TheEssen(alityandMysteryofPyrazinamide

OrenZimhonyMDInfec(ousDiseasesDepartmentKaplanMedicalCenter,affiliatedtotheSchoolof

MedicineHebrewUniversity

Content •  Historicnotes•  ThePZAparadox,thedifficul(es

•  Simula(onsinvivobyinvitrocondi(ons? •  PZAresistanceandsuscep(bility•  Theuniquesuscep(bilityofTBtoPZA•  PZAanalogs,whatdidwelearn?•  Insearchoftheactualsiteofac(on•  Summary

PZAhistory,thebeginning

•  1936 Dalmer O& Synthesis of PZA Walter E

•  1945 Chorine V Nicotinamide efficacy TB in mice

•  1952 Kushner S Testing PZA in mice

•  1952 Yeager et al. First Clinical Use

TheCornellgroupcontribu(on

•  1954 McDermott W& Activation of PZA in acidic Tompsett R environment

•  1956 Mackaness GB Activity of PZA in macrophages

•  1956 McCune RM The model of TB latency and sterilization.

•  1967 Konno K et al. Resistance to PZA loss of PZAse NAMase activity

Introduction of PZA into clinical studies and practice

•  1970s-1980s British PZA allows short Medical Research course chemotherapy Council Mitchison D

Formal guidelines (ATS) for TB therapy include PZA for first two month with INH and RIF

•  1988-1991 Salfinger M Standardized broth Heifets LB test for PZA

• 

PZAresistance,uniqueac(vityandprospect

•  1991‐1995WelchJ,Synthesisandtes(ngofCynamonMHseveralPZAanalogs

•  1996ScorpioA,Iden(fica(onofpncAgeneZhangY encodingpyrazinamidase

•  1999ZhangYRoleofacidicpHPZAScorpioA uniqueac(vityinMTB

ThemysteryofPZA?

WhyPZAissoMysteriousorwhatmakesitsstudysodifficult?

•  Discrepancybetweenthesterilizingac(vityinanimalmodelsandhumanandin–vitroac(vity.“ThePZAparadox“

•  Poorac(vity:Inoculumeffect,lackofbactericidalac(vity

•  Acidmediumdependentac(vity

•  Difficulttoassesssuscep(bility/resistance

TheImplica(onofpoorac(vityforPZAStudies.

DoesPZAisgivenandisreachingsufficientc%foranexpectedeffect?

Indrugsuscep(bilitytests

Inphysiologic/biochemicaltests

Animalandclinicalstudies

Sterilizing Effect of PZA in Murine Model (McCune R M, et al. JEM 1956)

“The fate of Mycobacterium tuberculosis in mouse tissues as determined …“ H isoniazid S streptomycin , P Para amino salicylic acid, Z pyrazinamide

TheCornellModelStudies(McCuneRM,McDermo`W1956‐1965).

•  PZAisindispensablefor(ssuesteriliza(on

•  Sterilized/curedmicerelapsed

•  Theremainingbacilli“Persisters”remainedfullysuscep(bletothedrugs.

•  Extensionofdrugtherapyfrom12wto26wresultedinlas(ngcure.

Interpreta(onoftheCornellModelResults

•  “ThiscompletedisappearanceofTBbacillimeetsthedefini(onofatrulylatentinfec(on…ishiddenbeyondthelimitsofdiagnos(creach”

•  Doesthestateoflatencyimplies“dormancy““semidormancy”?

•  Whatismeantbydormancyinvitro?

Howrelevantarein‐vitroCondi(onsandSimula(ontoExplainthePZAParadox? • WheredowefindacidicpHinthecontextofTB,thephagosome?casea(nggranuloma?

•  DoescausinglatencythroughPZAimplyac(vityagainst“dormant”orsemidormant“bacilli

•  Doestrea(nglatentTB(clinicalseeng)implyac(vityagainstsemi/dormantbacilli?

Ac(vityofPZAAgainstReplica(ng vs.NonReplica(ngBacilli“Semi‐Dormant”?.

InfavorofPZAEffectagainstReplicatingBacilli

•  PZAiseffectivetherapeuticallywhengivenearly.

•  InEx-vivomodel:AwindowofdrugsusceptibilitythatcoincideswiththeonsetoftheT-cell-mediatedimmuneresponse

•  ALLSusceptibilitytestsforPZArecommendusageoffreshlydilutedreplicatingbacilli.

Medium and specimen preparation: “cultures of M. tuberculosis should be freshly grown in …..should be used for this test when they are in an active growth phase. Do not use old, refrigerated cultures or cultures which have shown peak GI readings for more than one day.”

“Scrape with a sterile loop as many colonies as possible from growth no more than fourteen days old”

Suscep(bilityTests,replica(ngBacilliattherightInoculum

Suscep(bilityandResistancetoPZA.

•  PrinciplemechanismofresistancelackofPZAaseac(vityduetopncAlossoffunc(onmuta(ons

•  Reliable/reproducibletestforR/SischallengingFalseresistance,amajorproblem

•  TestsforPZAsuscep(bility/resistance:1.Culturemethodsplatesandbrothtests

2.Nico(namidesuscep(bilitytoNAMat5000µg/mlpH7!!!TanThiamHok’stest(1962)

3.Enzyma(ctestsforPZAseac(vity,Waynetest(1974)

Suscep(bilityandResistancetoPZA(2)

Suscep(bilityandResistancetoPZA(3)

4.SequenceofpncA

•  DiscrepancyinpncAmuta(onsratebetweenstudies

•  PZAse‐,nopncAmuta(ons

•  Alterna(vemechanism? PZAase+ Notexcludedrare

•  LackofdataonRrateevenfromdeveloped

PZA Conversion and Accumulation.

Studiesusing14CPZA

TheRoleofPncAinPOAAccumula(on

WhatUnderliestheUniqueSuscep(bilityofTBtoPZA?

•  MycobacteriaspeciesthatareproficientinPZAseyetPZAresistant.

•  MSMGpossesstwoPZAsesPncAandPzaAandisPZAR,MIC>2000µg.

Differences in POA accumulation between M. tuberculosis (M. tb and M. smegmatis (M. smeg.

TheUniqueSuscep(bilityofTB,morethanPZAse

Effect of reserpine and valinomycin on accumulation of POA in M. smegmatis (A) and M. tuberculosis (B).

TheUniqueSuscep(bilityofTBamongstMycobacteriatoPZA

Otherfactors?EffectofAera(ononPZAAc(vityagainstM.tuberculosis

H37Ra cells were treated with 100 g PZA ml1 (filled bars) or not (open bars), under aerobic, microaerobic or anaerobic conditions for 5 days prior to c.f.u. determination.

CanIncreasedPZAseAc(vityAffecttheSuscep(bilitytoPZA?

•  PZAdeamida(oncanbecatalyzedbyPncAandbyPzaA

•  IncreasedPZAseac(vityleadstoMSMGsuscep(bilitytoPZA

•  IncreasesMTBsuscep(bility

PZAAnalogs

•  Thera(onale?CircumventPZAseIncreasepotencyExpandac(vitytootherspecies

•  Pyrazinoates(POE),5‐Cl‐PZAanalogs,5‐Cl‐POE,5‐fluoropyrazinoates(5‐F‐POE).

SummaryandConclusionfromPZAAnalogsAc(vity

•  Insolubleinwater.

•  Broaderspectrumac(vitythatincludeM.avium,M.kansasii,M.smegama7s

•  Higherpotency(upto100folds)forMTB.

•  EsterhydrolysisofPAEsispossiblebutisnotrequired.

SummaryandconclusionfromPZAAnalogsAc(vity(2)

•  5‐Cl‐PZAdoesnotrequireconversionto5‐Cl‐POA(ac(veinM.bovislackingit).

•  5‐Cl‐POA,astrongeracidthanPOA,ismuchlessac(vethaneither5‐Cl‐PZAorPOA.

•  Neither5‐ClPZAorn’PPArequireacidicpH,yetsufferfrominoculumeffect.

MutuallyExclusiveGenotypesfor Pyrazinamideand5‐Chloro‐pyrazinamideResistance

BaughnA,DengJetal.AAC2010

MutuallyExclusiveGenotypesforPZAand5‐Cl‐PZAresistance

PZAseac(vityin5‐Cl‐PZAresistantstrainsMSMEG

IncreasedconversionofPZAdiminishtheRequirementforAcidicpH

AcidicpH

ConversiontoPOA Deficient

effluxofPOA

PZA/POAAc(vity

POAAccumula(on

?

Hypoxia“stress”

Accumula(onforac(va(on

HowPZAworks?InherentDifficul(esinIden(fyingtheActualMechanism

•  NoBona‐fidePOAresistantmutantAbsenceofPOAR??Morethanonemechanismorintolerablemuta(on.

•  Needtocorrelatean(mycobacterialac(vitywithbiochemicaleffect

Iden(fica(onoftheTargetof5‐Cl‐PZA

•  Selec(onof5‐Cl‐PZAresistantmutantsinMSMEG

•  Therangeofresistanceofthemutants(5‐Cl‐PZAR)isnarrow

•  Fa`yacidsynthase1(fas1)isthegenethatconfersthisphenotype(a9.3kbORF)

•  MTBdoesnottoleratemul(copyfas1orevenasinglecopyfromMSMEG.

Fa#yacidsynthesisinmycobacteriaandrelatedspecies

•  FASIsystemAllnon‐planteukaryotesandcertainprokaryotes

Mul(‐func(onalmul(‐domainproteincatalyzesthesynthesisoflongchainfa`yacidsfromC2units

•  FASIIMostprokaryotes,individualenzymes

•  Mycobacteria:BothFASIandFASII,FASIIgeneratesmycolicacidprecursors

PZA/POA Activity Correlates with Inhibition of FA Biosynthesis

APyrazinoateEster,n’PPAInhibitsFa`yAcidSynthesisinM.tuberculosis

POAInhibi(onofFa`yAcidBiosynthesisinTBComplexBacilliCorrelatestoAn(mycobacterial

Ac(vity

M. tuberculosis FAS I Inhibition in Cell Free System using NADPH Oxidation

Effect of increasing inhibitor concentration on NADPH oxidation. Blank runs with no enzyme are included for reference.

MinimalStructureofPyrazineRingwithanAcylGroup.

HPLC analysis of C16:0 to C26:0 fatty acids from PZA-treated MSMEG mc2155 and mc27031, treated with 2.5 mg/ml PZA for 2 h, and then pulsed with [1-14C]acetate for an additional 2 h.

Susceptibility to PZA Translates to Inhibition of FA Synthesis in MSMEG

Similari(esbetweenPOAand5‐Cl‐PZA,n’PPA

•  Rela(velypoorsimilarbacteriosta(cac(vity,andkillingcurvein‐vitro

•  NoresistantmutantsinMTB

•  Correla(onofan(‐mycobacterialac(vitytopalmitatebiosynthesisinhibi(on

Similari(esbetweenPOAand5‐Cl‐PZAn’PPA(2)

•  Invitroinhibi(onalbeitmarkeddifferenceinpotency

•  BindingtoFASINMRstudies

Dissimilari(esbetween5‐Cl‐PZAandPOANeedforacidicpH

EffectofFASIoverexpressiononPZAresistanceinMSMEG

Conclusions •  SufficiencyofPZA/POAisessen(alforcorrectinterpreta(onandreproducibility

•  PZAstudiesshouldbeconductedonreplica(ngbacilli(variouscondi(ons,exvivomodelsetc)

•  AcidicpHisacondi(onforPOAaccumula(oncanbepar(allysubs(tuted,no“mechanis(c”role

•  Anaccumulateddependentagentaffectsanintracellularsite

H+ + POA-

ATP dependent defective efflux

Passive diffusion

Deamidation

Passive diffusion

PZA POA

POA entrapment and “huge” accumulation

? Intracellular target, FAS I

Kill of replicating bacilli

PZA activity against MTB bacilli in media anoxic/acidic

POA

FurtherStudies?

•  Howdoesthepoorac(vityinvitro translateintosterilizingeffectin‐vivo?

Tissuelevels?

Synergismwithinflammatoryresponse?Promo(ngaccumula(onintothe bacilli

andorimprovedkilling?

Be`erinduc(onofcellularimmunity