Manuscript:2006-01-00990C
Journal:Nature
ArticleTitle:PlatensimycinisaselectiveFabFinhibitorwithpotentantibioticproperties
TypeIIfattyacidsynthesis(FASII)isessentialtobacterialcellviABIlity.Thesignificantdifferencesbetweenbacteriaandhumansinorganization,structureofenzymes,andtheroleplayedbyfattyacidsmakethispathwayanattractivetargetforantibacterialdrugdiscovery(refs.1,2).TwomarketedantibacterialagentsthattargettheFabIenzymearetriclosan(antiseptic)andisoniazid(ananti-Mycobacteriumtuberculosisagent)(refs3,4).Twonaturalproducts,cerulenin(ref.5)andthiolactomycin(ref.6)whichselectivelyinhibitthecondensationenzymesFabHandFabF/B,werediscoveredmorethantwodecadesago.Newscreeningeffortsandchemicalmodificationsofexistingcompoundshavebeenattemptedtoidentifymoreselectiveandpotentinhibitors.Todeterminetheselectivityoftheinhibitorsidentifiedduringscreeningeffortswedevelopedgel-elongationassayusingcrudebacteriallysatedirectlytodeterminethetargetspecificitiesoffattyacidsynthesisinhibitors(refs.7,8).
Reagents
DTTwasfromFisher(BP172-5);beta-mercaptoethanolwasfromBio-Rad(161-0710).[2-14C]Malonyl-CoA(60mCi/mmol,PerkinElmer,NEC612),ACP(Sigma,A7303)waspretreatedwith3mMDTTonicefor20min,aliquotedandstoredat-80°C.CeruleninandthiolactomycinwerepurchasedfromSigma.TriclosancouldbeobtainedfromVWR,003384.Urea,PVDFmembrane,10XTris/GlycinebufferandnativeproteinsamplebufferwerepurchasedfromBio-Rad.
Equipment
Gelelectrophoresisapparatus,PhosphorScreenandPhosphorImagerscanner
1)FASIIgel-elongationassaywasdonebypreincubating0.25-2µgofE.coliorS.aureuslysatewithaserialdilutionofinhibitorsatroomtemperaturefor20minin50µlofbuffercontaining100mMsodiumphosphate(pH7.0),5mMEDTA,1mMNADPH,1mMNADH,150µMDTT,5mMß-mercaptoethanol,20µMacetyl-CoAorn-octanoyl-CoAoranyotheracyl-CoAasneeded,4%DMSO,and8µMofACPpretreatedwithDTT.2)Thereactionwasinitiatedbyadditionof10µlofwater-diluted[C-14]malonyl-CoA,whichgaveafinalconcentrationof4µMmalonyl-CoA.3)Thereactionwasincubatedat37°Cfor30minforE.colilysateand60minforS.aureuslysate.4)Afterthereaction,10µl(plus10µl2Xnativesamplebuffer)ofeachsamplewasdirectlyappliedtoal6%polyacrylamidegelcontainingarangeof0.4Mto4Mureaasneeded.5)Thegelwasresolvedandtransferedtoapolyvinylidenedifluoride(PVDF)membrane,exposedtoPhosphorScreenandvisualizedbyusingaPhosphorImagerscanner.
Ureaconcentrationiscriticalforseparationofchainlengthsofacyl-ACPs.Theactivityofthelysatevariesamongbacterialstrains.So,therequiredFASIIactivityofthelysateneedstobeoptimizedbytitration.Acetyl-CoAisnotanidealsubstrateforS.aureus(ref.9).
Inbacteria,FabHcatalyzesthefirstcondensationreactionusingacetyl-CoAandmalonyl-ACPtoproduceacetoacetyl-ACP.ThisproductisreducedbyFabG,dehydratedbyFabA/ZtoproduceC4:1(Δ2)-ACP,andthenreducedsecondtimebyFabItomakebutyryl-ACP(C4:0-ACP)whichissubstrateforFabF/Bcondensingenzymes.Cerulenin,aselectiveFabF/Binhibitor,at200µg/mlaccumulatesbutyryl-ACPandthiolactomycin(333µg/ml)inhibitsbothFabHandFabF/Bcondensingenzymesresultinginaminoraccumulationofbutyryl-ACPwhileleavingamajorityofmalonyl-ACPunreacted.Triclosan(100µg/ml)treatmentleadstotheaccumulationofC4:1(Δ2)-ACPduetoitsinhibitionofFabI.InthegelelongationassayusingE.coliextracts(Fig.1),theinhibitionobservedwithplatensimycin(167µg/ml)wassimilartothatseenwithcerulenin,anddifferentfromthatobservedwiththiolactomycinandtriclosan,indicatingthatplatensimycinselectivelytargetsFabF/B.Similarly,titrationofplatensimycinusingaS.aureusgelelongationassay,withoctanoyl-CoA(C8:0)andmalonyl-CoAassubstrates,showedanIC~50~of0.2µg/ml(Fig2).Athigherconcentrationsofplatensimycinanaccumulationofmalonyl-ACPwasobserved,demonstratingthatthisinhibitordoesnotblockFaBD,amalonylCoA:ACPtransacylase.ThelowerconcentrationsofplatensimycinproducedpartialinhibitionofelongationactivityandaccumulationsofC2n:0-ACP(n>4),furtherindicatingthatthetargetofinhibitioninS.aureusistheFabFcondensingenzyme.
1.Heath,R.J.&Rock,C.O.Fattyacidbiosynthesisasatargetfornovelantibacterials.Curr.Opin.Investig.Drugs5,146–153(2004).2.White,S.W.,Zheng,J.,Zhang,Y.M.&Rock,C.O.ThestructuralBIOLOGyoftypeIIfattyacidbiosynthesis.Annu.Rev.Biochem.74,791–831(2005).3.Heath,R.J.,Yu,Y.-T.,Shapiro,M.A.,Olson,E.&Rock,C.O.BroadSpectrumAntimicrobialBiocidesTargettheFabIComponentofFattyAcidSynthesis.J.Biol.Chem.273,30316-30320(1998).4.Banerjee,A.etal.inhA,ageneencodingatargetforisoniazidandethionamideinMycobacteriumtuberculosis.Science263,227-230(1994).5.Matsumae,A.,Nomura,S.&Hata,T.Studiesoncerulenin.IV.Biologicalcharacteristicsofcerulenin.J.Antibiot.(Tokyo)17,1–7(1964).6.Noto,T.,Miyakawa,S.,Oishi,H.,Endo,H.&Okazaki,H.Thiolactomycin,anewantibiotic.III.Invitroantibacterialactivity.J.Antibiot.(Tokyo)35,401–410(1982).7.Kodali,S.etal.Determinationofselectivityandefficacyoffattyacidsynthesisinhibitors.J.Biol.Chem.280,1669–1677(2005).8.Young,K.etal.DiscoveryofFabH/FabFinhibitorsfromnaturalproducts.Antimicrob.AgentsChemother.50,519–526(2006).9.Qiu,X.etal.Crystalstructureandsubstratespecificityofthebeta-ketoacyl-acylcarrierproteinsynthaseIII(FabH)fromStaphylococcusaureus.ProteinSci.14,2087-2094(2005)
FabH,FabG,FabA,FabF,FabB,FabI,platensimycin,fattyacidsynthesis,Urea-PAGE,Gel-elongation,FASII
Determinationoftargetselectivity
E.colifattyacidsynthesis(FASII)gel-elongationassayused4µMof[2-14C]malonyl-CoA(60mCi/mmol)and20µMofacetyl-CoAassubstrates.Thereactionwascarriedoutat37°Cfor30minwithorwithoutaninhibitor,directlyappliedtoal6%polyacrylamidegelcontaining0.5Mureaandwasresolvedusingelectrophoresis(0.5Murea-PAGE).Withoutaninhibitorthereactionwascompletedandproducedfinalproductsoflongchain(>6C)acyl-ACPs.Withadditionofcerulenin(200µg/ml)orplatensimycin(167µg/ml),thereactionwasblockedshowingmajoraccumulationofbutyryl-ACP(4:0-ACP).Withthiolactomycin(333µg/ml),moremalonyl-ACPandlessbutyryl-ACPwereaccumulated.Inthepresenceoftriclosan(100µg/ml),aFabIinhibitor,C4:1(Δ2)-ACPwasaccumulated.Aweakbandshownabovebutyryl-ACPinthepresenceofceruleninorplatensimycinisnotdefined,butislikelyacetyl-ACP,anintermediateproductofmalonyl-ACPdecarboxylationcarriedoutbyFabH.
S.aureusFASIIgel-elongationassay
ThisassaywassimilartotheE.coliFASIIassayexceptthatacetyl-CoAwasreplacedwith20µMofoctonoyl-CoA(C8:0).Thereactionwascarriedoutfor60minwithaserialdilutionofplatensimycinandresolvedon4Murea-PAGE.PlatensimycininhibitedS.aureusFabF(saFabF)infattyacidsynthesispathwaywithanIC~50~of0.2µg/ml.SimilarexperimentswererepeatedatleastthreetimeswithreproducIBLeresults.
