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1、4906Biochemistry2004,43,4906-4912IdentificationofNovelInhibitorsoftheSARSCoronavirusMainProtease3CLpro²UsmanBacha,³JenniferBarrila,³AdrianVelazquez-Campoy,StephanieA.Leavitt,andErnestoFreire*DepartmentofBiology,JohnsHopkinsUniVersity,Baltimore,Maryland21218ReceiVedDecember3,2003
2、;ReVisedManuscriptReceiVedMarch11,2004ABSTRACT:SARS(severeacuterespiratorysyndrome)iscausedbyanewlydiscoveredcoronavirus.Akeyenzymeforthematurationofthisvirusand,therefore,atargetfordrugdevelopmentisthemainprotease3CLpro(alsotermedSARS-CoV3CLpro).WehaveclonedandexpressedinEscher
3、ichiacolithefull-lengthSARS-CoV3CLproaswellasatruncatedformcontainingonlythecatalyticdomains.Therecombinantproteinshavebeencharacterizedenzymaticallyusingafluorescentlylabeledsubstrate;theirstructuralstabilityinsolutionhasbeendeterminedbydifferentialscanningcalorimetry,andnoveli
4、nhibitorshavebeendiscovered.ExpressionofthecatalyticregionaloneyieldsaproteinwithareducedcatalyticefficiencyconsistentwiththeproposedregulatoryroleoftheR-helicaldomain.DifferentialscanningcalorimetryindicatesthattheR-helicaldomaindoesnotcontributetothestructuralstabilityofthecat
5、alyticdomains.Analysisoftheactivesitecavityrevealsthepresenceofsubsitesthatcanbetargetedwithspecificchemicalfunctionalities.Inparticular,aclusterofserineresidues(Ser139,Ser144,andSer147)wasidentifiedneartheactivesitecavityandwassusceptibletobeingtargetedbycompoundscontainingboro
6、nicacid.Thisclusterishighlyconservedinsimilarproteasesfromothercoronaviruses,defininganattractivetargetfordrugdevelopment.Itwasfoundthatbifunctionalarylboronicacidcompoundswereparticularlyeffectiveatinhibitingtheprotease,withinhibitionconstantsasstrongas40nM.Isothermaltitrationm
7、icrocalorimetricexperimentsindicatethattheseinhibitorsbindreversiblyto3CLproinanenthalpicallyfavorablefashion,implyingthattheyestablishstronginteractionswiththeproteasemolecule,thusdefiningattractivemolecularscaffoldsforfurtheroptimization.FromNovember2002throughJune2003,moretha
8、n8000personscontractedsevereacuterespiratorysyn
