Advancesinscreeningtechnologieshavemadeliganddiscoveryagainstbiologicaltargetsroutine,but convertingbindingligandsintospecificenzymeinhibitorsisextremelychallenging,evenformetalloproteinases andotherenzymeswithwell-definedactivesites.Thelackofspecificinhibitorspreventsfullelucidationof biologicalprocessesasbasicasextracellularmatrixremodeling.Proteinsandsmallmoleculeseachlackkey featuresofinhibitors.Antibodiesandotherproteinsrarelydisruptenzymefunction,butusuallyexhibithigh bindingspecificity.Smallmoleculesfrequentlylacksingle-enzymespecificity,butinterferewithenzymatic activity.Neitherofthesemodalitiesiswell-suitedforgeneratingpotent,specificenzymeinhibitors. Mylong-termgoalsareto1)establishgeneralprinciplesfordiscoveringpotent,specificinhibitorsagainst medicallyrelevantenzymes;?and2)utilizetheresultinginhibitorstounderstandtherolesofenzymessuchas metalloproteinasesinnormalphysiologyandpathologicalprocesses.Ihypothesizethatsimultaneously leveragingthecomplementarystrengthsofproteinsandsmallmoleculeswillgiverisetoentirelynewclasses ofpotent,specificinhibitors.Thegoalduringthisproposalperiodistoconvertyeastdisplay,apowerful liganddiscoveryplatform,intoacomprehensiveinhibitordiscoveryplatform.Mylabhasalreadyestablished strategiesforexpandingthechemicalfunctionalitythatcanbeutilizedincombinationwithyeastdisplay.Here, wewillenhanceourplatformfurtheranduseittoidentifyinhibitorsagainstatestsetofmetalloproteinase targets.Intheprocess,wewillgainfundamentalinsightsintohowtogenerateinhibitorsthatarenotaccessible usinganycurrentinhibitordiscoveryapproaches,settingthestagefor1)agreatlyexpandedtoolkitforstudying basicbiology;?and2)muchbroaderinhibitordiscoveryefforts.Theinitialdirectionswewillpursueare: Direction1.Expandtherangeofchemistriesthatcanbeencodedinyeast-displayedproteins. Proteinscontainingcanonicalaminoacidslackkeygroupsfoundinenzymeinhibitors.Wewillutilizeour quantitativereporterofncAAincorporationtoencodethesefunctionalitiesinyeast-displayedproteins. Direction2.Establishassaysforquantitativelyevaluatingenzymeinhibitionontheyeastsurface. Noexistingdisplaytechnologiessupportquantitativeevaluationsofenzymeinhibitionduringhigh throughputscreening.Wewillutilizedualyeastdisplaytechnologytoestablishthesecapabilities. Direction3.Usechemicallyaugmentedantibodylibrariestoevolvepotent,specificinhibitors. Antibodiesrarelyinhibitenzymes.Wewillgenerateandscreenlibrariesofantibodiescontainingadded chemicalgroupstoestablishgeneralprinciplesforinhibitorisolationinthisunexploreddiscoveryspace. Tofocusourdiscoveryefforts,weandourcollaboratorshaveidentifiedmetalloproteinasesfrommultiple familiesthatplayimportantrolesinhumanhealthanddisease.Thegeneraldiscoveryprinciplesweestablish herewillleaddirectlytonewclassesofinhibitorsforunderstandingandtreatinghumandisease.
Specific enzyme inhibitors are critical tools for understanding and treating disease, but discovery of these inhibitors is extremely challenging using current technologies. This program seeks to establish a comprehensive inhibitor discovery platform that will lead to new classes of enzyme inhibitors that combine the best properties of small molecules and biologics. The initial focus on enzymes known as metalloproteinases will lead immediately to new tools for studying how cells move and signal with one another while also revealing principles for generating inhibitors against any enzyme involved in disease progression.
