SheoSinghDr.
Manuscript:2006-01-00990C
Journal:Nature
ArticleTitle:PlatensimycinisaselectiveFabFinhibitorwithpotentantibioticproperties
TypeIIfattyacidsynthesis(FASII)isessentialtobacterialcellvi
ABIlity.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).
DTTwasfromFisher(BP172-5);beta-mercaptoethanolwasfrom
Bio-Rad(161-0710).[2-
14C]Malonyl-CoA(60mCi/mmol,PerkinElmer,
NEC612),
ACP(Sigma,
A7303)waspretreatedwith3mM
DTTonicefor20min,aliquotedandstoredat-80°C.CeruleninandthiolactomycinwerepurchasedfromSigma.Triclosancouldbeobtainedfrom
VWR,003384.Urea,
PVDFmembrane,10XTris/GlycinebufferandnativeproteinsamplebufferwerepurchasedfromBio-Rad.
Gelelectrophoresisapparatus,PhosphorScreenandPhosphorImagerscanner
Twodays.DayonetorunFASIIassay,resolveitonthegelandovernighttransferofproteinontoPVDFmembrane.DaytwotoexposethephosphorscreentoproteinboundtothePVDFmembraneandscanimageusingPhosphorImagerscanner
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.Inthegelelongationassayusing
E.coliextracts(Fig.1),theinhibitionobservedwithplatensimycin(167µg/ml)wassimilartothatseenwithcerulenin,anddifferentfromthatobservedwiththiolactomycinandtriclosan,indicatingthatplatensimycinselectivelytargetsFabF/B.Similarly,titrationofplatensimycinusinga
S.aureusgelelongationassay,withoctanoyl-CoA(C8:0)andmalonyl-CoAassubstrates,showedanIC~50~of0.2µg/ml(Fig2).Athigherconcentrationsofplatensimycinanaccumulationofmalonyl-ACPwasobserved,demonstratingthatthisinhibitordoesnotblockFa
BD,amalonylCoA:ACPtransacylase.ThelowerconcentrationsofplatensimycinproducedpartialinhibitionofelongationactivityandaccumulationsofC2n:0-ACP(n>4),furtherindicatingthatthetargetofinhibitionin
S.aureusistheFabFcondensingenzyme.
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FabH,FabG,FabA,FabF,FabB,FabI,platensimycin,fattyacidsynthesis,Urea-PAGE,Gel-elongation,FASII