420Co- enzyme 420
67