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
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?
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
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
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
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.
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
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