Development of Cyclic Peptide Inhibitors of Coagulation Factor XII and Matrix Metalloproteinase 2

Development of cyclic peptide inhibitors of coagulation factor XII and matrix metalloproteinase 2

THÈSE NO 8467 (2018)

PRÉSENTÉE LE 13 AVRIL 2018 À LA FACULTÉ DES SCIENCES DE BASE LABORATOIRE DE PROTÉINES ET PEPTIDES THÉRAPEUTIQUES PROGRAMME DOCTORAL EN CHIMIE ET GÉNIE CHIMIQUE

ÉCOLE POLYTECHNIQUE FÉDÉRALE DE LAUSANNE

POUR L'OBTENTION DU GRADE DE DOCTEUR ÈS SCIENCES

PAR

Jonas Alfred Karl WILBS

acceptée sur proposition du jury:

Prof. U. Röthlisberger, présidente du jury Prof. C. Heinis, directeur de thèse Prof. C. A. Olsen, rapporteur Dr E. Valeur, rapporteur Prof. M. Dal Peraro, rapporteur

Suisse 2018

$FNQRZOHGJHPHQWV

Firstȱofȱall,ȱIȱwouldȱlikeȱtoȱthankȱmyȱPhDȱsupervisorȱprofessorȱChristianȱHeinis.ȱThankȱ youȱforȱgivingȱmeȱtheȱpossibilityȱtoȱworkȱwithȱsomethingȱthatȱIȱamȱtrulyȱpassionateȱ about,ȱitȱhasȱbeenȱaȱprivilege.ȱAlsoȱthankȱyouȱforȱyourȱscientificȱadviceȱandȱgeneralȱdisȬ cussions,ȱandȱforȱsupportingȱmyȱattendanceȱtoȱAPS2017ȱandȱtheȱBenzonȱsymposium,ȱ twoȱhighlightsȱofȱtheȱlastȱfourȱyears.ȱ

Iȱwouldȱalsoȱlikeȱtoȱexpressȱmyȱthanksȱtoȱallȱtheȱjuryȱmembers,ȱUrsulaȱRöthlisbergerȱandȱ MatteoȱDalȱPeraroȱfromȱEPFL,ȱChristianȱA.ȱOlsenȱfromȱCopenhagenȱandȱEricȱValeurȱ fromȱGothenburg,ȱforȱdevotingȱtheirȱtimeȱtoȱmyȱPhDȱthesis.ȱIȱreallyȱappreciateȱyourȱefȬ fort.ȱ

Next,ȱIȱwouldȱlikeȱtoȱthankȱallȱtheȱlabȱmembersȱofȱLPPTȱwhomȱIȱhaveȱworkedȱwithȱdurȬ ingȱtheȱpastȱfourȱyears.ȱWhenȱIȱarrived,ȱIȱimmediatelyȱfeltȱwelcome,ȱthanksȱtoȱallȱtheȱ peopleȱthereȱatȱtheȱtime:ȱSangram,ȱRanga,ȱRaphael,ȱShiyu,ȱDavide,ȱMike,ȱVanessa,ȱCharȬ lotte,ȱKhan,ȱInma,ȱSilvia,ȱLisa,ȱCamille,ȱAlessandroȱandȱPhillippe.ȱThankȱyouȱforȱbeingȱ soȱhelpfulȱandȱaccommodating.ȱAȱspecialȱthanksȱtoȱtheȱpeopleȱinȱ“Ranga’sȱkindergarȬ den”,ȱitȱwasȱgreatȱtoȱshareȱtheȱlabȱwithȱyou!ȱIȱwouldȱalsoȱlikeȱtoȱespeciallyȱacknowledgeȱ KhanȱforȱmentoringȱmeȱduringȱtheȱbeginningȱofȱmyȱPhDȱandȱforȱintroducingȱmeȱtoȱtheȱ excitingȱMMPȬ2ȱproject.ȱFurther,ȱIȱwouldȱlikeȱtoȱthankȱtheȱpeopleȱwhoȱhaveȱjoinedȱtheȱ labȱduringȱmyȱtimeȱthere:ȱKaycie,ȱJoao,ȱPatrick,ȱVanessa,ȱXudong,ȱChristina,ȱGanesh,ȱ Simon,ȱLina,ȱMilan,ȱManh,ȱYuteng,ȱSevan,ȱCarlȱandȱGontran.ȱItȱhasȱbeenȱgreatȱtoȱworkȱ withȱallȱofȱyouȱandȱIȱamȱconvincedȱthatȱtheȱgoodȱspiritȱinȱtheȱlabȱwillȱremain.ȱAdditionȬ ally,ȱIȱwouldȱlikeȱtoȱgiveȱspecialȱthanksȱtoȱmyȱstudents,ȱLucaȱandȱGontran.ȱItȱwasȱaȱpleasȬ ureȱtoȱworkȱtogetherȱandȱthankȱyouȱsoȱmuchȱforȱyourȱvaluableȱcontributions.ȱ

Iȱwouldȱlikeȱtoȱaddressȱaȱparticularȱthanksȱtoȱallȱtheȱpeopleȱwhoȱhaveȱbeenȱinvolvedȱinȱ theȱFXIIȱproject,ȱtheȱmainȱprojectȱofȱmyȱPhDȱthesis.ȱInȱmyȱopinionȱthisȱhasȱbeenȱaȱtrueȱ teamworkȱandȱtheȱprojectȱwouldȱnotȱhaveȱyieldedȱsuchȱgreatȱresultsȱwithoutȱtheȱcontriȬ butionȱofȱeveryoneȱinvolved.ȱThanksȱtoȱVanessaȱforȱinitiatingȱtheȱproject,ȱandȱespeciallyȱ toȱSimonȱwhoȱletȱmeȱjoinȱhisȱprojectȱandȱwhomȱIȱhaveȱworkedȱwithȱinȱcloseȱcollaboraȬ tion.ȱItȱwasȱreallyȱniceȱtoȱworkȱwithȱyouȱandȱIȱwillȱalwaysȱrememberȱourȱfastȬpacedȱworkȱ whenȱsynthesizingȱtheȱJSȱanaloguesȱandȱwhenȱweȱfoundȱtheȱpicomolarȱinhibitor.ȱIȱalsoȱ wantȱtoȱthankȱChristina,ȱLucaȱandȱGontranȱforȱtheirȱcontributions.ȱThisȱprojectȱwouldȱ notȱhaveȱbeenȱpossibleȱwithoutȱexternalȱcollaborations.ȱIȱwantȱtoȱexpressȱmyȱthanksȱtoȱ

iȱ $FNQRZOHGJHPHQWV professorȱAnneȱAngelilloȬScherrerȱandȱherȱwholeȱlab,ȱwithȱaȱspecialȱmentionȱofȱRaja,ȱitȱ hasȱbeenȱaȱfantasticȱexperienceȱtoȱworkȱwithȱsuchȱaȱcompetentȱandȱpreciseȱresearcherȱasȱ youȱinȱtheȱFeCl3ȱproject.ȱTheȱmomentȱwhenȱweȱfoundȱthatȱJS33ȬRȱ(asȱweȱcalledȱitȱthen)ȱ inhibitedȱthrombosisȱwasȱpriceless!ȱFurtherȱIȱwouldȱlikeȱtoȱacknowledgeȱprofessorȱRobȬ ertȱRiebenȱandȱMaiȱforȱgivingȱusȱtheȱopportunityȱtoȱtestȱourȱinhibitorȱinȱanȱexcitingȱaniȬ malȱmodel.ȱIȱwasȱdeeplyȱimpressedȱbyȱtheȱprecisionȱandȱcomplexityȱofȱyourȱworkȱandȱIȱ amȱveryȱthankfulȱforȱthisȱexperience.ȱThisȱprojectȱalsoȱbenefitedȱgreatlyȱfromȱtheȱcontriȬ butionȱofȱprofessorȱKeithȱCook,ȱAlidaȱandȱtheirȱteamȱwhoȱtestedȱourȱcompoundȱinȱrabȬ bits.ȱItȱhasȱbeenȱreallyȱexcitingȱtoȱgetȱtheȱopportunityȱtoȱbeȱpartȱofȱsuchȱanȱexperiment.ȱ

InȱtheȱBCHȱatȱEPFLȱthereȱareȱmanyȱlabsȱandȱpersonsȱwhoȱhaveȱcontributedȱtoȱmyȱworkȱ andȱwhomȱIȱwouldȱlikeȱtoȱacknowledge,ȱbeȱitȱforȱscientificȱadvice,ȱborrowingȱofȱcomȬ poundsȱorȱjustȱpureȱentertainment.ȱFirstȱIȱwouldȱlikeȱtoȱthankȱallȱtheȱmembersȱofȱtheȱ LCBIM,ȱwithȱspecialȱthanksȱtoȱRic,ȱAnzhelikaȱandȱJens.ȱYouȱhaveȱcontributedȱgreatlyȱinȱ allȱtheȱways.ȱIȱfurtherȱthankȱallȱtheȱmembersȱofȱLIPȱwithȱspecialȱmentioningȱofȱSilvia,ȱ Helen,ȱFay,ȱNicoȱandȱYann.ȱIȱalsoȱwantȱtoȱmentionȱtwoȱpersonsȱfromȱotherȱlabs;ȱCKȱ–ȱ viceȱpresidentȱofȱtheȱBCHȱpeptideȱcommunityȱandȱNickȱ–ȱtheȱmanȱwhoȱtaughtȱmeȱthatȱ simulationsȱareȱmoreȱpreciseȱthanȱexperiments.ȱȱ

Forȱmakingȱmyȱlifeȱgreatȱalsoȱoutsideȱofȱtheȱlab,ȱIȱwouldȱlikeȱtoȱthankȱtheȱfollowingȱ persons:ȱRicȱ–ȱoneȱofȱtheȱmostȱamazingȱguysȱeverȱandȱoneȱofȱmyȱbestȱfriends.ȱThankȱyouȱ forȱallȱtheȱawesomeȱexperiencesȱweȱhaveȱsharedȱtogetherȱinȱtheȱAlps.ȱIȱhopeȱweȱwillȱ continueȱlivingȱtheȱgoodȱlife,ȱlet’sȱstartȱwithȱVättern!ȱAllȱmyȱfriendsȱfromȱAlingsås;ȱwithȱ specialȱmentionȱofȱJohan,ȱMarriz,ȱJohanna,ȱLovisa,ȱMalin,ȱJimmie,ȱFredric,ȱitȱisȱalwaysȱ greatȱtoȱhangȱoutȱwithȱyou,ȱbeȱitȱinȱSwedenȱorȱSwitzerland.ȱAȱspecialȱthanksȱtoȱJohan,ȱ forȱallȱtheȱscientificȱinputȱfromȱaȱMDȱperspective.ȱAllȱtheȱpeopleȱfromȱUppsala:ȱMartin,ȱ Håkan,ȱJessica,ȱKarro,ȱMimmi,ȱRobert,ȱAndreas,ȱAdam,ȱDavid,ȱTorbjörn,ȱMartin,ȱOlle,ȱ Kickanȱandȱeveryoneȱelse;ȱIȱalwaysȱenjoyȱveryȱmuchȱtoȱseeȱyouȱforȱskiingȱandȱotherȱ occasions.ȱIȱhopeȱweȱcanȱkeepȱtheȱtraditionȱofȱanȱannualȱtripȱtoȱtheȱAlps.ȱMyȱAustralianȱ community,ȱincluding:ȱOlof,ȱPhilip,ȱCarl,ȱBirger,ȱMarkus,ȱAgnes,ȱJorien,ȱLaura,ȱJosh,ȱBuȬ rak.ȱOurȱgetȬtogethersȱareȱalwaysȱaȱwelcomeȱbreakȱfromȱwork.ȱ

Aȱveryȱspecialȱthanksȱgoesȱtoȱmyȱfamily.ȱThankȱyouȱforȱalwaysȱbelievingȱinȱmeȱandȱforȱ helpingȱandȱsupportingȱmeȱforȱwhateverȱIȱamȱdoing.ȱȱ

Finally,ȱIȱwantȱtoȱthankȱyouȱCamilleȱforȱbeingȱsuchȱaȱgreatȱpartȱofȱmyȱlife.ȱThankȱyouȱforȱ allȱtheȱfunȱthingsȱweȱdoȱtogetherȱandȱforȱyourȱsupportȱbothȱprivateȱandȱscientifically.ȱ YouȱareȱaȱgreatȱscientistȱandȱIȱamȱluckyȱtoȱhaveȱyouȱsoȱcloseȱforȱimmediateȱfeedback.ȱ Andȱthankȱyouȱforȱbearingȱwithȱmeȱinȱallȱsituations,ȱsuchȱasȱwhenȱIȱjustȱhaveȱtoȱskiȱdownȱ hereȱorȱhikeȱupȱthere.ȱIȱhopeȱyouȱwillȱcontinueȱtoȱbearȱwithȱme.ȱ

ii $EVWUDFW

Peptidesȱrepresentȱaȱpromisingȱmolecularȱclassȱforȱdrugȱdevelopment.ȱTheyȱcombineȱ severalȱstrengthsȱofȱsmallȱmoleculesȱ(e.g.ȱefficientȱtissueȱdiffusion,ȱlowȱimmunogenicityȱ andȱaccessȱtoȱchemicalȱsynthesis)ȱandȱkeyȱpropertiesȱofȱbiologicsȱsuchȱasȱmonoclonalȱ antibodiesȱ(e.g.ȱhighȱaffinityȱandȱspecificity).ȱAroundȱ60ȱpeptidesȱareȱcurrentlyȱusedȱinȱ theȱclinic.ȱMostȱofȱtheȱpeptideȱdrugsȱareȱnaturalȱproductsȱorȱderivativesȱthereof,ȱasȱforȱ exampleȱhumanȱpeptideȱhormonesȱorȱantibacterialȱpeptides.ȱInȱrecentȱyears,ȱaȱrangeȱofȱ methodologiesȱwereȱestablishedȱtoȱdevelopȱpeptidesȱbindingȱtoȱtherapeuticȱtargetsȱdeȱ novo.ȱ Ourȱ laboratoryȱ isȱ specializedȱ onȱ theȱ inȱ vitroȱ evolutionȱ ofȱ bicyclicȱ peptidesȱ byȱ phageȱdisplay.ȱBicyclicȱpeptidesȱhaveȱtwoȱmacrocyclicȱringsȱthatȱallowȱforȱbindingȱproȬ teinȱtargetsȱwithȱhighȱaffinityȱandȱselectivity.ȱMembersȱofȱourȱresearchȱgroupȱhaveȱdeȬ velopedȱbicyclicȱpeptideȱligandsȱtoȱaȱrangeȱofȱimportantȱdiseaseȱtargets,ȱincludingȱtheȱ proteasesȱcoagulationȱfactorȱXIIȱ(FXII)ȱandȱmatrixȱmetalloproteinaseȱ2ȱ(MMPȬ2).ȱȱ

TheȱaimȱofȱmyȱthesisȱwasȱtoȱimproveȱtheȱpotencyȱandȱstabilityȱofȱphageȬselectedȱbicyclicȱ peptideȱinhibitorsȱofȱFXIIȱandȱMMPȬ2,ȱandȱtoȱtestȱtheirȱtherapeuticȱpotentialȱinȱanimalȱ diseaseȱmodels.ȱSpecifically,ȱIȱplannedȱatȱimprovingȱtheȱbindingȱaffinity,ȱtargetȱselectivȬ ityȱandȱproteolyticȱstabilityȱofȱbicyclicȱpeptidesȱbyȱsubstitutingȱnaturalȱaminoȱacidsȱwithȱ unnaturalȱones.ȱAsȱdescribedȱinȱtheȱfollowing,ȱtheȱsubstitutionsȱtoȱunnaturalȱaminoȱacidsȱ hadȱledȱtoȱsubstantialȱimprovementsȱinȱtheȱbicyclicȱpeptides,ȱandȱthisȱhadȱenabledȱmeȱ theȱevaluationȱofȱtheȱinhibitorsȱinȱdiseaseȱmodels.ȱ

Inȱmyȱfirstȱandȱsecondȱproject,ȱIȱaimedȱatȱimprovingȱtheȱpotencyȱandȱstabilityȱofȱaȱbicyȬ clicȱpeptideȱFXIIȱinhibitorȱthatȱwasȱpreviouslyȱdevelopedȱinȱourȱlabȱbyȱphageȱdisplay,ȱ andȱtoȱtestȱtheȱtherapeuticȱpotentialȱofȱtheȱresultingȱpeptideȱinȱvivo.ȱFXIIȱhasȱrecentlyȱ beenȱidentifiedȱasȱaȱpromisingȱtargetȱforȱsafeȱanticoagulationȱtherapy,ȱbasedȱonȱtheȱfindȬ ingȱthatȱmiceȱdeficientȱinȱtheȱcoagulationȱproteaseȱshowȱreducedȱthrombosisȱbutȱnoȱabȬ normalȱbleeding.ȱInȱtheȱfirstȱproject,ȱIȱinvestigatedȱifȱtheȱinsertionȱofȱaȱsingleȱcarbonȱatomȱ intoȱtheȱmacrocyclicȱbackboneȱofȱaȱphageȬselectedȱbicyclicȱpeptideȱFXIIȱinhibitorȱcanȱimȬ proveȱitsȱbindingȱaffinity.ȱMoleculesȱofȱthisȱtypeȱareȱnotȱpresentȱinȱtheȱbicyclicȱpeptideȱ phageȱdisplayȱlibrariesȱsinceȱeachȱcanonicalȱaminoȱacidȱcontributesȱthreeȱatomsȱtoȱtheȱ backbone.ȱPositionsȱwithinȱtheȱmacrocycleȱsusceptibleȱtoȱatomȱinsertionȱwereȱfirstȱidenȬ tifiedȱusingȱaȱscanningȱmethodologyȱwhereȱglycineȱmutantsȱwereȱcomparedȱtoȱEȬalanineȱ mutants.ȱUponȱinsertionȱofȱatomsȱinȱtheȱbackboneȱusingȱEȬaminoȱacidsȱorȱhomologatedȱ

iiiȱ $EVWUDFW

cysteineȱanalogues,ȱtwoȱpeptidesȱshowedȱ4.7Ȭȱandȱ2.5ȬfoldȱimprovedȱKiȱvalues.ȱTheȱbetȬ terȱoneȱblockedȱFXIIȱwithȱaȱKiȱofȱ1.5ȱ±ȱ0.1ȱnMȱandȱwasȱmoreȱpotentȱthanȱtheȱleadȱpeptideȱ inȱinhibitingȱtheȱactivationȱofȱtheȱintrinsicȱcoagulationȱpathway.ȱTheȱstrategyȱofȱringȱsizeȱ variationȱbyȱoneȱorȱseveralȱatomsȱshouldȱbeȱgenerallyȱapplicableȱforȱtheȱaffinityȱmaturaȬ tionȱofȱinȬvitroȬevolvedȱcyclicȱpeptides.ȱ

Inȱmyȱsecondȱproject,ȱIȱaimedȱatȱimprovingȱfurtherȱtheȱpotencyȱofȱtheȱbicyclicȱpeptideȱ FXIIȱinhibitorȱdescribedȱbefore,ȱandȱtoȱadditionallyȱimproveȱitsȱproteolyticȱstability.ȱIȱ achievedȱbothȱtheseȱgoalsȱbyȱreplacingȱfurtherȱnaturalȱaminoȱacidsȱtoȱunnaturalȱones.ȱ SubȬnanomolarȱactivityȱforȱhumanȱandȱmouseȱFXIIȱ(370ȱandȱ450ȱpMȱrespectively)ȱasȱwellȱ asȱaȱhighȱstabilityȱ(t1/2ȱ>ȱ128ȱ±ȱ8ȱhȱinȱplasma)ȱpermittedȱpreclinicalȱevaluationȱofȱtheȱpepȬ tide.ȱTheȱinhibitorȱefficientlyȱblockedȱintrinsicȱcoagulationȱinȱbloodȱplasmaȱfromȱhuman,ȱ mouseȱandȱrabbit.ȱIȱfurtherȱdemonstratedȱthatȱtheȱpeptideȱreducedȱexperimentalȱthromȬ bosisȱinducedȱbyȱferricȱchlorideȱinȱmiceȱandȱsuppressedȱbloodȱcoagulationȱinȱartificialȱ lungsȱinȱrabbits,ȱallȱwithoutȱincreasingȱtheȱriskȱofȱbleeding.ȱThisȱshowsȱthatȱFXIIȱactivityȱ isȱcontrollableȱwithȱaȱsyntheticȱinhibitorȱinȱvivo,ȱandȱthatȱtheȱoptimizedȱbicyclicȱpeptideȱ isȱaȱpromisingȱcandidateȱforȱthromboprotectionȱinȱvariousȱmedicalȱconditions.ȱ

Inȱaȱthirdȱproject,ȱIȱaimedȱatȱimprovingȱtheȱpotencyȱandȱstabilityȱofȱaȱbicyclicȱpeptideȱ MMPȬ2ȱinhibitorȱthatȱwasȱpreviouslyȱdevelopedȱinȱourȱlabȱbyȱphageȱdisplay.ȱMMPȬ2ȱisȱ implicatedȱinȱtumorȱgrowthȱandȱmetastasisȱformation.ȱDueȱtoȱtheȱstrongȱstructuralȱsimȬ ilaritiesȱsharedȱbyȱMMPȬ2ȱwithȱotherȱMMPs,ȱtheȱdevelopmentȱofȱselectiveȱinhibitorsȱofȱ MMPȬ2ȱhasȱsoȱfarȱnotȱbeenȱachieved.ȱByȱsubstitutingȱnaturalȱaminoȱacidsȱtoȱunnaturalȱ ones,ȱIȱsucceededȱatȱenhancingȱsubstantiallyȱboth,ȱtheȱbindingȱaffinityȱandȱstabilityȱofȱ theȱphageȬselectedȱbicyclicȱpeptide.ȱTheȱresultingȱMMPȬ2ȱinhibitorȱhasȱaȱhighȱaffinityȱ

(1.9ȱnM)ȱandȱstabilityȱinȱplasmaȱ(t1/2ȱ=ȱ4.4ȱh)ȱandȱthusȱprovidesȱaȱvaluableȱresearchȱtool.ȱ

Keyȱ words:ȱ Peptide,ȱ medicinalȱ chemistry,ȱ macrocycle,ȱ backbone,ȱ EȬaminoȱ acid,ȱ coagulationȱ factorȱ XII,ȱ anticoagulants,ȱ subȬnanomolar,ȱ thrombosis,ȱ ferricȱ chloride,ȱ artificialȱlung,ȱECMO,ȱMMP,ȱDȬaminoȱacid,ȱhydroxamateȱ

iv 5pVXPp

Lesȱpeptidesȱreprésententȱunȱformatȱmoléculaireȱprometteurȱpourȱleȱdéveloppementȱdeȱ nouveauȱprincipesȱactifs.ȱIlsȱcombinentȱàȱlaȱfoisȱlesȱavantagesȱdesȱpetitesȱmoléculesȱ(parȱ exempleȱlaȱdiffusionȱefficaceȱdansȱlesȱtissus,ȱuneȱimmunogenicitéȱfaibleȱetȱlaȱpossibilitéȱ d’êtreȱsynthétisésȱchimiquement)ȱetȱceuxȱdesȱprotéinesȱthérapeutiquesȱtellesȱqueȱlesȱanȬ ticorpsȱ(avecȱparȱexempleȱleurȱforteȱaffinitéȱetȱspécificité).ȱAujourd’hui,ȱenvironȱ60ȱpepȬ tidesȱfontȱl’objetȱd’essaisȱcliniques.ȱLaȱplupartȱsontȱdesȱcomposésȱnaturelsȱouȱbienȱenȱ sontȱdesȱdérivés,ȱcommeȱparȱexempleȱlesȱhormonesȱpeptidiquesȱhumainesȱouȱlesȱpepȬ tidesȱantimicrobiens.ȱToutefois,ȱcesȱdernièresȱannées,ȱdeȱnouvellesȱméthodologiesȱsontȱ apparuesȱpermettantȱleȱdéveloppementȱdeȱpeptidesȱliantȱdesȱciblesȱthérapeutiquesȱ«ȱdeȱ novoȱ».ȱ Notreȱ laboratoireȱ s’estȱ spécialiséȱ dansȱ l’uneȱ deȱ cesȱ méthodesȱ quiȱ consisteȱ enȱ l’évolutionȱinȱvitroȱdeȱpeptidesȱbicycliquesȱviaȱlaȱtechniqueȱduȱphageȱdisplay.ȱCesȱpepȬ tidesȱbicycliquesȱpossèdentȱdeuxȱchaînesȱmacrocycliquesȱquiȱleurȱpermettentȱdeȱseȱlierȱ auxȱciblesȱd’intérêtȱavecȱuneȱhauteȱaffinitéȱetȱspécificité.ȱDesȱmembresȱdeȱnotreȱlaboraȬ toireȱontȱgénéréȱdesȱligandsȱbicycliquesȱpourȱunȱgrandȱnombreȱdeȱciblesȱthérapeutiquesȱ comprenantȱdesȱinhibiteursȱduȱfacteurȱcoagulationȱXIIȱ(FXII)ȱetȱdeȱlaȱmetalloprotéinaseȱ matricielleȱ2ȱ(MMPȬ2).ȱȱ

L’objectifȱdeȱmaȱthèseȱaȱétéȱd’améliorerȱl’activitéȱetȱlaȱstabilitéȱdesȱpeptidesȱbicycliquesȱ sélectionnésȱparȱphageȱdisplayȱpourȱFXIIȱetȱMMPȬ2ȱainsiȱqueȱdeȱtesterȱleurȱpotentielȱ thérapeutiqueȱavecȱdesȱmodèlesȱanimaux.ȱPlusȱspécifiquement,ȱj’aiȱvouluȱaméliorerȱl’afȬ finitéȱdeȱliaison,ȱlaȱsélectivitéȱpourȱlaȱcibleȱetȱlaȱstabilitéȱprotéolytiqueȱdesȱpeptidesȱbicyȬ cliquesȱenȱsubstituantȱcertainsȱacidesȱaminésȱnaturelsȱparȱd’autres,ȱnonȬnaturels.ȱAinsiȱ queȱdécritȱdansȱlesȱparagraphesȱsuivants,ȱcesȱsubstitutionsȱontȱconduitȱàȱdesȱamélioraȬ tionsȱconséquentesȱdesȱpeptidesȱbicycliquesȱceȱquiȱaȱparȱlaȱsuiteȱpermisȱdeȱtesterȱcesȱ inhibiteursȱdansȱdesȱmodèlesȱthérapeutiquesȱinȱvivo.ȱȱ

Dansȱmesȱdeuxȱpremiersȱprojets,ȱj’avaisȱpourȱbutȱd’améliorerȱl’activitéȱetȱlaȱstabilitéȱd’unȱ peptideȱbicycliqueȱinhibiteurȱdeȱFXIIȱquiȱavaitȱétéȱauȱpréalableȱdéveloppéȱdansȱnotreȱ laboratoireȱafinȱdeȱparȱlaȱsuiteȱtesterȱsonȱpouvoirȱthérapeutiqueȱinȱvivo.ȱEnȱeffet,ȱFXIIȱaȱ récemmentȱétéȱidentifiéȱcommeȱuneȱcibleȱprometteuseȱpourȱleȱdéveloppementȱd’antiȬ coagulantsȱentraînantȱmoinsȱd’effetsȱsecondaires.ȱEnȱeffet,ȱcertainesȱétudesȱontȱmontréȱ queȱdesȱsouriesȱdéficientesȱpourȱFXIIȱprésentaientȱmoinsȱdeȱthrombosesȱtoutȱenȱneȱmaȬ nifestantȱpasȱdeȱsaignementsȱanormaux.ȱ

vȱ 5pVXPp

Auȱcoursȱduȱpremierȱprojet,ȱJ’aiȱtentéȱdeȱcomprendreȱsiȱl’insertionȱd’unȱatomeȱdeȱcarȬ boneȱdansȱleȱsqueletteȱmacrocycliqueȱd’unȱinhibiteurȱduȱFXIIȱpourraitȱaméliorerȱsonȱafȬ finitéȱpourȱlaȱcible.ȱDeȱtellesȱstructuresȱneȱsontȱpasȱprésentesȱdansȱlesȱlibrairiesȱdeȱphageȱ exposantȱdesȱpeptidesȱbicycliquesȱpuisqueȱcesȱderniersȱneȱsontȱconstituésȱqueȱd’acidesȱ aminésȱnaturelsȱayantȱuneȱbaseȱmoléculaireȱdeȱtroisȱatomesȱ(2ȱatomesȱdeȱcarboneȱetȱ1ȱ atomeȱd’azote).ȱDansȱlaȱchaîneȱpeptidiqueȱduȱmacrocycle,ȱlesȱpositionsȱsusceptiblesȱdeȱ tolérerȱl’insertionȱd’unȱatomeȱontȱétéȱidentifiéesȱgrâceȱàȱuneȱméthodeȱauȱcoursȱdeȱlaȬ quelleȱdesȱmutantsȱprésentantȱuneȱglycineȱàȱuneȱpositionȱdonnéeȱétaientȱcomparésȱàȱ leursȱhomologuesȱprésentantȱuneȱEȬalanine.ȱParȱlaȱsuite,ȱdesȱsubstitutionsȱavecȱunȱacideȱ aminéȱEȱouȱbienȱunȱhomologueȱdeȱcystéineȱontȱpermisȱl’identificationȱdeȱdeuxȱnouveauxȱ peptidesȱavecȱdesȱconstantesȱd’inhibitionȱ(Ki)ȱ4,7ȱetȱ2,5ȱfoisȱmeilleuresȱqueȱcelleȱduȱcomȬ poséȱdeȱdépart.ȱLeȱpeptideȱleȱplusȱefficaceȱbloqueȱl’activitéȱdeȱFXIIȱavecȱunȱKiȱdeȱ1,5ȱ±ȱ 0,1ȱnMȱetȱengendreȱuneȱinhibitionȱsupérieureȱdeȱl’activationȱdeȱlaȱcascadeȱdeȱcoagulationȱ intrinsèque.ȱDeȱplus,ȱcetteȱstratégieȱbaséeȱsurȱl’insertionȱd’unȱouȱplusieursȱatomesȱdansȱ leȱsqueletteȱd’unȱmacrocycleȱpeptidiqueȱdevraitȱpouvoirȱs’appliquerȱdeȱfaçonȱgénéraleȱ pourȱlaȱmaturationȱd’affinitéȱdeȱpeptidesȱcycliquesȱévoluéȱinȱvitro.ȱȱ

Dansȱmonȱdeuxièmeȱprojet,ȱj’aiȱvouluȱaméliorerȱencoreȱunȱpeuȱplusȱl’activitéȱdeȱl’inhiȬ biteurȱduȱFXIIȱdécritȱdansȱleȱparagrapheȱprécédentȱainsiȱqu’augmenterȱsaȱstabilitéȱproȬ téolytique.ȱJ’aiȱpuȱatteindreȱcesȱdeuxȱobjectifsȱenȱgénérantȱd’autresȱsubstitutionsȱavecȱ desȱacidesȱaminésȱnonȱnaturelsȱceȱquiȱaȱeuȱpourȱrésultatȱlaȱmiseȱàȱjourȱd’unȱpeptideȱayantȱ uneȱ activitéȱ àȱ desȱ concentrationsȱ subȬnanomolairesȱ contreȱ lesȱ protéasesȱ FXIIȱ chezȱ l’hommeȱetȱlaȱsourisȱ(respectivementȱ370ȱetȱ450ȱpM).ȱD’autreȱpart,ȱleȱnouveauȱpeptideȱaȱ uneȱtrèsȱgrandeȱstabilitéȱ(t1/2ȱ>ȱ128ȱ±ȱ8ȱhȱdansȱduȱplasma)ȱceȱquiȱaȱpermisȱsonȱévaluationȱ préclinique.ȱIlȱaȱétéȱprouvéȱqueȱl’inhibiteurȱbloqueȱefficacementȱlaȱcoagulationȱintrinȬ sèqueȱdansȱleȱplasmaȱsanguinȱdeȱl’homme,ȱlaȱsourisȱetȱleȱlapin.ȱJ’aiȱégalementȱdémontréȱ queȱleȱpeptideȱpouvaitȱréduireȱlesȱthrombosesȱinduitesȱexpérimentalementȱdansȱdesȱsouȬ risȱenȱutilisantȱleȱchlorureȱferriqueȱetȱqueȱlaȱcoagulationȱduȱsangȱétaitȱinhibéeȱdansȱunȱ modèleȱdeȱpoumonȱartificielȱchezȱleȱlapin,ȱleȱtoutȱsansȱentraînerȱdeȱrisqueȱdeȱsaignement.ȱ Cesȱdonnéesȱattestentȱqueȱl’activitéȱduȱFXIIȱpeutȱêtreȱmoduléeȱavecȱunȱinhibiteurȱsynȬ thétiqueȱetȱqueȱlaȱversionȱoptimiséeȱdeȱnotreȱpeptideȱbicycliqueȱestȱprometteuseȱpourȱlaȱ protectionȱcontreȱlesȱthrombosesȱainsiȱqueȱd’autresȱcomplicationsȱmédicales.ȱȱȱ

Dansȱleȱdernierȱprojet,ȱj’aiȱcherchéȱàȱaméliorerȱl’activitéȱetȱlaȱstabilitéȱd’unȱpeptideȱbicyȬ cliqueȱinhibiteurȱdeȱMMPȬ2.ȱMMPȬ2ȱestȱimpliquéeȱdansȱleȱdéveloppementȱdesȱtumeursȱ etȱdesȱmétastases.ȱAȱcauseȱdeȱlaȱgrandeȱsimilaritéȱstructuraleȱentreȱMMPȬ2ȱetȱd’autresȱ MMPs,ȱleȱdéveloppementȱd’unȱinhibiteurȱsélectifȱn’aȱjusqueȱmaintenantȱpasȱétéȱpossible.ȱ Enȱutilisantȱdeȱnouveauȱdesȱsubstitionsȱavecȱdesȱacidesȱaminésȱnonȱnaturels,ȱj’aiȱpuȱàȱlaȱ foisȱaugmenterȱl’affinitéȱetȱlaȱstabilitéȱdesȱpeptidesȱsélectionnésȱlorsȱdesȱcampagnesȱdeȱ sélectionȱdeȱphage.ȱLeȱmeilleurȱpeptideȱrésultantȱdeȱceȱtravailȱaȱunȱKiȱdeȱ1.9ȱnMȱetȱuneȱ stabilitéȱdeȱ4.4ȱhȱceȱquiȱenȱfaitȱunȱoutilȱdeȱrechercheȱprometteur.ȱȱ

vi 5pVXPp

Keyȱ words:ȱ Peptide,ȱ chimieȱ medicinale,ȱ macrocycle,ȱ acideȱ aminéȱΆ,ȱ Facteurȱ deȱ coagulationsȱ XII,ȱ anticoagulants,ȱ thrombose,ȱ Chlorureȱ ferrique,ȱ poumonȱ artificiel,ȱ ECMO,ȱMMP,ȱacideȱaminéȱD,ȱhydroxamateȱ

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Peptidesȱ areȱ definedȱ asȱ moleculesȱ consistingȱ ofȱ twoȱ orȱ moreȱ aminoȱ acidȱ monomersȱ linkedȱtogetherȱwithȱpeptideȱbonds.ȱThereȱisȱnotȱaȱclearȱupperȱlimitȱforȱtheȱnumberȱofȱ monomers,ȱbutȱinȱgeneralȱpeptidesȱareȱconsideredȱtoȱhaveȱlessȱthanȱ50ȱaminoȱacidȱresiȬ dues.ȱAsȱaȱmolecularȱformatȱforȱdrugȱdevelopment,ȱpeptidesȱrepresentȱaȱpromisingȱclassȱ asȱtheyȱpossessȱmanyȱdesirableȱinherentȱproperties.ȱPeptidesȱcanȱbindȱtheirȱtargetsȱwithȱ highȱaffinity,ȱdueȱtoȱtheȱrelativelyȱlargeȱcontactȱareaȱmadeȱwithȱtheȱtarget,ȱwhichȱalsoȱ rendersȱpeptideȱbindersȱhighlyȱtargetȱspecificȱasȱwellȱasȱenablingȱtheȱtargetingȱofȱproteinȱ proteinȱ interactionsȱ (PPI)ȱ (1).ȱ Moreover,ȱ peptidesȱ areȱ readilyȱ availableȱ throughȱ solidȱ phaseȱpeptideȱsynthesisȱ(SPPS)ȱwhichȱenablesȱanȱefficientȱdevelopment.ȱFurthermore,ȱ peptidesȱasȱwellȱasȱtheirȱmetabolites,ȱgenerallyȱhaveȱlowȱtoxicity,ȱminimizingȱtheȱriskȱofȱ negativeȱsideȬeffectsȱinȱtherapy.ȱȱ

Figureȱ1.ȱPeptidesȱgenerallyȱpossessȱpropertiesȱofȱbothȱsmallȱmoleculeȱdrugsȱandȱbiologics.ȱReprintedȱwithȱ permissionȱfromȱ(2),ȱ©ȱ2014ȱAmericanȱChemicalȱSociety.ȱ

PeptidesȱareȱoftenȱdescribedȱasȱaȱdrugȬclassȱfillingȱtheȱgapȱbetweenȱtraditionalȱsmallȱ moleculesȱandȱtheȱproteinȬbasedȱdrugsȱ(Figureȱ1)ȱ(2).ȱInȱoneȱaspectȱthisȱisȱrelatedȱtoȱtheirȱ molecularȱsize,ȱwhichȱrangesȱfromȱ500ȱtoȱ5000ȱDa,ȱhenceȱoccupyingȱtheȱspaceȱinȱbetweenȱ LipinskiȬ“ruleȱofȱfive”Ȭfollowingȱ(3)ȱsmallȱmoleculesȱandȱproteins.ȱOnȱtheȱotherȱhand,ȱ thisȱisȱalsoȱtrueȱforȱtheirȱinherentȱproperties.ȱPeptidesȱareȱindeedȱcombiningȱadvantagesȱ

3 ,QWURGXFWLRQ ofȱtheȱtwoȱclassesȱforȱexampleȱtheȱstrongȱandȱspecificȱbindingȱofȱproteinȬbasedȱdrugsȱ suchȱasȱmonoclonalȱantibodiesȱwithȱtheȱsyntheticȱavailabilityȱofȱsmallȱmoleculesȱ(4,ȱ5).ȱ Unfortunately,ȱalsoȱsomeȱlimitationsȱofȱtheȱtwoȱclassesȱareȱretained.ȱLikeȱsmallȱmoleȬ cules,ȱtheyȱhaveȱshortȱbiologicalȱhalfȬlives.ȱAsȱforȱantibodies,ȱpeptidesȱcanȱinȱgeneralȱnotȱ beȱusedȱforȱintracellularȱtargetsȱandȱare,ȱwithȱfewȱexceptions,ȱrequiringȱadministrationȱ byȱinjection.ȱȱ

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Manyȱpeptidesȱdrugsȱareȱnaturalȱproductsȱorȱderivativesȱthereof.ȱMostȱofȱtheȱapprovedȱ peptideȱdrugsȱcanȱbeȱbroadlyȱdividedȱintoȱtwoȱgroups,ȱpeptidesȱwithȱhumanȱhormonesȱ asȱleadȱcompoundsȱorȱpeptidesȱactingȱonȱotherȱtargetsȱforȱexampleȱtheȱantimicrobialȱ peptidesȱ (AMP).ȱ Thisȱ chapterȱ willȱ mainlyȱ focusȱ onȱ hormoneȱ derivedȱ peptideȱ drugsȱ whereasȱotherȱtypesȱwillȱbeȱdescribedȱinȱchaptersȱ1.1.3ȱandȱ1.2.1.ȱȱȱ

Inȱhumans,ȱmanyȱpeptidesȱfunctionȱasȱhormones,ȱbutȱcanȱalsoȱplayȱotherȱrolesȱsuchȱasȱ neurotransmitters,ȱgrowthȱfactorsȱandȱantimicrobialȱagentsȱ(4,ȱ6).ȱAsȱsuch,ȱpeptidesȱareȱ ligandsȱforȱmanyȱtypesȱofȱreceptorsȱsuchȱasȱGȬproteinȱcoupledȱreceptorsȱ(GPCR),ȱgrowthȱ factorȱreceptorsȱandȱionȱchannels.ȱPeptidesȱareȱoftenȱagonistsȱofȱvariousȱsignalȱtransducȬ tionȱpathways.ȱHormonesȱareȱproducedȱandȱsecretedȱinȱdifferentȱorgansȱsuchȱasȱtheȱpiȬ tuitaryȱglandȱandȱtheȱpancreasȱ(7).ȱCommonȱpeptideȱhormonesȱincludeȱoxytocin,ȱwhichȱ playsȱaȱroleȱinȱchildȱbirth,ȱvasopressin,ȱwhichȱcontrolsȱtheȱosmoticȱpressureȱofȱbodyȱfluȬ idsȱandȱsomatostatin,ȱaȱregulatorȱofȱtheȱendocrineȱsystemȱ(8–10).ȱ

Givenȱ theirȱ importantȱ rolesȱ inȱ humanȱ physiology,ȱ manyȱ naturallyȱ occurringȱ peptideȱ hormonesȱhaveȱbeenȱusedȱasȱleadȱcompoundsȱforȱtherapeuticȱdevelopmentȱ(Tableȱ1).ȱ Peptideȱdrugsȱhaveȱbeenȱdevelopedȱforȱmanyȱdifferentȱtherapeuticȱareas,ȱincludingȱonȬ cology,ȱ cardiovascularȱ diseases,ȱ multipleȱ sclerosisȱ andȱ diabetes.ȱ Theȱ mostȱ prominentȱ peptideȱdrugȱderivedȱfromȱaȱhormoneȱisȱinsulin,ȱtheȱbloodȱglucoseȱcontrollingȱhormone.ȱ Insulinȱisȱsometimesȱexcludedȱfromȱtheȱcategoryȱofȱpeptideȱdrugsȱdueȱtoȱitsȱsizeȱofȱ51ȱ aminoȱacids,ȱbutȱisȱincludedȱhereȱbecauseȱofȱitsȱimportance.ȱOverȱ400ȱmillionȱpatientsȱ worldwideȱsufferȱfromȱdiabetesȱandȱareȱtakingȱinsulinȱdailyȱ(11).ȱItȱwasȱoriginallyȱisoȬ latedȱfromȱpigȱpancreasȱandȱintroducedȱtoȱtheȱmarketȱasȱtheȱfirstȱpeptideȱtherapeuticȱinȱ theȱ 1920s.ȱ Inȱ theȱ 1980s,ȱ technologicalȱ advancementȱ hadȱmadeȱ itȱ possibleȱ toȱ recombiȬ nantlyȱproduceȱhumanȱinsulinȱwhichȱthenȱreplacedȱtheȱanimalȬderivedȱinsulins.ȱTodayȱ aȱnumberȱofȱdifferentȱinsulinȱanaloguesȱareȱonȱtheȱmarket.ȱTheȱnativeȱaminoȱacidȱseȬ quenceȱhasȱbeenȱalteredȱinȱorderȱtoȱengineerȱfastȬactingȱorȱbasalȱinsulinȱderivativesȱ(FigȬ ureȱ2).ȱTheȱfastȱactingȱanaloguesȱareȱconstructedȱwithȱtheȱpurposeȱofȱformingȱlessȱselfȬ aggregates,ȱandȱhenceȱgiveȱaȱmoreȱrapidȱresponseȱafterȱfoodȱintake.ȱExamplesȱofȱfastȱ actingȱinsulinȱanaloguesȱincludeȱlispro,ȱaspartȱandȱglulisineȱ(12).ȱInȱcontrast,ȱbasalȱinsuȬ linȱanaloguesȱareȱslowȱacting,ȱmimickingȱsteadyȱsecretionȱfromȱaȱfunctioningȱpancreas.ȱȱ

4 ,QWURGXFWLRQ

Tableȱ1.ȱApprovedȱpeptideȱtherapeuticsȱwhichȱhaveȱusedȱpeptideȱhormonesȱasȱleadȱcompoundsȱ

Hormoneȱleadȱ Peptideȱdrugȱ Insulinȱ Lisproȱ Aspartȱ Insulinȱ Glulisineȱ Glargineȱ Detemirȱ Degludecȱ Vasopressinȱ Felypressinȱ Vasopressinȱ Ornipressinȱ Desmopressinȱ Terlipressinȱ Oxytocinȱ Oxytocinȱ Atosibanȱ Carbetocinȱ Somatostatinȱ Octreotideȱ Somatostatinȱ Lanreotideȱ Pasireotideȱ Corticotropinȱ Adrenocorticotropicȱhormoneȱ Tetracosactideȱ Calcitoninȱ Calcitoninȱ Elcatoninȱ Busrelinȱ Leuprorelinȱ Triptorelinȱ Goserelinȱ Gonadorelinȱ Nafarelinȱ GonadotropinȬreleasingȱhormoneȱ Histrelinȱ Deslorelinȱ Cetrorelixȱ Ganirelixȱ Abarelixȱ Degarelixȱ Tesamorelinȱ

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ThyrotropinȬreleasingȱhormoneȱ Taltirelinȱ Vasoactiveȱintestinalȱpeptideȱ Aviptadilȱ Brainȱnatriureticȱpeptideȱ Nesiritideȱ Parathyroidȱhormoneȱ Teriparatideȱ Amylinȱ Pramlintideȱ Bradykininȱ Icatibantȱ GlucagonȬlikeȱpeptideȬ2ȱ Teduglutideȱ DȬmelanocyteȱstimulatingȱhormoneȱ Afamelanotideȱ Exenatideȱ Liraglutideȱ Lixisenatideȱ GlucagonȬlikeȱpeptideȬ1ȱ Albiglutideȱ Dulaglutideȱ Semaglutideȱ Uroguanylin Plecanatideȱ ȱ

Inȱ thisȱ caseȱ theȱ peptideȱ sequenceȱ hasȱ beenȱ alteredȱ inȱ orderȱ toȱ reduceȱ solubilityȱ andȱ therebyȱprolongȱtheȱreleaseȱofȱtheȱdrugȱfromȱtheȱinjectionȱsiteȱintoȱtheȱbloodȱstream.ȱ Insulinȱglargineȱisȱanȱexampleȱofȱthisȱapproach,ȱaȱhighlyȱsuccessfulȱtherapeuticȱamongȱ theȱtopȱ10ȱsellingȱdrugsȱworldwideȱ(2016ȱsales:ȱ6.3ȱbillionȱEURȱ=ȱ7.5ȱbillionȱUSD)ȱ(13).ȱ Insulinȱdetemirȱandȱdegludecȱinsteadȱuseȱfattyȱacidȱconjugationȱtoȱprolongȱtheȱplasmaȱ halfȬlife.ȱMoreȱrecently,ȱresearchȱhasȱfocusedȱonȱdevelopingȱaȱglucoseȱresponsiveȱinsulinȱ (14).ȱ

Figureȱ2.ȱSequencesȱofȱhumanȱinsulinȱandȱinsulinȱanaloguesȱthatȱhaveȱbeenȱapprovedȱandȱusedȱforȱtreatȬ mentȱofȱdiabetes.ȱSequenceȱdifferencesȱbetweenȱhumanȱinsulinȱandȱtheȱinsulinȱanaloguesȱareȱhighlightedȱinȱ yellow.ȱResiduesȱinvolvedȱinȱdisulfideȱbridgesȱareȱhighlightedȱinȱred.ȱReprintedȱwithȱpermissionȱfromȱfromȱ (12),ȱreprintingȱlicenseȱnr.ȱ4261840423681,ȱ©ȱ2016ȱMacmillanȱPublishersȱLimited.ȱȱ

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Besidesȱinsulin,ȱtheȱglucagonȬlikeȱpeptideȱ1ȱ(GLPȬ1)ȱanaloguesȱalsoȱaccountȱforȱaȱlargeȱ portionȱofȱtheȱpeptideȱdrugȱsales,ȱwithȱcombinedȱsalesȱofȱ4ȱbillionȱUSDȱinȱ2014ȱ(6).ȱGLPȬ 1ȱandȱanaloguesȱregulateȱbloodȱglucoseȱlevelsȱbyȱstimulatingȱinsulinȱsecretionȱ(15).ȱInȱ contrastȱtoȱinsulin,ȱthisȱhormoneȱcannotȱbeȱadministeredȱinȱitsȱnativeȱformȱdueȱtoȱrapidȱ proteolyticȱdegradationȱbyȱdipeptidylȱpeptidaseȱIVȱ(DPPȬIV).ȱInsteadȱpeptidesȱderivedȱ fromȱtheȱsalivaȱofȱtheȱGilaȱmonster,ȱaȱvenomousȱlizardȱnativeȱtoȱNorthȱAmerica,ȱwhichȱ actȱasȱGLPȬ1ȱagonistsȱandȱareȱresistantȱtoȱdegradationȱbyȱDPPȬIV,ȱareȱusedȱandȱmarketedȱ asȱexenatideȱandȱlixisenatideȱ(Figureȱ3)ȱ(16).ȱTheȱbestȬsellingȱGLPȬ1ȱanalogueȱisȱliragȬ lutide,ȱwithȱannualȱsaleȱofȱaroundȱ2.5ȱbillionȱUSD.ȱLiraglutideȱisȱtheȱnativeȱsequenceȱofȱ GLPȬ1ȱwithȱpointȱmutationsȱandȱfattyȱacidȱconjugation,ȱwhichȱimproveȱitsȱpharmacokiȬ neticȱpropertiesȱ(17).ȱAlbiglutideȱandȱdulaglutideȱareȱfusionȱproteinsȱofȱnativeȱorȱpointȬ mutatedȱGLPȬ1ȱfusedȱtoȱhumanȱalbuminȱandȱFcȱfragmentȱrespectively.ȱFormallyȱtheȱtwoȱ areȱnotȱclassifiedȱasȱpeptideȱdrugs.ȱAllȱGLPȬ1ȱanaloguesȱwereȱapprovedȱwithinȱtheȱlastȱ 12ȱyears.ȱTheȱlatestȱadditionȱtoȱtheȱGLPȬ1ȱanaloguesȱisȱsemaglutide,ȱwhichȱwasȱapprovedȱ byȱtheȱFDAȱinȱDecemberȱ2017ȱ(18).ȱThisȱpeptideȱincludesȱnonȬnaturalȱbuildingȱblocksȱ andȱisȱconjugatedȱwithȱaȱfattyȱacidȱ(19).ȱTheȱmodificationsȱincreaseȱbiologicalȱhalfȬlifeȱ evenȱfurtherȱcomparedȱtoȱliraglutideȱandȱwouldȱallowȱforȱonceȱweeklyȱadministration.ȱ

Figureȱ3.ȱSequencesȱofȱnativeȱGLPȬ1ȱandȱapprovedȱanaloguesȱforȱtreatmentȱofȱdiabetes.ȱȱȱ

Anotherȱexampleȱofȱaȱhormoneȱderivedȱpeptideȱdrugȱisȱoctreotide,ȱanȱanalogueȱofȱsoȬ mastostatinȱapprovedȱinȱ1988ȱusedȱtoȱtreatȱacromegalyȱandȱcancer.ȱOctreotideȱisȱoneȱofȱ theȱbestȬsellingȱpeptideȱdrugsȱwithȱanȱannualȱsaleȱofȱmoreȱthanȱ1.6ȱbillionȱUSDȱinȱ2016ȱ

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(20).ȱBesidesȱsomatostatin,ȱseveralȱotherȱpeptideȱhormonesȱhaveȱservedȱasȱleadȱcomȬ poundsȱforȱtherapeuticȱpeptidesȱincludingȱparathyroidȱhormone,ȱvasopressin,ȱgonadoȬ tropinȬreleasingȱhormoneȱandȱoxytocin.ȱExtensiveȱmedicinalȱchemistryȱapproachesȱareȱ oftenȱrequiredȱinȱorderȱtoȱyieldȱdrugȬlikeȱcompounds.ȱInterestingly,ȱtheȱtopȬsellingȱnonȬ insulinȱpeptideȱdrugȱisȱnotȱderivedȱfromȱaȱhormone.ȱGlatiramer,ȱmarketedȱbyȱTevaȱasȱ Copaxoneȱwasȱdiscoveredȱserendipitously.ȱItȱisȱaȱcomplexȱmixtureȱofȱpeptidesȱofȱvariousȱ lengthȱcontainingȱaminoȱacidȱresiduesȱalanine,ȱlysine,ȱglutamicȱacidȱandȱtyrosineȱ(21).ȱ Glatiramerȱisȱusedȱtoȱtreatȱmultipleȱsclerosisȱandȱitȱhadȱannualȱsalesȱinȱ2016ȱofȱ4.2ȱbillionȱ USDȱ(22).ȱȱ

Manyȱofȱtheȱapprovedȱpeptideȱtherapeuticsȱareȱanaloguesȱofȱnaturalȱhormonesȱwhichȱ haveȱundergoneȱmedicinalȱchemistryȱapproachesȱinȱorderȱtoȱtackleȱtheȱintrinsicȱlimitaȬ tionsȱofȱtheȱmolecularȱformat.ȱMuchȱworkȱisȱbeingȱperformedȱwithinȱthisȱfieldȱofȱpeptideȱ therapeuticsȱ andȱ innovativeȱ approachesȱ ofȱ chemicallyȱ modifyingȱ peptideȱ leadȱ comȬ poundsȱisȱthoughtȱtoȱbeȱcrucialȱforȱfutureȱdevelopmentȱofȱpeptidesȱaimingȱatȱotherȱtarȬ getsȱthanȱhormoneȱreceptorsȱ(23).ȱRecentȱdevelopmentȱofȱpeptideȱdrugsȱhasȱshiftedȱtheȱ focusȱfromȱhormoneȱderivedȱanaloguesȱtoȱpeptidesȱwithȱnovelȱmolecularȱtargets.ȱTheseȱ includeȱcellȱsurfaceȱreceptors,ȱantimicrobials,ȱionȱchannels,ȱstructuralȱproteinsȱandȱenȬ zymesȱ(24).ȱȱ

Asȱ highlightedȱ byȱ theȱ examplesȱ above,ȱ severalȱ peptideȱ drugsȱ areȱ soȱ calledȱ “blockȬ busters”ȱwithȱannualȱsalesȱexceedingȱ1ȱbillionȱUSD.ȱAsȱofȱ2016ȱthereȱareȱaboutȱ60ȱpeptideȱ drugsȱinȱtheȱclinic,ȱtheȱexactȱnumberȱdependingȱonȱtheȱdefinition.ȱMoreoverȱaroundȱ170ȱ areȱinȱclinicalȱdevelopmentȱandȱanotherȱ200ȱinȱpreclinicalȱdevelopmentȱ(6).ȱTheseȱnumȬ bersȱsuggestȱthatȱthereȱisȱsubstantialȱdevelopmentȱinȱtheȱfieldȱandȱthatȱpeptidesȱasȱdrugȱ formatȱisȱconsideredȱasȱpromising,ȱdespiteȱtheȱfactȱthatȱtheȱ“lowȬhangingȱfruit”ȱinȱtermsȱ ofȱhormoneȱbasedȱpeptideȱdrugsȱhaveȱalreadyȱbeenȱhighlyȱdevelopedȱ(25).ȱTheȱtotalȱsalesȱ ofȱpeptideȱbasedȱtherapeutics,ȱincludingȱinsulin,ȱisȱestimatedȱtoȱaroundȱ50ȱbillionȱUSDȱ inȱ2015,ȱwhichȱaccountsȱforȱ5%ȱofȱtheȱtotalȱworldwideȱpharmaceuticalȱmarketȱ(6).ȱTheȱ insulinȱderivativesȱareȱaccountingȱforȱmoreȱthanȱhalfȱofȱtheȱtotalȱsalesȱofȱpeptideȱdrugs.ȱ Theȱtwoȱdominantȱtherapeuticȱareasȱforȱwhichȱpeptideȱdrugsȱareȱusedȱareȱmetabolicȱ diseaseȱandȱoncologyȱ(4).ȱTheseȱareasȱareȱexpectedȱtoȱgrowȱfurtherȱinȱtheȱwesternȱworldȱ dueȱtoȱlifeȬstyleȱandȱagingȱpopulations.ȱTheȱannualȱgrowthȱrateȱofȱpeptideȱdrugȱsalesȱisȱ estimatedȱtoȱ9Ȭ10%.ȱTheȱnumberȱofȱpeptideȱmoleculesȱenteringȱclinicalȱdevelopmentȱhasȱ beenȱconstantlyȱincreasingȱsinceȱ1990ȱ(Figureȱ4)ȱ(24),ȱwithȱpeakȱvaluesȱofȱaroundȱ30ȱinȱ 2010.ȱHowever,ȱinȱtheȱlastȱyearsȱtheseȱnumbersȱareȱdecliningȱandȱwillȱprobablyȱsettleȱatȱ aȱvalueȱofȱaroundȱ20ȱpeptidesȱenteringȱclinicalȱdevelopmentȱeachȱyear.ȱThisȱtrendȱreflectsȱ theȱ saturationȱ inȱ theȱ developmentȱ ofȱ relativelyȱ straightȬforwardȱ hormoneȬbasedȱ anaȬ loguesȱforȱvalidatedȱtargetsȱbutȱisȱcounterbalancedȱbyȱnewȱinnovativeȱstrategiesȱofȱpepȬ tideȱdrugȱdevelopment.ȱȱȱȱ

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Figureȱ4.ȱȱCumulativeȱnumberȱofȱpeptidesȱapprovedȱinȱmajorȱpharmaceuticalȱmarketsȱandȱtheȱnumberȱofȱ peptidesȱenteringȱclinicalȱdevelopment.ȱEntryȱintoȱclinicalȱdevelopmentȱisȱdefinedȱasȱtheȱyearȱofȱtheȱfirstȱ Phaseȱ1ȱorȱpilotȱhumanȱstudy.ȱReprintedȱwithȱpermissionȱfromȱ(24),ȱ©ȱ2017ȱElsevierȱLtd.ȱȱȱ

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Cyclicȱpeptidesȱhaveȱaȱnumberȱofȱadvantagesȱoverȱlinearȱpeptides.ȱFirst,ȱcyclicȱpeptidesȱ canȱgenerallyȱbindȱtoȱtheirȱtargetsȱwithȱhigherȱaffinity.ȱTheȱcyclizationȱconstrainsȱtheȱ backbone,ȱwhichȱreducesȱconformationalȱflexibility.ȱTheȱpeptideȱcanȱthenȱbeȱpreȬorgaȬ nizedȱtowardȱitsȱbiologicallyȱactiveȱconformationȱandȱtherebyȱimprovingȱbindingȱaffinȬ ityȱbyȱreducingȱtheȱentropicȱpenaltyȱuponȱtargetȱbinding.ȱSecond,ȱcyclicȱpeptidesȱareȱ oftenȱlessȱproneȱtoȱproteolyticȱdegradationȱasȱcomparedȱtoȱlinearȱpeptidesȱ(1).ȱTheȱcyclicȱ structureȱmakesȱtheȱpeptideȱlessȱaccessibleȱtoȱproteases.ȱThird,ȱcyclicȱpeptidesȱbindȱmoreȱ specificȱtoȱtheirȱtargets.ȱThisȱisȱalsoȱrelatedȱtoȱtheirȱreducedȱconformationalȱflexibility.ȱ Cyclizationȱisȱalsoȱusedȱasȱaȱtoolȱinȱtheȱpursuitȱofȱmakingȱpeptidesȱbeyondȱtheȱruleȱofȱ fiveȱhavingȱoralȱavailabilityȱandȱcellȱpermeabilityȱ(26).ȱWhileȱoftenȱnativeȱcyclicȱpeptidesȱ areȱusedȱasȱleadȱcompoundsȱalsoȱtheȱconversionȱofȱlinearȱpeptidesȱtoȱcyclicȱanaloguesȱ hasȱbeenȱdemonstratedȱ(27).ȱȱ

Newȱcyclizationȱtechniquesȱasȱwellȱasȱvariousȱnovelȱscreeningȱplatforms,ȱtheȱlatterȱcovȬ eredȱinȱtheȱnextȱchapter,ȱpropelȱtheȱdiscoveryȱofȱnewȱcyclicȱpeptideȱtherapeuticsȱ(28).ȱ Generally,ȱcyclicȱpeptidesȱcanȱbeȱconstructedȱinȱfourȱdifferentȱformatsȱnamely:ȱheadȬtoȬ tail,ȱ headȬtoȬsideȱ chain,ȱ sideȱ chainȬtoȬtailȱ orȱ sideȱ chainȬtoȬsideȱ chainȱ (Figureȱ 5)ȱ (29).ȱ HeadȬtoȬtailȱcyclizationȱcanȱmostlyȱbeȱreadilyȱachievedȱviaȱpeptideȱbondȱformation,ȱbutȱ alsoȱ otherȱ reactionȱ typesȱ canȱ beȱ usedȱ suchȱ asȱ nativeȱ chemicalȱ ligationȱ (NCL)ȱ orȱ Staudingerȱligation.ȱȱForȱsideȱchainȱcyclizationȱaȱwideȱrangeȱofȱchemistriesȱhaveȱbeenȱ

9 ,QWURGXFWLRQ utilizedȱincludingȱdisulfideȱbridgeȱformation,ȱringȬclosingȱmetathesisȱandȱazideȬalkyneȱ cycloadditionsȱ(30).ȱAnȱinterestingȱapproachȱinȱorderȱtoȱrenderȱmoreȱstableȱdisulfideȱ cyclizedȱpeptidesȱbyȱintroducingȱaȱthioacetalȱwasȱrecentlyȱreportedȱ(31).ȱThisȱmodificaȬ tionȱisȱprolongingȱtheȱbiologicalȱhalfȬlifeȱofȱmodelȱpeptidesȱwithoutȱcompromisingȱtargetȱ binding.ȱȱ

Figureȱ5.ȱFourȱdifferentȱapproachesȱcanȱbeȱusedȱinȱorderȱtoȱconstructȱmacrocyclicȱpeptides.ȱTheȱdifferentȱ strategiesȱcanȱbeȱusedȱinȱcombinationȱforȱconstructingȱmulticyclicȱpeptides.ȱReprintedȱwithȱpermissionȱfromȱ fromȱ(29),ȱreprintingȱlicenseȱnr.ȱ4262470475054,ȱ©ȱ2011ȱMacmillanȱPublishersȱLimited.ȱ

Oxytocinȱisȱanȱexampleȱofȱaȱcyclicȱpeptideȱdrug,ȱwhichȱhasȱbeenȱusedȱinȱtheȱclinicȱforȱaȱ longȱtimeȱ(Figureȱ6).ȱItȱwasȱtheȱfirstȱpeptideȱhormoneȱtoȱhaveȱitsȱsequenceȱdeterminedȱ andȱtoȱbeȱsynthesizedȱ(32,ȱ33),ȱeffortsȱforȱwhichȱVincentȱduȱVigneaudȱwasȱawardedȱtheȱ Nobelȱprizeȱinȱ1955ȱ(34).ȱTheȱmainȱbiologicalȱfunctionȱofȱoxytocin,ȱwhichȱhasȱbeenȱusedȱ inȱtherapeuticȱpurposes,ȱisȱtheȱinductionȱofȱcontractionȱofȱsmoothȱmusclesȱinȱtheȱuterusȱ duringȱlabor.ȱThisȱfunctionȱhasȱbeenȱexploitedȱforȱpromotingȱcontractionsȱduringȱlateȱ stageȱofȱlaborȱandȱforȱtheȱpreventionȱofȱpostpartumȱhemorrhageȱ(35).ȱOxytocinȱisȱhowȬ everȱalsoȱactiveȱasȱaȱneuropeptide,ȱinfluencingȱbehavior.ȱItȱhasȱthereforeȱrecentlyȱbeenȱ evaluatedȱasȱaȱpotentialȱtreatmentȱforȱpsychiatricȱdisorders,ȱincludingȱautismȱ(36,ȱ37).ȱȱȱȱ

AnȱimportantȱclassȱofȱpeptideȱdrugsȱareȱtheȱAMPs.ȱAȱmajorityȱofȱtheseȱpeptidesȱhaveȱaȱ cyclicȱ structure.ȱ Theȱ mostȱ wellȬknownȱ examplesȱ includeȱ theȱ nonribosomallyȱ syntheȬ sizedȱpeptidesȱvancomycinȱandȱcyclosporineȱA.ȱTheȱformerȱwasȱfirstȱisolatedȱinȱ1957ȱ fromȱtheȱbacteriaȱStreptomycesȱorientalisȱandȱisȱusedȱtodayȱtoȱtreatȱinfectionsȱcausedȱbyȱ methicillinȬresistantȱStaphylococcusȱaureusȱ(MRSA)ȱ(38).ȱSeveralȱnonribosomallyȱsyntheȬ sizedȱAMPsȱhaveȱbeenȱisolated,ȱmainlyȱfromȱbacteria,ȱincludingȱapprovedȱdrugsȱsuchȱ asȱ polymyxinȱ Bȱ andȱ gramicidinȱ Cȱ (39).ȱ Moreȱ recentlyȱ alsoȱ ribosomallyȱ synthesizedȱ AMPsȱfromȱdifferentȱsourcesȱsuchȱasȱhuman,ȱpig,ȱfrogȱandȱplantsȱȱhaveȱbeenȱusedȱasȱ leadȱcompoundsȱandȱareȱinȱclinicalȱtrialsȱforȱdifferentȱapplicationsȱ(40,ȱ41).ȱNewȱdevelȬ opmentȱofȱAMPsȱuseȱvariousȱtypesȱofȱoptimizationȱofȱtheȱnaturallyȱderivedȱleadȱcomȬ poundsȱ(42).ȱCyclosporinȱAȱ(Figureȱ6)ȱwasȱisolatedȱ1970ȱfromȱtheȱfungusȱTolypocladiumȱ

10 ,QWURGXFWLRQ inflatum.ȱItȱwasȱfirstȱevaluatedȱforȱitsȱantifungalȱpropertiesȱbutȱisȱnowȱwidelyȱusedȱasȱanȱ immunosuppressantȱfollowingȱorganȱtransplanantation,ȱforȱwhichȱitȱwasȱapprovedȱbyȱ theȱFDAȱinȱ1983ȱ(43).ȱ

Figureȱ6.ȱChemicalȱstructuresȱofȱthreeȱapprovedȱcyclicȱpeptideȱtherapeuticsȱderivedȱfromȱdifferentȱsources.ȱ Oxytocinȱisȱtheȱnativeȱpeptideȱhormone,ȱcyclizedȱthroughȱaȱdisulfideȱbridge.ȱItȱwasȱtheȱfirstȱpeptideȱhorȬ moneȱtoȱhaveȱitsȱstructureȱdeterminedȱandȱtoȱbeȱchemicallyȱsynthesized.ȱOxytocinȱwasȱfirstȱapprovedȱinȱ 1962.ȱCyclosporinȱAȱisȱaȱnaturalȱproductȱfoundȱinȱfungus.ȱItȱisȱheadȬtoȬtailȱcyclizedȱandȱheavilyȱNȬmethylȬ ated,ȱwhichȱrendersȱitȱorallyȱavailable,ȱaȱveryȱrareȱpropertyȱofȱaȱpeptideȱtherapeutic.ȱLinaclotideȱisȱaȱderiȱ ativeȱofȱaȱbacterialȱtoxin,ȱcyclizedȱthroughȱthreeȱdifferentȱdisulfideȱbridges.ȱȱ

Severalȱcyclicȱpeptidesȱhaveȱbeenȱapprovedȱasȱdrugsȱinȱtheȱlastȱdecadeȱ(44).ȱThisȱincludesȱ theȱthreeȱantibacterialȱpeptidesȱtelavancin,ȱdalbavancinȱandȱoritavancin,ȱallȱbelongingȱtoȱ theȱsameȱclassȱofȱmoleculesȱasȱvancomycin.ȱPasireotide,ȱapprovedȱ2012,ȱisȱaȱsomastostaȬ tinȱanalogueȱsimilarȱtoȱlanreotideȱandȱoctreotide,ȱbutȱwithȱtailoredȱreceptorȱspecificityȱ forȱtheȱsst1ȱandȱsst5ȱreceptors,ȱrenderingȱitȱsuitableȱforȱtreatmentȱofȱdifferentȱtumorȱtypesȱ andȱCushing’sȱdiseaseȱ(10,ȱ45).ȱInterestingly,ȱduringȱtheȱmedicinalȱchemistryȱapproachȱ theȱdisulfideȱbondȱwasȱremovedȱandȱinsteadȱheadȬtoȬtailȱcyclizationȱisȱemployed.ȱThisȱ isȱlikelyȱcontributingȱtoȱtheȱlargelyȱincreasedȱhalfȬlifeȱofȱ12ȱh.ȱAnotherȱinterestingȱexamȬ pleȱisȱlinaclotide,ȱwhichȱwasȱapprovedȱinȱ2012ȱforȱtheȱtreatmentȱofȱconstipationȬdomiȬ

11 ,QWURGXFWLRQ nantȱirritableȱbowelȱsyndromeȱ(Figureȱ6)ȱ(46–48).ȱLinaclotideȱisȱaȱderivativeȱofȱtheȱbacȬ terialȱenterotoxinȱSTȱofȱE.coliȱwhichȱcausesȱdiarrhea.ȱSTȱisȱanȱagonistȱofȱguanylateȱcyclaseȱ Cȱ(GCȬC),ȱaȱmembraneȬspanningȱenzymeȱexpressedȱonȱtheȱluminalȱsurfaceȱofȱepithelialȱ cellsȱinȱtheȱintestine.ȱActivationȱofȱthisȱenzymeȱleadsȱtoȱchlorideȱsecretionȱandȱincreasedȱ colonicȱtransit.ȱSTȱandȱlinaclotideȱcontainȱthreeȱdisulfideȱbridgesȱconstrainingȱconforȬ mationȱandȱprovidingȱproteolyticȱstability.ȱLinaclotideȱisȱaȱtruncatedȱversionȱofȱSTȱalsoȱ containingȱaminoȱacidsȱsubstitutionsȱrenderingȱitȱevenȱmoreȱproteolytcallyȱstable.ȱTheȱ intestinalȱlocationȱofȱGCȬCȱfacilitatesȱoralȱadministrationȱofȱlinaclotideȱalthoughȱtheȱpepȬ tideȱisȱnotȱorallyȱavailable.ȱGCȬCȱisȱalsoȱactivatedȱbyȱtwoȱendogenousȱreceptorȱagonistȱ peptideȱhormonesȱguanylinȱandȱuroguanylin.ȱInȱcontrastȱtoȱSTȱandȱlinaclotideȱtheyȱconȬ tainȱonlyȱtwoȱdisulfideȱbonds.ȱUroguanylinȱwasȱusedȱasȱaȱleadȱpeptideȱinȱorderȱtoȱdeȬ velopȱplecanatideȱ(approvedȱinȱ2017),ȱdifferingȱonlyȱoneȱaminoȱacidȱformȱtheȱnativeȱseȬ quenceȱ(49,ȱ50).ȱPlecanatideȱisȱthoughtȱtoȱbeȱsuperiorȱtoȱlinaclotideȱinȱtheȱsenseȱthatȱitȱisȱ actuallyȱaȱlessȱpotentȱagonist,ȱthereforeȱresultingȱinȱlessȱsideȬeffectsȱsuchȱasȱdiarrhea.ȱAȱ wideȱrangeȱofȱcyclicȱpeptideȱareȱinȱclinicalȱdevelopmentȱandȱitȱcanȱbeȱexpectedȱthatȱmanyȱ moreȱwillȱbeȱapproved.ȱȱ

ȱȱ

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Historically,ȱtheȱmostȱcommonȱstrategyȱforȱdevelopingȱpeptideȱdrugsȱhasȱbeenȱtoȱselectȱ aȱpeptideȱwithȱknownȱbiologicalȱeffectȱasȱleadȱcompound.ȱThisȱstrategyȱhasȱbeenȱdeȬ scribedȱbyȱseveralȱexamplesȱinȱchaptersȱ1.1.2ȱandȱ1.1.3ȱofȱthisȱthesisȱandȱmedicinalȱchemȬ istryȱapproachesȱwillȱbeȱdiscussedȱinȱchapterȱ1.3.ȱInȱthisȱchapterȱexamplesȱofȱpeptidesȱ fromȱnaturalȱsourcesȱotherȱthanȱhormonesȱandȱantimicrobialȱpeptidesȱareȱdiscussed.ȱȱȱ

Aȱtraditionalȱsourceȱofȱbioactiveȱpeptidesȱisȱvenomousȱorganisms.ȱTheȱconotoxinsȱareȱ oneȱofȱtheȱmostȱprominentȱexamplesȱwithinȱthisȱclass.ȱTheyȱareȱhighlyȱconstrainedȱpepȬ tides,ȱcontainingȱseveralȱdisulfideȱbridgesȱandȱpostȬtranslationalȱmodifications,ȱderivedȱ fromȱconeȱsnailsȱ(51,ȱ52).ȱConotoxinsȱgenerallyȱtargetȱreceptorsȱandȱionȱchannelsȱinȱnervȬ ousȱandȱmuscleȱtissue.ȱZiconotideȱisȱaȱconotoxinȱapprovedȱbyȱtheȱFDAȱinȱ2004ȱforȱtheȱ treatmentȱofȱchronicȱpain.ȱOrallyȱavailableȱconotoxinsȱhaveȱbeenȱconstructedȱbyȱheadȬ toȬtailȱcyclizationȱandȱtheȱformatȱisȱconsideredȱpromisingȱforȱtherapeuticȱapplicationsȱ (53,ȱ54).ȱ

Otherȱ wellȬknownȱ peptidesȱ isolatedȱ fromȱ naturalȱ sourcesȱ includeȱ chlorotoxin,ȱ aȱ 36ȱ aminoȱacidȱcysteineȱknotȱpeptidesȱfoundȱinȱscorpionȱvenomȱ(55,ȱ56).ȱTheȱpeptideȱhasȱ foundȱapplicationȱdueȱtoȱitsȱtumorȱtargetingȱabilities.ȱBivalirudinȱisȱaȱ20ȱaminoȱacidȱdiȬ rectȱthrombinȱinhibitorȱwhichȱwasȱdevelopedȱusingȱtheȱhirudinȱpeptideȱfoundȱinȱtheȱ salivaȱofȱmedicalȱleachesȱasȱaȱleadȱcompoundȱ(57,ȱ58).ȱLinaclotideȱandȱ exenatide/lixȬ isenatideȱasȱdescribedȱaboveȱwereȱderivedȱfromȱaȱbacterialȱtoxinȱandȱsalivaȱfromȱtheȱGilaȱ monsterȱrespectively.ȱȱ

Anȱinterestingȱsourceȱwhichȱhasȱbeenȱexploitedȱisȱtheȱcyclotides.ȱTheseȱareȱaȱfamilyȱofȱ plantȱderivedȱheadȬtoȬtailȱcyclizedȱpeptidesȱofȱaroundȱ30Ȭ40ȱaminoȱacidsȱ(59,ȱ60).ȱTheȱ peptidesȱcontainȱsixȱcysteineȱresiduesȱwhichȱareȱoxidizedȱtoȱformȱthreeȱdisulfideȱbondsȱ whichȱformȱaȱcyclicȱcysteineȬknotȱtopologyȱ(Figureȱ7)ȱ(61).ȱThisȱmotifȱyieldsȱaȱveryȱrigidȱ structure,ȱwhichȱisȱstableȱtoȱthermalȱandȱchemicalȱdegradationȱasȱwellȱasȱtoȱproteolyticȱ cleavage.ȱNotablyȱtheȱhighlyȱstableȱstructureȱalsoȱhasȱtheȱpotentialȱofȱrenderingȱtheȱcyȬ clotidesȱorallyȱavailableȱ(62).ȱCyclotidesȱhenceȱpossessȱseveralȱintrinsicȱpropertiesȱwhichȱ overcomeȱsomeȱsevereȱlimitationsȱofȱpeptideȱdrugs.ȱThisȱmakesȱthemȱveryȱinterestingȱasȱ leadȱcompounds.ȱTheȱbiologicalȱfunctionȱofȱcyclotidesȱisȱnotȱyetȱfullyȱunderstoodȱbutȱtheȱ primaryȱintrinsicȱfunctionȱisȱhypothesizedȱtoȱbeȱhostȱdefenseȱ(63).ȱForȱexample,ȱtheȱfirstȱ identifiedȱcyclotide,ȱkalataȱB1,ȱisolatedȱfromȱOldenlandiaȱaffinisȱofȱtheȱRubiaceaeȱfamily,ȱ hasȱbeenȱshownȱtoȱcauseȱmortalityȱinȱbudworms.ȱKalataȱB1ȱhasȱbeenȱusedȱasȱindigenousȱ medicineȱinȱAfrica,ȱwereȱwomenȱdrankȱteaȱmadeȱfromȱOldenlandiaȱaffinisȱtoȱaccelerateȱ childbirth.ȱThisȱhighlightsȱtheȱthermalȱstabilityȱandȱoralȱavailabilityȱofȱtheȱcyclotides.ȱ

13 ,QWURGXFWLRQ

WhileȱsomeȱcyclotidesȱcanȱbeȱusedȱforȱpharmaceuticalȱapplicationsȱasȱtheirȱnativeȱseȬ quence,ȱmostȱrecentȱapplicationsȱfocusȱonȱgraftingȱbiologicallyȱactiveȱpeptideȱsequencesȱ identifiedȱbyȱotherȱtechniquesȱontoȱtheȱcyclotideȱscaffold.ȱTheȱmostlyȱutilizedȱcyclotidesȱ forȱ graftingȱ areȱ kalataȱ B1ȱ andȱ momordicaȱ cochinchinensisȱ trypsinȱ inhibitorȱ (MCoTI).ȱ Someȱrecentȱandȱinterestingȱexamplesȱincludeȱtheȱdevelopmentȱofȱaȱcoagulationȱfactorȱ XIIȱ(FXII)ȱinhibitorȱbyȱsubstrateȱscreeningȱandȱgraftingȱontoȱaȱMCoTIȱscaffoldȱ(64),ȱanȱ orallyȱactiveȱbradykininȱreceptorȱantagonistȱdevelopedȱbyȱgraftingȱaȱknownȱantagonistȱ peptideȱsequenceȱontoȱkalataȱB1ȱ(65)ȱandȱaȱMCoTIȱgraftȱwhichȱactsȱonȱintracellularȱtarȬ gets,ȱinhibitingȱp53ȱdegradationȱ(66).ȱȱ

Figureȱ7.ȱPrimaryȱandȱtertiaryȱstructuresȱofȱcyclotidesȱKalataȱB1ȱ(PDBȱID:1NB1)ȱandȱMCoTIȬIIȱ(PDBȱID:1IB9)ȱ belongingȱtoȱtheȱMöbiusȱandȱtrypsinȱinhibitorȱsubfamiliesȱrespectively.ȱTheȱsequenceȱofȱkalataȱB8,ȱaȱhybridȱ cyclotideȱisolatedȱfromȱtheȱplantȱO.ȱaffinis,ȱisȱalsoȱshown.ȱConservedȱCysȱandȱAsp/Asnȱ(requiredȱforȱcyclizaȬ tion)ȱresiduesȱareȱmarkedȱinȱyellowȱandȱlightȱblue,ȱrespectively.ȱDisulfideȱconnectivitiesȱandȱbackboneȱcyȬ clizationȱareȱshownȱinȱredȱandȱbyȱaȱdarkȱblueȱline,ȱrespectively.ȱAdaptedȱwithȱpermissionȱfromȱ(61),ȱreprintȬ ingȱlicenseȱnr.ȱ4262540872131,ȱ©ȱ2017ȱWileyȬVCHȱVerlagȱGmbH&Coȱ

3URWHLQHSLWRSHPLPHWLFV

InhibitorsȱofȱPPIȱcanȱbeȱdesignedȱrationallyȱbasedȱonȱpeptideȱsequencesȱatȱtheȱinterfaceȱ ofȱinteractingȱproteinsȱ(67).ȱAtȱtheȱinteractionȱsites,ȱproteinsȱfrequentlyȱhaveȱwellȱdefinedȱ secondaryȱstructures.ȱTheȱmimickingȱofȱtheseȱinteractingȱepitopesȱisȱoftenȱusefulȱinȱtheȱ developmentȱ ofȱ drugsȱ aimingȱ atȱ challengingȱ targetsȱ suchȱ asȱ PPI.ȱ Theȱ termȱ “peptidomimetics”ȱisȱsometimesȱusedȱtoȱdescribeȱsuchȱcompounds.ȱThereȱisȱnoȱclearȱ

14 ,QWURGXFWLRQ ruleȱtoȱdefineȱpeptidomimetics,ȱhoweverȱrecently,ȱaȱclassificationȱsystemȱwasȱproposed,ȱ inȱ orderȱ toȱ groupȱ theȱ differentȱ typesȱ togetherȱ (68).ȱ Inȱ thisȱ chapterȱ Iȱ willȱ describeȱ peptidomimeticsȱ havingȱ aȱ peptideȬbasedȱ scaffold,ȱ whichȱ areȱ mimickingȱ theȱ bindingȱ epitopesȱ ofȱ proteins,ȱ mainlyȱ withȱ aȱ focusȱ onȱ secondaryȱ structure.ȱ SmallȬmoleculeȱ peptidomimeticsȱ willȱ beȱ excluded.ȱ Theȱ chapterȱ willȱ includeȱ medicinalȱ chemistryȱ approachesȱwhichȱaimȱatȱintroducingȱorȱlockingȱsecondaryȱstructures.ȱSomeȱofȱthisȱworkȱ isȱ relatedȱ toȱ backboneȱ modifications,ȱ whichȱ isȱ aȱ broaderȱ fieldȱ alsoȱ servingȱ otherȱ purposes,ȱbutȱwillȱbeȱincludedȱinȱthisȱchapter.ȱȱȱ

AȱcommonȱbindingȱmotifȱwhichȱisȱoftenȱfoundȱasȱaȱkeyȱmediatorȱinȱPPIȱisȱtheȱDȬhelixȱ (69).ȱ Thereforeȱ peptideȱ sequencesȱ havingȱ anȱ DȬhelicalȱ conformation,ȱ derivedȱ fromȱ aȱ protein,ȱareȱattractiveȱasȱligandsȱofȱtherapeuticȱtargetsȱ(70).ȱShorterȱpeptidesȱareȱhoweverȱ notȱstableȱinȱtheirȱDȬhelicalȱconformationȱinȱsolutionȱ(71)ȱandȱmanyȱstrategiesȱhaveȱbeenȱ developedȱinȱorderȱtoȱlockȱthemȱinȱtheȱcorrectȱconformationȱforȱbinding.ȱOneȱofȱtheȱmostȱ wellȬknownȱ approachesȱ forȱ lockingȱ peptidesȱ inȱ aȱ DȬhelicalȱ conformationȱ isȱ peptideȱ stapling.ȱTheȱprincipleȱworksȱbyȱselectingȱtwoȱresiduesȱthatȱhaveȱsideȬchainsȱpointingȱ towardsȱtheȱsameȱfaceȱofȱtheȱhelixȱandȱconnectingȱthemȱcovalently.ȱTheȱresiduesȱwhichȱ areȱnormallyȱstapledȱtogetherȱareȱiȱandȱi+3,ȱi+4,ȱi+7ȱorȱi+11.ȱDifferentȱchemistriesȱhaveȱ beenȱutilizedȱinȱorderȱtoȱachieveȱtheȱcovalentȱlinkage,ȱwithȱtheȱmostȱcommonȱbeingȱanȱ allȬhydrocarbonȱcrossȱlinkȱachievedȱthroughȱtheȱincorporationȱofȱunnaturalȱaminoȱacidsȱ undergoingȱolefinȱringȱclosingȱmetathesis,ȱfirstȱestablishedȱbyȱVerdineȱandȱcoȬworkersȱ (Figureȱ8A)ȱ(72).ȱTheȱlengthȱofȱtheȱsideȬchainȱinȱtheȱstapleȱcanȱbeȱvariedȱdependingȱonȱ theȱdistanceȱofȱtheȱconnectedȱresidues.ȱTheȱpropertiesȱofȱtheȱstapledȱpeptideȱareȱfurtherȱ tunedȱbyȱvaryingȱparametersȱsuchȱasȱstapleȱposition,ȱstereochemistryȱandȱnumberȱofȱ staples.ȱLatelyȱcomputationalȱtoolsȱhaveȱbeenȱusedȱinȱtheȱdesignȱofȱstapledȱpeptidesȱ(73).ȱ HydrocarbonȬstapledȱ peptidesȱ canȱ beȱ generatedȱ readilyȱ onȱ automaticȱ peptideȱ synthesizersȱ andȱ haveȱ beenȱ developedȱ forȱ aȱ rangeȱ ofȱ targets,ȱ bothȱ intracellularȱ andȱ extracellularȱ (74,ȱ 75).ȱ Inȱ addition,ȱ theȱ stabilityȱ ofȱ peptidesȱ isȱ oftenȱ improvedȱ uponȱ hydrocarbonȱstapling.ȱOtherȱpeptideȱstaplingȱstrategiesȱinvolvingȱonlyȱtheȱsideȱchainsȱ ofȱ theȱ peptideȱ buildingȱ blocksȱ includeȱ amideȱ bondȱ formation,ȱ azideȬalkyneȱ cycloȬ addition,ȱdisulfideȱbridgeȱorȱthioetherȱbondȱ(Figureȱ8)ȱ(76).ȱȱ

AȱslightlyȱdifferentȱapproachȱinȱwhichȱtheȱNȬterminalȱintramolecularȱhydrogenȱbondȱisȱ replacedȱbyȱaȱstaple,ȱtermedȱ‘hydrogenȬbondȬsurrogate’ȱhasȱrecentlyȱbeenȱestablishedȱ andȱshownȱtoȱstabilizeȱhelicityȱ(77).ȱSomeȱstaplingȱtechniquesȱinvolveȱbuildingȱblocksȱ externalȱtoȱtheȱsideȱchainsȱsuchȱasȱphotoswitchableȱlinkersȱorȱdoubleȱazideȬalkyneȱcycloȬ additionsȱ(76,ȱ78).ȱOneȱinterestingȱapproachȱwasȱrecentlyȱreportedȱinȱwhichȱthiolȱreactiveȱ linkersȱwereȱusedȱtoȱgenerateȱpeptidesȱstapledȱthroughȱtwoȱcysteineȱresiduesȱinȱfixedȱ positionsȱ inȱ aȱ phageȱ displayȱ libraryȱ (79).ȱ Usingȱ thisȱ strategyȱ DȬhelicalȱ bindersȱ toȱ EȬ cateninȱwereȱdeveloped.ȱStapledȱpeptidesȱhaveȱalsoȱbeenȱshownȱcompatibleȱwithȱandȱ optimizedȱ usingȱ oneȬbeadȬoneȬcompoundȱ (OBOC)ȱ librariesȱ (80).ȱ Recentlyȱ theȱ tightȱ

15 ,QWURGXFWLRQ bindingȱofȱtheȱDȬhelixȱinȱaȱPPIȱwasȱexploitedȱbyȱHuhnȱandȱcoȬworkesȱtoȱdevelopȱcovalentȱ inhibitorsȱtoȱblockȱtheȱantiȬapoptoticȱinteractionȱofȱBH3ȱandȱBFLȬ1ȱ(81).ȱTheȱgroupȱofȱ FairlieȱhaveȱalsoȱutilizedȱpeptideȱstaplingȱforȱDȬhelixȱenhancementȱinȱtheirȱdevelopmentȱ ofȱmoreȱpotentȱanaloguesȱofȱGLPȬ1ȱandȱcovalentȱinhibitorsȱofȱBcl2ȱ(82,ȱ83).ȱȱȱȱ

Figureȱ8.ȱOverviewȱofȱdifferentȱchemistriesȱutilizingȱonlyȱaminoȱacidȱbuildingȱblocksȱforȱpeptideȱstapling.ȱ (A)ȱOlefinȱringȱclosingȱmetathesisȱ(B)ȱPeptideȱcouplingȱ(C)ȱazideȬalkyneȱcycloȬadditionȱ(clickȱchemistry)ȱ(D)ȱ Disuflideȱbridgeȱformationȱ(E)ȱThioetherȱformationȱȱ

AnotherȱwellȬknownȱclassȱofȱpeptidomimeticsȱisȱtheȱfoldamers,ȱwhichȱpossessȱinherentȱ propensitiesȱforȱformingȱsecondaryȱstructuresȱandȱareȱoftenȱusedȱtoȱmimicȱhelices.ȱInȱ thisȱcaseȱmainlyȱbackboneȱmodificationsȱleadȱtoȱaȱstabilizationȱofȱtheȱhelix.ȱTheȱconceptȱ wasȱintroducedȱinȱtheȱ1990s,ȱwhereȱitȱwasȱfoundȱthatȱpeptidesȱcomposedȱsolelyȱofȱEȬ aminoȱacidsȱfoldedȱintoȱhelicalȱstructuresȱ(84).ȱBasedȱonȱtheirȱsubstitutionȱpatternȱonȱtheȱ carbonȱatomsȱ2ȱandȱ3ȱtheȱbetaȱaminoȱacidsȱcanȱbeȱdividedȱintoȱtheȱtypesȱE2,ȱE3ȱandȱE2,3ȱ (Figureȱ 9).ȱ Theȱ introductionȱ ofȱ EȬaminoȱ acidsȱalsoȱ rendersȱ theȱ peptidesȱ highlyȱstableȱ towardsȱ proteolysis.ȱ However,ȱ theȱ EȬpeptidesȱ doȱ notȱ formȱ DȬhelicesȱ andȱ asȱ aȱ consequenceȱtheirȱsideȱchainsȱdoȱnotȱresembleȱtheȱsameȱpatternsȱasȱofȱDȬhelicalȱpeptides.ȱ WithȱtheȱpurposeȱofȱdevelopingȱpeptidesȱadoptingȱtheȱnativeȱDȬhelicalȱformationȱyetȱ stableȱtoȱproteolysis,ȱGellmanȱandȱcoȬworkersȱdesignedȱhybridȱpeptidesȱcomposedȱofȱEȬȱ andȱ DȬaminoȱ acidsȱ (85).ȱ Ifȱ theȱ distributionȱ andȱ proportionȱ betweenȱ theȱ twoȱ buildingȱ

16 ,QWURGXFWLRQ blocksȱ isȱ properlyȱ chosen,ȱ theȱ resultingȱ peptideȱ veryȱ closelyȱ resemblesȱ anȱ DȬhelix.ȱ PatternsȱsuchȱasȱDDE,ȱDDDE,ȱandȱDDEDDDEȱhaveȱbeenȱfoundȱbyȱcrystallographyȱtoȱformȱ DȬhelicalȱsecondaryȱstructures.ȱTheȱhybridȱformatsȱofȱD/Eȱ,ȱD/J,ȱE/Jȱpeptidesȱasȱwellȱasȱ theȱdifferentȱtypesȱofȱEȬaminoȱacidsȱtogetherȱwithȱtheȱintroductionȱofȱtheȱhelixȱinducingȱ unnaturalȱbuildingȱblockȱaminoȱisobutyricȱacidȱ(Aib,ȱFigureȱ9)ȱprovidesȱaȱhighȱlevelȱofȱ diversityȱ forȱ foldamersȱ (86).ȱ Peptideȱ basedȱ foldamersȱ haveȱ beenȱ generatedȱ againstȱ aȱ wideȱ rangeȱ ofȱ therapeuticȱ targetsȱ andȱ provideȱ aȱ promisingȱ formatȱ forȱ furtherȱ developmentȱ(87).ȱȱ

Figureȱ9.ȱ(A)ȱBackboneȱreplacementsȱofȱtheȱamideȱbondȱutilizedȱinȱfoldamers.ȱ(B)ȱModificationsȱofȱtheȱsideȱ chainsȱonȱbuildingȱblocksȱinȱfoldamers.ȱ(C)ȱConstrainedȱbuildingȱblocksȱandȱEȬaminoȱacidsȱforȱsecondaryȱ structureȱ stabilizationȱ Reprintedȱ withȱ permissionȱ fromȱ (88)ȱ reprintingȱ licenseȱ numberȱ 4270191028188,ȱ©ȱ 2014ȱTheȱRoyalȱSocietyȱofȱChemistry.ȱ

Inȱtheȱgenerationȱofȱfoldamers,ȱnotȱonlyȱEȬaminoȱacidsȱhaveȱbeenȱusedȱforȱbackboneȱ modifications.ȱ Theȱ replacementȱ ofȱ amideȱ bondsȱ inȱ theȱ peptideȱ backboneȱ withȱ ester,ȱ thioester,ȱ thioamide,ȱ alkeneȱ orȱ triazoleȱ heterocyclesȱ influenceȱ theȱ hydrogenȱ bondingȱ patternsȱofȱtheȱstructureȱandȱcanȱbeȱusedȱtoȱdesignȱfoldamersȱwithȱenhancedȱpropertiesȱ (Figureȱ9)ȱ(88).ȱConformationallyȱconstrainedȱcycloalkaneȱEȱorȱJȱaminoȱacidȱbuildingȱ blocksȱhaveȱalsoȱbeenȱusedȱinȱtheȱbackboneȱtoȱstabilizeȱeitherȱhelicalȱorȱsheetȱsecondaryȱ structuresȱ(87,ȱ88).ȱInȱoneȱrecentȱexample,ȱGrisonȱandȱcoȬworkersȱdesignȱaȱmimicȱofȱtheȱ p53ȱ helicalȱ structureȱ usingȱ anȱ alternatingȱ sequenceȱ ofȱ transȬ2Ȭ aminocyclobutanecarboxylicȱacidȱandȱJȬaminoȱacidsȱ(89).ȱTheȱpeptidesȱwereȱdesignedȱ soȱthatȱinteractionȱformingȱresiduesȱwouldȱremainȱinȱtheȱsameȱpositionȱasȱinȱtheȱnativeȱ p53.ȱTheȱconstructedȱpeptidesȱefficientlyȱmimickedȱtheȱDȬhelicalȱstructure,ȱwereȱresistantȱ againstȱ proteolysisȱ andȱ displayedȱ strongȱ targetȱ affinity.ȱȱInȱ aȱ similarȱ approachȱ 5Ȭ

17 ,QWURGXFWLRQ memberedȱthiazoleȬbasedȱJȬaminoȱacidsȱwereȱusedȱbyȱMathieuȱetȱal.ȱinȱanȱalternatingȱ fashionȱtoȱinduceȱhelicalȱconformationsȱ(90).ȱȱ

Whileȱoneȱfocusȱofȱbackboneȱmodificationȱhasȱbeenȱsecondaryȱstructureȱmimicking,ȱtheȱ strategyȱcanȱatȱtheȱsameȱtimeȱbeȱusedȱforȱimprovingȱproteolyticȱstabilityȱofȱpeptide,ȱasȱ highlightedȱ byȱ Gellmanȱ andȱ othersȱ (85).ȱ Inȱ aȱ systematicȱ studyȱ ofȱ 4ȱ commonlyȱ usedȱ backboneȱmodificationsȱ(NȬmethylation,ȱCȬDȬmethylation,ȱEȬaminoȱacidȱincorporationȱ andȱDȬaminoȱacidȱincorporation)ȱWernerȱetȱal.ȱfoundȱsignificantȱdifferencesȱbetweenȱtheȱ modificationsȱinȱtheȱabilityȱtoȱprotectȱaȱpeptideȱsequenceȱagainstȱdegradationȱ(91).ȱTheȱ mostȱefficientȱmodificationȱwasȱtheȱintroductionȱofȱDȬaminoȱacids.ȱAlsoȱtheȱpositionȱofȱ theȱ backboneȱ modificationȱ influencedȱ theȱ levelȱ ofȱ protection.ȱ Interestingly,ȱ theȱ CȬDȬ methylatedȱAibȱalsoȱprovidedȱaȱhighȱlevelȱofȱprotection,ȱhenceȱthereȱmightȱbeȱaȱdemandȱ ofȱ DȬmethylatedȱ aminoȱ acidsȱ bearingȱ naturalȱ (orȱ unnatural)ȱ sideȱ chainsȱ forȱ targetȱ interactions.ȱ However,ȱ theȱ studyȱ usedȱ anȱ artificialȱ peptideȱ sequenceȱ andȱ onlyȱ chymotrypsinȱ asȱ proteolyticȱ pressure,ȱ thereforeȱ theȱ resultsȱ areȱ notȱ fullyȱ representingȱ degradationȱofȱbiologicallyȱactiveȱpeptidesȱinȱbloodȱplasma,ȱbutȱcouldȱbeȱconsideredȱasȱ anȱ indicationȱ ofȱ howȱ toȱ effectivelyȱ improveȱ proteolyticȱ stability.ȱ Aȱ simpleȱ yetȱ veryȱ efficientȱbackboneȱmodificationȱisȱtheȱreplacementȱofȱaȱpeptideȱbondȱwithȱaȱthioamideȱ bondȱ nearȱ orȱ atȱ theȱ proteolyticȱ cleavageȱ site.ȱ Thisȱ approachȱ wasȱ recentlyȱ utilizedȱ toȱ improveȱtheȱstabilityȱofȱtherapeuticallyȱrelevantȱpeptidesȱGLPȬ1ȱandȱgastricȱinhibitoryȱ polypeptideȱ(GIP)ȱ(92).ȱAȱsingleȱbackboneȱmodificationȱimprovedȱtheȱstabilityȱofȱtheseȱ peptidesȱ 750Ȭfoldȱ asȱ comparedȱ toȱ theȱ nativeȱ sequences,ȱ withoutȱ compromisingȱ theȱ biologicalȱactivity.ȱȱ

TheȱgroupȱofȱCaiȱhaveȱusedȱextensiveȱbackboneȱmodificationȱforȱgeneratingȱaȱnewȱclassȱ ofȱpeptidomimetics;ȱJȬAAȬpeptidesȱ(Figureȱ10)ȱ(93,ȱ94).ȱTheȱconstructȱwasȱinspiredȱbyȱ theȱ structureȱ ofȱ peptideȱ nucleicȱ acidsȱ (PNA)ȱ inȱ whichȱ theȱ basesȱ areȱ attachedȱ toȱ theȱ nitrogenȱ atomȱ inȱ aȱ NȬ(2Ȭaminoethyl)ȱ glycineȱ backbone.ȱ Theȱ JȬAAȬpeptidesȱ consistȱ ofȱ suchȱbuildingȱblocksȱwithȱfunctionalitiesȱintroducedȱviaȱsideȬchainsȱatȱtheȱnitrogenȱatomȱ andȱatȱtheȱJȬcarbon.ȱTheȱpeptidesȱareȱbuiltȱupȱusingȱSPPSȱandȱmacrocyclicȱstructuresȱcanȱ beȱ achievedȱ throughȱ variousȱ approachesȱ suchȱ asȱ headȬtoȬtailȱ cyclizationȱ orȱ thioetherȱ linkageȱ(95).ȱThisȱmolecularȱformatȱisȱhighlyȱstableȱandȱhasȱbeenȱshownȱtoȱmimicȱvariousȱ propertiesȱofȱbiologicallyȱactiveȱpeptides,ȱsuchȱasȱcellȱpenetrationȱandȱhelicalȱstructures.ȱ Moreover,ȱ combinatorialȱ librariesȱ ofȱ JȬAAȬpeptidesȱ haveȱ beenȱ constructedȱ usingȱ theȱ OBOCȱapproachȱandȱpeptidomimeticsȱagainstȱseveralȱtargetsȱhaveȱbeenȱisolated.ȱȱ

Otherȱ notableȱ backboneȱ modificationsȱ ofȱ peptidesȱ includeȱ theȱ replacementȱ ofȱ theȱ DȬ carbonȱbyȱnitrogenȱwhichȱrendersȱazapeptides.ȱTheȱpeptidesȱcanȱbeȱconstructedȱhavingȱ aȱsideȬchainȱattachedȱtoȱtheȱnitrogenȱinȱDȬpositionȱorȱwithoutȱsideȬchain.ȱIntroductionȱofȱ azapeptideȱbondsȱinducesȱEȬturnȱmotifsȱandȱproperȱincorporationȱcanȱbeȱidentifiedȱbyȱ “azascan”ȱ (96).ȱ Aȱ numberȱ ofȱ otherȱ strategiesȱ forȱ backboneȱ extensionȱ incorporatingȱ

18 ,QWURGXFWLRQ heteroatomsȱ haveȱ alsoȱ beenȱ demonstratedȱ suchȱ asȱ DȬaminoxypeptides,ȱ DȬ hydrazionpeptidesȱandȱEȬaminoxypeptides.ȱ

EȬsheetȱmimeticsȱareȱlessȱcommonȱinȱtheȱliteratureȱasȱcomparedȱtoȱDȬhelixȱmimeticsȱbutȱ strategiesȱ toȱ induceȱ andȱ stabilizeȱ thisȱ secondaryȱ structureȱ inȱ peptidomimeticsȱ exist.ȱ Threeȱ generalȱ approachesȱ areȱ commonȱ namelyȱ theȱ useȱ ofȱ turnȱ mimetics,ȱ macrocyclizationȱandȱEȬstrandȱenforcingȱaminoȱacidsȱ(68).ȱTurnȱinducingȱaminoȱacidsȱ thatȱ haveȱ beenȱ usedȱ includeȱ LȬPro,ȱ DȬProȱ andȱ Gly.ȱ Alsoȱ conformationallyȱ restrictedȱ bicyclicȱdipeptideȱbuildingȱblocksȱhaveȱbeenȱusedȱforȱthisȱpurposeȱ(97).ȱ

Figureȱ10.ȱ(A)ȱTheȱsynthesisȱofȱtheȱJȬAAȬpeptidesȱisȱperformedȱusingȱmetalȱcatalysisȱandȱstandardȱpeptideȱ solidȱphaseȱchemistry.ȱTheȱdiversityȱofȱtheȱlibraryȱisȱintroducedȱviaȱtheȱsideȱchainȱofȱtheȱbuildingȱblockȱasȱ wellȱasȱthroughȱtheȱreactionȱwithȱaȱwideȱrangeȱofȱcommerciallyȱavailableȱcarboxylicȱacidsȱorȱacetylȱchlorides.ȱ (B)ȱTheȱNȬprotectedȱbuildingȱblocksȱareȱavailableȱthroughȱtwoȱsyntheticȱroutes.ȱReprintedȱwithȱpermissionȱ fromȱ(93),ȱ©ȱ2016ȱAmericanȱChemicalȱSocietyȱ ȱ

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Peptideȱtherapeuticsȱcanȱbeȱdevelopedȱdeȱnovoȱbyȱscreeningȱlargeȱlibrariesȱofȱpeptidesȱ withȱrandomȱsequences.ȱAȱnumberȱofȱdisplayȱsystemsȱhaveȱevolvedȱinȱorderȱtoȱfacilitateȱ theȱscreeningȱofȱlargeȱpeptideȱlibraries.ȱInȱgeneralȱanȱinȱvitroȱdisplayȱsystemȱconsistsȱofȱ threeȱsteps:ȱtheȱgenerationȱofȱaȱcombinatorialȱlibraryȱwhereȱphenotypeȱisȱlinkedȱtoȱgenȬ otype,ȱmultipleȱroundsȱofȱbiopanningȱandȱidentificationȱofȱtheȱselectedȱcompoundsȱ(98).ȱ Theȱsystemȱmostȱusedȱisȱphageȱdisplay.ȱInȱphageȱdisplayȱtheȱDNAȱofȱbacteriophagesȱisȱ modifiedȱsoȱthatȱaȱcombinatorialȱpeptideȱorȱproteinȱsequenceȱisȱfusedȱtoȱoneȱofȱtheȱcoatȱ proteinsȱonȱtheȱsurfaceȱofȱtheȱphageȱ(Figureȱ11).ȱAȱrandomȱlibraryȱcanȱbeȱconstructedȱ usingȱNNKȱcodons,ȱtheȱsizeȱandȱstructureȱofȱtheȱlibraryȱcanȱbeȱselectedȱaccordingȱtheȱ typeȱ ofȱ theȱ desiredȱ molecularȱ format.ȱ Inȱ thisȱ wayȱ phenotypeȱ (displayedȱ peptide)ȱ isȱ linkedȱtoȱgenotypeȱ(DNAȱofȱphage).ȱTheȱdiversityȱofȱaȱphageȱdisplayȱlibraryȱisȱtypicallyȱ aroundȱ109Ȭ1011,ȱandȱlibrariesȱareȱrequiredȱtoȱbeȱdesignedȱaccordinglyȱinȱtermsȱofȱsize.ȱ Twoȱdifferentȱcoatȱproteinsȱareȱnormallyȱused;ȱpVIIIȱwhichȱcanȱdisplayȱmultipleȱcopiesȱ ofȱtheȱcombinatorialȱpeptidesȱorȱPIIIȱwhichȱdisplaysȱ1Ȭ5ȱcopies.ȱTheȱphageȱlibraryȱisȱtypȬ icallyȱscreenedȱagainstȱanȱimmobilizedȱproteinȱtargetȱandȱseveralȱroundsȱofȱbiopanningȱ

19 ,QWURGXFWLRQ ensureȱtheȱenrichmentȱofȱbindingȱclones.ȱTheseȱareȱsubsequentlyȱDNAȱsequenced,ȱlatelyȱ byȱnextȱgenerationȱsequencingȱ(NGS)ȱwhichȱallowsȱforȱthoroughȱsequencingȱandȱanalyȬ sisȱofȱtheȱoutputȱphageȱpool.ȱPhageȱdisplayȱhasȱbeenȱusedȱextensivelyȱforȱtheȱidentificaȬ tionȱofȱbindersȱtoȱtherapeuticȱtargetsȱandȱhasȱyieldedȱoverȱ40ȱbiologicalȱdrugȱcandidatesȱ whichȱhaveȱenteredȱclinicalȱtrials,ȱoutȱofȱwhichȱmostȱareȱmonoclonalȱantibodiesȱ(99).ȱAȱ numberȱofȱtheȱphageȱdisplayȱidentifiedȱbiologicalsȱhaveȱbeenȱapprovedȱasȱdrugs,ȱincludȬ ingȱAdalimumab,ȱtheȱworld’sȱbestȬsellingȱdrugȱwithȱannualȱrevenueȱofȱ16.1ȱbillionȱUSDȱ inȱ2016ȱ(100).ȱPhageȱdisplayȱofȱpeptideȱlibrariesȱhaveȱalsoȱyieldedȱdrugsȱwhichȱareȱusedȱ inȱtheȱclinic,ȱforȱexampleȱromiplostim,ȱtrebananibȱandȱpeginesatide,ȱtheȱlatterȱhoweverȱ withdrawnȱ(44,ȱ99,ȱ101).ȱ

Figureȱ11.ȱInȱphageȱdisplayȱaȱcombinatorialȱlibraryȱofȱpeptidesȱisȱdisplayedȱonȱtheȱcoatȱproteinȱofȱaȱfilamenȬ tousȱphage.ȱTheȱpeptidesȱareȱencodedȱbyȱDNAȱencapsulatedȱinȱtheȱphage.ȱGenerally,ȱlibrariesȱareȱsubjectedȱ toȱseveralȱroundsȱofȱselectionȱagainstȱanȱimmobilizedȱtarget.ȱAfterȱeachȱround,ȱbindersȱareȱamplifiedȱbyȱ infectionȱofȱbacteria.ȱHitȱsequencesȱareȱidentifiedȱbyȱDNAȱsequencing.ȱ

WithȱtheȱpurposeȱofȱovercomingȱtheȱintrinsicȱlimitationsȱofȱpeptideȱdrugsȱandȱtoȱenȬ hanceȱbinding,ȱaȱnumberȱofȱstrategiesȱhaveȱbeenȱintroducedȱintoȱphageȱdisplay.ȱChemȬ icalȱmodificationsȱandȱvariousȱcyclizationȱtechniquesȱhaveȱbeenȱutilizedȱ(102).ȱSomeȱnoȬ tableȱinnovativeȱapproachesȱofȱusingȱphageȱdisplayȱincludeȱtheȱmodificationȱofȱpeptidesȱ withȱcarbohydratesȱ(103)ȱandȱencodingȱofȱpeptideȱmodificationsȱusingȱsilentȱbarcodesȱ (104).ȱMirrorȱimageȱphageȱdisplayȱisȱanotherȱtechniqueȱwhichȱhasȱbeenȱusedȱtoȱdevelopȱ DȬantagonistsȱtoȱHIVȱandȱcancerȱrelatedȱtargetsȱ(105,ȱ106).ȱAȱbicyclicȱpeptideȱformatȱhasȱ beenȱusedȱtogetherȱwithȱphageȱdisplayȱforȱdevelopingȱligandsȱforȱaȱwideȱrangeȱofȱtargetsȱ

20 ,QWURGXFWLRQ andȱwillȱbeȱreviewedȱinȱdetailȱinȱchapterȱ1.3.ȱOtherȱinȱvitroȱdisplayȱmethodsȱwhichȱutiȬ lizeȱaȱcellȱbasedȱsystemȱareȱbacteriaȱandȱyeastȱdisplay,ȱinȱwhichȱpeptideȱlibrariesȱareȱ displayedȱonȱtheȱcellȱsurface.ȱ

Inȱvitroȱdisplaysȱsystemsȱwhichȱomitȱtheȱuseȱofȱaȱcellȱbasedȱplatformȱareȱalsoȱavailable.ȱ InȱmRNAȱdisplayȱtheȱcombinatorialȱpeptidesȱareȱfusedȱtoȱtheirȱencodingȱmRNAȱdirectlyȱ (Figureȱ12A).ȱInȱcontrastȱtoȱphageȱdisplay,ȱtheseȱapproachesȱdoȱnotȱrequireȱaȱtransforȬ mationȱstep,ȱwhichȱisȱtheȱmainȱlimitationȱforȱdiversityȱ(98).ȱmRNAȱdisplayȱlibrariesȱcanȱ readilyȱreachȱaȱdiversityȱofȱ1013.ȱTheseȱsystemsȱalsoȱhaveȱanotherȱmajorȱadvantageȱoverȱ phageȱdisplay,ȱwhichȱisȱtheȱpotentialȱofȱincorporatingȱunnaturalȱbuildingȱblocksȱdirectlyȱ inȱtheȱcombinatorialȱlibrary.ȱThisȱpotentialȱhasȱbeenȱwidelyȱexploitedȱbyȱSugaȱandȱcoȬ workersȱbyȱtheȱdevelopmentȱofȱtheȱFITȱandȱRaPIDȱsystemsȱwhichȱallowȱforȱtheȱinȱvitroȱ selectionȱofȱpeptidesȱincorporatingȱvirtuallyȱanyȱnaturalȱorȱunnaturalȱaminoȱacidȱ(107,ȱ 108).ȱTheȱcoreȱofȱtheȱFITȱsystemȱareȱartificialȱribozymes,ȱ‘flexizymes’,ȱwhichȱallowȱtransȬ esterificationȱofȱtheȱ30ȬhydroxyȱgroupȱofȱtRNAȱwithȱanȱactivatedȱaminoȱacidȱofȱchoice.ȱ WhenȱcombinedȱwithȱanȱmRNAȱdisplayȱsystemȱitȱisȱcalledȱRaPID.ȱThisȱapproachȱhasȱ beenȱusedȱtoȱidentifyȱpeptidesȱtoȱdifferentȱtherapeuticȱtargets,ȱmacrocyclicȱpeptidesȱusȬ ingȱvariousȱcyclizationȱchemistriesȱandȱpeptidesȱlikelyȱtoȱbeȱcellȬpermeableȱ(109–111).ȱAȱ differentȱinȱvitroȱscreeningȱtechnologyȱtermedȱ‘splitȬinteinȱcircularȱligationȱofȱpeptidesȱ andȱproteins’ȱ(SICLOPPS),ȱutilizesȱtheȱsplitȬinteinȱmechanismȱtoȱgenerateȱlibrariesȱofȱcyȬ clicȱ peptidesȱ expressedȱ intracellularlyȱ (112).ȱ Thisȱ approachȱ isȱ particularlyȱ powerfulȱ whenȱusedȱinȱaȱreverseȬtwoȬhybridȱsystem,ȱforȱexampleȱinȱE.ȱcoli.ȱȱ

Oneȱapproachȱwhichȱinȱcontrastȱtoȱtheȱaboveȱmentionedȱinȱvitroȱscreeningȱplatformsȱ doesȱnotȱuseȱanyȱbiologicalȱsystemȱisȱtheȱOBOCȱlibraryȱ(Figureȱ12B)ȱ(113,ȱ114).ȱHereȱ combinatorialȱpeptideȱlibrariesȱareȱsynthesizedȱonȱsolidȱsupportȱusingȱtheȱsplitȱandȱpoolȱ technique.ȱThisȱallowsȱforȱaȱhighȱdiversityȱandȱforȱtheȱincorporationȱofȱunnaturalȱbuildȬ ingȱblocks.ȱSimilarȱtoȱtheȱinȱvitroȱtechniquesȱtheȱlibrariesȱareȱscreenedȱandȱbindersȱidenȬ tified.ȱThisȱcanȱtypicallyȱbeȱdoneȱeitherȱbyȱEdmanȱsequencing,ȱmassȱspectrometryȱorȱ latelyȱbyȱDNAȱencodingȱ(115).ȱForȱexampleȱtheȱOBOCȱtechniqueȱhasȱbeenȱusedȱtoȱdisȬ coverȱtumorȱtargetingȱpeptidesȱ(116).ȱȱȱȱȱȱȱȱ

Severalȱ deȱ novoȱ developedȱ cyclicȱ peptidesȱ areȱ present.ȱ Polyphorȱ hasȱ usedȱ aȱ proteinȱ epitopeȱmimickingȱ(PEM)ȱdiscoveryȱplatformȱtoȱdevelopȱcyclicȱpeptidesȱforȱvariousȱtarȬ getsȱ(117,ȱ118).ȱThisȱwasȱdoneȱbyȱgraftingȱpharmacophoresȱofȱnaturallyȱoccurringȱhostȬ defenseȱpeptidesȱontoȱPEMȱscaffoldsȱfollowedȱbyȱiterativeȱsynthesisȱandȱoptimization.ȱ MurepavadinȱandȱbalixafortideȱareȱantimicrobialȱandȱantiȬcancerȱcompoundsȱdevelopedȱ usingȱthisȱtechnology.ȱOtherȱdeȱnovoȱdevelopedȱcyclicȱpeptidesȱincludeȱAPLȬ2,ȱALRNȬ 6924ȱandȱRA101495ȱ(44).ȱTheȱpeptidesȱwereȱdevelopedȱbyȱphageȱdisplayȱandȱmRNAȱ displayȱtechnologies.ȱȱ

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Figureȱ12.ȱ(A)ȱOverviewȱofȱtheȱmRNAȬdisplayȱinȱvitroȱselectionȱprocess.ȱAfterȱtranscriptionȱofȱtheȱlibrary,ȱ theȱmRNAȱisȱmodifiedȱwithȱpuromycinȱatȱtheȱ3’ȱend.ȱUponȱtranslationȱofȱtheȱmRNA,ȱtheȱpeptideȱisȱcovaȬ lentlyȱboundȱtoȱtheȱmRNAȱthatȱencodedȱit.ȱȱTheȱmRNAȱisȱthenȱreverseȱtranscribedȱtoȱcDNAȱandȱselectionȱ isȱperformedȱagainstȱanȱimmobilizedȱtarget.ȱTheȱmRNAȱportionȱofȱthisȱdisplayȱconstructȱisȱthenȱreverseȱ transcribed.ȱTheȱboundȱpeptidesȱareȱelutedȱandȱtheirȱencodingȱDNAȱisȱthenȱamplifiedȱbyȱPCR,ȱthusȱcomȬ pletingȱtheȱfirstȱroundȱofȱselectionȱandȱamplification.ȱ(B)ȱOBOCȱlibrariesȱcanȱbeȱreadilyȱconstructedȱusingȱ traditionalȱcombinatorialȱchemistryȱemployingȱsplitȬandȬpoolȱsynthesis.ȱTheȱmodularȱnatureȱofȱaminoȱacidsȱ makesȱitȱidealȱforȱpeptideȱlibraries,ȱbutȱtheȱtechniqueȱcanȱbeȱusedȱforȱanyȱtypeȱofȱmodularȱchemistry.ȱTheȱ solidȱsupport,ȱconsistingȱofȱbeads,ȱisȱsplitȱintoȱequalȱpartsȱwhichȱisȱeachȱreactedȱwithȱaȱdifferentȱreagent.ȱ Theȱbeadsȱareȱthenȱpooledȱtogetherȱandȱsubsequentlyȱsplitȱagainȱandȱreactedȱwithȱanotherȱbuildingȱblock.ȱ Theseȱstepsȱareȱrepeatedȱuntilȱtheȱdesiredȱsizeȱofȱtheȱlibraryȱisȱreached.ȱTheȱlibraryȱisȱthenȱscreenȱagainstȱaȱ targetȱandȱbindingȱcompoundsȱareȱidentified,ȱforȱexampleȱusingȱaȱDNAȱtagȱorȱbyȱMS.ȱAȱreprintedȱwithȱ permissionȱfromȱ(98),ȱreprintingȱlicenseȱnumberȱ4264261154793ȱ©ȱ2016ȱTheȱRoyalȱSocietyȱofȱChemistry.ȱȱ

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Whileȱpeptidesȱhaveȱmanyȱpropertiesȱthatȱmakeȱthemȱsuitableȱforȱdrugȱdevelopment,ȱ theyȱ alsoȱ possessȱ severalȱ limitationsȱ whichȱ needȱ toȱ beȱ overcome.ȱ Peptideȱ leadȱ comȬ poundsȱoftenȱhaveȱtoȱbeȱimprovedȱbyȱchemicalȱmeansȱinȱorderȱtoȱcombatȱtheseȱshortȬ comings.ȱȱOneȱofȱtheȱmostȱpronouncedȱdrawbacksȱofȱtheȱpeptideȱformatȱisȱtheȱoftenȱhighȱ susceptibilityȱtoȱproteolyticȱcleavage,ȱwhichȱrendersȱpeptideȱdrugsȱmetabolicallyȱunstaȬ ble.ȱInȱaddition,ȱtheyȱareȱgenerallyȱrapidlyȱclearedȱfromȱtheȱcirculationȱdueȱtoȱexcretion.ȱ AnotherȱlimitationȱofȱpeptideȬbasedȱdrugsȱisȱtheirȱpoorȱmembraneȱpermeability.ȱThisȱ limitsȱtheȱuseȱofȱpeptidesȱforȱintracellularȱtargetsȱandȱforȱadministrationȱorally.ȱAsȱwithȱ smallȱmoleculeȱdrugs,ȱmodificationsȱcanȱalsoȱbeȱrequiredȱtoȱinfluenceȱtheȱaffinityȱofȱtheȱ peptide.ȱMedicinalȱchemistryȱapproachesȱareȱsometimesȱnecessaryȱinȱtheȱdevelopmentȱ ofȱhormoneȬbasedȱpeptideȱdrugsȱtoȱtailorȱtheȱaffinityȱforȱcertainȱreceptorȱsubtypesȱinȱ orderȱtoȱachieveȱtheȱdesiredȱpharmacologicalȱeffect.ȱInȱaȱsimilarȱmanner,ȱtheȱspecificityȱ ofȱ peptideȱ drugsȱ canȱ beȱ adjustedȱ withȱ theȱ purposeȱ ofȱ minimizingȱ offȬtargetȱ bindingȱ whichȱcanȱresultȱinȱsideȬeffects.ȱForȱdeȱnovoȱdevelopedȱpeptideȱleadȱcompounds,ȱitȱisȱ oftenȱrequiredȱtoȱimproveȱtheȱaffinityȱand/orȱspecificityȱofȱtheȱinitialȱhitȱcompounds.ȱȱ

Aȱnumberȱofȱdifferentȱstrategiesȱexistȱforȱtheȱimprovementȱofȱtheȱpharmacodynamicȱandȱ pharmacokineticȱpropertiesȱofȱpeptides.ȱTheȱperhapsȱmostȱwidelyȱusedȱapproachȱtoȱadȬ dressȱtheȱsusceptibilityȱtoȱproteolyticȱcleavageȱofȱpeptideȱdrugsȱisȱtheȱintroductionȱofȱDȬ aminoȱacids.ȱIfȱDȬaminoȱacidȱinsertionȱisȱtoleratedȱinȱtermsȱofȱaffinity,ȱspecificityȱetc.ȱitȱ canȱbeȱaȱusefulȱstrategyȱtoȱrenderȱpeptidesȱlessȱsusceptibleȱtoȱproteolyticȱcleavage.ȱTheȱ DȬaminoȱacidȱcanȱbeȱusedȱtoȱreplaceȱanȱaminoȱacidȱatȱaȱknownȱorȱidentifiedȱproteolyticȱ cleavageȱsite.ȱAsȱtheȱDȬaminoȱacidȱisȱnotȱrecognizedȱbyȱtheȱprotease,ȱtheȱpeptideȱremainsȱ intact.ȱAȱdifferentȱapproachȱtoȱprotectȱpeptidesȱfromȱexopeptidaseȱcleavageȱisȱtoȱprotectȱ theȱendȱofȱtheȱpeptides.ȱTheȱprincipleȱisȱagainȱtoȱmakeȱtheȱendsȱlessȱrecognizableȱbyȱtheȱ proteases.ȱThisȱcanȱbeȱachievedȱbyȱacetylationȱofȱtheȱNȬterminalȱand/orȱamidationȱofȱCȬ terminal.ȱAlsoȱotherȱalkylationȱchemistriesȱcanȱbeȱused.ȱAnotherȱstrategyȱwithȱtheȱsameȱ goalȱisȱtoȱintroduceȱorȱmodifyȱcyclizationȱofȱtheȱpeptideȱbackbone.ȱCyclicȱpeptidesȱareȱ generallyȱmoreȱstableȱthanȱlinearȱpeptides.ȱTherefore,ȱagainȱifȱallowedȱinȱtermsȱofȱtargetȱ binding,ȱcyclizationȱofȱaȱlinearȱpeptideȱleadȱcanȱbeȱusefulȱforȱimprovingȱstability.ȱAlsoȱ replacementȱofȱanȱexistingȱcyclicȱbackboneȱwithȱaȱdifferentȱcyclizationȱchemistryȱcanȱbeȱ used.ȱ Inȱ chapterȱ 1.2.2ȱ backboneȱ modificationsȱ ofȱ proteinȱ epitopeȱ mimeticsȱ wereȱ disȬ cussed.ȱManyȱbackboneȱmodificationsȱhaveȱbeenȱusedȱtoȱimproveȱtheȱstabilityȱofȱpeptideȱ leadȱcompounds.ȱThisȱincludesȱtheȱintroductionȱofȱEȬaminoȱacids.ȱSimilarlyȱtoȱtheȱ DȬ aminoȱacidsȱtheyȱareȱnotȱrecognizedȱbyȱproteases.ȱAdditionally,ȱpeptideȱbondȱsurrogatesȱ areȱusedȱwithȱtheȱsameȱpurposeȱ(Figureȱ9).ȱȱ

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Theȱpharmacokineticsȱofȱpeptidesȱcanȱalsoȱbeȱaddressedȱusingȱchemicalȱmodifications.ȱ Theseȱcanȱbeȱbasedȱonȱconjugations.ȱThisȱincludesȱtheȱconjugationsȱtoȱlargerȱproteinsȱ whichȱremainȱlongerȱinȱtheȱcirculationȱsuchȱasȱalbuminȱorȱFcȱfragments,ȱbutȱalsoȱtheȱ conjugationȱtoȱpolyȱethyleneȱglycolȱ(PEG)ȱofȱvariousȱchainȱlength,ȱwhichȱcanȱlargelyȱimȬ proveȱtheȱplasmaȱhalfȬlifeȱofȱpeptides.ȱInsteadȱofȱaȱcovalentȱconjugation,ȱaȱmoietyȱwhichȱ bindsȱalbumin,ȱforȱexampleȱaȱfattyȱacid,ȱcanȱbeȱintroducedȱinȱtheȱpeptideȱforȱprolongingȱ theȱhalfȬlife.ȱAsȱshownȱbyȱtheȱbasalȱinsulinȱanalogues,ȱmodificationsȱwhichȱaffectȱselfȬ aggregationȱcanȱalsoȱbeȱusedȱtoȱtailorȱpharmacokinetics.ȱȱ

DifferentȱtypesȱofȱmethylationȱpatternsȱhaveȱbeenȱappliedȱinȱpeptideȱmedicinalȱchemisȬ tryȱinȱorderȱtoȱimproveȱtheȱproperties,ȱmainlyȱmembraneȱpermeability,ȱofȱpeptideȱleadȱ compounds.ȱOneȱofȱtheȱmostȱcommonȱmethylationȱsitesȱwithinȱpeptidesȱisȱtheȱamideȱ bondȱnitrogenȱatomȱinȱtheȱbackboneȱofȱtheȱpeptide.ȱNȬmethylationȱfunctionalitiesȱareȱ presentȱinȱnature,ȱtheȱmostȱcommonȱbeingȱhistoneȱNȬmethylation,ȱwhichȱplaysȱanȱimȬ portantȱroleȱinȱepigeneticsȱ(119).ȱInȱthisȱcaseȱtheȱmethylationȱoccursȱonȱaȱlysineȱorȱargiȬ nineȱsideȱchain.ȱNoȱNȬmethylatedȱpeptideȱbondsȱhaveȱbeenȱobservedȱinȱmammals,ȱhowȬ everȱ nonȬribosomallyȱ synthesizedȱ peptidesȱ fromȱ differentȱnaturalȱ sourcesȱ containȱNȬ methylatedȱpeptideȱbonds.ȱTheseȱpeptidesȱhaveȱbeenȱfoundȱtoȱhaveȱaȱvarietyȱofȱbiologȬ icalȱ functions.ȱ Mostȱ ofȱ theȱ naturallyȱ occurringȱ NȬmethylatedȱ peptidesȱ areȱ foundȱ inȱ sourcesȱsuchȱasȱfungi,ȱbacteriaȱorȱplants.ȱExamplesȱincludeȱenniatins,ȱhemiasterlins,ȱechiȬ nomycinȱandȱbouvardin.ȱAȱwellȬknownȱexampleȱofȱaȱNȬmethylatedȱpeptideȱisȱcycloȬ sporineȱA,ȱwhichȱhasȱbeenȱmentionedȱearlierȱinȱtheȱchapterȱaboutȱcyclicȱpeptideȱtheraȬ peutics.ȱCyclosporineȱAȱhasȱaȱheadȬtoȬtailȱcyclizedȱscaffoldȱandȱ7ȱoutȱofȱ11ȱpeptideȱbondsȱ areȱ NȬmethylated.ȱ Theȱ peptideȱ isȱ orallyȱ availableȱ andȱ diffusesȱ passivelyȱ overȱ memȬ branesȱtargetingȱintracellularȱPPI.ȱNȬmethylationȱofȱamideȱbondsȱprovideȱaȱnumberȱofȱ propertiesȱforȱpeptidesȱwhichȱareȱattractiveȱinȱdrugȱdevelopment.ȱUponȱNȬmethylationȱ theȱpeptidesȱareȱlessȱsusceptibleȱtoȱproteolysisȱandȱmoreȱimportantlyȱitȱcanȱincreaseȱoralȱ availabilityȱandȱcellȱpermeabilityȱ(120).ȱHowever,ȱtheȱmodificationȱinfluencesȱconforȬ mationȱofȱtheȱpeptideȱbondȱasȱwellȱasȱtheȱdirectionsȱofȱtheȱsideȬchainsȱsurroundingȱtheȱ NȬmethylatedȱpeptideȱbond.ȱInȱaddition,ȱhydrogenȱbondingȱcapabilityȱisȱdemolished,ȱ influencingȱtheȱsecondaryȱstructureȱofȱtheȱpeptide.ȱBesidesȱNȬmethylation,ȱpeptidesȱareȱ sometimesȱalsoȱmethylatedȱatȱtheȱDȬcarbon.ȱHowever,ȱthisȱapproachȱisȱnotȱasȱcommon,ȱ likelyȱdueȱtoȱitsȱsyntheticȱcomplexityȱandȱlackȱofȱcommerciallyȱavailableȱbuildingȱblocksȱ (121).ȱOneȱDȬmethylatedȱaminoȱacidȱwhichȱisȱwidelyȱusedȱisȱAibȱ(alphaȱmethylatedȱAla).ȱ Asȱmentionedȱabove,ȱitȱisȱoftenȱusedȱtoȱinduceȱhelicesȱbutȱhasȱalsoȱbeenȱusedȱforȱproteȬ olyticȱstabilityȱimprovement.ȱȱ

AȱdifferentȱformȱofȱNȬalkylatedȱpeptideȱbackboneȱisȱtheȱpeptoidȱformat.ȱAȱpeptoidȱresiȬ dueȱisȱaȱNȬsubstitutedȱglycineȱwithȱaȱsideȱchainȱattachedȱtoȱtheȱbackboneȱnitrogenȱinȬ steadȱofȱtheȱDȬcarbonȱasȱinȱnormalȱpeptidesȱ(122).ȱPeptoidsȱareȱhighlyȱresistantȱtoȱproteȬ olyticȱcleavageȱdueȱtoȱtheȱlackȱofȱbackboneȱamideȱbonds.ȱTheyȱhaveȱalsoȱbeenȱshownȱtoȱ

24 ,QWURGXFWLRQ haveȱsuperiorȱmembraneȱpermeabilityȱasȱcomparedȱtoȱpeptides,ȱwithȱupȱtoȱ26ȬfoldȱinȬ creaseȱobservedȱ(123,ȱ124).ȱȱ

WhileȱmanyȱofȱtheȱmedicinalȱchemistryȱstrategiesȱmentionedȱaboveȱareȱusedȱforȱimprovȬ ingȱstability,ȱhalfȬlifeȱorȱpermeability,ȱtheyȱcanȱalsoȱbeȱusedȱforȱimprovingȱorȱtailoringȱ targetȱaffinityȱandȱspecificityȱofȱpeptideȱdrugs.ȱWhenȱworkingȱwithȱtheseȱpropertiesȱitȱ canȱalsoȱbeȱofȱgreatȱimportanceȱtoȱvaryȱtheȱsideȬchainsȱofȱtheȱpeptide.ȱByȱintroducingȱ unnaturalȱaminoȱacids,ȱtheȱinteractionsȱofȱtheȱpeptideȱwithȱitsȱtargetȱcanȱbeȱstronglyȱ influenced.ȱAȱwideȬrangeȱofȱprotectedȱunnaturalȱaminoȱacidȱbuildingȱblocksȱareȱcomȬ merciallyȱavailable,ȱallowingȱforȱfacileȱsynthesisȱandȱrapidȱscreeningȱofȱpeptideȱlibraries.ȱȱȱ

Figureȱ13.ȱAȱnumberȱofȱdifferentȱscanningȱstrategiesȱareȱutilizedȱinȱorderȱtoȱidentifyȱpositionsȱinȱtheȱpeptideȱ sequenceȱsusceptibleȱforȱoptimization,ȱforȱexampleȱinȱtermsȱofȱaffinityȱand/orȱstability.ȱ(A)ȱAlanineȱscanȱ(B)ȱ DȬaminoȱacidȱscanȱ(C)ȱProlineȱscanȱ(D)ȱNȬmethylationȱscanȱ(E)ȱStaplingȱscanȱ(F)ȱAzaȱpeptideȱscanȱ

Itȱisȱsometimesȱnecessaryȱtoȱidentifyȱpositionsȱwithinȱtheȱpeptideȱwhichȱcanȱbeȱtargetedȱ byȱmedicinalȱchemistryȱapproaches.ȱForȱexample,ȱproteolyticȱcleavageȱsitesȱorȱpharmaȬ cophoresȱcanȱbeȱidentified.ȱAȱnumberȱofȱscanningȱstrategiesȱhaveȱemergedȱwhereȱpepȬ tideȱleadsȱareȱvariedȱsystematicallyȱinȱorderȱtoȱidentifyȱimportantȱresiduesȱforȱbindingȱ butȱalsoȱpositionȱwereȱvariationsȱtoȱtheȱnaturalȱpeptideȱstructureȱareȱallowedȱinȱorderȱtoȱ increaseȱstabilityȱ(Figureȱ13)ȱ(125).ȱTheȱsimplestȱmethodȱisȱalanineȱscanning,ȱinȱwhichȱ singleȱresiduesȱareȱmutatedȱtoȱalanineȱinȱorderȱtoȱidentifyȱtheȱsideȱchainsȱresponsibleȱforȱ binding.ȱOtherȱscanningȱstrategiesȱincludeȱDȬaminoȱacidȱscan,ȱprolineȱscan,ȱNȬmethylȬ atedȱscan,ȱlactamȱscan,ȱpeptideȬstapleȱscanȱandȱazaȬaminoȱacidȱscan.ȱManyȱofȱtheseȱscanȬ ningȱtechniquesȱstriveȱtoȱallowȱforȱtheȱincorporationȱofȱunnaturalȱbuildingȱblocksȱand/orȱ changeȱtoȱtheȱbackboneȱstructureȱofȱtheȱpeptide.ȱTheȱtypeȱofȱscanȱutilizedȱisȱconstantlyȱ evolvingȱandȱdependsȱonȱfactorsȱsuchȱasȱleadȱpeptide,ȱtarget,ȱandȱtimeȱconstraints.ȱȱ

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([DPSOHVRIPRGLILHGSHSWLGHV

Anȱearlyȱexampleȱthatȱnicelyȱillustratesȱtheȱpowerȱofȱmedicinalȱchemistryȱappliedȱtoȱ peptidesȱisȱdesmopressin,ȱanȱanalogueȱofȱvasopressin,ȱtheȱplasmaȱosmolalityȱregulatingȱ hormone.ȱVasopressinȱisȱrapidlyȱdegradedȱinȱvivo,ȱwithȱaȱhalfȬlifeȱofȱonlyȱ4Ȭ20ȱminutesȱ (9).ȱDesmopressinȱwasȱdevelopedȱinȱtheȱ1960sȱandȱsubstitutingȱtheȱArgȬ8ȱresidueȱinȱvasȬ opressinȱtoȱDȬArgȱwasȱdoneȱinȱorderȱtoȱreduceȱproteolyticȱcleavageȱbyȱtrypsin.ȱInȱaddiȬ tion,ȱdesmopressinȱalsoȱhasȱaȱdeaminatedȱNȬterminal,ȱwhichȱprotectsȱfromȱexopeptidaseȱ cleavage.ȱTheseȱmodificationsȱresultedȱinȱaȱhighlyȱstableȱvasopressinȱanalogue,ȱwithȱanȱ eliminationȱhalfȬlifeȱofȱmoreȱthanȱ3ȱhȱ(126).ȱTheȱhighȱstabilityȱalsoȱprovidedȱaȱfoundationȱ forȱalternativeȱformulationsȱsuchȱasȱoralȱorȱnasalȱ(127).ȱItȱisȱworthȱmentioningȱthatȱtheȱ modificationsȱalsoȱresultedȱinȱaȱreceptorȱselectivity,ȱ(desmopressinȱisȱselectiveȱforȱV2,ȱ whereasȱvasopressinȱisȱanȱagonistȱofȱV1ȱandȱV2)ȱwhichȱrendersȱdesmopressinȱantidiuȬ reticȱbutȱwithȱlessȱvasoconstrictorȱactivityȱ(128).ȱOtherȱvasopressinȱanaloguesȱhaveȱbeenȱ developedȱinȱparallelȱwithȱdesmopressin,ȱsuchȱasȱterlipressin,ȱwhichȱretainsȱtheȱvasoȬ constrictorȱactivityȱbutȱhasȱincreasedȱstabilityȱthroughȱnaturalȱaminoȱacidȱsubstitutionȱ andȱadditions,ȱnamelyȱArg8ÆLysȱandȱGlyȬGlyȬGlyȱatȱNȬtermȱ(9,ȱ129).ȱExtensiveȱmedicȬ inalȱchemistryȱapproachesȱhaveȱbeenȱundertakenȱinȱorderȱtoȱtailorȱtheȱreceptorȱselectivȬ ityȱofȱvasopressinȱanalogues,ȱincludingȱtheȱuseȱofȱunnaturalȱaminoȱacidsȱ(130).ȱȱȱȱ

Anotherȱprominentȱexampleȱwhereȱpeptideȱengineering,ȱincludingȱtheȱuseȱofȱDȬaminoȱ acids,ȱwasȱappliedȱtoȱimproveȱproteolyticȱstabilityȱisȱtheȱdevelopmentȱofȱoctreotide,ȱanȱ analogueȱofȱsomatostatinȱ(Figureȱ14).ȱInȱthisȱcase,ȱtheȱpharmacophoreȱofȱtheȱpeptideȱwasȱ identifiedȱasȱresiduesȱ7Ȭ10ȱinȱtheȱcyclicȱ14Ȭmerȱ(131).ȱTheȱcyclicȱstructureȱwasȱkept,ȱwithȱ twoȱcysteineȱresiduesȱflankingȱtheȱpharmacophoreȱsequenceȱ(131,ȱ132).ȱInȱtheȱsequenceȱ TrpȱwasȱreplacedȱbyȱDȬTrp.ȱHowever,ȱthisȱpeptideȱwasȱinitiallyȱlessȱactiveȱthanȱnaturalȱ somatostatin.ȱTheȱactivityȱwasȱregainedȱbyȱintroducingȱDȬPheȱatȱtheȱNȬterminusȱandȱaȱ threonolȱbuildingȱblockȱatȱtheȱCȬterminusȱinȱorderȱtoȱmimicȱtheȱresiduesȱofȱnativeȱsomaȬ tostatin.ȱTheȱhalfȬlifeȱofȱsomatostatinȱisȱnormallyȱ2Ȭ3ȱminutesȱwhereasȱoctreotideȱhasȱaȱ halfȬlifeȱofȱ90Ȭ120ȱminȱ(133).ȱItȱshouldȱbeȱmentionedȱthoughȱthatȱsimilarlyȱtoȱdesmopresȬ sin,ȱoctreotideȱhasȱanȱalteredȱreceptorȱaffinityȱprofileȱasȱcomparedȱtoȱtheȱnaturalȱleadȱ peptide.ȱOtherȱsomatostatinȱanaloguesȱhaveȱalsoȱbeenȱdeveloped,ȱinȱ whichȱsyntheticȱ buildingȱblocksȱareȱincluded,ȱnamelyȱlanreotideȱandȱpasireotide.ȱLanreotideȱhasȱsimilarȱ pharmacodynamicsȱasȱoctreotide,ȱwhereasȱpasireotideȱwasȱengineeredȱtoȱhaveȱaȱmoreȱ universalȱbindingȱprofileȱsimilarȱtoȱnativeȱsomatostatinȱ(10,ȱ134,ȱ135).ȱInȱadditionȱtoȱtheȱ aboveȱmentionedȱpharmacophore,ȱtheȱdevelopersȱofȱpasireotideȱidentifiedȱLys4,ȱPhe6ȱ andȱPhe11ȱasȱimportantȱforȱreceptorȱactivity.ȱForȱimprovedȱstabilityȱitȱwasȱdecidedȱtoȱ useȱaȱhexamericȱpeptideȱscaffoldȱratherȱthanȱanȱoctamerȱasȱusedȱinȱoctreotideȱandȱlanreȬ otide.ȱTheȱLys4ȱisȱincorporatedȱasȱhydroxyprolineȱaminoethylurethaneȱresidue,ȱwhichȱ isȱalsoȱusedȱforȱreplacementȱofȱtheȱdisulfideȱbridgeȱwithȱaȱbackboneȱcyclization.ȱPhe6ȱisȱ notȱrepresented,ȱperhapsȱtheȱprolineȱringȱresemblesȱitsȱfunctionality.ȱPhe7ȱisȱrepresentedȱ

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byȱanȱoptimizedȱunnaturalȱphenylglycine,ȱTrp8ȱisȱreplacedȱbyȱDȬTrp,ȱLys9ȱandȱPhe11ȱ areȱkeptȱnative.ȱInterestingly,ȱThr10ȱisȱnotȱidentifiedȱasȱessentialȱforȱuniversalȱreceptorȱ binding.ȱThisȱresidueȱisȱhoweverȱreplacedȱbyȱOȬbenzylȬtyrosine.ȱMoreȱrecently,ȱworkȱ hasȱbeenȱundertakenȱtoȱreplaceȱtheȱPheȬresiduesȱinȱtheȱnativeȱsomatostatinȱsequenceȱbyȱ unnaturalȱanaloguesȱwithȱdecreasedȱorȱincreasedȱelectronȱdensityȱinȱtheȱaromaticȱringsȱ (136,ȱ137).ȱByȱdoingȱso,ȱtheȱauthorsȱmanagedȱtoȱtuneȱtheȱreceptorȱtypeȱaffinityȱasȱwellȱasȱ toȱimproveȱtheȱstabilityȱofȱtheȱpeptide.ȱAȱfurtherȱstudyȱonȱsomatostatinȱanaloguesȱcouldȱ adjustȱtheȱreceptorȱspecificityȱandȱantagonism/agonismȱbyȱtheȱincorporationȱofȱNȬimidȬ azolebenzylȬhistidineȱorȱnaphtylalanineȱinȱtheȱTrp8ȱpositionȱ(138).ȱInȱanotherȱstudy,ȱtheȱ authorsȱ incorporateȱ alphaȬdialkylatedȱ residuesȱ withȱ theȱ purposeȱ ofȱ constrainingȱ theȱ backboneȱofȱtheȱmacrocycleȱ(139).ȱȱ

Figureȱ14.ȱSeveralȱanaloguesȱhaveȱbeenȱdevelopedȱusingȱtheȱpeptideȱhormoneȱsomatostatinȱasȱleadȱsequenceȱ (upperȱleft).ȱTheȱpharmacophoreȱisȱgenerallyȱidentifiedȱasȱaminoȱacidsȱF7,ȱW8,ȱK9ȱandȱT10.ȱReprintedȱwithȱ permissionȱfromȱ(135)ȱreprintingȱlicenseȱnumberȱ4264280910391,ȱ©ȱ2012ȱGolorȱetȱal.ȱ

Thereȱareȱfurtherȱexamplesȱwhereȱunnaturalȱaminoȱacidsȱhaveȱbeenȱusedȱtoȱimproveȱ pharmacokinetics.ȱCarbetocinȱusedȱoxytocinȱasȱaȱleadȱpeptideȱandȱitȱwasȱfoundȱthatȱbyȱ deaminatingȱtheȱNȬterminusȱandȱsubstitutingȱtheȱdisulfideȱbridgeȱwithȱaȱthioetherȱtheȱ durationȱofȱactionȱwasȱincreasedȱ(140).ȱFurther,ȱtheȱhydroxylȱgroupȱofȱaȱtyrosineȱwasȱ

27 ,QWURGXFWLRQ changedȱtoȱaȱmethyloxyl.ȱTogetherȱtheseȱchangesȱimprovedȱtheȱhalfȬlifeȱmoreȱthanȱ10Ȭ foldȱtoȱ42ȱminutesȱ(141).ȱThereȱareȱhoweverȱsomeȱindicationsȱofȱchangedȱpharmacodyȬ namicsȱofȱcarbetocinȱasȱcomparedȱtoȱnativeȱoxytocinȱ(141,ȱ142).ȱSeveralȱanaloguesȱofȱtheȱ nineȱaminoȱacidȱinflammatoryȱmediatorȱbradykininȱhaveȱbeenȱdevelopedȱthroughȱexȬ tensiveȱmedicinalȱchemistryȱcampaignsȱforȱbothȱagonisticȱandȱantagonisticȱfunctionsȱasȱ wellȱasȱreceptorȱsubtypeȱspecificityȱ(143).ȱȱOfȱthese,ȱtheȱonlyȱapprovedȱdrugȱisȱicatibant,ȱ whichȱwasȱapprovedȱinȱ2008ȱforȱtheȱtreatmentȱofȱhereditaryȱangioedemaȱ(144).ȱTheȱpepȬ tideȱisȱcomposedȱoutȱofȱfiveȱnaturalȱresiduesȱandȱfiveȱunnatural,ȱincludingȱthreeȱprolineȱ derivativesȱ(145).ȱTheȱmodificationsȱrenderȱitȱproteolyticallyȱstableȱwithȱaȱhalfȬlifeȱofȱȱ1Ȭ 2ȱhoursȱ(146).ȱTheȱdevelopmentȱofȱsomatostatinȱandȱoxytocinȱanaloguesȱhighlightȱtheȱ risingȱimportanceȱofȱunnaturalȱaminoȱacidsȱandȱmuchȱworkȱisȱbeingȱundertakenȱinȱtheȱ developmentȱofȱtheirȱsynthesisȱandȱincorporationȱinȱdrugsȱ(147–149).ȱ

Systematicȱscanningȱapproachesȱhaveȱbeenȱusedȱtoȱimproveȱpropertiesȱofȱpeptidesȱandȱ aȱfewȱexamplesȱareȱdiscussedȱbelow.ȱMirandaȱandȱcoȬworkersȱwereȱableȱtoȱsubstantiallyȱ improveȱtheȱstabilityȱofȱcalcitoninȱgeneȬrelatedȱpeptideȱreceptorȱantagonistsȱbyȱsystemȬ aticallyȱreplacingȱlabileȱresiduesȱbyȱcitrulline,ȱhomoarginineȱandȱotherȱunnaturalȱbuildȬ ingȱblocksȱ(150).ȱFreyȱetȱalȱstudiedȱtheȱSARȱofȱBakȱderivedȱpeptidesȱandȱwereȱableȱtoȱ improveȱtheȱpotencyȱbyȱintroducingȱnonȬcanonicalȱaminoȱacidsȱ(151).ȱMurrayȱandȱcoȬ workersȱusedȱanȱenhancedȱformȱofȱtheȱalanineȱscanȱtermedȱmultiȱattributeȱpositionalȱ scan,ȱsubstitutingȱresiduesȱofȱtheȱleadȱpeptideȱnotȱonlyȱbyȱalanineȱbutȱalsoȱbyȱrepresentaȬ tiveȱbasic,ȱacidicȱandȱhydrophobicȱresiduesȱ(152).ȱTheȱauthorsȱwereȱtherebyȱableȱtoȱimȬ proveȱpropertiesȱofȱpeptideȱtoxinȱleadȱcompoundsȱtargetingȱionȱchannels.ȱȱȱȱȱȱȱ

InȱanȱinterestingȱapproachȱBironȱandȱcoȬworkersȱappliedȱaȱNȬmethylȱscanȱusingȱtheȱsoȬ matostatinȱanalogueȱVeberȬHirschmannȱcyclicȱhexapeptideȱasȱaȱleadȱcompoundȱ(Figureȱ 15)ȱ(153).ȱȱ

Figureȱ15.ȱTheȱVeberȬHirschmannȱpeptideȱ(right)ȱisȱaȱsomatostatinȱanalogueȱselectiveȱtowardsȱreceptorȱsubȬ typesȱsst2ȱandȱsst5,ȱadministeredȱbyȱinjection.ȱTheȱpharmacophoreȱofȱsomatostatinȱisȱindicatedȱinȱred.ȱAȱ libraryȱscanȱofȱNȬmethylatedȱleadȱcompoundsȱshowedȱthatȱNȬmethylationȱinȱtheȱindicatedȱpositionsȱreȬ sultedȱinȱcompoundsȱwithȱretainedȱaffinityȱandȱoralȱavailability.ȱAdaptedȱwithȱpermissionȱfromȱ(154),ȱ©ȱ 2008ȱAmericanȱChemicalȱSocietyȱ

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TheȱobjectiveȱwasȱtoȱmakeȱanȱorallyȱavailableȱcompoundȱwithȱretainedȱbiologicalȱactivȬ ity,ȱwhichȱcouldȱpotentiallyȱbeȱusedȱasȱaȱsubstituteȱofȱoctreotide.ȱByȱincorporatingȱ3ȱNȬ methylationsȱ aȱ cyclicȱ peptidomimeticȱ wasȱ obtainedȱ havingȱ 10%ȱ oralȱ availability.Theȱ leadȱpeptideȱwithoutȱNȬmethylationsȱshowedȱnoȱoralȱavailability.ȱInȱaddition,ȱitȱwasȱ demonstratedȱ thatȱ theȱ NȬmethylationsȱ increasedȱ eliminationȱ halfȬlifeȱ fivefoldȱ toȱ 74ȱ minutes.ȱTheȱsameȱgroup,ȱledȱbyȱprofȱKessler,ȱhaveȱperformedȱgroundbreakingȱworkȱ relatedȱtoȱoptimizationȱofȱtheȱsynthesisȱofȱNȬmethylatedȱpeptidesȱandȱaminoȱacidsȱasȱ wellȱasȱinȱtheȱinvestigationȱofȱtheȱconformationalȱinfluenceȱofȱmultipleȱNȬmethylationȱinȱ cyclicȱpeptidesȱ(154).ȱȱ

TheȱgroupȱofȱLokeyȱareȱperformingȱextensiveȱresearchȱinȱtheȱfieldȱofȱmembraneȱpermeȬ abilityȱandȱdiffusionȱofȱcyclicȱpeptides.ȱTheyȱhaveȱusedȱaȱlibraryȱbasedȱapproachȱsynȬ thesizingȱ1200ȱderivativesȱofȱnaturalȱmembraneȱpermeableȱpeptidesȱinȱorderȱtoȱidentifyȱ newȱscaffoldsȱforȱcellȱpermeabilityȱ(155).ȱInȱtheȱstudyȱitȱwasȱfoundȱthatȱasȱexpectedȱNȬ methylationȱstronglyȱincreasesȱpermeability,ȱbutȱonlyȱwhenȱappliedȱonȱsolventȱexposedȱ amideȱ bonds.ȱ Alsoȱ theȱ importanceȱ ofȱ sideȬchainsȱ isȱ highlighted,ȱ permeabilityȱ canȱ beȱ achievedȱbyȱintroducingȱlipophilicityȱviaȱtheȱsideȬchains.ȱSimilarȱconclusionsȱwereȱmadeȱ aboutȱanaloguesȱofȱtheȱorallyȱavailableȱSanguinamideȱA,ȱwhereȱitȱwasȱfoundȱthatȱNȬ methylationȱofȱaȱsolventȱexposedȱpeptideȱbondȱincreasedȱpermeabilityȱand,ȱsurprisingly,ȱ solubilityȱ(156).ȱItȱshouldȱbeȱnotedȱthatȱtheȱincreaseȱinȱmembraneȱpermeabilityȱisȱhighlyȱ dependentȱonȱtheȱpositionȱofȱtheȱNȬmethylation.ȱInterestingly,ȱpermeabilityȱwasȱalsoȱ influencedȱbyȱtheȱstructureȱofȱaȱsideȬchainȱadjacentȱtoȱtheȱNȬmethylationȱsite.ȱRemovalȱ ofȱaȱEȬbranchȱinȱtheȱsideȱchainȱstronglyȱincreasedȱpermeability,ȱlikelyȱdueȱtoȱconformaȬ tionalȱflexibility.ȱȱ

Theȱtraditionalȱmodelȱofȱaȱnaturallyȱoriginatingȱpeptideȱbeingȱcellȱpermeableȱandȱorallyȱ availableȱisȱcyclosporineȱA.ȱHowever,ȱmanyȱotherȱnaturallyȱoccurringȱcyclicȱpeptidesȱofȱ differentȱscaffoldsȱhaveȱalsoȱrecentlyȱbeenȱidentifiedȱasȱmembraneȱpermeableȱbyȱpassiveȱ diffusion,ȱhavingȱinȱcommonȱwithȱcyclosporineȱtheȱheavyȱNȬmethylationȱpatternȱ(157).ȱ Theȱabilityȱofȱtheȱformationȱofȱintramolecularȱhydrogenȱbondsȱwhichȱareȱnotȱsolventȱ exposedȱinȱnonpolarȱsolventsȱisȱcrucialȱforȱmembraneȱpermeability.ȱTheȱmostȱpermeableȱ compoundsȱhadȱeveryȱamideȱbondȱinvolvedȱinȱeitherȱintramolecularȱhydrogenȱbondsȱorȱ NȬmethylated.ȱTheȱinvestigationsȱbyȱLokeyȱandȱcoȬworkersȱhighlightȱtheȱpotentialȱofȱNȬ methylationsȱforȱincreasingȱmembraneȱpermeability,ȱhoweverȱtheȱpositionȱisȱhighlyȱimȬ portantȱandȱotherȱparametersȱsuchȱasȱtheȱlipophilicityȱandȱconformationȱofȱsideȬchainsȱ alsoȱhaveȱaȱstrongȱinfluence.ȱTheȱsideȬchainsȱcanȱbeȱusedȱtoȱshieldȱpolarȱregionsȱofȱtheȱ peptideȱandȱflexibilityȱcanȱbalanceȱhydrophobicityȱwithȱsolubilityȱ(158).ȱTheseȱmodifiȬ cationsȱtogetherȱwithȱmacrocyclizationȱhasȱtheȱpotentialȱofȱgeneratingȱorallyȱavailableȱ peptidesȱbeyondȱtheȱruleȱofȱ5.ȱȱ

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Naturally,ȱmodificationsȱsuchȱasȱNȬmethylationȱcanȱalsoȱalterȱbindingȱpropertiesȱofȱpepȬ tides.ȱ Scanlonȱ andȱ coȬworkersȱ usedȱ aȱ systematicȱ NȬmethylationȱ scanȱ toȱ improveȱ theȱ pharmacokineticȱpropertiesȱofȱaȱphageȱdisplayȱderivedȱlinearȱpeptideȱbindingȱtoȱmalariaȱ relatedȱtargetȱAMAȬ1ȱ(159,ȱ160).ȱMultipleȱNȬmethylationsȱindeedȱimprovedȱtheȱproteoȬ lyticȱstabilityȱ30Ȭfold,ȱbutȱalsoȱincreasedȱtheȱpotencyȱofȱtheȱinhibitor.ȱAsȱdescribedȱearlier,ȱ variousȱ medicinalȱ chemistryȱ approachesȱ haveȱ resultedȱ inȱ theȱ alternationȱ ofȱ receptorȱ specificityȱofȱsomatostatinȱanalogues.ȱToȱthisȱendȱNȬmethylationȱhasȱalsoȱbeenȱusedȱtoȱ increaseȱtheȱactivityȱforȱtheȱsst5ȱreceptorȱ(161).ȱInȱanotherȱstudy,ȱSkoghȱetȱal.ȱperformedȱ aȱNȬmethylȱscanȱusingȱsubstanceȱPȱ1Ȭ7,ȱaȱpainȱrelivingȱneuroactiveȱpeptide,ȱasȱaȱleadȱ compoundȱ(162).ȱWhileȱNȬmethylation,ȱespeciallyȱwhenȱpresentȱatȱseveralȱsites,ȱstronglyȱ increasedȱtheȱstabilityȱofȱtheȱcompound,ȱitsȱpotencyȱwasȱdiminished.ȱTheseȱstudiesȱshowȱ thatȱNȬmethylationȱisȱaȱusefulȱstrategyȱforȱimprovingȱpermeabilityȱandȱstabilityȱofȱpepȬ tideȱleadȱcompounds,ȱbutȱcareȱhasȱtoȱbeȱtakenȱsinceȱtargetȱbindingȱcanȱbeȱcompromisedȱ orȱaltered.ȱȱȱ

ThereȱhaveȱbeenȱcasesȱreportedȱwhereȱsystematicȱcarbonȬDȱmethylationȱscanȱhasȱbeenȱ used,ȱbutȱwithȱlimitedȱsuccessȱ(163).ȱAȱnumberȱofȱDȬcarbonȱmethylatedȱaminoȱacidsȱwithȱ sideȬchainsȱlongerȱthanȱalanineȱhaveȱbeenȱusedȱinȱpeptidesȱtoȱstabilizeȱhelicalȱstructuresȱ (164).ȱTheȱtypeȱofȱhelixȱisȱaffectedȱbyȱtheȱsideȬchainȱofȱtheȱaminoȱacid.ȱInȱseveralȱstudiesȱ cyclicȱquaternaryȱaminoȱacidsȱwereȱusedȱtoȱinduceȱhelicalȱconformationsȱ(165–167).ȱȱȱȱ

ByȱsystematicallyȱsubstitutingȱpeptideȱresiduesȱwithȱpeptoidȱresiduesȱinȱaȱcyclicȱhexȬ apeptideȱ theȱ cellȱ permeabilityȱ wasȱ enhancedȱ (Figureȱ 16)ȱ (168).ȱ However,ȱ asȱ withȱ NȬ methylation,ȱtheȱpositionȱisȱhighlyȱimportant.ȱTheȱintrinsicȱpropertiesȱrenderȱpeptoidsȱ highlyȱflexibleȱinȱtheirȱstructure,ȱwhichȱcanȱimpairȱtargetȱselectivityȱand/orȱbinding.ȱTheȱ inherentȱstructuralȱflexibilityȱisȱrelatedȱtoȱtheȱlackȱofȱsideȬchainsȱinȱDȬpositionȱandȱlackȱ ofȱhydrogenȱbondingȱthroughȱtheȱbackboneȱnitrogen.ȱPeptoidsȱhaveȱbeenȱstudiedȱforȱ variousȱtherapeuticȱapplications.ȱAȱnumberȱofȱexamplesȱexitsȱwhereȱpeptoidsȱareȱindeedȱ mimickingȱsecondaryȱstructuresȱsuchȱasȱhelices,ȱloopsȱandȱturnsȱ(169).ȱInȱsomeȱcases,ȱtheȱ flexibilityȱofȱtheȱpeptoidsȱisȱevenȱbeneficialȱforȱtargetȱbinding.ȱDespiteȱtheȱstructuralȱflexȬ ibility,ȱpeptoidsȱwereȱdesignedȱasȱDȬhelicalȱmimeticsȱincorporatingȱnaturalȱandȱunnatuȬ ralȱsideȬchainsȱ(170).ȱKnownȱantimicrobialȱpeptidesȱwereȱusedȱasȱleadȱcompounds.ȱTheȱ peptoidsȱdemonstratedȱhelicityȱandȱantimicrobialȱactivity.ȱAsȱhighlightedȱinȱtheȱexamȬ pleȱabove,ȱreplacingȱaȱpeptideȱbackboneȱwithȱaȱpeptoidȱcanȱhaveȱbeneficialȱeffectsȱonȱ membraneȱpermeability,ȱbutȱalsoȱstabilityȱcanȱbeȱimproved.ȱParkȱetȱal.ȱhaveȱreportedȱanȱ elegantȱwayȱofȱidentifyingȱpeptoidȱreplaceableȱresiduesȱwithinȱaȱleadȱpeptideȱbyȱapplyȬ ingȱaȱsarcosineȱscanȱcombinedȱwithȱalanineȱandȱprolineȱscansȱ(171).ȱTheȱresultingȱpepȬ tideȬpeptoidȬhybridȱshowedȱonlyȱslightȱlossȱinȱactivity.ȱLikelyȱthisȱapproachȱcouldȱalsoȱ beȱusedȱonȱotherȱpeptideȱleadȱmoleculesȱinȱorderȱtoȱidentifyȱpositionsȱwereȱpeptoidȱsideȬ chainsȱcanȱincreaseȱbindingȱaffinity.ȱMoreoverȱnewȱsideȱchainsȱcouldȱeasilyȱbeȱintroȬ

30 ,QWURGXFWLRQ ducedȱinȱsuchȱaȱposition.ȱInȱanotherȱstudyȱaȱmacrocyclicȱpeptideȬpeptoidȱhybridȱisȱdeȬ velopedȱthroughȱextensiveȱmedicinalȱchemistryȱapproaches,ȱallowedȱthroughȱtheȱhighȱ diversityȱofȱpeptoidȱbuildingȱblocksȱ(172).ȱByȱreplacingȱaminoȱacids,ȱpeptoidȱsideȱchainsȱ andȱNȬmethylation,ȱtheȱpotencyȱofȱaȱCXCR7ȱinhibitorȱwasȱimprovedȱbyȱmoreȱthanȱ230Ȭ fold.ȱInȱaddition,ȱpassiveȱpermeabilityȱandȱoralȱavailabilityȱofȱ18%ȱwasȱachieved.ȱȱȱȱ

Figureȱ16.ȱSubstitutionȱofȱNȬmethylatedȱpeptideȱresiduesȱintoȱpeptoidȱresiduesȱincreasedȱmembraneȱperȬ meabilityȱofȱaȱhexapeptide.ȱAsȱindicatedȱinȱtheȱlowerȱrightȱpanel,ȱtheȱpositionȱisȱimportant.ȱReprintedȱwithȱ permissionȱfromȱ(168),ȱ©ȱ2015ȱAmericanȱChemicalȱSocietyȱ

Theȱrelativelyȱfacileȱsynthesisȱofȱpeptoidsȱhasȱalsoȱbeenȱutilizedȱinȱtheȱgenerationȱandȱ screeningȱofȱlargeȱsyntheticȱlibraries.ȱMuchȱworkȱhasȱbeenȱundertakenȱbyȱtheȱgroupȱofȱ Kodadek.ȱTraditionallyȱtheyȱhaveȱusedȱOBOCȱpeptoidȱlibrariesȱencodedȱbyȱMS.ȱLatelyȱ theyȱhaveȱbeenȱutilizingȱdifferentȱapproachesȱinȱorderȱtoȱfacilitateȱscreeningȱofȱtheseȱliȬ braries.ȱByȱscreeningȱseveralȱlibrariesȱwithȱderivativesȱfromȱprimaryȱhitsȱandȱbyȱobtainȬ ingȱSARȱinȱvariousȱwaysȱincludingȱusingȱanȱequivalentȱofȱalanineȱscanningȱ(peptoidȱÆȱ

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NȬmethylȱscan)ȱtheyȱwereȱableȱtoȱdevelopȱsubȬmicromolarȱbindersȱtoȱMMPȬ14ȱ(173).ȱOneȱ limitationȱofȱsuchȱlibrariesȱisȱtheȱencodingȱstepȱbyȱMS,ȱwhichȱsometimesȱisȱnotȱableȱtoȱ identifyȱtheȱstructureȱofȱhitȱcompoundsȱdueȱtoȱpoorȱorȱcomplicatedȱfragmentation.ȱThisȱ canȱlimitȱtheȱnumberȱandȱtypeȱofȱbuildingȱblocksȱusedȱinȱtheȱlibrary.ȱToȱovercomeȱthisȱ drawbackȱDNAȱencodedȱlibrariesȱofȱpeptoidsȱwereȱrecentlyȱdevelopedȱ(115,ȱ174).ȱToȬ getherȱwithȱNGSȱandȱFluorescenceȬactivatedȱcellȱsortingȱ(FACS)ȱthisȱallowsȱforȱfastȱandȱ deepȱanalysisȱofȱhitȱstructuresȱandȱforȱincorporationȱofȱaȱrangeȱofȱbuildingȱblocksȱwhichȱ wereȱpreviouslyȱfoundȱtoȱbeȱdifficultȱtoȱencode,ȱsuchȱasȱheterocycleȱcontainingȱhaloacidsȱ andȱchloropentenoicȱacids.ȱȱȱȱȱ

Asȱ highlightedȱ byȱ theȱ studiesȱ above,ȱ peptoidsȱ canȱ beȱ usedȱ inȱ librariesȱ toȱ achieveȱ anȱ enourmousȱdiversityȱfarȱexceedingȱtheȱdiversityȱofȱaȱpeptideȱlibrary.ȱThisȱisȱdueȱtoȱtheȱ highȱavailabilityȱofȱprimaryȱamines.ȱPeptoidsȱcanȱalsoȱbeȱusedȱinȱaȱsimilarȱfashionȱasȱNȬ methylation,ȱinȱorderȱtoȱimproveȱstabilityȱandȱpermeabilityȱofȱleadȱcompounds.ȱPeptideȬ peptoidȱhybridsȱisȱaȱpromisingȱformatȱwhichȱcanȱovercomeȱintrinsicȱlimitationȱofȱbothȱ classesȱofȱmolecules.ȱȱȱȱ

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Asȱdescribedȱinȱpreviousȱsectionsȱofȱthisȱthesisȱthereȱareȱmanyȱdifferentȱstrategiesȱofȱ developingȱtherapeuticȱpeptidesȱandȱaȱnumberȱofȱdifferentȱformatsȱexist.ȱOneȱsuccessfulȱ approachȱhasȱbeenȱcyclization,ȱwhichȱprovidesȱmanyȱadvantagesȱoverȱlinearȱpeptides.ȱ Withinȱthisȱpeptideȱformatȱthereȱisȱaȱspecialȱbranch;ȱbicyclicȱpeptides,ȱonȱwhichȱweȱareȱ focusingȱinȱourȱlaboratory.ȱȱ

Asȱcyclicȱpeptidesȱinȱgeneral,ȱbicyclicȱpeptidesȱareȱalsoȱpresentȱinȱnature,ȱandȱhaveȱbeenȱ foundȱtoȱbeȱbioactiveȱinȱvariousȱfunctionsȱ(175).ȱTheȱformatȱisȱattractiveȱasȱaȱnewȱmodalȬ ityȱinȱdrugȱdiscoveryȱdueȱtoȱitsȱincreasedȱstabilityȱandȱrigidityȱasȱcomparedȱtoȱlinearȱandȱ monocyclicȱpeptides.ȱTheȱbicyclicȱstructureȱallowsȱforȱaȱlargerȱinteractionȱsurface,ȱwithȬ outȱinducingȱtooȱmuchȱflexibilityȱinȱtheȱscaffoldȱandȱwithoutȱcompromisingȱtheȱproteoȬ lyticȱstability.ȱSeveralȱformatsȱandȱcyclizationȱstrategiesȱhaveȱbeenȱreportedȱforȱtheȱgenȬ erationȱ ofȱ bicyclicȱ peptides,ȱ suchȱ asȱ theȱ combinationȱ ofȱ headȬtoȬtailȬamide/thioether,ȱ NCLȬamide/disulfide,ȱringȬclosingȱmetathesis/ringȬclosingȱmetathesisȱandȱothers.ȱBicyȬ clicȱpeptideȱleadȱcompoundsȱhaveȱbeenȱdevelopedȱusingȱaȱnumberȱofȱapproachesȱinȬ cludingȱrationalȱdesign,ȱphageȱdisplay,ȱmRNAȱdisplay,ȱSICLOPPSȱandȱoneȬbeadȬtwoȬ compoundsȱlibraries.ȱȱȱ

TheȱbicyclicȱpeptideȱformatȱusedȱinȱourȱlaboratoryȱwasȱpioneeredȱbyȱTimmermanȱetȱal.ȱ whoȱusedȱthiolȱreactiveȱscaffoldsȱtoȱachieveȱmonoȬ,ȱbiȬ,ȱorȱtricyclicȱpeptidesȱviaȱcyclizaȬ tionȱthroughȱcysteineȱresiduesȱ(176).ȱTheȱcyclizationȱchemistryȱwasȱhighlyȱefficientȱandȱ couldȱbeȱperformedȱinȱaqueousȱconditions.ȱHeinisȱetȱal.ȱdevelopedȱaȱstrategyȱforȱdisȬ playingȱaȱlibraryȱofȱrandomȱpeptidesȱcontainingȱthreeȱcysteineȱresiduesȱonȱphageȱ(deȬ scribedȱinȱsectionȱ1.1.3)ȱandȱcyclizingȱthemȱusingȱsuchȱaȱtrifunctionalȱthiolȱreactiveȱlinkerȱ molecule,ȱgeneratingȱbicyclicȱpeptidesȱ(177).ȱTheȱreactionȱtakesȱplaceȱatȱroomȱtemperaȬ tureȱandȱwasȱfoundȱtoȱnotȱimpairȱphageȱinfectivity,ȱhenceȱallowingȱforȱtheȱscreeningȱofȱ largeȱlibrariesȱofȱbicyclicȱpeptidesȱagainstȱimmobilizedȱtargets.ȱHavingȱconstructedȱtheȱ phageȱlibraryȱsuchȱthatȱtheȱcysteineȱresiduesȱareȱplacedȱinȱtheȱmiddleȱandȱoneȱresidueȱ awayȱfromȱeachȱend,ȱwithȱ6ȱrandomȱaminoȱacidsȱbetween,ȱtheȱresultingȱpeptideȱisȱcomȬ posedȱofȱtwoȱmacrocyclicȱringsȱconnectedȱthroughȱtheȱorganicȱlinkerȱ(Figureȱ17).ȱTheȱ bicyclicȱpeptidesȱareȱthoughtȱtoȱbeȱmoreȱrigidȱthanȱmonocyclicȱpeptides,ȱhavingȱtwoȱ bondsȱconstrainingȱeachȱringȱ(102).ȱMoreover,ȱbothȱringsȱcanȱpotentiallyȱinteractȱwithȱ theȱtargetȱsimultaneously,ȱallowingȱtightȱbindingȱandȱinȱaȱwayȱmimickingȱtheȱcompleȬ mentarityȬdeterminingȱregionsȱ(CDR)ȱofȱanȱantibodyȱ(178).ȱTheȱrecentlyȱreportedȱdevelȬ opmentȱofȱaȱpotentȱcyclicȱpeptideȱasȱanȱinfluenzaȱhemagglutininȱinhibitor,ȱderivedȱfromȱ aȱCDR,ȱfurtherȱsupportsȱtheȱtheoryȱthatȱcyclicȱpeptidesȱcanȱfunctionallyȱmimicȱCDRsȱ

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(179).ȱBicyclicȱpeptidesȱofȱthisȱformatȱwereȱindeedȱshownȱtoȱhaveȱlargeȱinteractionȱsurȬ facesȱwithȱtheirȱtargetȱproteins,ȱwhichȱisȱalsoȱlikelyȱcausingȱtheirȱgenerallyȱhighȱtargetȱ specificityȱ(180).ȱTheyȱwereȱalsoȱdemonstratedȱtoȱbeȱmoreȱstableȱthanȱlinearȱorȱmonocyȬ clicȱpeptidesȱ(181).ȱȱ

Whenȱscreeningȱcompoundȱlibrariesȱwithȱtheȱpurposeȱofȱidentifyingȱtargetȱbinders,ȱaȱ highȱdiversityȱisȱcrucial.ȱTheȱgenerationȱofȱrandomȱpeptideȱlibrariesȱcanȱindeedȱprovideȱ anȱalmostȱunlimitedȱdiversity,ȱhoweverȱtheȱphageȱdisplayȱformatȱlimitsȱtheȱnumberȱofȱ compoundsȱtoȱaroundȱ1010ȱdueȱtoȱinherentȱlimitationsȱofȱtheȱtechnologyȱ(aȱlimitedȱnumȬ berȱofȱbacteriaȱcanȱbeȱtransformedȱwithȱDNAȱplasmid).ȱToȱfurtherȱincreaseȱtheȱdiversityȱ ofȱbicyclicȱpeptideȱlibraries,ȱwithȱthisȱlimitationȱinȱmind,ȱourȱlaboratoryȱhasȱundertakenȱ twoȱstrategies:ȱtheȱusageȱofȱdifferentȱlinkerȱmoleculesȱandȱtheȱalternationȱofȱlibraryȱforȬ mat.ȱ

Figureȱ17.ȱ(A)ȱBicyclicȱpeptidesȱareȱformedȱbyȱreactingȱaȱpeptideȱcontainingȱthreeȱcysteineȱresiduesȱwithȱaȱ trifunctionalȱlinkerȱmoleculeȱ(blue).ȱ(B)ȱThreeȱdifferentȱlinkerȱmoleculesȱareȱusedȱinȱtheȱbicyclicȱpeptideȱ libraries.ȱȱ

Theȱ initialȱ linkerȱ usedȱ inȱ theȱ proofȬofȬconceptȱ studyȱ wasȱ trisȬ(bromomethyl)benzeneȱ (TBMB),ȱwhichȱwasȱselectedȱdueȱtoȱitsȱthreefoldȱrotationalȱsymmetryȱensuringȱtheȱforȬ mationȱofȱaȱuniqueȱstructuralȱisomer.ȱItȱwasȱthoughtȱthatȱbyȱintroducingȱhydrogenȱbindȬ ingȱmoietiesȱinȱtheȱlinkerȱmolecule,ȱadditionalȱtargetȱinteractionsȱcouldȱbeȱformedȱandȱ theȱbackboneȱconformationȱofȱtheȱbicyclicȱpeptideȱcouldȱbeȱinfluencedȱ(182).ȱTwoȱnewȱ linkerȱmoleculesȱwereȱdesigned,ȱhavingȱtheȱsameȱsymmetryȱasȱTBMB:ȱ1,3,5ȬtriacryloylȬ 1,3,5Ȭtriazinaneȱ (TATA)ȱ andȱ N,N’,N’’Ȭ(benzeneȬ1,3,5Ȭtriyl)tris(2Ȭbromoacetamide)ȱ (TBAB).ȱIndeed,ȱtheȱbindingȱaffinityȱofȱaȱbicyclicȱpeptideȱwasȱstronglyȱcompromisedȱ

34 ,QWURGXFWLRQ whenȱcyclizedȱwithȱaȱdifferentȱlinkerȱmolecule,ȱindicatingȱaȱstructuralȱroleȱofȱtheȱlinker.ȱ Inȱaȱfurtherȱstudyȱitȱwasȱshownȱthatȱwhenȱphageȱselectionsȱwereȱperformedȱagainstȱtheȱ sameȱ targetȱ butȱ usingȱ differentȱ linkerȱ molecules,ȱ specificȱ consensusȱ sequencesȱ wereȱ foundȱforȱeachȱlinkerȱ(183).ȱAȱmajorityȱofȱconsensusȱsequencesȱwereȱfoundȱexclusivelyȱ inȱselectionsȱwithȱoneȱofȱtheȱthreeȱlinkers.ȱTheȱreplacementȱofȱtheȱlinkerȱwithȱanotherȱ yieldedȱpeptidesȱseveralȱorderȱofȱmagnitudesȱlessȱpotent,ȱconfirmingȱtheȱresultsȱofȱtheȱ previousȱ study.ȱ Crystalȱ structuresȱ ofȱ theȱ bicyclicȱ peptidesȱ bindingȱ toȱ theirȱ targetȱ reȬ vealedȱ nonȱ covalentȱ interactionsȱ betweenȱ theȱ linkerȱ moleculeȱ andȱ theȱ peptideȱ sidechains,ȱ inducingȱ aȱ specificȱ conformationȱ ofȱ theȱ backbone.ȱ Theȱ linkerȱ isȱ thereforeȱ thoughtȱtoȱactȱasȱaȱstructuralȱscaffoldȱofȱtheȱbicyclicȱpeptideȱandȱhenceȱintroducingȱdiȬ versityȱtoȱtheȱlibrary.ȱȱ

Anotherȱapproachȱofȱgeneratingȱdiversityȱofȱbicyclicȱpeptideȱlibraries,ȱwhichȱisȱrelatedȱ toȱtheȱcyclizationȱstrategy,ȱisȱtheȱselectionȱofȱdisulfideȱlinkedȱpeptidesȱ(184).ȱInȱtheȱ6x6ȱ libraryȱmentionedȱaboveȱthereȱisȱaȱprobabilityȱofȱaȱfourthȱcysteineȱtoȱoccurȱinȱanyȱofȱtheȱ randomȱpositions.ȱTherefore,ȱtheȱformationȱofȱtwoȱdisulfideȱbridgesȱinȱsuchȱaȱlibraryȱ generatesȱaȱhighȱtopologicalȱdiversity.ȱTheseȱpeptidesȱareȱconstructedȱinȱtheȱsameȱwayȱ asȱaboveȱduringȱaȱphageȱselectionȱbutȱbyȱoxidationȱratherȱthanȱcyclizationȱbyȱaȱreactionȱ usingȱaȱlinker.ȱInȱadditionȱtoȱtheȱtopologicalȱdiversityȱintroducedȱbyȱtheȱpositionȱofȱtheȱ fourthȱ cysteine,ȱ aȱ singleȱ peptideȱ sequenceȱ couldȱ leadȱ toȱ threeȱ differentȱ regioisomersȱ whichȱfurtherȱexpandsȱtheȱvarietyȱofȱpossibleȱconformationsȱ(Figureȱ18).ȱȱ

Figureȱ18.ȱInȱtheȱbicyclicȱpeptideȱlibrariesȱwithȱthreeȱfixedȱcysteines,ȱaȱfourthȱcysteineȱcanȱoccurȱinȱanyȱofȱ theȱrandomȱpositionsȱ(left).ȱInȱsuchȱaȱpeptide,ȱaȱbicyclicȱstructureȱcanȱbeȱobtainedȱbyȱdisulfideȱformationȱ betweenȱtheȱcysteineȱresiduesȱ(middle).ȱThreeȱdifferentȱregioisomersȱcanȱbeȱformed,ȱfurtherȱincreasingȱtheȱ diversityȱofȱtheȱlibrary.ȱReprintedȱwithȱpermissionȱfromȱ(184),ȱ©ȱ2013ȱAmericanȱChemicalȱSociety.ȱ

Theȱsecondȱstrategyȱtoȱincreaseȱdiversityȱinȱbicyclicȱpeptideȱphageȱdisplayȱlibrariesȱisȱtoȱ changeȱtheȱformatȱofȱtheȱpeptides.ȱInȱthisȱcaseȱtheȱlengthȱofȱtheȱrandomȱregionsȱbetweenȱ theȱ cysteineȱ residuesȱ (theȱ twoȱ macrocyclicȱ rings)ȱ wasȱ variedȱ (Figureȱ 19)ȱ (185).ȱ Loopȱ lengthȱdiversityȱhadȱalreadyȱbeenȱappliedȱinȱtheȱtailoringȱofȱmolecularȱrecognitionȱsurȬ facesȱinȱproteinȱscaffolds,ȱandȱwasȱshownȱtoȱbeȱreadilyȱapplicableȱtoȱbicyclicȱpeptides.ȱ

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Bicyclicȱpeptideȱphageȱlibrariesȱwereȱgeneratedȱwithȱcombinationsȱofȱdifferentlyȱsizedȱ macrocyclicȱringsȱofȱtheȱformatȱCysȬ(Xaa)mȬCysȬ(Xaa)nȬCys,ȱwhereinȱtheȱnumberȱ‘m’ȱandȱ ‘n’ȱofȱrandomȱaminoȱacidsȱbetweenȱtheȱcysteineȱresiduesȱwasȱ3,ȱ4,ȱ5ȱorȱ6.ȱWhenȱscreenedȱ againstȱtheȱsameȱtargetȱasȱtheȱ6x6ȱlibrary,ȱtheȱdiverseȱformatsȱyieldedȱnovelȱconsensusȱ sequences,ȱ presumablyȱ bindingȱ differentlyȱ toȱ theȱ activeȱ siteȱ ofȱ theȱ enzyme.ȱ Oneȱ adȬ vantageȱofȱisolatingȱdifferentlyȱbindingȱpeptidesȱisȱtheȱincreasedȱsuccessȱrateȱofȱaffinityȱ maturationȱlibrariesȱconstructedȱbasedȱonȱtheȱinitialȱhit(s).ȱTheseȱlibrariesȱareȱcreatedȱinȱ aȱsemiȬrandomizedȱmannerȱtoȱensureȱthatȱhighȱdiversityȱisȱreachedȱforȱallȱresidues.ȱInȬ teractionȱformingȱresiduesȱofȱtheȱinitialȱhitȱpeptideȱareȱidentifiedȱusingȱalanineȱscanningȱ asȱdescribedȱinȱaȱpreviousȱchapter.ȱSubsequently,ȱaȱnewȱlibraryȱisȱgeneratedȱinȱwhichȱ theseȱresiduesȱareȱkeptȱconstantȱandȱlessȱimportantȱaminoȱacidsȱareȱrandomized.ȱThisȱ libraryȱisȱthenȱpannedȱagainstȱtheȱtargetȱinȱoneȱorȱtwoȱroundsȱofȱaffinityȱselectionȱwithȱ increasedȱstringency,ȱoftenȱachievedȱbyȱreducingȱtheȱamountȱofȱimmobilizedȱtarget.ȱInȱ orderȱtoȱfacilitateȱtheȱidentificationȱofȱconsensusȱsequencesȱandȱtheȱfollowingȱconstrucȬ tionȱofȱaffinityȱmaturationȱlibrariesȱRebolloȱetȱal.ȱestablishedȱaȱmethodȱutilizingȱNGSȱforȱ theȱidentificationȱofȱbicyclicȱpeptideȱhitȱcompoundsȱ(186).ȱUsingȱthisȱtechniqueȱenabledȱ theȱidentificationȱofȱrareȱtargetȬbindingȱpeptideȱmotifs,ȱasȱwellȱasȱtheȱdefinitionȱofȱmoreȱ preciselyȱconsensusȱsequencesȱandȱsubȬgroupsȱofȱconsensusȱsequences.ȱThisȱapproachȱ isȱnowȱestablishedȱandȱroutinelyȱusedȱinȱourȱlaboratory.ȱ

Figureȱ19.ȱBicyclicȱpeptideȱlibrariesȱwithȱdifferentȱloopȱlengthsȱwereȱconstructedȱinȱorderȱtoȱincreaseȱdiverȬ sity.ȱByȱvaryingȱtheȱloopȱlength,ȱtheȱsizeȱofȱtheȱbindingȱinterfaceȱofȱtheȱpeptideȬtargetȱinteractionȱisȱadjusted.ȱ (A)ȱAȱrepresentativeȱpeptideȱofȱsizeȱ4x5ȱisȱindicatedȱinȱgreen.ȱ(B)ȱTheȱschemeȱshowsȱtheȱloopȱcombinationsȱ inȱtheȱdifferentȱlibrariesȱ(darkȱgreen,ȱlightȱgreenȱandȱblack)ȱandȱofȱtheȱformerȱ6x6ȱlibraryȱ(red).ȱȱReprintedȱ withȱpermissionȱfromȱ(185)ȱreprintingȱlicenseȱnumberȱ4265410782621,ȱ©ȱ2013ȱTheȱRoyalȱSocietyȱofȱChemisȬ tryȱ

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([DPSOHVRIELF\FOLFSHSWLGHV

Usingȱtheȱstrategiesȱdetailedȱabove,ȱourȱlaboratoryȱhasȱgeneratedȱbicyclicȱpeptideȱinhibȬ itorsȱandȱligandsȱtoȱseveralȱimportantȱtherapeuticȱtargets.ȱInȱgeneral,ȱinitialȱbindersȱisoȬ latedȱfromȱphageȱdisplayȱdemonstrateȱtargetȱaffinitiesȱinȱtheȱnanomolarȱtoȱmicromolarȱ range.ȱ Theseȱ hitȱ compoundsȱ canȱ thenȱ beȱ optimizedȱ usingȱ variousȱ strategiesȱ asȱ highȬ lightedȱinȱtheȱpreviousȱchapterȱandȱasȱexemplifiedȱbelow.ȱ

InȱtheȱfirstȱproofȬofȬprincipleȱstudy,ȱbicyclicȱpeptideȱinhibitorsȱofȱtheȱ6x6ȱformatȱcyclizedȱ withȱTBMBȱwereȱgeneratedȱagainstȱtheȱproteasesȱcathepsinȱGȱandȱplasmaȱkallikreinȱ(PK)ȱ (177).ȱTheȱlatterȱisȱaȱserineȱproteaseȱinvolvedȱinȱcontactȱactivationȱandȱtheȱkallikreinȬ kininogenȱsystemȱ(187).ȱOverȬactivationȱofȱPKȱcanȱleadȱtoȱincreasedȱinflammationȱandȱ vascularȱleakageȱultimatelyȱresultingȱinȱtheȱswellingȱdisorderȱhereditaryȱangioedemaȱ (HAE),ȱwhichȱoccursȱinȱpatientsȱlackingȱfunctionalȱC1ȱinhibitor,ȱtheȱendogenousȱinhibiȬ torȱofȱtheȱprotease.ȱCompaniesȱsuchȱasȱCSLȱBehringȱandȱShireȱhaveȱapproachedȱPKȱasȱaȱ therapeuticȱtargetȱwithȱapprovedȱinhibitorsȱasȱwellȱasȱaȱnumberȱofȱnewȱmoleculesȱinȱlateȱ stageȱdevelopment.ȱTheȱbicyclicȱpeptideȱinhibitorȱPK15ȱinhibitsȱPKȱwithȱaȱKiȱofȱ2ȱnMȱ andȱwasȱdevelopedȱbyȱphageȱselectionȱandȱsubsequentȱaffinityȱmaturationȱ(177).ȱPK15ȱ wasȱshownȱtoȱbeȱhighlyȱspecificȱandȱdidȱnotȱinhibitȱtheȱhomologousȱproteinsȱfactorȱXIaȱ (FXIa)ȱandȱthrombin.ȱHowever,ȱtheȱhighȱspecificityȱalsoȱresultedȱinȱPK15ȱnotȱefficientlyȱ inhibitingȱorthologousȱproteases,ȱsuchȱasȱmouseȱPKȱorȱratȱPK,ȱpreventingȱtheȱevaluationȱ ofȱtheȱcompoundȱinȱsmallȱlaboratoryȱanimals.ȱTheȱspecificityȱofȱtheȱPKȱinhibitorȱcouldȱ beȱtailoredȱbyȱreducingȱtheȱinteractionȱsurfaceȱofȱtheȱbicyclicȱpeptideȱ(Figureȱ20)ȱ(188).ȱ Usingȱaȱ3x3ȱlibrary,ȱbicyclicȱpeptidesȱinhibitingȱhumanȱandȱratȱPK,ȱbutȱalsoȱhumanȱFXIaȱ wereȱisolated.ȱThisȱresultȱindicatedȱthatȱtheȱsmallerȱbicyclicȱpeptidesȱbindȱtoȱregionsȱthatȱ areȱidenticalȱinȱhPK,ȱrPKȱandȱhfXIa.ȱȱ

Figureȱ20.ȱ(A)ȱTheȱstrategyȱofȱBaeriswylȱetȱal.ȱtoȱmakeȱaȱPKȱbinderȱwithȱcrossȬspeciesȱaffinityȱwasȱtoȱreduceȱ theȱbindingȱinterfaceȱofȱtheȱpeptide.ȱThisȱwasȱdoneȱbyȱusingȱlibrariesȱwithȱsmallerȱloopȱsizeȱinȱtheȱphageȱ selection.ȱInsteadȱofȱ6x6ȱlibrariesȱusedȱpreviously,ȱ3x3ȱandȱ5x5ȱlibrariesȱwereȱused.ȱRandomȱaminoȱacidsȱareȱ indicatedȱasȱX,ȱAlanineȱasȱAȱandȱtheȱfixedȱcysteinsȱasȱC.ȱ(B)ȱStructureȱofȱtheȱbicyclicȱpeptidesȱhavingȱloopsȱ ofȱ3,ȱ5ȱorȱ6ȱaminoȱacids.ȱReprintedȱwithȱpermissionȱfromȱ(188)ȱreprintingȱlicenseȱnumberȱ4270220768830,ȱ©ȱ 2012ȱWileyȬVCHȱVerlagȱGmbH&Coȱ

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Next,ȱpeptideȱlibrariesȱwithȱloopsȱofȱfiveȱaminoȱacidsȱwereȱgeneratedȱinȱorderȱtoȱhaveȱ slightlyȱlargerȱbindingȱinterfaceȱandȱthereforeȱnotȱinhibitȱFXIa.ȱThisȱstrategyȱresultedȱinȱ severalȱpeptidesȱinhibitingȱPKȱatȱsubȬnanomolarȱconcentrationsȱwhileȱalsoȱinhibitingȱratȱ andȱmonkeyȱPK.ȱTheȱ5x5ȱpeptidesȱwereȱmoreȱspecificȱthanȱtheȱ3x3ȱpeptidesȱandȱinhibȬ itedȱFXIaȱonlyȱinȱtheȱmicromolarȱrange.ȱȱ

AnotherȱexampleȱofȱaȱbicyclicȱpeptideȱserineȱproteaseȱinhibitorȱisȱtheȱurokinaseȬtypeȱ plasminogenȱactivatorȱ(uPA)ȱinhibitorȱUK18ȱ(180).ȱuPAȱisȱinvolvedȱinȱcancerȱmetastasisȱ andȱextracellularȱmatrixȱdegradationȱandȱisȱthereforeȱaȱrelevantȱtherapeuticȱtargetȱ(189).ȱ AsȱwithȱtheȱselectionsȱagainstȱPK,ȱtheȱ6x6ȱlibraryȱcyclizedȱwithȱTBMBȱwasȱusedȱforȱtheȱ phageȱselection.ȱTheȱinitialȱpeptideȱhitȱisolatedȱafterȱtwoȱroundsȱofȱselection,ȱUK18,ȱpoȬ tentlyȱinhibitsȱuPAȱwithȱaȱKiȱofȱ53ȱnM,ȱwhichȱrepresentsȱaroundȱ200ȬfoldȱstrongerȱbindȬ ingȱthanȱtheȱbestȱmonocyclicȱpeptide.ȱAsȱwithȱPK15,ȱUK18ȱwasȱhighlyȱspecificȱtoȱhumanȱ uPAȱandȱdidȱnotȱbindȱanyȱrelatedȱproteasesȱ(Figureȱ21A).ȱȱ

Figureȱ21.ȱ(A)ȱTheȱspecificityȱofȱUK18ȱwasȱdeterminedȱinȱenzymaticȱinhibitionȱassays.ȱ(B)ȱPharmacokineticsȱ ofȱfluoresceinȱconjugatedȱUK18ȱ(8)ȱandȱUK18ȬSA21ȱ(13)ȱafterȱi.v.ȱinjectionȱinȱmice.ȱPeptideȱconcentrationȱ wasȱdeterminedȱbyȱliquidȱchromatography.ȱ(C)ȱPharmacokineticsȱofȱaȱbicyclicȱpeptideȱUK18ȬH12ȬFcȬstrepȬ tag/FcȬhis6tagȱinȱfemaleȱBALB/cȱmice.ȱSerumȱconcentrationsȱofȱtheȱheterodimerȱwereȱmeasuredȱwithȱtwoȱ differentȱELISAȱassaysȱatȱtheȱindicatedȱtimeȱpoints.ȱ(D)ȱNewmanȱprojectionsȱofȱGly11ȱandȱGly13ȱinȱUK18ȱ withȱtheȱIȱandȱ\ȱtorsionȱanglesȱfoundȱinȱtheȱcrystalȱstructure.ȱDȬaminoȱacidȱsubstitutionsȱinȱGly11ȱwouldȱ createȱaȱclashȱbetweenȱtheȱsideȱchainȱofȱtheȱaminoȱacidȱandȱtheȱcarbonylȱgroupȱofȱArg10;ȱ DȬaminoȱacidȱ substitutionsȱofȱGly13ȱwereȱnotȱexpectedȱtoȱcreateȱstericȱconflictsȱȱReprintedȱwithȱpermissionȱfromȱ(180,ȱ181,ȱ 190,ȱ191),ȱreprintingȱlicenseȱ4270240211893,ȱ©ȱ2012ȱAmericanȱChemicalȱSociety,ȱ©ȱ2013ȱWileyȬVCHȱVerlagȱ GmbH&Co.ȱȱ

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TheȱpharmacokineticsȱofȱUK18ȱwereȱsubsequentlyȱexamined.ȱItȱwasȱfoundȱthatȱtheȱbicyȬ clicȱpeptideȱwasȱclearedȱrapidlyȱinȱmiceȱhavingȱaȱhalfȬlifeȱofȱ30ȱminȱ(181).ȱTwoȱstrategiesȱ wereȱevaluatedȱforȱimprovingȱtheȱhalfȬlife,ȱnamelyȱconjugationȱwithȱanȱalbuminȱbindingȱ peptideȱSA21ȱandȱFcȱfusionȱ(181,ȱ190).ȱTheȱpeptideȱconjugatesȱshowedȱretainedȱactivityȱ andȱtheirȱhalfȬlivesȱwereȱsuccessfullyȱprolongedȱtoȱ24ȱhȱandȱ36ȱhȱrespectivelyȱ(Figureȱ 21BȱandȱC).ȱInȱanȱadditionalȱmouseȱstudy,ȱitȱwasȱshownȱthatȱtheȱSA21ȱconjugateȱcouldȱ efficientlyȱdiffuseȱintoȱsolidȱtumorsȱ(192).ȱMoreȱrecently,ȱUK18ȱwasȱusedȱasȱaȱmodelȱpepȬ tideȱfusedȱtoȱaȱdifferentȱalbuminȱbindingȱtagȱforȱhalfȬlifeȱimprovementȱ(193).ȱTheȱUK18ȱ peptideȱhasȱalsoȱbeenȱsubjectedȱtoȱmedicinalȱchemistryȱapproachesȱwithȱtheȱpurposeȱofȱ improvingȱbindingȱaffinity.ȱInȱtheȱcrystalȱstructureȱofȱUK18ȱboundȱtoȱuPAȱaȱglycineȱresȬ idueȱwasȱobservedȱthatȱhasȱaȱpositiveȱIȱdihedralȱangleȱwhenȱboundȱtoȱtheȱtarget.ȱReȬ placementȱbyȱaȱDȬaminoȱacid,ȱwhichȱfavorsȱpositiveȱIȱdihedralȱangles,ȱincreasedȱbothȱ bindingȱaffinityȱandȱproteolyticȱstabilityȱofȱUK18ȱ(Figureȱ21D)ȱ(191).ȱ

OtherȱmedicinalȱchemistryȱapproachesȱhaveȱalsoȱbeenȱusedȱinȱbicyclicȱpeptideȱleadȱcomȬ pounds.ȱOneȱexampleȱisȱtheȱinclusionȱofȱaȱsyntheticȱdiȬthiolȱaminoȱacidȱintoȱaȱphageȱ selectedȱhitȱ(194).ȱInȱtheȱselectionsȱmentionedȱabove,ȱofȱfourȱcysteineȱcontainingȱpeptidesȱ cyclizedȱthroughȱaȱdisulfideȱbridge,ȱaȱmajorityȱofȱtheȱidentifiedȱbindersȱcontainedȱaȱpairȱ ofȱadjacentȱcysteineȱresidues.ȱByȱsubstitutingȱtwoȱadjacentȱcysteinsȱinȱsuchȱaȱpeptideȱ bindingȱaffinityȱcouldȱbeȱincreasedȱ40Ȭfold,ȱsomewhatȱunexpectedlyȱlargelyȱbyȱenthalpicȱ ratherȱthanȱentropicȱeffects.ȱȱ

Inȱ aȱ finalȱ example,ȱ bicyclicȱ peptidesȱ wereȱ selectedȱ againstȱ theȱ therapeuticȱ targetȱ EȬ cateninȱ(195).ȱThisȱproteinȱplaysȱaȱkeyȱroleȱinȱtheȱWntȱgrowthȱfactorȱsignalingȱpathwayȱ andȱitsȱupregulationȱisȱassociatedȱwithȱseveralȱtypesȱofȱhumanȱcancerȱ(196).ȱAgain,ȱtheȱ bicyclicȱpeptideȱ6x6ȱlibraryȱwasȱusedȱforȱselection,ȱcyclizedȱwithȱtheȱthreeȱdifferentȱlinkȬ ersȱTBMB,ȱTATAȱandȱTBAB.ȱThisȱstudyȱalsoȱhighlightsȱtheȱstructuralȱdiversityȱintroȬ ducedȱbyȱtheȱdifferentȱlinkerȱmoleculesȱasȱspecificȱconsensusȱsequencesȱwereȱfoundȱforȱ each.ȱFluorescenceȱpolarizationȱexperimentsȱshowedȱthatȱtheȱisolatedȱpeptidesȱboundȱtoȱ multipleȱsitesȱofȱEȬcatenin.ȱSeveralȱpeptidesȱwereȱfoundȱtoȱinterfereȱwithȱtheȱnativeȱligȬ andȱICATȱandȱthusȱboundȱtoȱEȬcateninȱatȱaȱprimeȱtargetȱsiteȱforȱtherapeuticȱinterventionȱ toȱwhichȱnoȱsyntheticȱligandȱwasȱpreviouslyȱidentified.ȱȱ

Inȱadditionȱtoȱtheȱthreeȱexamplesȱabove,ȱbicyclicȱpeptidesȱhaveȱbeenȱselectedȱagainstȱ severalȱotherȱtargetsȱsuchȱasȱtheȱcancerȱrelatedȱreceptorsȱHER2ȱandȱNotch1ȱ(197,ȱ198).ȱ BicyclicȱpeptidesȱinhibitingȱS.ȱaureusȱSortaseȱAȱwereȱalsoȱidentifiedȱandȱcouldȱpotentiallyȱ beȱdevelopedȱfurtherȱintoȱantimicrobialȱagentsȱ(199).ȱ

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Peptideȱdrugsȱareȱusedȱforȱtheȱtreatmentȱofȱaȱwideȱrangeȱofȱdiseases.ȱTheyȱareȱtypicallyȱ developedȱbasedȱonȱbioactiveȱpeptidesȱfoundȱinȱnatureȱorȱpeptideȱligandsȱgeneratedȱdeȱ novoȱbyȱinȱvitroȱevolution.ȱOften,ȱpeptidesȱfromȱtheseȱtwoȱsourcesȱdoȱnotȱreadilyȱhaveȱ theȱrequiredȱpropertiesȱasȱsuchȱasȱsufficientlyȱgoodȱbindingȱaffinity,ȱselectivityȱorȱstabilȬ ity,ȱandȱtheyȱneedȱtoȱbeȱengineered.ȱThisȱappliesȱalsoȱtoȱmanyȱofȱtheȱbicyclicȱpeptidesȱ developedȱinȱourȱlaboratoryȱbyȱphageȱdisplay.ȱTheȱaimȱofȱmyȱPhDȱprojectȱwasȱtoȱdeȬ velopȱnewȱstrategiesȱandȱapplyȱexistingȱonesȱtoȱimproveȱtheȱpotency,ȱselectivityȱandȱ proteolyticȱstabilityȱofȱphageȬselectedȱbicyclicȱpeptides.ȱAȱfurtherȱobjectiveȱwasȱtoȱcharȬ acterizeȱtheȱdevelopedȱpeptidesȱinȱvitroȱandȱinȱvivoȱinȱrelevantȱpreclinicalȱanimalȱmodȬ els.ȱTheȱultimateȱgoalȱofȱmyȱthesisȱwasȱtoȱdevelopȱaȱpeptideȱdrugȱcandidateȱthatȱhasȱtheȱ potentialȱtoȱbeȱtranslatedȱintoȱaȱtherapeutic.ȱ

FormerȱmembersȱofȱourȱlaboratoryȱhadȱappliedȱphageȱdisplayȱtoȱdevelopȱbicyclicȱpepȬ tideȱinhibitorsȱofȱtheȱproteasesȱcoagulationȱfactorȱXIIȱ(FXII)ȱandȱmatrixȱmetalloproteinȬ aseȱ 2ȱ (MMPȬ2).ȱ Theȱ bicyclicȱ peptidesȱ showedȱ inhibitoryȱ constantsȱ inȱ theȱ nanomolarȱ range.ȱTheȱbicyclicȱpeptidesȱdidȱnotȱhaveȱtheȱrequiredȱaffinity,ȱselectivityȱandȱstabilityȱ toȱbeȱevaluatedȱinȱanimalȱdiseaseȱmodels.ȱInȱmyȱthesis,ȱIȱaimedȱatȱsubstitutingȱnaturalȱ aminoȱacidsȱinȱtheȱbicyclicȱpeptidesȱtoȱunnaturalȱones,ȱinȱorderȱtoȱimproveȱsaidȱproperȬ ties.ȱThisȱthesisȱisȱsplitȱintoȱthreeȱdifferentȱparts,ȱwhereȱtheȱfirstȱtwoȱareȱrelatedȱtoȱtheȱ engineeringȱofȱtheȱFXIIȱinhibitorȱandȱtheȱlatterȱtoȱtheȱMMPȬ2ȱinhibitor.ȱTheȱspecificȱaimsȱ ofȱtheȱprojectsȱareȱasȱfollows:ȱ

i) Inȱtheȱfirstȱproject,ȱmyȱaimȱwasȱtoȱtestȱifȱinsertionȱofȱoneȱorȱtwoȱcarbonȱatomsȱ atȱdifferentȱpositionsȱintoȱtheȱmacrocycleȱbackboneȱofȱtheȱphageȬselectedȱbiȬ cyclicȱpeptideȱFXIIȱinhibitorȱcanȱimproveȱitsȱbindingȱaffinity.ȱThisȱprojectȱhadȱ aȱdualȱaim,ȱnamelyȱtheȱtestingȱofȱaȱnewȱaffinityȱmaturationȱstrategyȱandȱtheȱ improvementȱofȱtheȱpotencyȱofȱtheȱinhibitorȱtowardsȱitsȱpotentialȱdevelopȬ mentȱintoȱaȱdrugȱlead.ȱ ȱ ii) TheȱsecondȱprojectȱconstitutesȱtheȱlargestȱpartȱofȱmyȱPhDȱthesisȱandȱseveralȱ intermediateȱgoalsȱwereȱdefined,ȱwithȱtheȱfinalȱaimȱofȱtestingȱanȱoptimizedȱ bicyclicȱpeptideȱFXIIȱinhibitorȱinȱdifferentȱanimalȱdiseaseȱmodels.ȱAȱfirstȱinȬ termediateȱgoalȱwasȱtoȱfurtherȱimproveȱtheȱaffinityȱofȱtheȱinhibitorȱbyȱinsertȬ ingȱadditionalȱunnaturalȱaminoȱacidsȱthatȱenhanceȱtheȱbindingȱaffinity.ȱAȱsecȬ ondȱobjectiveȱwasȱtoȱimproveȱtheȱaffinityȱforȱmouseȱFXII,ȱsinceȱtheȱleadȱpepȬ tideȱhadȱlowȱaffinityȱforȱtheȱmurineȱhomologueȱandȱwasȱthusȱlimitingȱpossiȬ bleȱinȱvivoȱstudiesȱinȱthisȱspecies.ȱAȱthirdȱobjectiveȱwasȱtoȱimproveȱtheȱproȬ teolyticȱstabilityȱofȱtheȱinhibitor.ȱAfterȱtheȱoptimizationȱofȱtheȱinhibitor,ȱtheȱ nextȱgoalȱwasȱtoȱcharacterizeȱit,ȱfirstȱexȱvivo,ȱtoȱidentifyȱsuitableȱspeciesȱforȱ inȱvivoȱexperimentationȱandȱlaterȱinȱseveralȱtherapeuticallyȱrelevantȱpreclinȬ icalȱinȱvivoȱmodels.ȱȱ

43 $LPRIWKHVLV

iii) Theȱaimȱofȱmyȱthirdȱprojectȱwasȱtoȱimproveȱtheȱbindingȱaffinity,ȱtargetȱselecȬ tivityȱandȱproteolyticȱstabilityȱofȱtheȱaboveȱmentionedȱphageȬselectedȱbicyclicȱ peptideȱMMPȬ2ȱinhibitor.ȱTheȱdevelopmentȱofȱselectiveȱMMPȱinhibitorsȱhasȱ beenȱaȱlongȬstandingȱchallenge.ȱForȱMMPȬ2,ȱnoȱsyntheticȱinhibitorsȱareȱavailȬ ableȱthatȱefficientlyȱblockȱtheȱactivityȱofȱthisȱproteaseȱbutȱnotȱthatȱofȱotherȱ MMPs.ȱItȱwasȱourȱhypothesisȱthatȱtheȱbicyclicȱpeptideȱstructureȱwouldȱformȱ aȱlargeȱenoughȱcontactȱareaȱwithȱtheȱproteaseȱinȱorderȱtoȱachieveȱhighȱtargetȱ specificity.ȱMyȱnextȱaimȱinȱthisȱprojectȱwasȱtoȱfullyȱcharacterizeȱtheȱspecificityȱ ofȱtheȱbicyclicȱpeptideȱMMPȬ2ȱinhibitor.ȱAȱfurtherȱgoalȱwasȱtoȱoptimizeȱtheȱ specificityȱinȱorderȱtoȱachieveȱlessȱinhibitionȱofȱcloselyȱrelatedȱproteases.ȱAȱ nearȬtermȱgoalȱofȱthisȱworkȱwasȱtoȱestablishȱaȱpowerfulȱtoolȱthatȱallowsȱseȬ lectiveȱinhibitionȱofȱtheȱproteaseȱinȱvivo,ȱallowingȱstudyȱofȱtheȱproteaseȱinȱ vivo.ȱ

44 ,PSURYLQJWKHELQGLQJDIILQLW\RI LQYLWURHYROYHGF\FOLFSHSWLGHVE\ LQVHUWLQJDWRPVLQWRWKHPDFURF\ FOHEDFNERQH

Thisȱchapterȱisȱbasedȱonȱtheȱfollowingȱresearchȱarticle:ȱȱ

J.ȱWilbs,ȱS.ȱJ.ȱMiddendorp,ȱC.ȱHeinis,ȱImprovingȱtheȱBindingȱAffinityȱofȱinȬVitroȬEvolvedȱ Cyclicȱ Peptidesȱ byȱ Insertingȱ Atomsȱ intoȱ theȱ Macrocycleȱ Backbone,ȱ Chembiochemȱ 17,ȱ 2299–2303ȱ(2016).ȱReprintingȱlicenseȱnumber:ȱ4274090904498ȱ©ȱ2017ȱWileyȬVCHȱVerlagȱ GmbH&Co.ȱ

Authorȱcontributions:ȱJ.W.ȱandȱC.H.ȱconceivedȱtheȱstrategy.ȱJ.WȱsynthesizedȱandȱcharȬ acterizedȱtheȱpeptides.ȱJ.W.,ȱS.J.M.ȱandȱC.H.ȱdesignedȱandȱperformedȱtheȱbloodȱcoaguȬ lationȱexperiments.ȱJ.W.,ȱS.J.M.ȱandȱC.H.ȱwroteȱtheȱmanuscript.ȱȱ

,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

$EVWUDFW

Cyclicȱpeptidesȱbindingȱtoȱtargetsȱofȱinterestȱcanȱbeȱgeneratedȱefficientlyȱwithȱpowerfulȱ inȱvitroȱdisplayȱtechniques,ȱsuchȱasȱphageȱdisplayȱorȱmRNAȱdisplay.ȱTheȱcyclicȱpeptideȱ librariesȱscreenedȱwithȱtheseȱmethodsȱareȱgeneratedȱbyȱalteringȱinȱaȱcombinatorialȱfashȬ ionȱtheȱaminoȱacidȱsequenceȱofȱtheȱpeptides,ȱtheȱnumberȱofȱaminoȱacidsȱinȱtheȱmacrocyȬ cleȱrings,ȱandȱtheȱcyclizationȱchemistry.ȱAȱstructuralȱelementȱthatȱcannotȱeasilyȱbeȱvariedȱ inȱtheȱcyclicȱpeptidesȱisȱtheȱbackbone,ȱwhichȱisȱbuiltȱfromȱaminoȱacids,ȱeachȱofȱwhichȱ contributesȱthreeȱatomsȱtoȱtheȱmacrocyclicȱringȱstructure.ȱHere,ȱweȱproposedȱtoȱimproveȱ theȱaffinityȱofȱaȱphageȬselectedȱbicyclicȱpeptideȱinhibitorȱofȱcoagulationȱfactorȱXIIȱ(FXII)ȱ byȱscreeningȱvariantsȱwithȱoneȱorȱtwoȱcarbonȱatomsȱinsertedȱintoȱdifferentȱpositionsȱofȱ theȱbackbone,ȱandȱthusȱtappingȱintoȱaȱstructuralȱspaceȱthatȱwasȱnotȱsampledȱbyȱphageȱ display.ȱ Twoȱ mutantsȱ showedȱ 4.7Ȭȱ andȱ 2.5Ȭfoldȱ improvedȱ Kiȱ values.ȱ Theȱ betterȱ oneȱ blockedȱFXIIȱwithȱaȱKiȱofȱ1.5ȱ±ȱ0.1ȱnMȱandȱinhibitedȱactivationȱofȱtheȱintrinsicȱcoagulationȱ pathwayȱ (EC2xȱ 1.7ȱΐM).ȱ Theȱ strategyȱ ofȱ ringȱ sizeȱ variationȱ byȱ oneȱ orȱ severalȱ atomsȱ shouldȱbeȱgenerallyȱapplicableȱforȱtheȱaffinityȱmaturationȱofȱinȬvitroȬevolvedȱcyclicȱpepȬ tides.ȱ ,QWURGXFWLRQ

Peptideȱmacrocyclesȱhaveȱaȱnumberȱofȱfavorableȱpropertiesȱthatȱmakeȱthemȱattractiveȱ forȱtheȱdevelopmentȱofȱtherapeutics.ȱTheyȱcanȱbindȱwithȱhighȱaffinityȱandȱselectivityȱtoȱ proteinȱtargets,ȱtheirȱdegradationȱproductsȱareȱnontoxic,ȱtheyȱofferȱdifferentȱadministraȬ tionȱoptions,ȱandȱtheyȱcanȱbeȱchemicallyȱsynthesized.ȱAnotherȱadvantageȱisȱtheirȱfastȱ development:ȱtheyȱcanȱbeȱisolatedȱfromȱlargeȱcombinatorialȱlibrariesȱgeneratedȱbyȱeitherȱ chemicalȱ(200)ȱorȱribosomalȱsynthesisȱ(102,ȱ107,ȱ201).ȱInȱvitroȱdisplayȱtechniquesȱsuchȱasȱ phageȱdisplayȱorȱmRNAȱdisplayȱallowȱtheȱgenerationȱandȱscreeningȱofȱbillionsȱofȱpepȬ tideȱmacrocyclesȱinȱaȱshortȱtimeȱandȱforȱaȱmoderateȱcost.ȱChemicalȱandȱstructuralȱdiverȬ sityȱofȱpeptideȱmacrocycleȱlibrariesȱisȱobtainedȱbyȱvaryingȱtheȱaminoȱacidȱsequence,ȱtheȱ numberȱofȱaminoȱacids,ȱ(185,ȱ202)ȱandȱtheȱcyclizationȱchemistryȱ(102,ȱ182).ȱ

Forȱsomeȱproteinȱtargets,ȱitȱhasȱbeenȱdifficultȱtoȱgenerateȱhighȬaffinityȱpeptideȱmacrocyȬ clesȱ byȱ inȱ vitroȱ displayȱ techniques,ȱ despiteȱ theȱ enormousȱ sizeȱ andȱ diversityȱ ofȱ theȱ screenedȱ library.ȱ Forȱ therapeuticȱ application,ȱ peptideȱ macrocyclesȱ typicallyȱ requireȱ aȱ dissociationȱ constantȱ inȱ theȱ lowȱ nanomolarȱ orȱ picomolarȱ range.ȱ Aȱ frequentlyȱ chosenȱ strategyȱtoȱfurtherȱimproveȱinȱvitroȬevolvedȱcyclicȱpeptideȱligandsȱisȱtoȱmodifyȱtheȱsideȱ chainsȱthatȱmakeȱinteractionsȱwithȱtheȱtarget.ȱTheȱmodularȱarchitectureȱofȱpeptides,ȱtheȱ commercialȱavailabilityȱofȱmanyȱunnaturalȱaminoȱacids,ȱandȱautomatedȱsynthesisȱandȱ purificationȱmethodsȱallowȱefficientȱpreparationȱofȱmediumȬsizedȱlibrariesȱofȱsuchȱpepȬ tides.ȱWeȱrecentlyȱappliedȱsuchȱanȱapproachȱtoȱimproveȱtheȱbindingȱaffinityȱofȱtheȱbicyȬ clicȱpeptideȱFXII618ȱ(1,ȱFigureȱ22A),ȱ(203)ȱaȱnanomolarȱinhibitorȱofȱcoagulationȱfactorȱXIIȱ

47 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

(FXII)ȱisolatedȱbyȱphageȱdisplay.ȱSynthesisȱandȱtestingȱofȱaroundȱ50ȱbicyclicȱpeptidesȱ withȱalteredȱaminoȱacidȱsideȱchainsȱledȱtoȱtheȱidentificationȱofȱoneȱvariantȱwithȱsubstanȬ tiallyȱimprovedȱinhibitoryȱactivityȱ(204).ȱ

Figureȱ22.ȱ(A)ȱChemicalȱstructureȱofȱFXII618ȱ(1).ȱ(B)ȱAffinityȱmaturationȱstrategy.ȱCarbonȱatomsȱareȱinsertedȱ intoȱtheȱbackboneȱbyȱreplacingȱDȬaminoȱacidsȱwithȱEȬaminoȱacids,ȱorȱbyȱreplacingȱcysteinesȱconnectedȱtoȱ theȱcyclizationȱlinkerȱbyȱcysteineȱhomologuesȱwithȱlongerȱsideȱchains.ȱ

48 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

Inȱthisȱwork,ȱweȱaimedȱtoȱidentifyȱadditionalȱmodificationsȱthatȱimproveȱtheȱinhibitoryȱ activityȱofȱ1ȱbyȱanȱapproachȱthatȱisȱbasedȱonȱtheȱmodificationȱofȱtheȱpeptideȱbackboneȱ insteadȱofȱtheȱsideȱchains.ȱSpecifically,ȱweȱinsertedȱoneȱorȱtwoȱcarbonȱatomsȱatȱdifferentȱ positionsȱofȱtheȱmacrocyclicȱrings.ȱPeptidesȱscreenedȱbyȱphageȱdisplayȱareȱcomposedȱofȱ canonicalȱaminoȱacids,ȱeachȱofȱwhichȱcontributesȱthreeȱatomsȱtoȱtheȱmacrocyclicȱring.ȱ Moreȱprecisely,ȱtheyȱcontainȱ3×n+mȱbackboneȱatoms,ȱwhereȱȈnȈȱisȱtheȱnumberȱofȱaminoȱ acidsȱandȱȈmȈȱtheȱnumberȱofȱatomsȱcontributedȱbyȱtheȱcyclizationȱlinker.ȱInȱtheȱphageȱ displayȱscreenȱinȱwhichȱ1ȱwasȱisolated,ȱcyclicȱpeptidesȱwithȱ3×n+m+1ȱorȱ3×n+m+2ȱbackȬ boneȱatomsȱwereȱthusȱnotȱsampled.ȱWeȱspeculatedȱthatȱinsertingȱcarbonȱatomsȱinȱsomeȱ sitesȱofȱtheȱbackboneȱcouldȱleadȱtoȱsmallȱchangesȱinȱconformation,ȱandȱthatȱthisȱinȱturnȱ couldȱstrengthenȱexistingȱmolecularȱinteractionsȱorȱallowȱtheȱformationȱofȱnewȱnoncovaȬ lentȱcontactsȱofȱ1ȱwithȱFXII.ȱ

TheȱstrategyȱofȱinsertingȱatomsȱintoȱtheȱbackboneȱhasȱbeenȱappliedȱtoȱseveralȱnatureȬ derivedȱcyclicȱpeptidesȱforȱstudyingȱstructureȬactivityȱrelationshipsȱorȱenhancingȱbioȬ logicalȱactivity.ȱForȱexampleȱGhadiriȱandȱcoȬworkersȱinsertedȱsingleȱatomsȱintoȱhistoneȱ deacetylaseȱ(HDAC)ȱinhibitors,ȱmeasuredȱtheirȱcytotoxicȱactivityȱtowardsȱtumorȱcells,ȱ andȱdeterminedȱtheirȱstructuresȱ(205).ȱTheȱMuirȱgroupȱvariedȱtheȱmacrocycleȱsizeȱofȱauȬ toinducingȱpeptidesȱ(AIPs)ȱsecretedȱbyȱStaphylococcusȱaureus,ȱinȱorderȱtoȱstudyȱtheirȱ structureȬactivityȱrelationshipsȱ(206).ȱHansenȱandȱcoȬworkersȱextendedȱtheȱringȱsizeȱofȱ amphipathicȱcyclicȱpeptidesȱbyȱdifferentȱnumbersȱofȱatomsȱtoȱinvestigateȱtheȱeffectsȱonȱ antimicrobialȱandȱhemolyticȱactivityȱ(207).ȱInsertingȱorȱdeletingȱsingleȱcarbonȱatomsȱatȱ differentȱpositionsȱinȱmacrocyclicȱringsȱofȱpeptideȱligandsȱhasȱnotȱbeenȱusedȱasȱaȱsysȬ tematicȱapproachȱforȱimprovingȱtheȱbindingȱaffinityȱofȱinȬvitroȬevolvedȱcyclicȱpeptides.ȱ

Aȱsyntheticallyȱefficientȱwayȱofȱgeneratingȱcyclicȱpeptidesȱwithȱadditionalȱcarbonȱatomsȱ inȱtheȱringȱisȱreplacingȱDȬaminoȱacidsȱwithȱEȬaminoȱacidsȱthatȱcontainȱanȱadditionalȱcarȬ bonȱatomȱbetweenȱtheȱaminoȱandȱcarboxylȱgroupsȱ(Figureȱ22B).ȱSubstitutingȱEȬȱforȱȱDȬ aminoȱacidȱhasȱbeenȱappliedȱextensivelyȱtoȱDȬhelicalȱpeptides,ȱasȱEȬaminoȱacidsȱcanȱsubȬ stantiallyȱimproveȱstabilityȱ(208–211).ȱInȱsomeȱDȬhelicalȱpeptides,ȱEȬaminoȱacidsȱhaveȱ alsoȱimprovedȱbindingȱaffinity,ȱasȱforȱexampleȱinȱanȱanalogueȱofȱparathyroidȱhormoneȱ receptorȬ1ȱagonistȱ(212)ȱorȱanȱengineeredȱVEGFȱsignalingȱinhibitorȱbasedȱonȱtheȱZȬdoȬ mainȱ(213).ȱTheȱstrategyȱofȱDȬtoȬEȬaminoȱacidȱsubstitutionȱhasȱalsoȱbeenȱappliedȱtoȱaȱ handfulȱofȱnatureȬderivedȱcyclicȱpeptides,ȱincludingȱtheȱaboveȱmentionedȱHDACȱinhibȬ itorsȱandȱAIPsȱ(205,ȱ206).ȱInȱthisȱwork,ȱweȱchoseȱtoȱapplyȱEȬaminoȱacidsȱtoȱaccessȱvariantsȱ ofȱ1ȱwithȱaȱsingleȱcarbonȱatomȱinsertedȱinȱdifferentȱpositionsȱofȱtheȱbackbone.ȱ 5HVXOWVDQGGLVFXVVLRQ

InȱorderȱtoȱidentifyȱaminoȱacidȱpositionsȱwhereȱinsertionȱofȱaȱcarbonȱatomȱcouldȱpotenȬ tiallyȱimproveȱtheȱbindingȱaffinityȱofȱ1,ȱweȱsynthesizedȱtwoȱseriesȱofȱpeptideȱvariants:ȱ

49 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH oneȱwithȱindividualȱaminoȱacidsȱreplacedȱbyȱEȬalanine,ȱandȱtheȱotherȱwithȱglycineȱ(Tableȱ 2).ȱComparisonȱofȱtheȱresultingȱpeptidesȱallowedȱanȱunderstandingȱofȱwhetherȱanȱaddiȬ tionalȱcarbonȱatomȱatȱaȱspecificȱpositionȱenhancesȱbindingȱtoȱFXII,ȱindependentȱofȱtheȱ aminoȱacidȱsideȱchain.ȱUnmodifiedȱ1ȱinhibitedȱFXIIȱwithȱaȱKiȱofȱ7ȱ±ȱ0.4ȱnM.ȱSubstitutionȱ ofȱPhe3,ȱArg4,ȱorȱLeu5ȱtoȱEȬalanineȱ(2,ȱ4,ȱ6)ȱorȱglycineȱ(3,ȱ5,ȱ7)ȱreducedȱtheȱinhibitoryȱ activityȱsignificantlyȱ(>ȱ300Ȭfold).ȱForȱallȱthreeȱpositions,ȱsubstitutionȱtoȱEȬalanineȱgaveȱaȱ largerȱdropȱinȱactivityȱthanȱsubstitutionȱtoȱglycine,ȱthusȱindicatingȱthatȱanȱadditionalȱ carbonȱatomȱinȱtheseȱthreeȱpositionsȱofȱtheȱmainȱchainȱnegativelyȱaffectedȱbinding.ȱSubȬ stitutionȱofȱPro6ȱtoȱEȬalanineȱ(8)ȱorȱglycineȱ(9)ȱreducedȱaffinityȱ(~threeȱandȱ15Ȭfold,ȱreȬ spectively).ȱTheȱsmallerȱlossȱinȱaffinityȱforȱtheȱEȬalanineȱvariantȱsuggestsȱthatȱinsertionȱ ofȱoneȱcarbonȱatomȱinȱthisȱpositionȱenhancesȱbindingȱtoȱFXII.ȱAminoȱacidȱsubstitutionȱatȱ Arg8ȱhadȱtheȱoppositeȱeffect:ȱaȱlargerȱaffinityȱdropȱforȱEȬalanineȱ(19Ȭȱversusȱtwofold;ȱ10,ȱ 11).ȱAtȱGln9ȱandȱLeu10,ȱsubstitutionȱtoȱEȬalanineȱandȱglycineȱreducedȱtheȱinhibitoryȱacȬ tivityȱmoreȱthanȱ100Ȭfoldȱ(12,ȱ13,ȱ14,ȱ15).ȱFinally,ȱatȱArg11,ȱreplacementȱwithȱbothȱEȬalaȬ nineȱandȱglycineȱreducedȱbindingȱbyȱaȱsmallȱfactorȱ(threefold;ȱ16ȱandȱ17).ȱ

Tableȱ2.ȱEȬalanineȱandȱglycineȱscreen.ȱStandardȱdeviationsȱwereȱdeterminedȱforȱpeptidesȱwithȱKiȱ<ȱ1ȱPMȱandȱ areȱindicated.ȱForȱpeptidesȱwithȱKiȱ>ȱ10ȱPM,ȱtheȱremainingȱproteaseȱactivityȱatȱaȱconcentrationȱofȱ10ȱPMȱisȱ indicatedȱinȱbrackets.ȱ

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SHSWLGH KL Q0 SHSWLGH KL Q0 3KH ! $UJ ! /HX ! ! 3UR $UJ *OQ /HX $UJ

Basedȱonȱtheseȱresults,ȱweȱconsideredȱPro6ȱandȱArg11ȱasȱtheȱmostȱpromisingȱsitesȱforȱDȬ toȬEȬaminoȱacidȱsubstitutions.ȱEȬAminoȱacidsȱcanȱhaveȱsideȱchainsȱatȱeitherȱtheȱDȱ(C2)ȱorȱ Eȱ(C3)ȱcarbonȱ(E2ȬȱandȱE3Ȭresidues,ȱrespectively).ȱGivenȱthatȱtheseȱcarbonȱatomsȱcanȱhaveȱ RȱorȱSȱconfiguration,ȱfourȱdiastereoisomericȱEȬaminoȱacidsȱexistȱforȱeachȱsideȱchain.ȱWeȱ firstȱsubstitutedȱPro6ȱwithȱaȱrangeȱofȱcyclicȱEȬaminoȱacidsȱ(18Ȭ22;ȱTableȱ3).ȱAllȱtheseȱsubȬ stitutionsȱreducedȱinhibitoryȱactivityȱ80Ȭfoldȱorȱmore.ȱItȱisȱlikelyȱthatȱtheȱcyclicȱEȬaminoȱ acidsȱimposedȱconformationalȱconstraints,ȱthusȱhinderingȱefficientȱbindingȱtoȱFXII.ȱWeȱ subsequentlyȱsynthesizedȱpeptidesȱinȱwhichȱPro6ȱwasȱsubstitutedȱwithȱacyclicȱEȬaminoȱ

50 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH acids:ȱmethylȱgroupsȱlinkedȱtoȱeitherȱC3,ȱC2,ȱorȱtheȱaminoȱgroupȱ(23Ȭ27;ȱTableȱ3).ȱWeȱ reasonedȱthatȱtheȱmethylȱgroupsȱcouldȱpotentiallyȱreplaceȱinteractionsȱformedȱbyȱtheȱ prolineȱring.ȱThreeȱofȱtheseȱ(23,ȱ26,ȱ27)ȱshowedȱdoubleȬdigitȱKiȱvalues,ȱbutȱallȱhadȱweakerȱ valuesȱthanȱtheȱpeptideȱwithȱEȬalanineȱinȱthisȱposition.ȱ

Tableȱ3.ȱSubstitutionȱofȱPro6ȱandȱArg11ȱbyȱEȬaminoȱacids.ȱStandardȱdeviationsȱwereȱdeterminedȱforȱpepȬ tidesȱwithȱKiȱ<ȱ1ȱPM.ȱForȱpeptidesȱwithȱKiȱ>ȱ10ȱPM,ȱtheȱremainingȱproteaseȱactivityȱatȱaȱconcentrationȱofȱ10ȱ PMȱisȱindicatedȱinȱbrackets.ȱ

SRVLWLRQ SHSWLGH EDPLQRDFLG KL Q0 1DPH VWUXFWXUH 3UR 6 ȕKRPRSUROLQH

3UR 5 ȕKRPRSUROLQH !

3UR 6 ȕSUROLQH 2

+1 2+ 3UR 6 ȕKRPRSUROLQH !

3UR 5 ȕKRPRSUROLQH

3UR 6 ȕKRPRDODQLQH

3UR 5 ȕKRPRDODQLQH 3UR 6 ȕKRPRDODQLQH

3UR 5 ȕKRPRDODQLQH

3UR 10HȕDODQLQH

$UJ 6 ȕKRPRDUJLQLQH

51 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

AnotherȱpositionȱthatȱtoleratedȱtheȱEȬalanineȱsubstitutionȱreasonablyȱwellȱwasȱArg11.ȱ WeȱreplacedȱthisȱwithȱtheȱE3ȬaminoȱacidȱstructurallyȱbestȱresemblingȱLȬArgȱ(SȱconfiguȬ rationȱinȱC3;ȱ(S)ȬE3Ȭhomoarginine;ȱ28).ȱThisȱsubstitutionȱyieldedȱaȱpeptideȱwithȱaȱ4.7Ȭ foldȱimprovedȱKiȱ(1.5ȱ±ȱ0.1ȱnM;ȱweȱnamedȱthisȱFXII700).ȱGivenȱthatȱ16ȱandȱ17ȱ(EȬalanineȱ andȱglycineȱatȱArg11)ȱhadȱcomparableȱaffinity,ȱitȱisȱlikelyȱthatȱtheȱaffinityȱimprovementȱ achievedȱwithȱtheȱ(S)ȬE3ȬhomoarginineȱsubstitutionȱresultedȱfromȱinteractionsȱofȱtheȱarȬ ginineȱsideȱchainȱwithȱFXII;ȱtheseȱareȱapparentlyȱbetterȱforȱtheȱE3ȬaminoȱacidȱthanȱforȱLȬ Arg.ȱ

Anotherȱefficientȱstrategyȱforȱinsertingȱcarbonȱatomsȱintoȱtheȱmacrocyclicȱringsȱofȱ1ȱisȱ substitutionȱofȱcysteinesȱwithȱhomocysteineȱorȱ5Ȭmercaptonorvalineȱ(oneȱorȱtwoȱaddiȬ tionalȱcarbonsȱinȱtheȱsideȱchains,ȱrespectively;ȱFigureȱ22B).ȱTheȱthiolȱgroupsȱofȱhomocysȬ teineȱandȱ5ȬmercaptonorvalineȱhaveȱslightlyȱhigherȱpKaȱvaluesȱthanȱforȱcysteine,ȱ(214)ȱ andȱwereȱthusȱexpectedȱtoȱreactȱoptimallyȱwithȱtheȱacrylamideȱfunctionalȱgroupsȱofȱtheȱ cyclizationȱreagentȱ1,3,5ȬtriacryloylȬ1,3,5Ȭtriazinaneȱ(TATA)ȱatȱaȱslightlyȱhigherȱpHȱthanȱ forȱcysteine.ȱWeȱfoundȱthatȱpeptidesȱcontainingȱtwoȱcysteinesȱandȱeitherȱhomocysteineȱ orȱ5ȬmercaptonorvalineȱreactedȱefficientlyȱwithȱTATA,ȱevenȱwhenȱtheȱpHȱwasȱnotȱinȬ creased.ȱMostȱlikely,ȱTATAȱreactsȱfirstȱwithȱaȱcysteineȱ(intermolecularly)ȱandȱonlyȱthenȱ withȱhomocysteineȱorȱ5Ȭmercaptonorvalineȱ(intramolecularly).ȱTheȱfirst,ȱintermolecularȱ eventȱisȱlikelyȱtheȱrateȬlimitingȱstep,ȱandȱtheȱslowerȱreactionȱofȱhomocysteineȱorȱ5ȬmerȬ captonorvalineȱcomparedȱtoȱcysteineȱthereforeȱdoesȱnotȱslowȱtheȱformationȱofȱtheȱbicyȬ clicȱpeptide.ȱWeȱsynthesizedȱsixȱvariantsȱofȱ1ȱ(eachȱofȱtheȱthreeȱcysteinesȱreplacedȱbyȱ eitherȱofȱtheȱtwoȱcysteineȱanalogues;ȱTableȱ4).ȱȱ

Tableȱ4.ȱSubstitutionȱofȱcysteinesȱwithȱhomocysteine,ȱ5ȬmercaptoȬnorvalineȱandȱ4ȬmercaptoȬproline.ȱStandȬ ardȱdeviationsȱwereȱdeterminedȱforȱpeptidesȱwithȱKiȱ<ȱ1ȱPM.ȱForȱpeptidesȱwithȱKiȱ>ȱ10ȱPM,ȱtheȱremainingȱ proteaseȱactivityȱatȱaȱconcentrationȱofȱ10ȱPMȱisȱindicatedȱinȱbrackets.ȱ

SRVLWLRQ KRPRF\VWHLQH PHUFDSWR SS SR QRUYDOLQH PHUFDSWRSUROLQH PHUFDSWRSUROLQH

SHSWLGH KL Q0 SHSWLGH KL Q0 SHSWLGH KL Q0 SHSWLGH KL Q0 &\V ! ! &\V &\V ȱ

ReplacementȱofȱCys12ȱwithȱhomocysteineȱandȱ5Ȭmercaptonorvalineȱimprovedȱbindingȱ byȱ2.5Ȭȱandȱ1.2Ȭfold,ȱrespectivelyȱ(Kiȱ=ȱ2.8ȱ±ȱ0.5ȱnMȱ(31)ȱandȱ6ȱ±ȱ1ȱnMȱ(34)).ȱReplacementȱ

52 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH ofȱtheȱotherȱtwoȱcysteinesȱretainedȱbindingȱaffinityȱinȱtheȱcaseȱofȱhomocysteineȱ(29ȱandȱ 30)ȱandȱslightlyȱreducedȱbindingȱforȱ5Ȭmercaptonorvalineȱ(32ȱandȱ33).ȱWeȱnamedȱbicyȬ clicȱpeptideȱ31ȱ(withȱtheȱhighestȱaffinity)ȱFXII701.ȱHavingȱfoundȱthatȱcysteineȱanaloguesȱ withȱlongerȱsideȱchainsȱcanȱimproveȱaffinity,ȱweȱalsoȱtriedȱtheȱconformationallyȱconȬ strainedȱcyclicȱaminoȱacidȱ4Ȭmercaptoprolineȱ(2S,4Sȱorȱ2S,4Rȱconfiguration;ȱTableȱ4).ȱInȱ theseȱtwoȱaminoȱacids,ȱtheȱsulfurȱandȱcarbonȱDȱatomsȱareȱseparatedȱbyȱtwoȱcarbonȱatȬ oms,ȱasȱinȱhomocysteine.ȱReplacementȱofȱCys12ȱwithȱ4Ȭmercaptoprolineȱ(2S,4Sȱorȱ2S,4R)ȱ improvedȱbindingȱ2.8Ȭȱandȱ1.7Ȭfold,ȱrespectivelyȱ(Kiȱ=ȱ2.5ȱ±ȱ0.4ȱnMȱ(37)ȱandȱ4ȱ±ȱ1ȱnMȱ(40)).ȱ ReplacementȱofȱCys7ȱwithȱ4ȬmercaptoprolineȱimprovedȱtheȱbindingȱforȱoneȱofȱtheȱdiaȬ stereomersȱ(2S,4R,ȱ39;ȱKiȱ=ȱ6ȱ±ȱ1ȱnM)ȱbutȱnotȱforȱtheȱotherȱ(2S,4S,ȱ36;ȱKiȱ=ȱ1230ȱnM).ȱReȬ placementȱofȱCys2ȱwithȱbothȱ4Ȭmercaptoprolineȱdiastereomersȱreducedȱbindingȱmoreȱ thanȱ1000Ȭfoldȱ(35,ȱ38)ȱ

Weȱcombinedȱtheȱtwoȱsubstitutionsȱthatȱimprovedȱtheȱinhibitoryȱactivityȱofȱ1ȱbyȱlargeȱ factors:ȱArg11ȱtoȱ(S)ȬE3ȬhomoarginineȱandȱCys12ȱtoȱhomocysteine.ȱTheȱresultingȱpeptideȱ

(41)ȱshowedȱKiȱ=ȱ2.7ȱ±ȱ0.3ȱnM,ȱthusȱdemonstratingȱthatȱtheȱaffinityȱenhancementȱofȱtheȱ twoȱmodificationsȱisȱnotȱadditive.ȱThisȱisȱnotȱsurprisingȱgivenȱtheȱcloseȱproximityȱofȱ Arg11ȱandȱCys12.ȱInsertionȱofȱaȱcarbonȱatomȱatȱoneȱofȱtheȱtwoȱpositionsȱpresumablyȱ allowsȱaȱsmallȱconformationalȱchangeȱinȱthisȱregionȱofȱtheȱmacrocycle,ȱtherebyȱresultingȱ inȱstrongerȱmolecularȱinteractionsȱofȱanȱaminoȱacidȱsideȱchain,ȱsuchȱasȱforȱArg11ȱwithȱ FXIIa.ȱ

Finally,ȱweȱtestedȱwhetherȱtheȱmodifiedȱbicyclicȱpeptidesȱ28ȱandȱ31ȱblockȱtheȱintrinsicȱ coagulationȱpathwayȱmoreȱefficientlyȱthanȱtheȱleadȱpeptideȱ1.ȱAllȱthreeȱpeptidesȱdoseȬ dependentlyȱprolongedȱtheȱintrinsicȱcoagulationȱtimeȱ(aPTT)ȱofȱhumanȱplasma,ȱwithȱ28ȱ andȱ31ȱsuperiorȱtoȱ1ȱ(Figureȱ23A).ȱTheȱinhibitorȱwithȱtheȱhighestȱaffinity,ȱ28,ȱshowedȱtheȱ bestȱprolongationȱ(EC2x=1.7ΐM,ȱcomparedȱtoȱ3.1ȱΐMȱforȱ1,ȱwhereȱEC2xȱisȱtheȱinhibitorȱ concentrationȱatȱwhichȱtheȱcoagulationȱtimeȱisȱdoubled).ȱExtrinsicȱcoagulationȱ(PT)ȱwasȱ similarȱforȱtheȱthreeȱcompounds,ȱthusȱshowingȱthatȱtheȱhighȱtargetȱselectivityȱwasȱreȬ tainedȱ(Figureȱ23).ȱ

53 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

Figureȱ23.ȱ(A)ȱaPTTȱandȱ(B)ȱPTȱcoagulationȱdataȱaPTTȱforȱbicyclicȱpeptidesȱ1ȱ(FXII618),ȱ28ȱ(FXII700)ȱandȱ31ȱ (FXII701).ȱȱ

Inȱsummary,ȱweȱhaveȱimprovedȱtheȱbindingȱaffinityȱofȱinȬvitroȬevolvedȱcyclicȱpeptideȱ ligandsȱbyȱvaryingȱtheȱsizeȱofȱtheȱmacrocyclicȱringsȱbyȱoneȱorȱtwoȱcarbonȱatoms.ȱThisȱ chemicalȱspaceȱisȱnotȱsampledȱbyȱscreeningȱgeneticallyȱencodedȱcyclicȱpeptideȱlibraries,ȱ andȱpotentiallyȱoffersȱaȱrichȱsourceȱforȱslightlyȱimprovedȱligands.ȱIndeed,ȱsynthesisȱandȱ screeningȱofȱaȱsmallȱlibraryȱofȱbackboneȬmodifiedȱbicyclicȱpeptideȱvariantsȱofȱFXIIȱinȬ hibitorȱ1ȱyieldedȱseveralȱinhibitorsȱwithȱsubstantiallyȱimprovedȱKiȱvalues.ȱ ([SHULPHQWDOVHFWLRQ

Materials:ȱFmocȬLȬ΅Ȭaminoȱacids,ȱOȬ(benzotriazolȬ1Ȭyl)ȬN,N,Nȇ,NȇȬtetramethyluroniumȱ hexafluorophosphateȱ(HBTU),ȱ1Ȭhydroxybenzotriazoleȱhydrateȱ(HOBt),ȱandȱRinkȱAmȬ ideȱAMȱresinȱwereȱpurchasedȱfromȱGLȱBiochemȱ(Shanghai,ȱChina).ȱFmocȬEȬaminoȱacidsȱ wereȱpurchasedȱfromȱChemȬImpexȱ(WoodȱDale,ȱIL),ȱPolyPeptideȱ(Malmo,ȱSweden),ȱandȱ TCIȱ(Tokyo,ȱJapan).ȱFmocȱcysteineȱderivativesȱwereȱpurchasedȱfromȱPolyPeptide.ȱ

54 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

Peptideȱ synthesis:ȱ SolidȬphaseȱ peptideȱ synthesisȱ wasȱ performedȱ onȱ anȱ Advancedȱ ChemTechȱ348ȱ:ȱpeptideȱsynthesizerȱ(AAPPTec,ȱLouisville,ȱKY)ȱbyȱstandardȱFmocȱproȬ cedures.ȱRinkȱAmideȱAMȱresinȱwasȱtheȱsolidȱsupportȱ(0.03ȱmmolȱscale),ȱandȱDMFȱwasȱ theȱ solvent.ȱ Eachȱ aminoȱ acidȱ wasȱ coupledȱ twiceȱ (4ȱ equiv,ȱ 0.2ȱ Mȱ inȱ DMF)ȱ byȱ usingȱ HBTU/HOBtȱ(4ȱequiv,ȱ0.45ȱMȱinȱDMF)ȱandȱDIPEAȱ(6ȱequiv,ȱ0.5ȱMȱinȱDMF;ȱRT,ȱ30ȱmin,ȱ 400ȱrpm).TheȱresinȱwasȱwashedȱfourȱtimesȱwithȱDMFȱafterȱtheȱcouplingȱreaction.ȱTheȱNȬ terminalȱFmocȱprotectingȱgroupȱwasȱremovedȱwithȱpiperidineȱ(20%ȱv/v)ȱinȱDMFȱ(RT,ȱ 2×5ȱmin,ȱ400ȱrpm).ȱTheȱresinȱwasȱwashedȱfiveȱtimesȱwithȱDMFȱafterȱdeprotection.ȱ

Peptideȱcleavageȱfromȱtheȱresin:ȱTheȱsideȱchainsȱwereȱdeprotected,ȱandȱtheȱpeptideȱwasȱ cleavedȱfromȱtheȱRinkȱAmideȱAMȱresinȱbyȱincubationȱwithȱcleavageȱsolutionȱ(5ȱmL;ȱTFAȱ

(90%ȱv/v),ȱ1,2Ȭethanedithiolȱ(2.5%ȱv/v),ȱphenolȱ(2.5%ȱw/v),ȱthioanisoleȱ(2.5%ȱv/v),ȱH2Oȱ (2.5%ȱv/v))ȱforȱ2ȱhȱwithȱshaking.ȱTheȱresinȱwasȱremovedȱbyȱvacuumȱfiltration,ȱandȱtheȱ peptidesȱwereȱprecipitatedȱwithȱiceȬcoldȱdiethylȱetherȱ(50ȱmL),ȱincubatedȱforȱ30ȱminȱatȱȬ 20ȱ°C,ȱandȱpelletedȱbyȱcentrifugationȱ(2700ȱg,ȱ5ȱmin).ȱTheȱdiethylȱetherȱwasȱdiscarded,ȱ andȱtheȱprecipitateȱwasȱthenȱwashedȱtwiceȱwithȱdiethylȱether.ȱTheȱremainingȱsolventȱ wasȱevaporatedȱatȱRT.ȱ

PeptideȱcyclizationȱwithȱTATA:ȱTheȱcrudeȱpeptideȱ(typicallyȱ~50ȱmg)ȱwasȱdissolvedȱ

(~3.5ȱmM)ȱinȱMeCN/H2Oȱ(33:67,ȱ6ȱmL).ȱTATAȱ(10ȱmM,ȱ1.2ȱequiv)ȱinȱMeCNȱwasȱadded.ȱ

TheȱreactionȱwasȱstartedȱbyȱtheȱadditionȱofȱdegassedȱaqueousȱNH4HCO3ȱbufferȱ(60ȱmM,ȱ pHȱ8.0)ȱforȱaȱfinalȱpeptideȱconcentrationȱofȱ1ȱmMȱandȱincubatedȱatȱ30°Cȱforȱ1ȱhȱinȱaȱwaterȱ bath.ȱTheȱpeptideȱwasȱlyophilized.ȱ

Peptideȱ purificationȱ byȱ reversedȬphaseȱ HPLC:ȱ Modifiedȱ peptideȱ powderȱ wasȱ disȬ solvedȱinȱDMSOȱ(1ȱmL)ȱwithȱMeCNȱ(2ȱmLȱcontainingȱTFAȱ(0.1%))ȱandȱH2Oȱ(7ȱmLȱconȬ tainingȱTFAȱ(0.1%),ȱandȱpurifiedȱonȱaȱpreparativeȱVydacȱTP1022ȱ250ȱC18ȱcolumnȱ(22ȱ mm,ȱ10ȱmm)ȱwithȱaȱlinearȱgradientȱofȱsolventȱBȱ(MeCNȱwithȱTFAȱ(0.1%))ȱoverȱsolventȱAȱ

(H2OȱwithȱTFAȱ(0.1%):ȱ13ȱmin,ȱ15Ȭ28%ȱsolventȱB,ȱ20ȱmLminȬ1.ȱFractionsȱcontainingȱtheȱ desiredȱpeptideȱproductȱwereȱidentifiedȱbyȱESIȬMSȱanalysisȱandȱlyophilized.ȱTheȱpurityȱ ofȱpeptidesȱwasȱassessedȱbyȱRPȬHPLC,ȱandȱtheȱidentityȱwasȱconfirmedȱbyȱmassȱspecȬ trometryȱ(FigureȱS3.1ȱandȱTableȱS3.1ȱinȱtheȱSupportingȱInformation).ȱ

Proteaseȱinhibitionȱassays:ȱTheȱinhibitoryȱactivityȱofȱtheȱsynthesizedȱbicyclicȱpeptidesȱ wasȱdeterminedȱbyȱincubationȱwithȱproteaseȱandȱquantificationȱofȱtheȱresidualȱactivityȱ atȱvariousȱpeptideȱconcentrationsȱusingȱaȱfluorogenicȱsubstrate.ȱPeptidesȱwereȱpreparedȱ asȱ2ȱmMȱstockȱsolutionsȱinȱH2O.ȱResidualȱenzymaticȱactivityȱwasȱmeasuredȱinȱTrisȬHClȱ

(150ȱPL,ȱ10ȱmM,ȱpHȱ7.4)ȱcontainingȱNaClȱ(150ȱmM),ȱMgCl2ȱ(10ȱmM),ȱCaCl2ȱ(1ȱmM),ȱTriȬ tonȱXȬ100ȱ(0.01%ȱv/v),ȱandȱBSAȱ(0.1%ȱw/v).ȱTheȱfinalȱconcentrationȱofȱhumanȱEȬFXIIaȱ (#HFXIIAB;ȱMolecularȱInnovations,ȱNovi,ȱMI)ȱwasȱ0.5ȱorȱ1ȱnM,ȱwithȱpeptideȱatȱ10ȱPMȱtoȱ 0.1ȱnM.ȱTheȱfinalȱconcentrationȱofȱtheȱfluorogenicȱsubstrateȱBocȬQGRȬAMCȱ(Bachem,ȱ

55 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

Bubendorf,ȱSwitzerland)ȱwasȱ50ȱPM.ȱFluorescenceȱwasȱrecordedȱinȱanȱInfiniteȱM200ȱProȱ plateȱreaderȱ(Oex=368ȱnm,ȱOem=467ȱnm;ȱTecan,ȱMännedorf,ȱSwitzerland)ȱeveryȱminuteȱforȱ 60ȱminȱatȱ25°C.ȱSigmoidalȱcurvesȱwereȱfittedȱtoȱtheȱdataȱbyȱusingȱtheȱfollowingȱdoseȬȬ responseȱequation.ȱ

ȱ whereȱxȱ=ȱpeptideȱconcentration,ȱyȱ=ȱ%ȱactivityȱofȱreactionȱwithoutȱpeptide,ȱA1ȱ=ȱ100%,ȱ

A2ȱ=ȱ0%,ȱpȱ=ȱ1.ȱIC50ȱvaluesȱwereȱderivedȱfromȱtheȱfittedȱcurveȱinȱGraphPadȱPrismȱ5ȱsoftȬ ware.ȱȱ

TheȱinhibitoryȱconstantȱKiȱwasȱcalculatedȱaccordingȱtoȱChengȱandȱPrusoff;ȱKiȱ=ȱIC50ȱ/(1ȱ+ȱ

([S]0/Km),ȱwhereinȱIC50ȱisȱtheȱfunctionalȱstrengthȱofȱtheȱinhibitor,ȱ[S]0ȱisȱtheȱtotalȱsubstrateȱ concentration,ȱandȱKmȱisȱtheȱMichaelisȬȬMentenȱconstant.ȱTheȱKmȱforȱBocȬQGRȬAMCȱwasȱ determinedȱtoȱbeȱ256ȱ±ȱ42ȱPMȱ(meanȱ±ȱSD,ȱn=4).ȱ

Coagulationȱ assays:ȱ Coagulationȱ timesȱ (aPTTȱ andȱ PT)ȱ wereȱ determinedȱ inȱ humanȱ plasmaȱbyȱusingȱaȱSTart4ȱcoagulationȱanalyzerȱ(DiagnosticaȱStago,ȱAsnieresȱsurȱSeine,ȱ France).ȱȱHumanȱsingleȱdonorȱplasmaȱwasȱusedȱ(InnovativeȱResearch,ȱNovi,ȱMI).ȱForȱ extrinsicȱcoagulation,ȱplasmaȱ(50ȱPL)ȱwasȱplacedȱinȱtheȱincubatingȱchamberȱofȱtheȱinstruȬ mentȱforȱ2ȱminȱatȱ37°C.ȱInnovinȱ(100ȱPL;ȱrecombinantȱhumanȱtissueȱfactor,ȱsyntheticȱ phospholipids,ȱandȱcalciumȱinȱstabilizedȱHEPESȱbufferȱsystem;ȱDadeȱBehring/Siemensȱ B4212Ȭ50)ȱwasȱaddedȱbyȱusingȱtheȱpipetteȱconnectedȱtoȱtheȱinstrument.ȱUponȱadditionȱ ofȱthisȱreagentȱtheȱelectromagneticallyȱinducedȱmovementȱofȱaȱsteelȱballȱinȱtheȱplasmaȱisȱ monitored.ȱTheȱtimeȱuntilȱtheȱballȱstopsȱmovingȱisȱrecordedȱasȱcoagulationȱtime.ȱForȱtheȱ intrinsicȱcoagulation,ȱplasmaȱ(100ȱPL)ȱwasȱincubatedȱwithȱ100ȱPLȱofȱPathromtin*ȱSLȱ(silȬ iconȱdioxideȱparticles,ȱplantȱphospholipidsȱinȱHEPESȱbufferȱsystem,ȱSiemensȱOQGS29)ȱ forȱ2ȱminȱatȱ37°C.ȱCoagulationȱwasȱtriggeredȱbyȱadditionȱofȱCaCl2ȱ(100ȱPL,ȱ25ȱmM;ȱSieȬ mens).ȱ

ȱȱ

56 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

6XSSRUWLQJLQIRUPDWLRQ

FigureȱS3.1.ȱAnalysisȱofȱimportantȱbicyclicȱpeptideȱFXIIȱinhibitorsȱbyȱreversedȱphaseȱchromatogȬ raphyȱandȱmassȱspectrometry.ȱ ȱ ȱ

57 ,PSURYLQJWKHELQGLQJDIILQLW\RILQYLWURHYROYHGF\FOLFSHSWLGHVE\LQVHUWLQJDWRPVLQWRWKHPDFURF\FOHEDFNERQH

TableȱS3.1.ȱIdentityȱandȱpurityȱofȱimportantȱbicyclicȱpeptideȱFXIIȱinhibitorsȱanalysedȱbyȱreversedȱphaseȱ chromatographyȱandȱmassȱspectrometry.ȱ

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Thisȱchapterȱisȱbasedȱonȱaȱmanuscriptȱforȱsubmissionȱbyȱtheȱfollowingȱauthors:ȱ

JonasȱWilbs,ȱSimonȱJ.ȱMiddendorp,ȱAlidaȱCooke,ȱCaitlinȱT.ȱDemarest,ȱRajaȱPrince,ȱMaiȱ M.ȱAbdelhafez,ȱChristinaȱLamers,ȱKaycieȱDeyle,ȱRobertȱRieben,ȱAnneȱAngelilloȬScherȬ rer,ȱKeithȱE.ȱCook,ȱandȱChristianȱHeinisȱ

Authorȱcontributions:ȱJ.W.,ȱS.J.M,ȱC.L.,ȱandȱC.H.ȱdevelopedȱandȱcharacterizedȱtheȱFXIIaȱ inhibitor.ȱJ.W.,ȱS.J.M.,ȱandȱC.H.ȱdesignedȱandȱperformedȱtheȱpharmacokineticȱexperiȬ mentsȱinȱrabbits.ȱM.M.A.,ȱJ.W.,ȱR.R.,ȱandȱC.H.ȱdesignedȱandȱperformedȱtheȱpharmacokiȬ neticȱexperimentsȱinȱpigs.ȱR.P.,ȱJ.W.,ȱA.A.S.,ȱandȱC.H.ȱdesignedȱandȱperformedȱtheȱpharȬ macokineticȱ andȱ thrombosisȱ experimentsȱ inȱ mice.ȱ A.C.,ȱ C.T.D.,ȱ K.E.C.,ȱ andȱ J.W.ȱ deȬ signedȱandȱperformedȱtheȱartificialȱlungȱexperimentsȱinȱrabbits.ȱJ.W.ȱandȱC.H.ȱwroteȱtheȱ manuscript.ȱAllȱauthorsȱcontributedȱtoȱeditingȱandȱcriticalȱproofȬreadingȱofȱtheȱmanuȬ script.ȱ

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Inhibitingȱthrombosisȱwithoutȱinherentlyȱgeneratingȱbleedingȱrisksȱisȱaȱmajorȱchallengeȱ inȱmedicine.ȱTheȱdiscoveryȱthatȱmiceȱdeficientȱinȱcoagulationȱfactorȱXIIȱ(FXII)ȱshowȱreȬ ducedȱthrombosisȱwithoutȱabnormalȱbleedingȱsuggestedȱthatȱtargetingȱFXIIȱcouldȱofferȱ aȱsolutionȱtoȱthisȱlongstandingȱproblem.ȱSeveralȱstrategiesȱtoȱcontrolȱFXIIȱactivity,ȱinȬ cludingȱmonoclonalȱantibodies,ȱwereȱdevelopedȱandȱsuccessfullyȱevaluatedȱinȱprecliniȬ calȱmodels.ȱInȱcontrast,ȱtheȱdevelopmentȱofȱsmallȱmoleculeȱinhibitorsȱprovedȱmoreȱchalȬ lenging.ȱHerein,ȱweȱhaveȱengineeredȱaȱpeptideȱmacrocycleȱinhibitorȱofȱactivatedȱFXIIȱ

(FXIIa)ȱwithȱsubȬnanomolarȱactivityȱ(Kiȱ=ȱ370ȱ±ȱ40ȱpM)ȱandȱaȱhighȱstabilityȱ(t1/2ȱ>ȱ128ȱ±ȱ8ȱ hȱinȱplasma),ȱallowingȱforȱtheȱpreclinicalȱevaluationȱofȱaȱfirstȱsyntheticȱFXIIaȱinhibitor.ȱ Theȱ1899ȱDaȱinhibitor,ȱtermedȱFXII900,ȱefficientlyȱblockedȱFXIIaȱinȱmice,ȱrabbits,ȱandȱ pigs.ȱWeȱshowȱthatȱitȱreducedȱexperimentalȱthrombosisȱinducedȱbyȱferricȱchlorideȱinȱ miceȱ andȱ suppressedȱ bloodȱ coagulationȱ inȱ anȱ extracorporealȱ membraneȱ oxygenationȱ (ECMO)ȱsettingȱinȱrabbits,ȱallȱwithoutȱincreasingȱtheȱriskȱofȱbleeding.ȱThisȱshowsȱthatȱ FXIIaȱactivityȱisȱcontrollableȱwithȱaȱsyntheticȱinhibitorȱinȱvivo,ȱandȱthatȱtheȱinhibitorȱ FXII900ȱpresentedȱhereinȱisȱaȱpromisingȱcandidateȱforȱsafeȱthromboprotectionȱinȱvariousȱ medicalȱconditions ,QWURGXFWLRQ

Coagulationȱisȱrequiredȱtoȱstopȱtheȱbleedingȱatȱsitesȱofȱvesselȱwallȱinjuries,ȱbutȱexcessȱ coagulationȱcanȱalsoȱleadȱtoȱthrombosis.ȱBloodȱvesselȱblockages,ȱcanȱoccurȱinȱtheȱarterialȱ andȱvenousȱcirculationȱtoȱcauseȱmyocardialȱinfarctions,ȱischemicȱstrokes,ȱandȱpulmoȬ naryȱembolisms,ȱwhichȱcollectivelyȱareȱtheȱleadingȱcausesȱofȱdisabilityȱandȱdeathȱinȱtheȱ industrializedȱworldȱ(215).ȱInȱallȱofȱtheȱanticoagulantsȱwidelyȱusedȱforȱtheȱacuteȱandȱ prophylacticȱtreatmentȱofȱthrombosisȱ(216,ȱ217),ȱbleedingȱisȱaȱcommonȱsideȱeffect,ȱandȱ theȱinitiationȱofȱanyȱtherapyȱtoȱtreatȱcoagulationȱdeficienciesȱmustȱalwaysȱweighȱtheseȱ risksȱandȱbenefits,ȱconsideringȱtheȱseverityȱofȱtheȱpotentialȱsideȱeffects.ȱThus,ȱthereȱisȱaȱ growingȱneedȱforȱtheȱdevelopmentȱofȱeffectiveȱanticoagulantsȱthatȱideallyȱwouldȱnotȱ impairȱhemostaticȱabilityȱ(218).ȱ

Aȱpromisingȱnovelȱperspectiveȱforȱdevelopingȱsafeȱanticoagulantsȱwithȱreducedȱorȱnoȱ bleedingȱrisksȱisȱtheȱinhibitionȱofȱcoagulationȱfactorsȱXIIȱ(FXII)ȱandȱXIȱ(FXI),ȱbothȱbeingȱ proteasesȱofȱtheȱinstrinsicȱcoagulationȱpathwayȱ(219).ȱFXIIȱisȱtheȱinitiatingȱproteaseȱofȱ theȱprocoagulantȱandȱproinflammatoryȱcontactȱsystem,ȱandȱitȱdrivesȱbothȱtheȱintrinsicȱ pathwayȱofȱcoagulationȱandȱtheȱkallikreinȬkininȱsystemȱ(220).ȱMiceȱlackingȱFXIIȱhaveȱaȱ highlyȱreducedȱriskȱofȱinjuryȬinducedȱarterialȱandȱvenousȱthrombosisȱ(221,ȱ222)ȱandȱareȱ protectedȱfromȱpathologicalȱthrombosisȱinȱcerebralȱischemiaȱ(223).ȱAtȱtheȱsameȱtime,ȱhuȬ mansȱnaturallyȱlackingȱFXIIȱandȱFXIIȬknockoutȱmiceȱhaveȱaȱnormalȱhemostaticȱcapacityȱ

61 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ andȱdoȱnotȱbleedȱabnormallyȱ(221,ȱ224),ȱwhichȱindicatedȱthatȱdrugsȱtargetingȱtheȱproteȬ aseȱcouldȱpotentiallyȱproduceȱantithromboticȱeffectsȱwithoutȱsignificantlyȱcompromisȬ ingȱhemostasis.ȱConcordantly,ȱtheȱreductionȱofȱFXIIȱexpressionȱinȱmiceȱbyȱantisenseȱoliȬ gonucleotidesȱsuppressedȱthrombosisȱ(225,ȱ226).ȱTheȱinhibitionȱofȱFXIIȱbyȱproteinȬbasedȱ inhibitorsȱsuchȱasȱantibodiesȱ(227,ȱ228)ȱorȱinsectȬȱandȱplantȬderivedȱproteinsȱ(229–231)ȱ alsoȱreducedȱthrombosisȱinȱanimalȱmodelsȱandȱshowedȱpotentialȱavenuesȱforȱtherapeuticȱ anticoagulation.ȱȱ

FXIIȬdrivenȱbloodȱcoagulationȱisȱaȱmajorȱchallengeȱinȱcardiopulmonaryȱbypassȱ(CPB)ȱ surgeriesȱinȱwhichȱaȱheartȬlungȱmachineȱtemporarilyȱsupportsȱtheȱcirculation.ȱContactȱ betweenȱFXIIȱandȱartificialȱsurfaces,ȱsuchȱasȱtheȱoxygenatorȱmembraneȱorȱtubing,ȱinȬ ducesȱaȱconformationalȱchangeȱthatȱleadsȱtoȱtheȱproteolyticȱactivationȱofȱtheȱFXIIȱzymoȬ genȱthatȱturnsȱonȱtheȱcontactȱsystem.ȱActivationȱofȱtheȱprocoagulantȱintrinsicȱcoagulationȱ pathwayȱandȱtheȱproinflammatoryȱkallikreinȬkininȱsystemȱleadsȱtoȱbloodȱclottingȱandȱ inflammationȱ(187).ȱTheȱriskȱofȱcontactȱactivationȱisȱparticularlyȱhighȱinȱextracorporealȱ membraneȱoxygenationȱ(ECMO),ȱaȱformȱofȱCPBȱinȱwhichȱanȱartificialȱlungȱsystemȱisȱusedȱ forȱlongerȱperiodsȱofȱtimeȱasȱaȱlifeȱsupportȱforȱpatientsȱwithȱsevereȱcardiacȱand/orȱpulȬ monaryȱfailureȱ(232).ȱTheȱstandardȱstrategyȱforȱsuppressingȱcontactȱsystemȱactivationȱinȱ extracorporealȱcircuitsȱreliesȱonȱhighȱdosesȱofȱheparin,ȱwhichȱinhibitsȱalsoȱproteasesȱofȱ theȱextrinsicȱandȱcommonȱcoagulationȱpathwayȱandȱthusȱbearsȱanȱinherentȱriskȱofȱbleedȬ ingȱ(233).ȱSeveralȱstrategiesȱforȱsuppressingȱcontactȱactivationȱwereȱestablishedȱinȱexperȬ imentalȱmodels,ȱbutȱheparinȱremainsȱtheȱanticoagulationȱstrategyȱofȱchoiceȱ(234).ȱInȱaȱ recentȱstudy,ȱRenneȱandȱcoȬworkersȱshowedȱthatȱaȱhumanȱFXIIaȬinhibitingȱantibody,ȱ 3F7,ȱpreventedȱclottingȱandȱthrombosisȱinȱaȱcardiopulmonaryȱbypassȱsystemȱinȱrabbitsȱ withoutȱincreasedȱtherapyȬassociatedȱbleedingȱ(227),ȱindicatingȱtheȱusefulnessȱofȱtargetȬ ingȱFXII.ȱ

FXIIȱisȱimplicatedȱinȱseveralȱotherȱmedicalȱconditionsȱ(235),ȱincludingȱhereditaryȱangiȬ oedemaȱ(HAE)ȱ(236),ȱreperfusionȱinjuryȱ(237),ȱAlzheimerȇsȱdiseaseȱ(238,ȱ239),ȱandȱmulȬ tipleȱsclerosisȱ(240),ȱmeaningȱthatȱpotentialȱFXIIȱinhibitorsȱcouldȱbeȱusedȱinȱaȱvarietyȱofȱ treatmentsȱnotȱlimitedȱtoȱthrombosisȱprevention.ȱInȱTypeȱIIIȱHAE,ȱforȱexample,ȱaȱmutaȬ tionȱ inȱ FXIIȱ increasesȱ itsȱ activity,ȱ leadingȱ toȱ excessiveȱ bradykininȱ releaseȱ thatȱ causesȱ edemaȱ(241).ȱAȱderivativeȱofȱtheȱaboveȬmentionedȱFXIIaȱinhibitoryȱantibodyȱ3F7ȱhasȱenȬ teredȱaȱphaseȱIȱclinicalȱevaluationȱforȱtheȱtreatmentȱofȱHAEȱ(242).ȱ

WhileȱhighȬaffinityȱproteinȬbasedȱFXIIaȱinhibitorsȱwereȱsuccessfullyȱgeneratedȱoverȱtheȱ lastȱyearsȱ(243),ȱtheȱdevelopmentȱofȱsynthetic,ȱsmallȱmoleculeȱinhibitorsȱhasȱbeenȱmoreȱ challenging.ȱSmallȱmoleculesȱhaveȱaȱnumberȱofȱstrengthsȱthatȱmakeȱthemȱattractiveȱforȱ drugȱdevelopment,ȱincludingȱaȱuniformȱcomposition,ȱefficientȱtissueȱpenetration,ȱhighȱ stability,ȱlowȱimmunoreactivity,ȱandȱeaseȱofȱproductionȱbyȱchemicalȱsynthesis.ȱTheȱbestȱ smallȱmoleculeȱFXIIaȱinhibitorsȱreportedȱtoȱdateȱareȱtheȱcoumarinȱderivativeȱ44ȱ(IC50ȱ=ȱ

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4.4ȱ PM)ȱ (244)ȱ andȱ HȬDȬProȬPheȬArgȬchloromethylketoneȱ (PCK;ȱ IC50ȱ =ȱ 0.18ȱ PM)ȱ (223).ȱ WhileȱtheyȱhaveȱprovenȱtoȱbeȱusefulȱasȱresearchȱcompoundsȱinȱFXIIaȱinhibitionȱstudies,ȱ theirȱcovalentȱinhibitionȱmechanismȱandȱtheirȱmoderateȱpotencyȱandȱselectivityȱlimitȱ theirȱdrugȱdevelopmentȱpotential.ȱ

WeȱhaveȱrecentlyȱidentifiedȱhighȬaffinityȱFXIIaȱinhibitorsȱbasedȱonȱpeptideȱmacrocyclesȱ usingȱphageȱdisplay,ȱincludingȱtheȱbicyclicȱpeptideȱFXII618ȱ(Kiȱ=ȱ8.1ȱnM)ȱ(203).ȱInȱpreviȬ ousȱwork,ȱtheȱsubstitutionȱofȱindividualȱaminoȱacidsȱinȱFXII618ȱtoȱunnaturalȱonesȱfurtherȱ improvedȱtheȱaffinityȱ(245,ȱ246),ȱthoughȱdueȱtoȱtheirȱlimitedȱbindingȱtoȱanimalȱFXIIaȱ homologsȱandȱlowȱproteolyticȱstabilityȱinȱplasma,ȱtheseȱparticularȱpeptideȱmacrocycleȱ inhibitorsȱcouldȱnotȱbeȱevaluatedȱinȱvivo.ȱForȱthisȱreason,ȱweȱhaveȱnowȱfurtherȱimprovedȱ theȱinhibitorȱtoȱachieveȱpicomolarȱpotencyȱtowardsȱhumanȱandȱmouseȱFXIIa,ȱandȱweȱ enhancedȱtheȱstabilityȱinȱplasmaȱtoȱseveralȱdaysȱ(halfȬlifeȱinȱhumanȱplasmaȱexȱvivo).ȱ TheseȱpropertiesȱallowedȱforȱtheȱpreȬclinicalȱevaluationȱofȱtheȱinhibitorȱinȱvariousȱanimalȱ models.ȱWeȱshowȱhereinȱthatȱsyntheticȱFXIIaȱinhibitorsȱcanȱbeȱdevelopedȱtoȱefficientlyȱ preventȱthrombosisȱinȱmiceȱandȱsuppressȱcoagulationȱinȱartificialȱlungsȱinȱrabbitsȱwithȬ outȱincreasingȱtheȱriskȱofȱbleeding.ȱ 5HVXOWV

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TheȱNȬterminalȱarginineȱinȱFXII618ȱ(Arg1)ȱisȱrapidlyȱcleavedȱbyȱplasmaȱproteasesȱ(t1/2ȱ aroundȱ4ȱhȱinȱhumanȱplasma)ȱandȱitsȱlossȱreducesȱtheȱKiȱofȱtheȱinhibitorȱ40Ȭfoldȱ(204).ȱInȱ aȱ previousȱ studyȱ substitutingȱ Arg1ȱ withȱ diverseȱ unnaturalȱ aminoȱ acids,ȱ theȱ stabilityȱ couldȱbeȱimprovedȱatȱtheȱpriceȱofȱaȱweakerȱinhibition,ȱwhereinȱaminoȱacidsȱwithȱposiȬ tivelyȱchargedȱsideȱchainsȱshowedȱtheȱsmallestȱaffinityȱlossesȱ(204).ȱInȱaȱnewȱattempt,ȱ weȱreplacedȱArg1ȱwithȱaȱpanelȱofȱdiȬpeptidesȱthatȱcouldȱpotentiallyȱreachȱaȱlargerȱsurfaceȱ areaȱonȱFXIIaȱtoȱformȱproductiveȱinteractionsȱ(Figureȱ24A,ȱTableȱS4.1).ȱBothȱaminoȱacidsȱ inȱtheȱdiȬpeptidesȱwereȱDȬaminoȱacidsȱtoȱpreventȱproteolysis,ȱandȱoneȱofȱtheȱtwoȱwasȱDȬ Argȱtoȱmaintainȱaȱpositiveȱcharge.ȱWeȱdevelopedȱaȱsynthesisȱandȱscreeningȱapproachȱ basedȱ onȱ coumarinȬlabeledȱ peptidesȱ thatȱ allowedȱ theȱ comparisonȱ ofȱ theȱ activitiesȱ ofȱ crudeȱpeptidesȱtoȱavoidȱtheȱneedȱforȱpurifyingȱlargeȱnumbersȱofȱpeptidesȱinȱtheȱinitialȱ screeningȱtestsȱ(supplementaryȱdata).ȱSeveralȱdiȬpeptidesȱimprovedȱtheȱKiȱvaluesȱaroundȱ twoȬfoldȱoverȱFXII618ȱ(Figureȱ24A).ȱAȱstabilityȱassayȱshowedȱthatȱtheȱdiȬpeptidesȱbasedȱ onȱDȬaminoȱacidsȱwereȱresistantȱtoȱproteolysis,ȱprolongingȱtheȱplasmaȱhalfȬlifeȱaroundȱ fiveȬfoldȱtoȱ20ȱhoursȱ(FigureȱS4.1).ȱTheȱbestȱinhibitor,ȱArg1ÆDȬArgȬDȬSerȱ(FXII850)ȱwasȱ synthesizedȱwithoutȱtheȱcoumarinȱlabelȱandȱshowedȱaȱKiȱofȱ5ȱ±ȱ2ȱnM,ȱwhichȱwasȱaȱ1.6Ȭ foldȱimprovementȱcomparedȱtoȱFXII618ȱ(Kiȱ=ȱ8.1ȱ±ȱ0.7ȱnM;ȱFigureȱ24BȱandȱC,ȱFigureȱS4.2).ȱ WeȱalsoȱtestedȱaȱpanelȱofȱdiverseȱaminoȱacidȱsubstitutionsȱforȱArg8,ȱaȱpositionȱinȱFXII618ȱ

63 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ thatȱhadȱnotȱbeenȱsystematicallyȱoptimized,ȱusingȱtheȱsameȱscreeningȱstrategyȱbasedȱonȱ coumarinȬlabeledȱpeptidesȱ(Figureȱ24A,ȱTableȱS4.1).ȱTheȱsubstitutionȱArg8ÆHisȱshowedȱ improvedȱinhibition,ȱparticularlyȱofȱmouseȱFXIIa,ȱwhichȱallowedȱforȱtheȱconsiderationȱ ofȱpreȬclinicalȱstudiesȱinȱmice.ȱTheȱpeptideȱwithoutȱtheȱcoumarinȱlabelȱ(FXII851)ȱinhibȬ itedȱhumanȱandȱmouseȱFXIIaȱwithȱKiȱvaluesȱofȱ6.5ȱ±ȱ0.3ȱnMȱandȱ36ȱ±ȱ3ȱnM,ȱrespectively,ȱ whichȱcorrespondedȱtoȱ1.2Ȭȱandȱ2.4ȬfoldȱimprovementsȱoverȱFXII618ȱ(Figureȱ24BȱandȱC,ȱ Figure.ȱS4.2).ȱ

Weȱnextȱcombinedȱtheȱbeneficialȱaminoȱacidȱsubstitutionsȱidentifiedȱinȱthisȱwork,ȱtheȱ Arg1ÆDȬArgȬDȬSerȱ(FXII850)ȱandȱArg8ÆHisȱ(FXII851)ȱwithȱtheȱtwoȱpreviouslyȱidentiȬ fiedȱsubstitutions,ȱArg11Æ(S)ȬE3Ȭhomoarginineȱ(EhArg;ȱFXII700;ȱKiȱ=ȱ1.5ȱ±ȱ0.1ȱnM)ȱ(246)ȱ andȱPhe3Æ4Ȭfluorophenylalanineȱ(Phe4F;ȱFXII800;ȱKiȱ=ȱ0.84ȱ±ȱ0.03ȱnM)ȱ(204)ȱ(Figureȱ24Bȱ andȱC,ȱTableȱS4.2).ȱInȱaddition,ȱweȱdeletedȱtheȱCȬterminalȱArg13ȱthatȱweȱhadȱpreviouslyȱ foundȱtoȱnotȱcontributeȱtoȱtheȱbindingȱaffinityȱ(unpublishedȱresult).ȱTheȱresultingȱinhibȬ itor,ȱFXII900,ȱblockedȱhumanȱandȱmouseȱFXIIaȱwithȱKiȱvaluesȱofȱ0.37ȱ±ȱ0.04ȱnMȱandȱ0.45ȱ ±ȱ0.11ȱnM,ȱrespectively,ȱandȱwasȱthusȱsubstantiallyȱmoreȱpotentȱthanȱanyȱofȱitsȱprecurȬ sorsȱ(Figureȱ24C).ȱAȱspecificityȱprofileȱagainstȱaȱpanelȱofȱhomologousȱplasmaȱproteasesȱ showedȱthatȱFXII900ȱisȱhighlyȱselectiveȱ(Figureȱ24D,ȱTableȱS4.3).ȱAllȱphysiologicallyȱrelȬ evantȱproteasesȱdisplayedȱ100,000Ȭfoldȱorȱhigherȱselectivityȱ(Kiȱvaluesȱ>ȱ40ȱPM),ȱwithȱ onlyȱ trypsinȱ beingȱ inhibitedȱ atȱ lowȱ micromolarȱ concentrationsȱ (Kiȱ =ȱ 1.46ȱ ±ȱ 0.12ȱ PM).ȱ FXII900ȱcouldȱbeȱefficientlyȱsynthesizedȱinȱgramȬscale,ȱbyȱsolidȬphaseȱpeptideȱsynthesisȱ ofȱtheȱlinearȱprecursorȱandȱsubsequentȱmacrocyclizationȱofȱtheȱcrudeȱpeptideȱwithȱtheȱ 1,3,5ȬtriacryloylȬ1,3,5Ȭtriazinaneȱ linkerȱ (TATA).ȱ Onlyȱ aȱ singleȱ HPLCȱ purificationȱ stepȱ wasȱrequiredȱtoȱobtainȱ>ȱ95%ȱpureȱproductȱwithȱanȱisolatedȱyieldȱgreaterȱthanȱ50%ȱ(FigȬ ureȱS4.3).ȱȱ

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IncubatingȱFXII900ȱinȱhumanȱplasmaȱatȱ37°CȱforȱextendedȱtimeȱperiodsȱandȱthenȱquanȬ tifyingȱtheȱremainingȱFXIIaȱinhibitoryȱactivityȱwasȱusedȱtoȱmeasureȱtheȱstabilityȱofȱtheȱ inhibitorȱinȱplasmaȱ(Figureȱ24E).ȱMassȱspectrometricȱanalysisȱofȱtheȱvariousȱplasmaȱsamȬ plesȱshowedȱonlyȱFXII900ȱandȱnoȱdegradationȱproducts,ȱwhereinȱtheȱquantityȱofȱtheȱinȬ tactȱinhibitorȱwasȱreducedȱoverȱtimeȱ(Figureȱ24F,ȱrightȱpanel).ȱThisȱdataȱsuggestedȱthatȱ afterȱanȱinitialȱcleavageȱeventȱoccurred,ȱtheȱinhibitorȱwasȱrapidlyȱdegradedȱand,ȱthereȬ fore,ȱnotȱdetectable.ȱTheȱhalfȬlifeȱofȱFXII900ȱwasȱ128ȱ±ȱ8ȱh,ȱaroundȱ25Ȭfoldȱlongerȱthanȱ thatȱofȱFXII618.ȱItȱwasȱalsoȱmuchȱlongerȱthanȱthatȱofȱFXII850ȱcarryingȱonlyȱtheȱArg1ÆDȬ

ArgȬDȬSerȱmodificationȱ(t1/2ȱ=ȱ18.5ȱ±ȱ1.3ȱh;ȱFigureȱ24E),ȱindicatingȱthatȱmoreȱthanȱoneȱofȱ theȱ introducedȱ mutationsȱ contributedȱ toȱ theȱ improvedȱ stability.ȱ Inȱ orderȱ toȱ identifyȱ whichȱmodification(s)ȱwereȱimportantȱforȱstability,ȱweȱsynthesizedȱFXII900ȱvariantsȱinȱ whichȱtheȱaminoȱacidȱsubstitutionsȱwereȱindividuallyȱrevertedȱ(FigureȱS4.4,ȱTableȱS4.4).ȱȱ

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Figureȱ24.ȱImprovingȱaffinityȱandȱstabilityȱofȱbicyclicȱpeptideȱFXIIaȱinhibitor.ȱ(A)ȱAminoȱacidsȱArg1ȱandȱ Arg8ȱofȱFXII618ȱwereȱindividuallyȱsubstitutedȱtoȱtwoȱDȬaminoȱacidsȱorȱnaturalȱaminoȱacids,ȱrespectively.ȱ

TheȱrelativeȱKiȱvaluesȱforȱhumanȱandȱmouseȱFXIIaȱareȱindicatedȱasȱcomparedȱtoȱtheȱleadȱpeptideȱFXII618.ȱ Averageȱvaluesȱofȱthreeȱmeasurementsȱareȱshown.ȱ(B)ȱSchematicȱrepresentationȱofȱtheȱleadȱpeptideȱFXII618.ȱ Aminoȱacidȱsubstitutionsȱthatȱimproveȱtheȱactivityȱand/orȱstabilityȱareȱindicated.ȱ(C)ȱInhibitionȱofȱhumanȱ FXIIaȱbyȱFXII618,ȱvariantsȱofȱFXII618ȱcontainingȱaȱsingleȱaminoȱacidȱsubstitution,ȱandȱFXII900ȱinȱwhichȱfourȱ beneficialȱmodificationsȱwereȱcombinedȱandȱArg13ȱwasȱdeleted.ȱResidualȱFXIIaȱactivityȱwasȱmeasuredȱatȱ leastȱthreeȱtimes.ȱMeansȱ±ȱSDȱareȱindicated.ȱ(D)ȱSpecificityȱprofilingȱofȱFXII900.ȱResidualȱactivitiesȱofȱhumanȱ FXIIaȱandȱ11ȱhomologousȱhumanȱproteasesȱwereȱmeasuredȱthreeȱtimes.ȱMeansȱ±ȱSDȱareȱindicated.ȱ(E)ȱProȬ teolyticȱstabilityȱofȱFXII900ȱandȱtwoȱprecursors.ȱBicyclicȱpeptideȱwasȱincubatedȱinȱhumanȱplasmaȱatȱ37°Cȱforȱ theȱindicatedȱtimeȱperiods,ȱandȱtheȱremainingȱinhibitorȱactivityȱwasȱquantifiedȱinȱaȱFXIIaȱactivityȱassayȱusingȱ fluorogenicȱsubstrate.ȱMeansȱ±ȱSDȱareȱindicated.ȱ(F)ȱProteolyticȱstabilityȱofȱFXII618ȱandȱFXII900.ȱBicyclicȱ peptideȱwasȱincubatedȱinȱplasmaȱatȱ37°CȱforȱtheȱindicatedȱtimeȱperiodsȱandȱanalyzedȱbyȱLCȬMS.ȱȱ ȱȱ

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Massȱspectrometricȱanalysisȱofȱtheȱvariantsȱincubatedȱinȱhumanȱplasmaȱrevealedȱthatȱ theȱArg11ÆEhArgȱcontributedȱtheȱmostȱtoȱtheȱstabilityȱimprovementȱ(FigureȱS4.5).ȱ

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ToȱassessȱtheȱabilityȱofȱFXII900ȱtoȱblockȱFXIIȬdrivenȱbloodȱplasmaȱcoagulationȱandȱtoȱ evaluateȱfurtherȱitsȱselectivity,ȱweȱperformedȱcoagulationȱtestsȱtoȱmeasureȱactivatedȱparȬ tialȱthromboplastinȱtimeȱ(aPTT)ȱandȱprothrombinȱtimeȱ(PT).ȱaPTTȱandȱPTȱmeasureȱtheȱ timeȱuntilȱcoagulationȱuponȱinitiationȱofȱtheȱintrinsicȱandȱextrinsicȱpathways,ȱrespecȬ tively,ȱmeaningȱthatȱselectiveȱFXIIaȱinhibitionȱwouldȱprolongȱaPTTȱbutȱnotȱPT.ȱFXII900ȱ prolongedȱaPTTȱinȱhumanȱplasmaȱwithȱanȱEC1.5xȱofȱ0.79ȱ±ȱ0.08ȱPM,ȱthusȱ4.3Ȭfoldȱbetterȱ thanȱitsȱprecursorȱFXII618ȱ(EC1.5xȱofȱ3.4ȱ±ȱ0.3ȱPM;ȱFigureȱ25A).ȱFXII900ȱdidȱnotȱaffectȱPT,ȱ evenȱatȱtheȱhighestȱconcentrationȱtestedȱ(120ȱPM),ȱconfirmingȱtheȱselectivityȱforȱtheȱFXIIȱ targetȱ(Figureȱ25B).ȱȱ

Figureȱ25.ȱInhibitionȱofȱtheȱintrinsicȱcoagulationȱpathwayȱinȱhuman,ȱmouse,ȱrabbit,ȱandȱpigȱplasmaȱexȱ vivo.ȱ(A)ȱProlongationȱofȱtheȱaPTTȱinȱtheȱpresenceȱofȱdifferentȱconcentrationsȱofȱFXII900.ȱForȱhumanȱandȱ mouseȱplasma,ȱtheȱaPTTȱofȱFXII618ȱwasȱmeasuredȱforȱcomparison.ȱ(B)ȱPTȱinȱtheȱpresenceȱofȱdifferentȱconȬ centrationsȱofȱFXII900ȱandȱFXII618.ȱAllȱcoagulationȱtimesȱwereȱmeasuredȱinȱtriplicateȱandȱmeansȱ±ȱSDȱareȱ indicatedȱȱ

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ThisȱcontrastsȱwithȱFXII618ȱthatȱshowedȱaȱslightȱprolongationȱofȱPTȱatȱtheȱhighestȱconȬ centrationȱtestedȱ(1.2Ȭfoldȱatȱ120ȱPM),ȱindicatingȱanȱimprovedȱselectivityȱofȱFXII900ȱoverȱ FXII618.ȱTheȱinhibitorȱalsoȱefficientlyȱprolongedȱtheȱaPTTȱinȱplasmaȱofȱallȱotherȱspeciesȱ tested,ȱnamelyȱmouseȱ(EC1.5xȱ=ȱ2.9ȱ±ȱ0.5ȱPM),ȱrabbitȱ(EC1.5xȱ=ȱ0.102ȱ±ȱ0.001ȱPM)ȱandȱpigȱ

(EC1.5xȱ=ȱ12.2ȱ±ȱ0.7ȱPM;ȱFigureȱ25A).ȱInȱmouseȱplasma,ȱtheȱaPTTȱimprovementȱwasȱmoreȱ thanȱ10ȬfoldȱcomparedȱtoȱFXII618ȱ(EC1.5xȱ>ȱ30ȱPM).

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Weȱassessedȱtheȱpharmacokineticȱpropertiesȱinȱthreeȱspecies,ȱmice,ȱrabbits,ȱandȱpigs,ȱasȱ suitableȱmodelsȱforȱthromboticȱdiseasesȱareȱavailableȱforȱtheseȱanimals.ȱInȱmice,ȱweȱdeȬ terminedȱtheȱpharmacokineticsȱfollowingȱsubcutaneousȱadministrationȱ(5ȱmg/kg,ȱnȱ=ȱ2ȱ perȱ timeȱ point).ȱ FXII900ȱ reachedȱ aȱ plasmaȱ concentrationȱ ofȱ aroundȱ 1ȱ PMȱ afterȱ threeȱ minutesȱandȱremainedȱaboveȱthisȱconcentrationȱforȱ30ȱminutesȱasȱdeterminedȱbyȱLCȬMSȱ (Figureȱ26A,ȱupperȱpanel).ȱTheȱaPTTȱwasȱprolongedȱasȱexpectedȱbasedȱonȱtheȱplasmaȱ concentrations,ȱnamelyȱitȱwasȱextendedȱbyȱtwoȬfoldȱatȱ15ȱminutesȱandȱremainedȱproȬ longedȱbyȱoverȱ1.5Ȭfoldȱuntilȱ30ȱminutesȱ(Figureȱ26A,ȱlowerȱpanel).ȱ

Inȱrabbits,ȱweȱdeterminedȱtheȱpharmacokineticȱpropertiesȱforȱintravenousȱandȱsubcutaȬ neousȱ administration.ȱ Uponȱ intravenousȱ administrationȱ (nȱ =ȱ 3,ȱ 3.7ȱ mg/kg),ȱ FXII900ȱ showedȱanȱeliminationȱhalfȬlifeȱofȱ12ȱ±ȱ2ȱminȱ(Figureȱ26B,ȱupperȱpanel;ȱpharmacokineticȱ parametersȱareȱprovidedȱinȱTableȱS4.5).ȱTheȱaPTTȱwasȱinitiallyȱmoreȱthanȱeightȬfoldȱproȬ longedȱandȱremainedȱmoreȱthanȱthreeȬfoldȱprolongedȱforȱtheȱentireȱtimeȱmonitoredȱ(40ȱ min;ȱFigureȱ26B,ȱlowerȱpanel).ȱWhenȱapplyingȱtheȱsameȱdoseȱsubcutaneouslyȱ(nȱ=ȱ4,ȱ3.7ȱ mg/kg),ȱtheȱpeptideȱremainedȱinȱaȱnarrowerȱconcentrationȱrange,ȱstayingȱaboveȱ100ȱnMȱ andȱbelowȱ300ȱnMȱforȱbetweenȱ10ȱandȱ80ȱminutesȱafterȱadministrationȱ(Figureȱ26C,ȱupȬ perȱpanel).ȱTheȱaPTTȱwithȱtheȱsubcutaneousȱadministrationȱwasȱprolongedȱbyȱmoreȱthanȱ twoȬfoldȱforȱ40ȱminutesȱ(Figureȱ26C,ȱlowerȱpanel).ȱ

WeȱfurtherȱdeterminedȱtheȱpharmacokineticsȱofȱFXII900ȱinȱpigs,ȱasȱthisȱspeciesȱoffersȱ modelsȱforȱindicationsȱinȱwhichȱFXIIȱplaysȱaȱrole,ȱsuchȱasȱischemicȱreperfusionȱinjuryȱ (237,ȱ247).ȱForȱthisȱstudy,ȱweȱappliedȱconditionsȱthatȱareȱusedȱinȱtheȱdiseaseȱmodelȱforȱ reperfusionȱ injury,ȱ whichȱ includedȱ heparinȱ anticoagulationȱ toȱ preventȱ inductionȱ ofȱ thrombosisȱthroughȱtheȱcathetersȱthatȱisȱusedȱtoȱinduceȱischemiaȱorȱtoȱmeasureȱbloodȱ pressureȱȱ(2500ȱIUȱwhenȱACTȱfallȱbelowȱ180ȱseconds).ȱAfterȱintravenousȱadministrationȱ (nȱ=ȱ3,ȱ4ȱmg/kg)ȱtheȱinhibitorȱreachedȱaȱmaximalȱconcentrationȱofȱaroundȱ7ȱPMȱandȱwasȱ clearedȱwithȱaȱt1/2ȱofȱ36ȱ±ȱ5ȱminȱ(Figureȱ26D,ȱupperȱpanel).ȱTheȱclearanceȱrateȱandȱvolumeȱ ofȱdistributionȱwereȱ11.8ȱ±ȱ1.8ȱml/kg/minȱandȱ610ȱ±ȱ140ȱml/kgȱ(TableȱS4.6).ȱDespiteȱtheȱ applicationȱofȱheparinȱthatȱboreȱtheȱriskȱofȱshieldingȱtheȱeffectȱofȱFXII900,ȱaȱtwoȬfoldȱ prolongationȱofȱtheȱaPTTȱwasȱseenȱinȱplasmaȱsamplesȱtakenȱaȱshortȱtimeȱafterȱtheȱadȬ ministrationȱofȱtheȱinhibitorȱ(Figureȱ26E).ȱTheȱsameȱeffectȱwasȱnotȱseenȱforȱtheȱnegativeȱ

67 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ controlȱpeptide,ȱFXII901,ȱaȱvariantȱofȱFXII900ȱwithȱthreeȱmodificationsȱ(Phe4F3ÆPhe,ȱ Arg4ÆAla,ȱΆhArg11ÆArg,ȱusingȱtheȱaminoȱacidȱnumberingȱofȱFXII618;ȱFigureȱS4.6)ȱthatȱ reduceȱitsȱinhibitoryȱconstantȱ100,000ȬfoldȱforȱFXIIaȱ(Kiȱ=ȱ39ȱ±ȱ14ȱΐM),ȱthoughȱalsoȱlowerȱ itsȱplasmaȱhalfȬlifeȱdueȱtoȱproteolyticȱdegradationȱ(Figureȱ26D,ȱlowerȱpanel).ȱTheȱsmallȱ andȱshortȱeffectȱofȱFXII900ȱonȱaPTTȱcanȱbeȱexplainedȱwithȱtheȱcomparativelyȱhighȱinhibȬ itorȱconcentrationsȱrequiredȱtoȱprolongȱtheȱaPTTȱinȱpigȱplasmaȱ(EC1.5xȱ=ȱ12.2ȱ±ȱ0.7ȱPM).ȱ

Figureȱ26.ȱPharmacokineticsȱofȱFXII900ȱinȱmouse,ȱrabbit,ȱandȱpigsȱandȱinhibitionȱofȱFXIIȬdrivenȱcoaguȬ lation.ȱ (A)ȱ Pharmacokineticsȱ inȱ mouseȱ afterȱ subcutaneousȱ administrationȱ (5ȱ mg/kg).ȱ Concentrationȱ ofȱ FXII900ȱinȱplasmaȱ(upperȱpanel)ȱandȱaPTTȱ(lowerȱpanel)ȱareȱindicated.ȱTwoȱmiceȱwereȱusedȱforȱeachȱtimeȱ point,ȱandȱtheȱsamplesȱwereȱanalyzedȱinȱtriplicate.ȱMeansȱ±ȱSDȱwereȱcombined.ȱ(B)ȱPharmacokineticsȱinȱ rabbitȱafterȱintravenousȱadministrationȱ(3.7ȱmg/kg,ȱnȱ=ȱ3).ȱConcentrationȱofȱFXII900ȱinȱplasmaȱ(upperȱpanel)ȱ andȱaPTTȱ(lowerȱpanel)ȱareȱindicated.ȱPlasmaȱsamplesȱwereȱanalyzedȱinȱtriplicate.ȱMeansȱ±ȱSDȱareȱindicatedȱ forȱeachȱrabbit.ȱ(C)ȱPharmacokineticsȱinȱrabbitȱafterȱsubcutaneousȱadministrationȱ(3.7ȱmg/kg,ȱnȱ=ȱ4).ȱConȬ centrationȱofȱFXII900ȱinȱplasmaȱ(upperȱpanel)ȱandȱaPTTȱ(lowerȱpanel)ȱwasȱmeasured.ȱPlasmaȱsamplesȱwereȱ analyzedȱinȱtriplicate.ȱMeansȱ±ȱSDȱareȱindicatedȱforȱeachȱrabbit.ȱ(D)ȱPharmacokineticsȱinȱpigȱafterȱintraveȬ nousȱadministrationȱ(4ȱmg/kg,ȱnȱ=ȱ3).ȱConcentrationsȱofȱFXII900ȱandȱinactiveȱpeptideȱFXII901ȱinȱplasmaȱwereȱ

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determinedȱinȱtriplicate,ȱandȱmeansȱ±ȱSDȱareȱindicated.ȱ(E)ȱProlongationȱofȱaPTTȱinȱpig.ȱPigsȱanticoagulatedȱ withȱheparinȱ(injectionsȱofȱ2500ȱIUȱwhenȱACTȱwasȱbelowȱ180ȱs;ȱinjectionsȱareȱindicatedȱbyȱarrows)ȱwereȱ treatedȱwithȱFXII900ȱorȱtheȱnegativeȱcontrolȱFXII901ȱatȱtimeȱ0ȱ(indicatedȱbyȱdashedȱline;ȱnȱ=ȱ3ȱinȱeachȱgroup,ȱ 4ȱmg/kg),ȱandȱtheȱaPTTȱwasȱmeasuredȱinȱplasmaȱsamplesȱtakenȱoverȱtime.ȱaPTTȱwasȱmeasuredȱinȱtriplicate.ȱ Meansȱ±ȱSDȱareȱindicatedȱ

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Weȱ nextȱ evaluatedȱ theȱ thromboprotectiveȱ propertiesȱ ofȱ FXII900ȱ inȱ aȱ ferricȱ chlorideȱ

(FeCl3)Ȭinducedȱarterialȱthrombosisȱmouseȱmodel.ȱWhenȱappliedȱdirectlyȱontoȱtheȱbloodȱ vessels,ȱFeCl3ȱinducesȱthrombosisȱthroughȱaggregationȱofȱtheȱredȱbloodȱcells,ȱwhichȱinȱ turnȱactivatesȱplateletsȱatȱtheȱapplicationȱsiteȱ(248,ȱ249).ȱFXII900,ȱorȱanȱinactiveȱcontrolȱ peptideȱFXII901,ȱwereȱadministeredȱsubcutaneouslyȱ(nȱ=ȱ10ȱforȱbothȱgroups,ȱ5ȱmg/kg),ȱ withȱ7.5%ȱFeCl3ȱappliedȱtoȱtheȱmesentericȱarteriolesȱwhileȱtheȱbloodȱflowȱwasȱmonitoredȱ byȱintravitalȱmicroscopy.ȱMiceȱthatȱwereȱinjuredȱbyȱFeCl3ȱcouldȱbeȱclearlyȱidentifiedȱdueȱ toȱaȱspeckledȱpatternȱthatȱsupposedlyȱrepresentedȱanȱaccumulationȱofȱplateletsȱ(Figureȱ 27A)ȱ(250).ȱSeventeenȱoutȱofȱtheȱ20ȱmiceȱshowedȱthisȱpatternȱ(nineȱtreated,ȱeightȱcontrol)ȱ andȱwereȱtakenȱforȱfurtherȱanalysisȱ(FigureȱS4.7ȱandȱS4.8).ȱTheȱarteriolesȱofȱtheȱmiceȱ receivingȱtheȱinactiveȱcontrolȱpeptideȱFXII901ȱrapidlyȱformedȱclots,ȱandȱinȱaȱmajorityȱofȱ theȱanimals,ȱtheȱvesselsȱwereȱcompletelyȱoccludedȱafterȱaroundȱ10ȱminutes,ȱasȱexempliȬ fiedȱinȱtheȱlowerȱmicrographsȱinȱFigureȱ27AȱandȱshownȱforȱallȱmiceȱinȱFigureȱS4.7.ȱInȱ contrast,ȱmiceȱthatȱwereȱtreatedȱwithȱtheȱinhibitorȱFXII900ȱshowedȱonlyȱtheȱcharacteristicȱ speckledȱpatternȱwithȱtheȱoccasionalȱformationȱofȱaȱbloodȱclotȱ(Figureȱ27A,ȱupperȱmicroȬ graphs;ȱFigureȱS4.8).ȱInȱthisȱgroup,ȱnoȱcompleteȱocclusionȱwasȱobserved.ȱWeȱquantifiedȱ theȱ anticoagulantȱ effectȱ ofȱ FXII900ȱ byȱ comparingȱ theȱ ratesȱ forȱ bothȱ clotȱ formationȱ (aȱ clearlyȱvisibleȱbloodȱclot;ȱFigureȱ27B,ȱleftȱpanel)ȱandȱbloodȱvesselȱocclusionȱ(aȱbloodȱclotȱ withȱaȱdiameterȱofȱtheȱbloodȱvessel;ȱFigureȱ27B,ȱrightȱpanel).ȱOfȱtheȱeightȱmiceȱreceivingȱ theȱcontrolȱpeptide,ȱsevenȱshowedȱbloodȱclottingȱandȱfourȱshowedȱfullȬvesselȱocclusion.ȱ Inȱcontrast,ȱinȱtheȱFXII900Ȭtreatedȱmice,ȱonlyȱthreeȱshowedȱbloodȱclotting,ȱandȱinȱnoȱmiceȱ wereȱtheȱvesselsȱfullyȱoccludedȱ(pȱ=ȱ0.02ȱandȱ0.005,ȱrespectively).ȱInȱaddition,ȱtheȱfewȱ miceȱinȱtheȱtreatedȱgroupȱthatȱshowedȱsignsȱofȱcoagulationȱdevelopedȱtheseȱbloodȱclotsȱ inȱaverageȱaroundȱ10ȱminutesȱlaterȱthanȱtheȱcontrolȱgroupȱ(pȱ=ȱ0.006;ȱTableȱS4.7).ȱȱ

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Figureȱ27.ȱThromboprotectionȱofȱFXII900ȱinȱaȱferricȱchlorideȬinducedȱthrombosisȱmouseȱmodel.ȱ(A)ȱInȬ travitalȱfluorescenceȱmicroscopyȱimagesȱshowingȱmesentericȱarteriolesȱinȱwhichȱthrombosisȱwasȱinducedȱ byȱtopicalȱapplicationȱofȱFeCl3ȱ(7.5%,ȱ1ȱmin).ȱPlateletsȱwereȱfluorescentlyȱlabeledȱwithȱRhodamineȱ6Gȱforȱ visualization.ȱRepresentativeȱimagesȱareȱshownȱforȱtwoȱmice,ȱoneȱtreatedȱwithȱFXII900ȱ(upperȱpanels)ȱandȱ oneȱ withȱ inactiveȱ controlȱ peptideȱ FXII901ȱ (lowerȱ panels)ȱ 15ȱ minȱ beforeȱ applicationȱ ofȱ ferricȱ chlorideȱ (5ȱ mg/kg,ȱsubcutaneousȱinjection).ȱVesselȱwallsȱatȱtheȱFeCl3ȱapplicationȱsiteȱareȱindicatedȱwithȱyellowȱmarkers.ȱ Threeȱdistinctȱmorphologicalȱchanges,ȱaȱcharacteristicȱspeckledȱpatternȱ(1),ȱclotȱformationȱ(2),ȱandȱvesselȱ occlusionȱ(3),ȱareȱindicated.ȱ(B)ȱTheȱpercentageȱofȱmiceȱshowingȱeitherȱclotȱformationȱorȱfullȱocclusionȱatȱ differentȱtimeȱpointsȱafterȱferricȱchlorideȱapplicationȱisȱindicatedȱoverȱtime.ȱClotȱformationȱwasȱdefinedȱasȱ theȱappearanceȱofȱanȱaggregateȱwithȱaȱdiameterȱofȱaroundȱ10ȱΐm.ȱFullȱocclusionȱofȱtheȱbloodȱvesselȱwasȱ definedȱasȱaȱbloodȱclotȱhavingȱtheȱsameȱdiameterȱasȱtheȱbloodȱvessel

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ToȱtestȱtheȱclinicalȱpotentialȱofȱFXII900ȱforȱinhibitingȱFXIIȬdrivenȱcoagulationȱinȱECMO,ȱ weȱtestedȱtheȱinhibitorȱinȱanȱartificialȱlungȱrabbitȱmodel.ȱNewȱZealandȱWhiteȱrabbitsȱ wereȱanesthetized,ȱeitherȱtreatedȱintravenouslyȱwithȱFXII900ȱ(nȱ=ȱ4,ȱ2ȱmg/kgȱbolusȱandȱ 0.075ȱmg/kg/minȱinfusionȱforȱfourȱhours)ȱorȱleftȱuntreatedȱ(controlȱgroup,ȱnȱ=ȱ3),ȱandȱ wereȱmountedȱforȱaȱfourȬhourȱperiodȱtoȱaȱvenous/venousȱECMOȱconfigurationȱusingȱaȱ polymethylpenteneȱ(PMP)ȱfiberȱartificialȱlungȱsystemȱ(251).ȱCoagulationȱparametersȱandȱ theȱ resistanceȱ ofȱ theȱ lungȱ device,ȱ aȱ measureȱ forȱ occlusiveȱ thrombusȱ formation,ȱ wereȱ

70 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ measuredȱoverȱtheȱentireȱdurationȱofȱtheȱexperiment,ȱandȱclotȱformationȱinȱtheȱlungȱdeȬ viceȱwasȱanalyzedȱafterȱtheȱfourȱhoursȱwereȱcompletedȱ(Figureȱ28;ȱdataȱforȱtheȱindividualȱ rabbitsȱisȱshownȱinȱFigureȱS4.9).ȱ

TreatmentȱwithȱFXII900ȱprolongedȱtheȱcoagulationȱparametersȱaPTTȱandȱactivatedȱclotȬ tingȱtimeȱ(ACT)ȱaroundȱ10Ȭfoldȱduringȱtheȱentireȱcourseȱofȱtheȱexperimentȱ(Figureȱ28Aȱ andȱB).ȱForȱmostȱaPTTȱmeasurements,ȱnoȱcoagulationȱwasȱobservedȱatȱ240ȱseconds,ȱbeȬ ingȱtheȱmaximalȱaPTTȱvalueȱmeasurementȱwithȱtheȱdeviceȱusedȱ(FigureȱS4.9A).ȱInȱunȬ treatedȱrabbits,ȱtheȱcoagulationȱtimesȱremainedȱatȱbaselineȱvaluesȱofȱaroundȱ20ȱsecondsȱ (aPTT)ȱandȱ170ȱsecondsȱ(ACT)ȱthroughoutȱtheȱexperiment.ȱIncreasedȱbloodȱpressureȱatȱ theȱinletȱofȱtheȱartificialȱlungȱindicatesȱcloggingȱofȱtheȱdeviceȱthatȱcanȱbeȱcausedȱbyȱbloodȱ coagulation.ȱWeȱthereforeȱmeasuredȱtheȱresistanceȱatȱtheȱartificialȱlung,ȱindicatedȱbyȱtheȱ differenceȱinȱpressureȱatȱtheȱinletȱandȱoutletȱofȱtheȱlungȱdividedȱbyȱtheȱflow.ȱInȱallȱrabbitsȱ treatedȱwithȱFXII900,ȱtheȱresistanceȱremainedȱatȱtheȱbaselineȱofȱ50ȱmmHg/L/minȱduringȱ theȱentireȱexperiment,ȱexceptȱforȱoneȱrabbitȱinȱwhichȱtheȱresistanceȱstartedȱtoȱincreaseȱ stronglyȱafterȱtwoȱhoursȱatȱbaselineȱ(Figureȱ28CȱandȱFigureȱS4.9C).ȱThisȱwasȱinȱstarkȱ contrastȱtoȱtheȱuntreatedȱanimalsȱinȱwhichȱtheȱresistanceȱdoubledȱfromȱ50ȱmmHg/L/minȱ toȱ100ȱmmHg/L/minȱorȱhigher.ȱȱ

Withȱexcessiveȱbleedingȱasȱtheȱmainȱconcernȱwhenȱapplyingȱstandardȱanticoagulationȱ inȱ ECMOȱ systems,ȱ weȱ analyzedȱ ifȱ FXII900ȱ affectedȱ theȱ hematologicalȱ parametersȱ ofȱ bleedingȱtimeȱandȱplateletȱcount.ȱRabbitsȱtreatedȱwithȱtheȱinhibitorȱshowedȱcompletelyȱ normalȱ bleedingȱ timesȱ throughoutȱ theȱ experimentȱ (4–5ȱ min;ȱ Figureȱ 28Dȱ andȱ Figureȱ S4.9D).ȱTheȱplateletȱcountȱwasȱessentiallyȱtheȱsameȱinȱtheȱtwoȱgroups,ȱdecreasingȱaroundȱ twoȬfoldȱfromȱanȱinitialȱlevelȱofȱ2ȱxȱ108ȱcells/mlȱ(beforeȱconnectingȱtheȱlung)ȱandȱremainȬ ingȱconstantȱoverȱtheȱentireȱcourseȱofȱtheȱexperimentȱ(Figureȱ28EȱandȱFigureȱS4.9E).ȱȱ

Atȱtheȱendȱofȱtheȱexperiment,ȱweȱdeterminedȱtheȱextentȱofȱbloodȱclotȱformationȱinsideȱofȱ theȱartificialȱlungsȱbyȱmeasuringȱtheȱtotalȱvolumeȱofȱtheȱbloodȱclotȱinȱeachȱlung.ȱAȱvisualȱ inspectionȱofȱtheȱmembranesȱshowedȱaȱsubstantiallyȱreducedȱamountȱofȱclottedȱbloodȱinȱ theȱinhibitorȬtreatedȱrabbits.ȱThisȱfindingȱwasȱconfirmedȱbyȱquantifyingȱvolumeȱofȱtheȱ clotȱinȱrelationȱtoȱtheȱdeviceȱvolume,ȱwhichȱwasȱ10ȱ±ȱ6%ȱforȱtheȱFXII900Ȭtreatedȱandȱ37ȱ ±ȱ10%ȱforȱtheȱuntreatedȱrabbitsȱ(pȱ=ȱ0.03;ȱFigureȱ28FȱandȱFigureȱS4.9F).ȱ

ȱȱ

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Figureȱ28.ȱBleedingȬfreeȱanticoagulationȱinȱanȱartificialȱlungȱmodelȱinȱrabbits.ȱRabbitsȱwereȱconnectedȱ venoȬvenousȱtoȱanȱartificialȱlungȱsystemȱforȱfourȱhours.ȱFXII900ȱwasȱinjectedȱasȱaȱbolusȱ(2ȱmg/kg)ȱbeforeȱtheȱ startȱofȱtheȱextracorporealȱcirculationȱandȱasȱaȱconstantȱinfusionȱ(0.075ȱmg/kg/min)ȱoverȱtheȱfullȱtimeȱcourseȱ ofȱtheȱexperimentȱ(nȱ=ȱ4;ȱdataȱshownȱinȱblue).ȱRabbitsȱinȱtheȱcontrolȱgroupȱwereȱuntreatedȱ(nȱ=ȱ3,ȱdataȱshownȱ inȱred).ȱValuesȱindicatedȱforȱtheȱtimeȱpointȱ0ȱreferȱtoȱmeasurementsȱmadeȱbeforeȱconnectionȱofȱtheȱartificialȱ lung.ȱMeanȱ±ȱSDȱisȱindicatedȱforȱtheȱrabbitsȱinȱeachȱgroupȱunlessȱindicated.ȱDataȱofȱindividualȱrabbitsȱisȱ shownȱinȱFigureȱS4.9.ȱ(A)ȱaPTT.ȱCoagulationȱtimesȱofȱ240ȱsȱindicateȱthatȱplasmaȱdidȱnotȱcoagulateȱatȱthisȱ time.ȱ(B)ȱACT.ȱClottingȱtimesȱofȱ1500ȱsȱindicateȱthatȱbloodȱdidȱnotȱcoagulateȱatȱthisȱtime.ȱ(C)ȱResistanceȱ calculatedȱbasedȱonȱpressureȱatȱtheȱinletȱandȱoutletȱofȱtheȱdeviceȱandȱtheȱflowȱrate.ȱ(D)ȱBleedingȱtimeȱmeasȬ uredȱbyȱincisionȬprovokedȱinjuriesȱatȱtheȱears.ȱ(E)ȱPlateletȱcount.ȱ(F)ȱVolumeȱofȱbloodȱclotsȱindicatedȱinȱ%ȱofȱ volumeȱofȱtheȱartificialȱlungs.ȱForȱtheȱcontrolȱgroup,ȱmeanȱ±ȱSDȱisȱindicatedȱonlyȱforȱthreeȱoutȱofȱtheȱfourȱ rabbits,ȱasȱforȱoneȱanimalȱtheȱvolumeȱwasȱnotȱdetermined ȱ

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TheȱroleȱofȱFXIIȱinȱthrombosisȱobservedȱinȱexperimentalȱanimalȱmodels,ȱbutȱnotȱinȱheȬ mostasisȱoffersȱanȱexcitingȱpotentialȱstrategyȱforȱsafeȱanticoagulationȱbasedȱonȱinterferȬ enceȱwithȱtheȱplasmaȱprotease.ȱVariousȱproteinȬbasedȱinhibitorsȱhaveȱbeenȱdevelopedȱ andȱshowedȱthatȱsuppressionȱofȱFXIIȱactivityȱprovidesȱefficientȱanticoagulationȱwithoutȱ increasingȱbleedingȱrisks.ȱTheȱdevelopmentȱofȱhighȬaffinityȱsmallȱmoleculeȱFXIIaȱinhibȬ itorsȱhasȱbeenȱlaggingȱbehind.ȱGivenȱtheȱattractionȱofȱsmall,ȱsyntheticȱmoleculesȱasȱdrugȱ modality,ȱweȱhaveȱimprovedȱinȱthisȱworkȱtheȱbindingȱaffinityȱandȱstabilityȱofȱFXII618,ȱaȱ peptideȱmacrocycleȱFXIIaȱinhibitorȱthatȱwasȱpreviouslyȱdevelopedȱbyȱphageȱdisplay.ȱTheȱ generatedȱinhibitor,ȱFXII900,ȱblocksȱFXIIaȱwithȱaȱKiȱofȱ370ȱpM,ȱshowsȱ100,000ȬfoldȱselecȬ tivityȱoverȱotherȱplasmaȱproteases,ȱandȱisȱstableȱinȱblood.ȱ

TheȱimprovementȱofȱtheȱbindingȱaffinityȱofȱFXII618ȱbyȱ21Ȭfoldȱandȱproteolyticȱstabilityȱ byȱ25Ȭfoldȱwasȱachievedȱbyȱcombiningȱrandomȱscreeningȱandȱrationalȱdesignȱstrategies.ȱ Twoȱaminoȱacidȱsubstitutionsȱthatȱenhanceȱtheȱaffinityȱand/orȱstabilityȱofȱtheȱinhibitorȱ wereȱidentifiedȱbyȱscreeningȱaȱpanelȱofȱsinglyȱmutatedȱpeptides,ȱandȱwereȱcombinedȱ withȱ twoȱ previouslyȱ identifiedȱ beneficialȱ mutations.ȱ Individualȱ reversionȱ ofȱ theȱ fourȱ aminoȱacidȱsubstitutionsȱinȱFXII900ȱshowedȱthatȱeachȱsingleȱoneȱcontributesȱtoȱimprovȬ ingȱtheȱbindingȱaffinityȱandȱthatȱmainlyȱtheȱmodificationȱofȱArg11ȱtoȱEhArgȱimprovesȱ theȱstability.ȱTheȱhigherȱbindingȱaffinityȱtranslatesȱintoȱaȱprolongationȱofȱtheȱaPTTȱinȱallȱ species.ȱInȱhumanȱplasma,ȱtheȱEC1.5xȱwasȱreducedȱ4.3Ȭfold,ȱandȱinȱmouseȱplasmaȱevenȱ 10Ȭfold.ȱTheȱmuchȱenhancedȱpotencyȱinȱmouseȱplasmaȱwasȱofȱinterestȱinȱviewȱofȱtheȱ plannedȱpharmacologicȱstudyȱinȱmice.ȱFXII900ȱdidȱnotȱprolongȱPTȱatȱ120ȱPMȱatȱall,ȱwhichȱ wasȱinȱcontrastȱtoȱFXII618ȱthatȱprolongedȱPTȱslightlyȱatȱthisȱhighestȱconcentrationȱtested,ȱ indicatingȱthatȱmodificationȱofȱtheȱfourȱaminoȱacidsȱhadȱalsoȱimprovedȱtheȱselectivityȱofȱ theȱinhibitor.ȱTheȱenhancementȱofȱaffinity,ȱselectivityȱandȱstabilityȱachievedȱinȱthisȱworkȱ isȱowedȱmostlyȱtoȱunnaturalȱaminoȱacidsȱthatȱcouldȱbeȱincorporatedȱdueȱtoȱtheȱsyntheticȱ natureȱofȱtheȱinhibitor,ȱwhichȱisȱanȱadvantageȱoverȱproteinȬbasedȱinhibitorsȱthatȱneedȱtoȱ beȱproducedȱbyȱrecombinantȱexpressionȱandȱdoȱnotȱeasilyȱallowȱtheȱincorporationȱofȱ unnaturalȱbuildingȱblocks.ȱ

FXII900ȱisȱcomposedȱofȱnineȱnaturalȱaminoȱacids,ȱfourȱnonȬnaturalȱones,ȱandȱaȱcentralȱ coreȱstructureȱthatȱconnectsȱtheȱsideȱchainsȱofȱtheȱthreeȱcysteines.ȱTheȱinhibitorȱcanȱbeȱ convenientlyȱsynthesizedȱbyȱautomatedȱsolidȬphaseȱpeptideȱsynthesisȱinȱlargeȱquantiȬ ties.ȱAllȱappliedȱnonȬnaturalȱaminoȱacidsȱareȱinexpensive,ȱallowingȱforȱproductionȱofȱ inhibitorȱforȱaȱmoderateȱcost.ȱCrudeȱlinearȱpeptideȱwasȱcyclizedȱefficientlyȱbyȱtheȱchemȬ icalȱlinkerȱTATAȱandȱonlyȱaȱsingleȱHPLCȱpurificationȱstepȱwasȱrequiredȱtoȱobtainȱ>ȱ95%ȱ pureȱproductȱwithȱanȱisolatedȱyieldsȱgreaterȱthanȱ70%.ȱWeȱprovideȱaȱsupplementaryȱ figureȱthatȱdescribesȱtheȱexactȱpeptideȱsequenceȱandȱtheȱconditionsȱrecommendedȱforȱ

73 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ peptideȱcyclization,ȱthatȱmayȱbeȱusedȱforȱorderingȱtheȱpeptideȱfromȱcompaniesȱofferingȱ customȱpeptideȱsynthesisȱ(FigureȱS4.3).ȱ

FXII900ȱisȱrapidlyȱclearedȱbyȱrenalȱfiltration,ȱasȱcouldȱbeȱexpectedȱbasedȱonȱtheȱsmallȱ sizeȱandȱtheȱpolarȱstructure.ȱTheȱeliminationȱhalfȬlifeȱrangesȱfromȱ12ȱminutesȱinȱrabbitsȱ toȱ36ȱminutesȱinȱpigs.ȱBasedȱonȱallometricȱscaling,ȱthisȱhalfȬliveȱinȱhumanȱcanȱbeȱexpectedȱ inȱtheȱrangeȱofȱoneȱhour.ȱNoȱdegradationȱproductsȱwereȱdetectedȱinȱplasmaȱsamplesȱofȱ allȱspecies,ȱsuggestingȱthatȱtheȱinhibitorȱfullyȱresistsȱproteasesȱinȱtheȱbloodȱcirculationȱ andȱthatȱitȱisȱfilteredȱoutȱasȱanȱintactȱmolecule,ȱwhichȱisȱinȱlineȱwithȱtheȱhighȱstabilityȱ andȱlongȱhalfȬlifeȱinȱplasmaȱfoundȱexȱvivo.ȱDespiteȱtheȱfastȱclearance,ȱsingleȬdoseȱinjecȬ tionȱofȱFXII900ȱprolongedȱaPTTȱinȱallȱthreeȱspeciesȱtested.ȱInȱpig,ȱtheȱeffectsȱwereȱsmallȱ andȱseenȱonlyȱforȱaȱfewȱminutesȱafterȱinjection.ȱTheȱsmallȱeffectȱwasȱexpectedȱgivenȱtheȱ relativelyȱhighȱEC1.5xȱforȱaPTTȱinȱpigȱplasma,ȱcausedȱmostȱlikelyȱfromȱaȱweakerȱinhibitionȱ ofȱtheȱporcineȱFXIIa.ȱTheȱrelativelyȱshortȱcirculationȱtimeȱallowsȱflexibleȱadjustmentȱofȱ theȱdurationȱforȱwhichȱtheȱbodyȱisȱexposedȱtoȱtheȱinhibitorȱinȱtherapeuticȱapplications.ȱ Indeed,ȱcontinuousȱinfusionȱinȱtheȱrabbitȱECMOȱstudyȱallowedȱforȱmaintainingȱtheȱaPTTȱ aboveȱ200ȱsecȱoverȱtheȱfourȱhoursȱofȱtheȱexperiment,ȱdespiteȱtheȱrelativelyȱfastȱclearanceȱ ofȱ16.1ȱml/kg/min.ȱTheȱpharmacokineticȱprofileȱofȱFXII900ȱmakesȱitȱattractiveȱforȱacuteȱ diseaseȱtreatmentȱsuchȱasȱCPB,ȱincludingȱECMO,ȱorȱanticoagulationȱforȱshortȱtimeȱperiȬ odsȱafterȱischemicȱstrokeȱorȱafterȱsurgery.ȱ

WeȱhaveȱevaluatedȱtheȱpharmacologicȱactivityȱofȱFXII900ȱinȱaȱferricȱchlorideȬinducedȱ thrombosisȱmouseȱmodel.ȱFXIIȬdeficientȱmiceȱshowȱstrongȱreductionȱofȱcoagulationȱinȱ thisȱmodel,ȱindicatingȱthatȱFXIIȱplaysȱaȱcentralȱroleȱinȱthisȱprocess.ȱWeȱfoundȱthatȱaȱbolusȱ injectionȱofȱFXII900ȱefficientlyȱprotectedȱmiceȱfromȱthrombosis.ȱAtȱtheȱsingleȱdoseȱofȱ5ȱ mg/kgȱtested,ȱnoneȱofȱtheȱnineȱtreatedȱmiceȱshowedȱthromboticȱvesselȱocclusionȱwhereasȱ fiveȱofȱtheȱeightȱinȱtheȱuntreatedȱgroupȱformedȱaȱthrombus.ȱItȱhasȱtoȱbeȱstatedȱthatȱthromȬ bosisȱinȱmyocardialȱinfarction,ȱischemicȱstroke,ȱorȱpulmonaryȱembolismȱisȱmoreȱcomplexȱ thanȱinȱtheȱappliedȱmouseȱthrombosisȱmodel,ȱasȱthrombosisȱisȱalsoȱcausedȱbyȱtriggersȱ thatȱareȱnotȱdependentȱonȱFXIIȱactivation.ȱIndependentȱofȱthis,ȱtheȱresultsȱobtainedȱshowȱ thatȱaȱsyntheticȱFXIIaȱinhibitorȱcanȱefficientlyȱsuppressȱcoagulationȱinȱcasesȱwhereȱcoagȬ ulationȱisȱmediatedȱviaȱFXIIȱactivation.ȱ

WeȱhaveȱfurtherȱtestedȱifȱFXII900ȱisȱsuitedȱtoȱsuppressȱcoagulationȱactivatedȱbyȱtheȱmemȬ braneȱofȱartificialȱlungs,ȱusingȱaȱrabbitȱmodel.ȱHeartȬlungȱmachinesȱareȱusedȱforȱheartȱ surgeryȱbecauseȱofȱtheȱdifficultyȱofȱoperatingȱonȱtheȱbeatingȱheart.ȱApproximatelyȱhalfȱ aȱmillionȱofȱtheȱcardiacȱoperationsȱperformedȱperȱyearȱinȱtheȱUSȱuseȱCPB.ȱHeartȬlungȱ machinesȱareȱadditionallyȱusedȱtoȱsupportȱpatientsȱwithȱcardiorespiratoryȱdysfunction.ȱ Moreȱthanȱ7000ȱpatientsȱareȱtreatedȱannuallyȱwithȱextracorporealȱlifeȱsupportȱandȱtheȱ heartȬlungȱ machineȱ isȱ rescuingȱ manyȱ livesȱ byȱ supportingȱ oxygenationȱ andȱ systemicȱ bloodȱcirculationȱ(252).ȱContactȱofȱbloodȱwithȱnonphysiologicalȱsurfacesȱinducesȱFXIIȱ

74 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ contactȱactivationȱandȱthusȱcoagulation,ȱthatȱisȱcurrentlyȱsuppressedȱbyȱhighȱdoseȱofȱ heparin,ȱwhichȱbearsȱbleedingȱrisks.ȱAnalysisȱofȱnearlyȱ80,000ȱECMOȱpatientsȱshowedȱ thatȱhemorrhageȱisȱtheȱmajorȱadverseȱevent,ȱwithȱbleedingȱfromȱsurgicalȱsitesȱhappeningȱ inȱ6.3%ȱtoȱ29.3%ȱpatients,ȱdependingȱonȱtheȱpatientȱgroupȱ(252).ȱWeȱfoundȱthatȱFXII900ȱ substantiallyȱreducedȱcoagulationȱinȱanȱartificialȱlung,ȱasȱmeasuredȱbyȱtheȱresistanceȱofȱ theȱdeviceȱwhichȱremainedȱlowȱandȱindicatedȱreducedȱcloggingȱbyȱcoagulation,ȱandȱtheȱ volumeȱofȱtheȱbloodȱclotsȱinȱtheȱdeviceȱwhichȱwasȱmoreȱthanȱthreeȬfoldȱsmallerȱinȱtheȱ treatedȱrabbits.ȱImportantly,ȱallȱrabbitsȱtreatedȱwithȱFXII900ȱshowedȱnormalȱbleedingȱ times.ȱTheseȱfindingsȱindicateȱthatȱaȱsmallȱmoleculeȱFXIIaȱinhibitorȱisȱsuitableȱtoȱreduceȱ bloodȱcoagulationȱinȱCPB.ȱWhileȱheparinȱmayȱstillȱbeȱrequiredȱtoȱsuppressȱcoagulationȱ triggeredȱthroughȱotherȱroutesȱthanȱFXII,ȱapplicationȱofȱaȱsmallȱmoleculeȱFXIIaȱinhibitorȱ mayȱallowȱreductionȱofȱtheȱhighȱheparinȱdose,ȱreducingȱbleedingȱrisks.ȱInȱadditionȱtoȱitsȱ roleȱinȱthrombosisȱinȱECMOȱandȱCPBȱinȱgeneral,ȱFXIIaȱinitiatesȱtheȱinflammatoryȱkalȬ likreinȬkinninȱsystemȱ(187).ȱBradykininȱplasmaȱconcentrationsȱareȱlargelyȱelevatedȱinȱ patientsȱonȱCPBȱandȱparticularlyȱtheȱlongerȱlastingȱECMO.ȱTargetingȱFXIIaȱmayȱprovideȱ anȱadditionalȱantiȬinflammatoryȱbenefitȱtoȱtheseȱpatients,ȱtoȱreduceȱorȱpreventȱadverseȱ effectsȱsuchȱasȱdamageȱofȱorgans.ȱ

Inȱsummary,ȱweȱshowȱthatȱaȱsyntheticȱFXIIaȱinhibitorȱwithȱsubȬnanomolarȱaffinity,ȱhighȱ selectivityȱandȱgoodȱstabilityȱcanȱbeȱdeveloped.ȱWeȱshowȱthatȱcontrolȱofȱFXIIaȱwithȱsuchȱ aȱsyntheticȱinhibitorȱallowsȱforȱefficientȱanticoagulationȱinȱrelevantȱanimalȱdiseaseȱmodȬ elsȱwithoutȱincreasingȱrisksȱofȱbleeding.ȱTheȱsyntheticȱnatureȱandȱtheȱsmallȱsizeȱpromiseȱ aȱ minimalȱ riskȱ ofȱ immunogenicityȱ andȱ allowȱ efficientȱ production.ȱ Withȱ itsȱ excellentȱ bindingȱpropertiesȱandȱstability,ȱtheȱinhibitorȱmayȱbeȱreadilyȱapplicableȱinȱitsȱcurrentȱ formȱforȱapplicationȱinȱacuteȱproceduresȱorȱconditionsȱassociatedȱwithȱincreasedȱriskȱofȱ thrombosisȱsuchȱasȱCPBȱduringȱheartȱsurgeryȱorȱextracorporealȱmembraneȱoxygenation,ȱ withoutȱcompromisingȱhemostasisȱatȱwoundȱsite.ȱ 0DWHULDOVDQG0HWKRGV

Studyȱdesign:ȱTheȱobjectivesȱofȱthisȱstudyȱwereȱtoȱimproveȱtheȱpharmacologicallyȱreleȬ vantȱpropertiesȱofȱaȱpeptideȱmacrocycleȱinhibitorȱofȱFXIIa,ȱtoȱassessȱtheȱpharmacokineticȱ propertiesȱofȱtheȱresultingȱinhibitorȱinȱthreeȱspecies,ȱandȱtoȱevaluateȱtheȱinhibitorȱinȱclinȬ icallyȱrelevantȱanimalȱmodels.ȱTheȱinhibitorȱwasȱengineeredȱbyȱsynthesizingȱvariantsȱofȱ theȱleadȱpeptideȱandȱtestingȱtheirȱinhibitoryȱactivity,ȱspecificityȱandȱstabilityȱinȱvariousȱ assaysȱinȱvitro.ȱInhibitorsȱshowingȱimprovedȱactivitiesȱwereȱpreparedȱwithȱgreaterȱthanȱ 95%ȱpurityȱandȱtheirȱactivitiesȱwereȱtestedȱatȱleastȱinȱtriplicate.ȱTheȱaffinityȱofȱtheȱfinalȱ peptideȱFXII900ȱwasȱdeterminedȱinȱquadruplicateȱfromȱthreeȱdifferentȱsyntheticȱbatches.ȱ Theȱpharmacokineticȱpropertiesȱinȱmice,ȱrabbits,ȱandȱpigsȱwereȱassessedȱbyȱquantifyingȱ theȱinhibitorȱinȱplasmaȱsamplesȱusingȱliquidȱchromatographyȱandȱmassȱspectrometry,ȱ andȱbyȱmeasuringȱaPTTȱinȱplasmaȱsamplesȱinȱtriplicate.ȱTheȱpharmacologicȱeffectsȱofȱtheȱ

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inhibitorȱwereȱtestedȱinȱaȱFeCl3Ȭinducedȱthrombosisȱmodelȱinȱmiceȱandȱinȱanȱartificialȱ lungȱmodelȱinȱrabbits.ȱInȱallȱanimalȱexperiments,ȱsubjectsȱwereȱrandomlyȱassignedȱtoȱ groups.ȱ

Peptideȱ Synthesis:ȱ SolidȬphaseȱ peptideȱ synthesisȱ wasȱ performedȱ onȱ anȱ Advancedȱ ChemTechȱ348ȱ:ȱpeptideȱsynthesizerȱ(AAPPTec)ȱusingȱstandardȱFmocȱprocedures.ȱRinkȱ AmideȱAMȱresinȱwasȱusedȱasȱsolidȱsupportȱandȱdimethylformamideȱ(DMF)ȱasȱsolvent.ȱ Peptideȱvariantsȱwereȱsynthesizedȱatȱaȱ0.03ȱmmolȱscaleȱtoȱobtainȱaroundȱ10–20ȱmgȱofȱ pureȱpeptide.ȱPeptideȱcouplingsȱwereȱperformedȱtwiceȱforȱeachȱnaturalȱaminoȱacidȱbyȱ reactingȱaminoȱacidȱ(4ȱeq.,ȱ600ȱPlȱofȱ0.2ȱMȱinȱDMF),ȱHBTU/HOBtȱ(4ȱeq.ȱeach,ȱ267ȱPlȱofȱ 0.45ȱMȱinȱDMF),ȱandȱDIPEAȱ(6ȱeq.,ȱ360ȱPlȱofȱ0.5ȱMȱinȱDMF)ȱatȱRTȱforȱ30ȱminutesȱatȱ400ȱ rpm.ȱTheȱcouplingȱofȱunnaturalȱaminoȱacidsȱ(2ȱeq.,ȱ600ȱPlȱofȱ0.1ȱMȱinȱDMF)ȱwasȱperȬ formedȱonceȱusingȱHATUȱ(2ȱeq.,ȱ650ȱPlȱofȱ0.1ȱMȱinȱDMF)ȱandȱDIPEAȱ(4ȱeq.,ȱ250ȱPlȱofȱ0.5ȱ MȱinȱDMF).ȱTheȱresinȱwasȱwashedȱfourȱtimesȱwithȱDMFȱafterȱtheȱcouplingȱreaction.ȱTheȱ NȬterminalȱFmocȱprotectingȱgroupȱwasȱremovedȱwithȱpiperidineȱ(20%ȱv/v)ȱinȱDMFȱ(RT,ȱ 2ȱ×ȱ5ȱmin,ȱ400ȱrpm).ȱTheȱresinȱwasȱwashedȱfiveȱtimesȱwithȱDMFȱafterȱFmocȱremoval.ȱTheȱ peptidesȱwereȱsynthesizedȱfromȱtheȱCȬterminalȱendȱtoȱtheȱNȬterminusȱbyȱfirstȱcouplingȱ aȱglycineȱtoȱtheȱresinȱfollowedȱbyȱtheȱcoumarinȬcontainingȱaminoȱacidȱFmocȬEȬ(7ȬmethȬ oxyȬcoumarinȬ4Ȭyl)ȬAlaȬOHȱ(Bachem).ȱTheseȱtwoȱcouplingȱstepsȱasȱwellȱasȱHATUȱcouȬ plingsȱwereȱfollowedȱbyȱanȱacetylationȱcappingȱstepȱusingȱaceticȱanhydrideȱ(1ȱmlȱofȱ10%ȱ [v/v]ȱinȱDMF)ȱandȱDIPEAȱ(1ȱmlȱofȱ15%ȱ[v/v]ȱinȱDMF)ȱatȱRTȱforȱ30ȱminutesȱatȱ400ȱrpm.ȱ FmocȬLȬDȬaminoȱacids,ȱHBTU,ȱHOBt,ȱandȱRinkȱAmideȱAMȱresinȱwereȱpurchasedȱfromȱ GLȱBiochem.ȱNonȬcanonicalȱFmocȬaminoȱacidsȱwereȱpurchasedȱfromȱBachem,ȱChemȬ Impex,ȱPolyPeptide,ȱandȱTCI.ȱPeptidesȱwereȱcleavedȱfromȱtheȱsolidȱsupportȱandȱprotectȬ ingȱ groupsȱ wereȱ removedȱ byȱ incubationȱ inȱ 5ȱ mlȱ cleavageȱ cocktailȱ (90%ȱ TFA,ȱ 2.5%ȱ ethaneȬ1,2Ȭdithiol,ȱ2.5%ȱphenol,ȱ2.5%ȱthioanisole,ȱ2.5%ȱH2O)ȱforȱtwoȱhoursȱwithȱshaking.ȱ Theȱresinȱwasȱremovedȱbyȱvacuumȱfiltration,ȱandȱtheȱpeptidesȱwereȱprecipitatedȱwithȱ iceȬcoldȱdiethylȱetherȱ(50ȱml),ȱincubatedȱforȱ30ȱminutesȱatȱȬ20°C,ȱandȱpelletedȱbyȱcentrifȬ ugationȱ(2700ȱg,ȱ5ȱminutes).ȱTheȱdiethylȱetherȱwasȱdiscarded,ȱtheȱprecipitateȱwashedȱ twiceȱwithȱdiethylȱether,ȱandȱtheȱremainingȱsolventȱevaporatedȱatȱRT.ȱTheȱlinearȱpeptideȱ ofȱFXII900ȱwasȱsynthesizedȱatȱaȱoneȱgramȬscaleȱbyȱGLȱBiochem.ȱ

Peptidesȱcyclizationȱandȱpurification:ȱCrudeȱpeptideȱatȱaȱconcentrationȱofȱ1ȱmMȱwasȱ reactedȱwithȱ1.2ȱmMȱofȱTATAȱinȱ70%ȱNH4HCO3ȱbufferȱ(60ȱmM,ȱpHȱ8.0)ȱandȱ30%ȱMeCNȱ forȱoneȱhourȱatȱ30°C.ȱTheȱpeptideȱandȱTATAȱwereȱpreparedȱandȱmixedȱusingȱtheȱsolȬ vents,ȱconcentrations,ȱandȱvolumesȱexemplifiedȱforȱtheȱcyclizationȱofȱ~ȱ50ȱmgȱcrudeȱpepȬ tide.ȱToȱ6ȱmlȱofȱ3.5ȱmMȱpeptideȱinȱMeCN/H2Oȱ(1:2),ȱ2.7ȱmlȱofȱ10ȱmMȱTATAȱinȱMeCNȱ andȱ 2ȱ mlȱ ofȱ MeCNȱ wereȱ addedȱ andȱ theȱ reactionȱ startedȱ byȱ theȱ additionȱ ofȱ 12ȱ mlȱ

NH4HCO3ȱbufferȱ(60ȱmM,ȱpHȱ8.0).ȱTheȱcyclizedȱpeptidesȱsynthesizedȱonȱaȱ50ȱmgȱscaleȱ wereȱpurifiedȱbyȱreversedȬphaseȱHPLCȱ(WatersȱPrepȱLCȱ4000ȱsystem)ȱusingȱaȱpreparaȬ tiveȱC18ȱRPȱcolumnȱ(VydacȱC18ȱTP1022ȱcolumn,ȱ250ȱ×ȱ22ȱmm,ȱ10ȱPm,ȱ300ȱÅ)ȱandȱaȱlinearȱ

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gradientȱofȱ15–28%ȱsolventȱBȱ(MeCN,ȱ0.1%ȱ[v/v)]ȱTFA)ȱinȱsolventȱAȱ(H2O,ȱ0.1%ȱ[v/v]ȱ TFA)ȱinȱ19ȱminutesȱatȱaȱflowȱofȱ20ȱml/min.ȱAbsorbanceȱwasȱdetectedȱatȱ220ȱnm.ȱTheȱ cyclizedȱpeptidesȱwereȱinjectedȱdirectlyȱasȱtheȱreactionȱmixtureȱorȱafterȱlyophilizationȱofȱ theȱreactionȱbyȱdissolvingȱtheȱpeptideȱinȱ10ȱmlȱofȱH2Oȱcontainingȱ10%ȱ(v/v)ȱMeCNȱandȱ 0.1%ȱ(v/v)ȱTFA.ȱFractionsȱcontainingȱtheȱdesiredȱpeptideȱwereȱlyophilized.ȱȱ

HPLCȱandȱmassȱspectrometricȱanalysis:ȱTheȱmolecularȱmassȱofȱtheȱpurifiedȱpeptidesȱ wasȱdeterminedȱonȱaȱsingleȱquadrupoleȱmassȱspectrometerȱinȱpositiveȱionȱmodeȱusingȱ electrosprayȱionizationȱ(LCMSȬ2020,ȱShimadzu).ȱTheȱpurityȱofȱtheȱpeptidesȱwasȱdeterȬ minedȱbyȱanalyticalȱRPȬHPLCȱ(Agilentȱ1260ȱHPLCȱsystem)ȱusingȱanȱanalyticalȱC18ȱcolȬ umnȱ(AgilentȱZorbaxȱ300SBȬC18,ȱ4.6ȱmmȱ×ȱ250ȱmm,ȱ5ȱPm)ȱandȱaȱlinearȱgradientȱofȱ0–

50%ȱsolventȱBȱ(MeCN,ȱ0.1%ȱ[v/v]ȱTFA)ȱinȱsolventȱAȱ(H2O,ȱ5%ȱ[v/v]ȱMeCN,ȱ0.1%ȱ[v/v]ȱ TFA)ȱinȱ15ȱminutesȱatȱaȱflowȱofȱ1ȱml/min.ȱAbsorbanceȱwasȱdetectedȱatȱ220ȱnm.ȱ

Peptideȱconcentrationȱdetermination:ȱLyophilizedȱpeptideȱwasȱweighedȱandȱdissolvedȱ inȱwaterȱtoȱobtainȱpeptideȱstocksȱofȱ1ȱmM.ȱTheȱconcentrationsȱofȱcrudeȱpeptidesȱcontainȬ ingȱtheȱcoumarinȱaminoȱacidȱwereȱdeterminedȱbyȱmeasuringȱtheȱabsorptionȱatȱ325ȱnmȱ (Ήȱ=ȱ12000ȱMȬ1ȱcmȬ1).ȱTheȱpeptidesȱwereȱtypicallyȱdilutedȱseveralȱfoldȱwithȱwaterȱtoȱmeasȬ ureȱtheȱconcentrationȱinȱanȱabsorptionȱrangeȱofȱ0.1–1.ȱ

ScreeningȱmethodȱbasedȱonȱcoumarinȬlabeledȱpeptides:ȱInȱorderȱtoȱtestȱtheȱinhibitoryȱ activityȱofȱpeptidesȱwithoutȱpriorȱpurification,ȱweȱdevelopedȱtheȱfollowingȱmethod.ȱVarȬ iantsȱofȱtheȱleadȱFXII618ȱpeptideȱwereȱsynthesizedȱwithȱtheȱCȬterminalȱaminoȱacidȱFmocȬ EȬ(7ȬmethoxyȬcoumarinȬ4Ȭyl)ȬAlaȬOH.ȱ Theȱ fluorophoreȱ 7ȬmethoxyȬcoumarinȱ allowedȱ preciseȱquantificationȱofȱtheȱcrudeȱpeptide.ȱAfterȱsolidȬphaseȱpeptideȱsynthesis,ȱcleavȬ age,ȱandȱetherȱprecipitation,ȱtheȱpeptidesȱwereȱchemicallyȱcyclizedȱandȱtheirȱKiȱvaluesȱ wereȱdeterminedȱinȱaȱFXIIaȱactivityȱassayȱusingȱaȱfluorogenicȱsubstrate.ȱPeptidesȱsynȬ thesizedȱandȱcharacterizedȱwithȱthisȱstrategyȱdisplayedȱanȱaroundȱ2.5ȬfoldȱhigherȱapȬ parentȱKiȱthanȱanalogousȱHPLCȬpurifiedȱpeptidesȱwithoutȱtheȱtag.ȱForȱexample,ȱtheȱrefȬ erenceȱpeptideȱFXII618ȱcarryingȱtheȱcoumarinȱaminoȱacidȱhadȱanȱaroundȱ2.5Ȭfoldȱweakerȱ

Kiȱ(20ȱ±ȱ3ȱnM)ȱcomparedȱtoȱtheȱpurifiedȱFXII618ȱwithoutȱtheȱtagȱ(8.1ȱ±ȱ0.7).ȱDespiteȱtheȱ difference,ȱthisȱmethodȱallowedȱforȱaȱcomparisonȱbetweenȱpeptidesȱandȱtheȱidentificaȬ tionȱofȱtheȱmostȱactiveȱvariantsȱofȱFXII618.ȱTheȱbestȱpeptidesȱfromȱtheȱscreenȱwereȱsynȬ thesizedȱwithoutȱtheȱcoumarinȱlabel,ȱpurified,ȱandȱcharacterized.ȱ

Proteaseȱinhibitionȱassays:ȱTheȱinhibitoryȱactivityȱofȱbicyclicȱpeptidesȱwasȱdeterminedȱ byȱmeasuringȱtheȱresidualȱproteaseȱactivityȱwithȱaȱfluorogenicȱsubstrate.ȱTheȱassayȱwasȱ performedȱinȱbufferȱcontainingȱ10ȱmMȱTrisȬCl,ȱpHȱ7.4,ȱ150ȱmMȱNaCl,ȱ10ȱmMȱMgCl2,ȱ1ȱ mMȱCaCl2,ȱ0.1%ȱw/vȱBSA,ȱ0.01%ȱv/vȱTritonȬX100,ȱandȱ1%ȱv/vȱDMSOȱinȱaȱvolumeȱofȱ150ȱ Pl.ȱFXIIaȱactivityȱwasȱmeasuredȱwithȱtheȱfluorogenicȱsubstrateȱBocȬGlnȬGlyȬArgȬAMCȱ (Bachem)ȱatȱaȱfinalȱconcentrationȱofȱ50ȱPM.ȱMouseȱDȬFXIIaȱ(MolecularȱInnovationsȱcatȱ#ȱ

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MFXIIA)ȱandȱhumanȱEȬFXIIaȱ(MolecularȱInnovationsȱcatȱ#ȱHFXIIAB)ȱwereȱusedȱatȱfinalȱ concentrationsȱofȱ0.4ȱnM.ȱTheȱreactionȱwasȱpipettedȱasȱfollows:ȱ50ȱPlȱofȱbicyclicȱpeptideȱ inȱassayȱbufferȱwereȱaddedȱtoȱ50ȱPlȱofȱFXIIaȱinȱassayȱbufferȱandȱincubatedȱforȱ10ȱminutesȱ atȱ25°Cȱbeforeȱ50ȱPlȱofȱ150ȱPMȱfluorogenicȱsubstrateȱinȱassayȱbufferȱwereȱaddedȱtoȱmeasȬ ureȱtheȱresidualȱproteaseȱactivityȱwithȱanȱInfiniteȱM200Proȱfluorescenceȱplateȱreaderȱ(exȬ citationȱatȱ368ȱnm,ȱemissionȱatȱ467ȱnm;ȱTecan)ȱforȱaȱperiodȱofȱ30ȱminutesȱwithȱaȱreadȱ everyȱminuteȱatȱ25°C.ȱStockȱsolutionsȱofȱ1ȱmMȱbicyclicȱpeptideȱwereȱpreparedȱbyȱeitherȱ dissolvingȱtheȱunpurifiedȱcyclizationȱreactionȱwithȱwaterȱorȱbyȱdissolvingȱpurifiedȱandȱ lyophilizedȱpeptideȱinȱwater.ȱTheȱbicyclicȱpeptidesȱwereȱseriallyȱdilutedȱtwoȬfoldȱusingȱ assayȱbuffer.ȱSigmoidalȱcurvesȱwereȱfittedȱtoȱtheȱdataȱusingȱtheȱfollowingȱdoseȱresponseȱ equation,ȱwhereinȱxȱ=ȱpeptideȱconcentration,ȱyȱ=ȱ%ȱproteaseȱactivity,ȱandȱpȱ=ȱHillȱslope.ȱ

IC50ȱvaluesȱwereȱderivedȱfromȱtheȱfittedȱcurve:ȱ

ͳͲͲ ݕൌ ͳ൅ͳͲሺ୪୭୥ ூ஼ହ଴ ି௫ሻ௣

Theȱinhibitionȱconstantsȱ(Ki)ȱwereȱcalculatedȱaccordingȱtoȱtheȱequationȱofȱChengȱandȱ

Prusoff,ȱKiȱ=ȱIC50/(1+([S]0/Km),ȱwhereinȱIC50ȱisȱtheȱfunctionalȱstrengthȱofȱtheȱinhibitor,ȱ[S]0ȱ isȱtheȱtotalȱsubstrateȱconcentration,ȱandȱKmȱisȱtheȱMichaelisȬMentenȱconstant.ȱAȱKmȱofȱ260ȱ PMȱwasȱusedȱforȱtheȱsubstrateȱBocȬGlnȬGlyȬArgȬAMC.ȱ

Forȱtestingȱtheȱspecificityȱofȱtheȱbicyclicȱpeptides,ȱtheȱfollowingȱfinalȱconcentrationsȱofȱ humanȱserineȱproteasesȱwereȱused:ȱtPAȱ(MolecularȱInnovations)ȱ7.5ȱnM,ȱuPAȱ(Molecularȱ Innovations)ȱ1.5ȱnM,ȱfactorȱXIaȱ(InnovativeȱResearch)ȱ6ȱnM,ȱplasmaȱkallikreinȱ(InnovaȬ tiveȱResearch)ȱ0.25ȱnM,ȱthrombinȱ(MolecularȱInnovations)ȱ1ȱnM,ȱplasminȱ(MolecularȱInȬ novations)ȱ2.5ȱnM,ȱtrypsinȱ(MolecularȱInnovations)ȱ0.05ȱnM,ȱfactorȱVIIaȱ(Haematologicȱ Technologies)ȱ50ȱnM,ȱandȱfactorȱXaȱ(HaematologicȱTechnologies)ȱ6ȱnM.ȱTwoȬfoldȱdiluȬ tionsȱofȱtheȱpeptideȱwereȱpreparedȱthatȱrangedȱfromȱ0.04ȱtoȱ40ȱPM.ȱTheȱfollowingȱfluoȬ rogenicȱ substratesȱ wereȱ usedȱ atȱ aȱ finalȱ concentrationȱ ofȱ 50ȱ PM:ȱ ZȬPheȬArgȬAMCȱ (Bachem)ȱforȱplasmaȱkallikrein;ȱBocȬPheȬSerȬArgȬAMCȱ(Bachem)ȱforȱfactorȱXia;ȱZȬGlyȬ GlyȬArgȬAMCȱ(Bachem)ȱforȱtPA,ȱuPA,ȱthrombin,ȱandȱtrypsin;ȱHȬDȬValȬLeuȬLysȬAMCȱ (Bachem)ȱforȱplasmin;ȱandȱDȬPheȬProȬArgȬANSNHȬC4H9ȱ(HaematologicȱTechnologies)ȱ forȱFVIIaȱandȱFXa.ȱȱ

Plasmaȱstabilityȱassays:ȱPeptideȱ(2ȱPlȱofȱ2ȱmMȱinȱH2O)ȱwasȱaddedȱtoȱ398ȱPlȱofȱcitratedȱ humanȱplasmaȱ(InnovativeȱResearch)ȱtoȱobtainȱaȱfinalȱpeptideȱconcentrationȱofȱ10ȱPM.ȱ Theȱmixtureȱwasȱincubatedȱinȱaȱwaterȱbathȱatȱ37°C.ȱAtȱdifferentȱtimeȱpointsȱ(0,ȱ0.5,ȱ1,ȱ2,ȱ 4,ȱ8,ȱ12,ȱ24,ȱ48,ȱandȱ96ȱhours),ȱsamplesȱofȱ30ȱPlȱwereȱremoved,ȱdilutedȱtoȱ200ȱPlȱwithȱ aqueousȱbufferȱ(10ȱmMȱTrisȬHCl,ȱpHȱ7.4,ȱ150ȱmMȱNaCl,ȱ10ȱmMȱMgCl2,ȱ1ȱmMȱCaCl2),ȱ andȱincubatedȱforȱ20ȱminutesȱatȱ65°Cȱtoȱinactivateȱplasmaȱproteases.ȱTheȱpeptide/plasmaȱ samplesȱwereȱstoredȱatȱȬ20°Cȱuntilȱtheȱresidualȱinhibitoryȱactivityȱofȱtheȱpeptidesȱwasȱ measuredȱinȱaȱFXIIaȱinhibitionȱassay.ȱForȱtheȱactivityȱassay,ȱtheȱpeptide/plasmaȱsamplesȱ

78 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ wereȱcentrifugedȱforȱfiveȱminutesȱatȱ16,000ȱg,ȱserialȱtwoȬfoldȱdilutionsȱofȱtheȱsupernatantȱ wereȱpreparedȱ(peptideȱconcentrationȱrangesȱfromȱ0.5ȱnMȱtoȱ0.5ȱPM),ȱandȱtheȱresidualȱ activityȱofȱ0.5ȱnMȱhumanȱDȬFXIIaȱwasȱmeasuredȱusingȱ50ȱPMȱBocȬGlnȬGlyȬArgȬAMCȱ substrate.ȱIC50ȱvaluesȱwereȱderivedȱfromȱtheȱfittedȱcurveȱusingȱtheȱequationȱindicatedȱ above.ȱ Residualȱ inhibitionȱ inȱ %ȱ wasȱ calculatedȱ usingȱ theȱ equationȱ IC50,0h/IC50,xh*100,ȱ whereinȱIC50,0hȱisȱtheȱfunctionalȱstrengthȱofȱtheȱinhibitorȱatȱtimeȱpointȱ0ȱandȱIC50,xhȱtheȱ functionalȱstrengthȱofȱtheȱinhibitorȱafterȱoneȱofȱtheȱdifferentȱplasmaȱincubationȱperiodsȱ mentionedȱabove.ȱȱ

Plasmaȱdegradationȱassays:ȱPlasmaȱstabilityȱandȱpeptideȱcleavageȱsitesȱwereȱassessedȱ byȱincubatingȱtheȱbicyclicȱpeptidesȱinȱmouseȱplasmaȱandȱanalyzingȱtheȱproductsȱwithȱanȱ

LCȬMSȱsystemȱ(LCMSȬ2020,ȱShimadzu).ȱPeptideȱ(2ȱPlȱofȱ2ȱmMȱinȱH2O)ȱwasȱaddedȱtoȱ48ȱ ΐlȱofȱcitratedȱmouseȱplasmaȱ(InnovativeȱResearch)ȱtoȱobtainȱaȱfinalȱpeptideȱconcentrationȱ ofȱ80ȱPM.ȱTheȱmixtureȱwasȱincubatedȱinȱaȱwaterȱbathȱatȱ37°C.ȱAtȱdifferentȱtimeȱpointsȱ(0,ȱ 15,ȱ24,72,ȱ96,ȱ120,ȱandȱ144ȱhours),ȱsamplesȱofȱ5ȱPlȱwereȱremoved,ȱmixedȱwithȱ5ȱPlȱofȱ6ȱMȱ guanidiniumȱhydrochloride,ȱandȱincubatedȱforȱ30ȱminutesȱatȱRT.ȱPlasmaȱproteinsȱwereȱ precipitatedȱbyȱincubationȱwithȱ200ȱPlȱofȱiceȱcoldȱEtOHȱandȱ0.1ȱ%ȱ(v/v)ȱformicȱacidȱforȱ 30ȱminutesȱandȱcentrifugedȱatȱ9000ȱgȱforȱ20ȱminutesȱatȱ4°C.ȱTheȱsupernatantȱwasȱevapȬ oratedȱinȱaȱSpeedvacȱatȱ50°Cȱandȱreducedȱpressure,ȱthenȱtheȱresidueȱwasȱdissolvedȱinȱ40ȱ Plȱofȱdeionizedȱwaterȱcontainingȱ0.1%ȱ(v/v)ȱCHOOHȱandȱanalyzedȱbyȱLCȬMS.ȱTheȱsamȬ plesȱwereȱanalyzedȱusingȱanȱanalyticalȱC18ȱcolumnȱ(PhenomenexȱC18ȱKinetexȱcolumn,ȱ 50ȱ×ȱ2.1ȱmm,ȱ2.6ȱPm,ȱ100ȱÅ)ȱandȱaȱlinearȱgradientȱofȱ5–35%ȱsolventȱBȱ(MeCN,ȱ0.05%ȱ[v/v]ȱ

CHOOH)ȱinȱsolventȱAȱ(H2O,ȱ0.05%ȱ[v/v]ȱCHOOH)ȱinȱ5.5ȱminutesȱatȱaȱflowȱofȱ1ȱml/min.ȱ Theȱmassesȱofȱtheȱintactȱpeptideȱandȱdegradationȱproductsȱwereȱmeasuredȱonȱaȱsingleȱ quadrupoleȱmassȱspectrometerȱinȱpositiveȱionȱmodeȱusingȱelectrosprayȱionization.ȱPepȬ tidesȱwereȱquantifiedȱbasedȱonȱtheȱabsoluteȱintensitiesȱofȱtheȱdetectedȱmassȱpeaksȱ(M3+ȱ andȱM4+).ȱ

Coagulationȱassays:ȱTheȱcoagulationȱparametersȱaPTTȱandȱPTȱwereȱdeterminedȱinȱbloodȱ plasmaȱ usingȱ aȱ STAGOȱ STart4ȱ Coagulationȱ analyzerȱ (Diagnostica).ȱ Citratedȱ plasmaȱ fromȱhumanȱ(singleȱdonor),ȱmouseȱ(CD1,ȱInnovativeȱgrade),ȱrabbitȱ(NZW,ȱInnovativeȱ grade),ȱandȱpigȱ(Innovativeȱgrade)ȱwereȱsuppliedȱbyȱInnovativeȱResearch.ȱCoagulationȱ wasȱ monitoredȱ byȱ anȱ electromagneticallyȱ inducedȱ movementȱ ofȱ aȱ steelȱ ballȱ inȱ theȱ plasma.ȱTheȱtimeȱuntilȱtheȱballȱstoppedȱmovingȱwasȱrecordedȱasȱtheȱcoagulationȱtime.ȱ Frozenȱplasmaȱsamplesȱwereȱthawedȱforȱaroundȱ30ȱminutesȱatȱRTȱbeforeȱuse.ȱForȱaPPTȱ measurementsȱinȱhumanȱplasma,ȱ100ȱPlȱofȱplasmaȱwasȱplacedȱintoȱtheȱcuvette,ȱ100ȱPlȱofȱ Pathromtin*ȱSLȱ(siliconȱdioxideȱparticles,ȱplantȱphospholipidsȱinȱHEPESȱbufferȱsystem;ȱ Siemens)ȱwasȱaddedȱandȱincubatedȱforȱtwoȱminutesȱatȱ37°Cȱinȱtheȱdeviceȱbeforeȱtheȱ coagulationȱwasȱtriggeredȱbyȱtheȱadditionȱofȱ100ȱPlȱofȱpreȬwarmedȱ(37°C)ȱCaCl2ȱsolutionȱ (25ȱmM,ȱSiemens).ȱForȱaPTTȱmeasurementsȱinȱmouse,ȱrabbit,ȱandȱpigȱplasma,ȱ100ȱPlȱofȱ plasmaȱ wasȱ placedȱ intoȱ theȱ cuvette,ȱ 100ȱ Plȱ ofȱ Dadeȱ Actinȱ (Cephalin,ȱ Ellagicȱ acidȱ inȱ

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HEPESȱbufferȱsystem;ȱSiemens)ȱwasȱaddedȱandȱincubatedȱforȱthreeȱminutesȱatȱ37°Cȱinȱ theȱdeviceȱbeforeȱcoagulationȱwasȱtriggeredȱbyȱadditionȱofȱ100ȱPlȱofȱpreȬwarmedȱ(37°C)ȱ

CaCl2ȱsolution.ȱForȱPTȱmeasurementsȱinȱhumanȱplasma,ȱ50ȱPlȱofȱplasmaȱwasȱplacedȱintoȱ theȱcuvetteȱandȱincubatedȱforȱoneȱminuteȱatȱ37°Cȱbeforeȱcoagulationȱwasȱtriggedȱbyȱtheȱ additionȱofȱ100ȱPlȱofȱpreȬwarmedȱ(37°C)ȱInnovinȱ(recombinantȱhumanȱtissueȱfactor,ȱsynȬ theticȱphospholipids,ȱandȱcalciumȱinȱstabilizedȱHEPESȱbufferȱsystem;ȱDadeȱBehring/SieȬ mens).ȱȱ

Animalȱstudies:ȱAllȱexperimentsȱinȱmiceȱandȱpigsȱwereȱconductedȱinȱaccordanceȱwithȱ theȱtermsȱofȱtheȱSwissȱanimalȱprotectionȱlawȱandȱwereȱapprovedȱbyȱtheȱanimalȱexperiȬ mentationȱcommitteeȱofȱtheȱcantonalȱveterinaryȱserviceȱ(CantonȱofȱBerne,ȱSwitzerland).ȱ TheȱpharmacokineticȱstudiesȱinȱrabbitsȱwereȱperformedȱatȱWashingtonȱBiotechȱInc.ȱfolȬ lowingȱethicalȱstandardsȱforȱanimalȱstudiesȱofȱtheȱOfficeȱforȱLaboratoryȱAnimalȱWelfareȱ (OLAW),ȱaȱdivisionȱofȱtheȱUSȱPublicȱHealthȱServiceȱasȱadministeredȱbyȱtheȱNationalȱ InstitutesȱforȱHealth.ȱTheȱextracorporealȱcirculationȱstudiesȱinȱrabbitsȱwereȱperformedȱ inȱcomplianceȱwithȱtheȱAlleghenyȱHealthȱNetworkȱInstitutionalȱAnimalȱCareȱandȱUseȱ Committee.ȱ

Pharmacokineticsȱ studyȱ inȱ mice:ȱ C57BL/6Jȱ wildȬtypeȱ miceȱ (male,ȱ 25–30ȱ g,ȱ Charlesȱ River)ȱwereȱinjectedȱsubcutaneouslyȱoverȱtheȱshouldersȱwithȱ5ȱmg/kgȱofȱFXII900ȱ(0.5ȱ mg/mlȱinȱPBS,ȱpHȱ7.4).ȱTheȱmiceȱwereȱanaesthetizedȱthreeȱminutesȱbeforeȱtheȱscheduledȱ bloodȱ collectionȱ timeȱ pointȱ (40ȱ mg/kgȱ pentobarbital).ȱ Anȱ abdominalȱ midlineȱ incisionȱ wasȱperformedȱandȱ450ȱPlȱofȱwholeȱbloodȱwasȱdrawnȱfromȱtheȱinferiorȱvenaȱcavaȱintoȱaȱ syringeȱcontainingȱsodiumȱcitrateȱ(50ȱPlȱ3.2%ȱ[w/v]ȱsodiumȱcitrate).ȱTheȱmiceȱwereȱeuȬ thanizedȱbyȱcervicalȱdislocation.ȱTheȱbloodȱwasȱimmediatelyȱprocessedȱtoȱplasmaȱbyȱ centrifugationȱforȱ10ȱminutesȱatȱ2000ȱgȱatȱ4°C,ȱandȱstoredȱatȱȬ80°C.ȱTheȱconcentrationȱofȱ FXII900ȱinȱtheȱplasmaȱsamplesȱwasȱdeterminedȱbyȱLCȬMSȱasȱdescribedȱbelow.ȱTwoȱmiceȱ wereȱusedȱforȱeachȱtimeȱpoint.ȱ

Pharmacokineticȱstudyȱinȱrabbits:ȱTheȱpharmacokineticȱpropertiesȱofȱFXII900ȱappliedȱ intravenouslyȱ wereȱ determinedȱ asȱ describedȱ previouslyȱ (193).ȱ Femaleȱ Newȱ Zealandȱ Whiteȱrabbitsȱwereȱinjectedȱwithȱ3.7ȱmg/kgȱFXII900ȱdissolvedȱinȱ1ȱmlȱPBS,ȱpHȱ7.4ȱviaȱtheȱ earȱvein.ȱBloodȱsamplesȱ(2.7ȱml)ȱwereȱcollectedȱatȱdifferentȱtimeȱpointsȱintoȱsodiumȱcitȬ rateȱtubesȱ(BDȱVacutainerȱrefȱ#ȱ363083)ȱandȱimmediatelyȱprocessedȱtoȱplasmaȱbyȱcentrifȬ ugationȱatȱ1400ȱgȱforȱ15ȱminutes.ȱTheȱconcentrationȱofȱFXII900ȱinȱtheȱplasmaȱsamplesȱ wasȱ determinedȱ byȱ LCȬMSȱ asȱ describedȱ below.ȱ Theȱ pharmacokineticȱ propertiesȱ ofȱ FXII900ȱ appliedȱ subcutaneouslyȱ wereȱ determinedȱ asȱ follows.ȱ Femaleȱ Newȱ Zealandȱ Whiteȱ rabbitsȱ (2.5–2.9ȱ kg)ȱ wereȱ injectedȱ subcutaneouslyȱ overȱ theȱ shouldersȱ withȱ 3.7ȱ mg/kgȱofȱFXII900ȱdissolvedȱinȱ1ȱmlȱPBSȱpHȱ7.4.ȱBloodȱsamplesȱ(1.8ȱml)ȱwereȱcollectedȱatȱ differentȱtimeȱpointsȱintoȱsodiumȱcitrateȱtubesȱ(BDȱVacutainerȱrefȱ#ȱ363080)ȱandȱimmeȬ diatelyȱprocessedȱtoȱplasmaȱbyȱcentrifugationȱatȱ1800ȱgȱforȱ10ȱminutes.ȱTheȱplasmaȱwasȱ

80 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ storedȱatȱȬ80°C.ȱTheȱconcentrationȱofȱFXII900ȱinȱtheȱplasmaȱsamplesȱwasȱdeterminedȱbyȱ LCȬMSȱasȱdescribedȱbelow.ȱ

Pharmacokineticȱstudyȱinȱpigs:ȱPigsȱ(30ȱ±ȱ5ȱkg)ȱwereȱanesthetizedȱandȱpreparedȱfollowȬ ingȱproceduresȱusedȱtoȱstudyȱanȱischemia/reperfusionȱinjuryȱ(253).ȱInȱthisȱprocedure,ȱtheȱ ACTȱisȱmonitoredȱandȱ2500ȱIUȱofȱheparinȱareȱinjectedȱwhenȱACTȱvaluesȱfallȱbelowȱ180ȱ s.ȱPigsȱwereȱinjectedȱintravenouslyȱwithȱ4ȱmg/kgȱofȱFXII900ȱdissolvedȱinȱ1ȱmlȱPBS,ȱpHȱ 7.4.ȱBloodȱsamplesȱ(2.7ȱml)ȱwereȱcollectedȱatȱdifferentȱtimeȱpointsȱintoȱsodiumȱcitrateȱ tubesȱ(BDȱVacutainerȱrefȱ#ȱ363083)ȱandȱimmediatelyȱprocessedȱtoȱplasmaȱbyȱcentrifugaȬ tionȱatȱ1400ȱgȱforȱ15ȱminutesȱatȱRT.ȱTheȱaPTTȱwasȱmeasuredȱasȱdescribedȱabove.ȱTheȱ concentrationȱofȱFXII900ȱinȱtheȱplasmaȱsamplesȱwasȱdeterminedȱbyȱLCȬMSȱasȱdescribedȱ below.ȱȱ

Quantificationȱ ofȱ inhibitorȱ inȱ plasmaȱ samples:ȱ Theȱ concentrationȱ ofȱ FXII900ȱ inȱ theȱ plasmaȱsamplesȱwasȱquantifiedȱbasedȱonȱpeakȱintensitiesȱofȱtotalȱionȱcurrentȱ(TIC)ȱchroȬ matogramsȱacquiredȱbyȱLCȬMSȱ(LCMSȬ2020,ȱShimadzu).ȱToȱ15ȱPlȱofȱplasmaȱsample,ȱ1ȱ PMȱofȱinternalȱstandardȱpeptideȱandȱ5ȱPlȱofȱ6ȱMȱguanidiniumȱhydrochlorideȱsolutionȱ wereȱaddedȱandȱmixed.ȱPlasmaȱproteinsȱwereȱprecipitatedȱbyȱtheȱadditionȱofȱ400ȱPlȱofȱ iceȱcoldȱethanolȱ(99.9%ȱv/vȱEtOH,ȱ0.1%ȱv/vȱTFA)ȱandȱincubatedȱonȱiceȱforȱoneȱhour.ȱ Precipitateȱwasȱremovedȱbyȱcentrifugationȱ(9000ȱg,ȱ20ȱminutes,ȱ4°C)ȱandȱtheȱsupernatantȱ driedȱbyȱcentrifugalȱevaporationȱunderȱvacuum.ȱDriedȱsamplesȱwereȱdissolvedȱbyȱseȬ quentiallyȱaddingȱ2ȱPlȱofȱDMSOȱandȱ18ȱPlȱofȱH2Oȱcontainingȱ0.1%ȱ(v/v)ȱCHOOHȱandȱ analyzedȱbyȱLCȬMS.ȱTheȱsamplesȱwereȱanalyzedȱusingȱanȱanalyticalȱC18ȱcolumnȱ(PheȬ nomenexȱC18ȱKinetexȱcolumn,ȱ50ȱ×ȱ2.1ȱmm,ȱ2.6ȱPm,ȱ100ȱÅ)ȱandȱaȱlinearȱgradientȱofȱ5–

30%ȱsolventȱBȱ(MeCN,ȱ0.05%ȱ[v/v]ȱCHOOH)ȱinȱsolventȱAȱ(H2O,ȱ0.05%ȱ[v/v]ȱCHOOH)ȱinȱ 4.5ȱminutesȱatȱaȱflowȱofȱ1ȱml/min.ȱTheȱmassȱwasȱmeasuredȱonȱaȱsingleȱquadrupoleȱmassȱ spectrometerȱinȱpositiveȱionȱmodeȱusingȱelectrosprayȱionization.ȱPeptidesȱwereȱquantiȬ fiedȱbasedȱonȱtheȱabsoluteȱintensitiesȱofȱtheȱdetectedȱmassȱpeaksȱ(M3+ȱandȱM4+).ȱȱ

Ferricȱ chlorideȬinducedȱ thrombosisȱ inȱ mice:ȱ 57BL/6Jȱ wildȬtypeȱ miceȱ (male,ȱ 25–30ȱ g,ȱ CharlesȱRiver)ȱwereȱinjectedȱintravenouslyȱwithȱRhodamineȱ6Gȱ(100ȱPl,ȱ1ȱmM,ȱACROSȱ Organicsȱ productȱ 41902)ȱ toȱ fluorescentlyȱ labelȱ theȱ plateletsȱandȱ leucocytes.ȱ Theȱ miceȱ wereȱinjectedȱsubcutaneouslyȱoverȱtheȱshouldersȱwithȱFXII900ȱorȱtheȱnegativeȱcontrolȱ FXII901ȱ(0.5ȱmg/mlȱinȱPBS,ȱpHȱ=ȱ7.4)ȱandȱsubsequentlyȱanesthetizedȱwithȱketamineȱ(80ȱ mg/kg)ȱandȱxylazineȱ(16ȱmg/kg)ȱviaȱintraperitonealȱinjection.ȱAnȱabdominalȱmidlineȱinȬ cisionȱwasȱmadeȱtoȱexposeȱtheȱmesentericȱarteriolesȱwhichȱwereȱimagedȱbyȱintravitalȱ microscopyȱusingȱaȱMikronȱIVM500ȱmicroscopeȱ(MikronȱInstruments)ȱcoupledȱwithȱaȱ 50ȱWȱmercuryȱlampȱ(HBOȱ50ȱmicroscopeȱilluminator,ȱZeiss)ȱattachedȱtoȱcombinedȱblueȱ (exciterȱ455DF70,ȱdichroicȱ515DRLP,ȱandȱemitterȱ515ALP)ȱandȱgreenȱ(exciterȱ525DF45,ȱ dichroicȱ560DRLP,ȱandȱemitterȱ565ALP)ȱfilterȱblocks.ȱThrombusȱformationȱwasȱinducedȱ byȱtheȱapplicationȱofȱaȱ1ȱ×ȱ2ȱmmȱfilterȱpaperȱsaturatedȱwithȱFeCl3ȱsolutionȱ(7.5%ȱw/v,ȱ

81 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ

Roth,ȱartȱnoȱ5192.1)ȱontoȱtheȱbloodȱvesselȱforȱoneȱminute.ȱTheȱbloodȱflow,ȱclotȱformationȱ andȱ vesselȱ occlusionȱ wereȱ monitoredȱ forȱ 30ȱ minutesȱ whereinȱ imagesȱ wereȱ recordedȱ everyȱminuteȱusingȱaȱdigitalȱvideoȱcassetteȱrecorderȱ(DSRȬ11,ȱSony)ȱandȱanalyzedȱusingȱ ImageJȱsoftware.ȱMiceȱwereȱeuthanizedȱbyȱfinalȱbleedingȱandȱcervicalȱdislocation.ȱTimeȱ toȱclotȱformationȱandȱfullȱocclusionȱofȱtheȱbloodȱvesselȱwereȱdeterminedȱasȱfollows.ȱClotȱ formationȱwasȱdefinedȱasȱtheȱformationȱofȱaȱclearȱaggregationȱwithȱaȱdiameterȱofȱaroundȱ 10ȱPmȱorȱlarger.ȱOcclusionȱwasȱdefinedȱasȱaȱclotȱcoveringȱtheȱfullȱdiameterȱofȱtheȱvessel.ȱ

MiceȱshowingȱnoȱspeckledȱpatternȱatȱtheȱsiteȱofȱFeCl3ȱapplicationȱwereȱassumedȱtoȱbeȱ notȱinjuredȱatȱtheȱbloodȱvesselȱandȱwereȱexcludedȱfromȱtheȱanalysis.ȱ

Artificialȱlungs:ȱMiniȬlungsȱwereȱcreatedȱthatȱallowedȱforȱbloodȱexposureȱtoȱtheȱfibersȱ inȱ theȱ artificialȱ lung,ȱ butȱ forȱ constructionȱ simplicity,ȱ doȱ notȱ transferȱ gasȱ (254).ȱ Polymethylpenteneȱ(PMP)ȱfiberȱ(3M)ȱwithȱ50%ȱporosity,ȱaȱ380ȱPmȱouterȱdiameter,ȱandȱ twoȱlayersȱatȱ30°ȱcrossȱanglesȱwereȱcutȱintoȱ1.78ȱ×ȱ1.78ȱcmȱsquares.ȱFiveȱofȱtheseȱlayersȱ wereȱputȱtogether,ȱmakingȱsureȱtheȱfibersȱallȱranȱinȱtheȱsameȱdirection,ȱandȱtheyȱwereȱ sealedȱtogetherȱintoȱaȱfiberȱbundleȱusingȱaȱhotȱplateȱonȱeachȱsideȱofȱtheȱsquareȱsheets.ȱ Theseȱfiberȱbundlesȱwereȱmeltedȱsuchȱthatȱtheyȱhadȱaȱ1.57ȱ×ȱ1.57ȱcmȱsquareȱfrontalȱareaȱ perpendicularȱ toȱ theȱ flow.ȱ Eightȱ ofȱ theseȱ 10Ȭlayerȱ “chiclets”ȱ wereȱ placedȱ togetherȱ toȱ makeȱupȱtheȱfiberȱbundleȱofȱoneȱminiȱlungȱdevice,ȱgivingȱaȱfinalȱsurfaceȱareaȱofȱ526ȱcm2.ȱ Theseȱfiberȱbundlesȱwereȱplacedȱinȱaȱsquareȱplasticȱhousingȱofȱ3.05ȱcmȱinȱlength,ȱmakingȱ sureȱtheȱfiberȱbundlesȱwereȱinȱtheȱdeviceȱtightlyȱsoȱthatȱnoȱshuntingȱcouldȱoccurȱaroundȱ theȱfiberȱbundle.ȱThisȱhousingȱwasȱattachedȱtoȱplasticȱendȱcapsȱthatȱwereȱplacedȱonȱeachȱ endȱwithȱaȱ1/8ȞȬbarbedȱtubeȱfittingȱforȱ3/16ȞȬIDȱtubingȱtoȱallowȱtheȱdeviceȱtoȱbeȱconȬ nectedȱtoȱtubingȱinȱtheȱcircuit.ȱTheseȱendȱconnectorsȱwereȱcoatedȱwithȱTeflonȱtapeȱbeforeȱ connectingȱthemȱtoȱtheȱplasticȱendȱcapsȱtoȱpreventȱleakage.ȱTheȱdeviceȱwasȱheldȱtogetherȱ withȱtwoȱscrewsȱgoingȱfromȱendȱcapȱtoȱendȱcap.ȱTheȱentireȱdeviceȱwasȱsecuredȱwithȱ siliconeȱtoȱeliminateȱleakage.ȱTheȱsiliconeȱwasȱleftȱtoȱdryȱforȱ24ȱhours,ȱandȱtheȱdeviceȱ leakȱtestedȱasȱfollows.ȱFilteredȱdeionizedȱwaterȱwasȱrunȱthroughȱtheȱdeviceȱtoȱcheckȱforȱ leaksȱwithinȱtheȱdevice.ȱIfȱnoȱleaksȱwereȱfound,ȱtheȱdeviceȱwasȱleftȱtoȱdryȱwithȱfilteredȱ airȱrunningȱthroughȱtheȱdeviceȱforȱ24ȱhours.ȱ

Extracorporealȱcirculationȱinȱrabbits:ȱNewȱZealandȱwhiteȱrabbitsȱofȱ3.2ȱ–ȱ4.2ȱkgȱ(Charlesȱ River)ȱ wereȱ anesthetizedȱ viaȱ intramuscularȱ injectionsȱ ofȱ ketamineȱ (30ȱ mg/kg)ȱ andȱ xylazineȱ(5ȱmg/kg).ȱOneȱofȱtheȱearȱveinsȱwasȱcatheterizedȱviaȱaȱ24Gȱwingedȱcatheter,ȱandȱ theȱ rabbitsȱ wereȱ intubatedȱ withȱ aȱ 3.0ȱ ETȱ tube.ȱ Theȱ animalsȱ wereȱ keptȱ anesthetizedȱ throughoutȱtheȱfourȬhourȱexperimentȱviaȱinhaledȱisofluraneȱ(2%)ȱandȱwereȱventilatedȱ withȱaȱpeakȱinspiratoryȱpressureȱ(PIP)ȱofȱ<ȱ20ȱcmH2O,ȱpositiveȱendȱexpiratoryȱpressureȱ ofȱ5ȱmmHg,ȱtidalȱvolumeȱofȱ4–6ȱml/kg,ȱandȱaȱrespiratoryȱrateȱofȱ22–60ȱbreaths/min.ȱTidalȱ volumeȱandȱrespiratoryȱrateȱwereȱadjustedȱtoȱmaintainȱnormalȱarterialȱbloodȱgasesȱandȱ theȱlistedȱPIP.ȱPhenylephrineȱwasȱappliedȱintravenouslyȱatȱaȱrateȱofȱ0.5–5ȱPg/kgȱminȱtoȱ maintainȱbloodȱpressure.ȱForȱmonitoringȱbloodȱpressureȱandȱcollectingȱbloodȱsamples,ȱ

82 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ theȱrabbits’ȱrightȱorȱleftȱcarotidȱarteriesȱwereȱcannulatedȱusingȱaȱ16Ȭgaugeȱangiocatheterȱ (BectonȱDickinson)ȱandȱwereȱsecuredȱwithȱsilkȱties.ȱTheȱdeviceȱandȱcircuitȱwereȱfirstȱ primedȱwithȱfilteredȱCO2ȱandȱthenȱwithȱsalineȱ(NaCl,ȱ0.9%ȱ[w/v])ȱcontainingȱsolumedrolȱ (30ȱ mg/kg).ȱ Atȱ thisȱ point,ȱ rabbitsȱ ofȱ theȱ inhibitorȬtreatedȱ groupȱ wereȱ injectedȱ withȱ 2ȱ mg/kgȱofȱFXII900ȱasȱaȱ2ȱmMȱsolutionȱinȱPBSȱ(pHȱ7.4)ȱviaȱtheȱearȱveinȱandȱtheȱcircuitȱwasȱ connectedȱtoȱtheȱrabbitsȱviaȱaȱvenovenousȱECMOȱconfigurationȱusingȱaȱ14ȬgaugeȱangiȬ ocatheterȱ(BectonȱDickinson)ȱandȱaȱ6Ȟȱpressureȱtubingȱthatȱwasȱcutȱtoȱlengthȱfromȱaȱ24Ȟȱ pressureȱtubingȱ(EdwardsȱLifesciences)ȱinȱtheȱrightȱandȱleftȱinternalȱjugularȱveins,ȱreȬ spectively.ȱTheȱcircuitȱwasȱplacedȱinȱaȱrollerȱpumpȱ(Cobe),ȱandȱtheȱbloodȱflowȱwasȱsetȱtoȱ 45ȱml/min.ȱAfterȱconnectingȱtheȱextracorporealȱcircuit,ȱrabbitsȱofȱtheȱinhibitorȬtreatedȱ groupȱwereȱinfusedȱwithȱ0.075ȱmg/kg/minȱFXII900ȱviaȱtheȱearȱveinȱforȱtheȱentireȱdurationȱ ofȱtheȱstudy.ȱAfterȱfourȱhours,ȱtheȱanimalsȱwereȱeuthanizedȱbyȱpentobarbitalȱinjectionȱ (FatalȬPlus,ȱVortech,ȱ1ȱml/10ȱlbs).ȱAtȱtheȱendȱofȱtheȱexperiment,ȱ5ȱmlȱofȱheparinȱwasȱrunȱ throughȱtheȱartificialȱlungȱwhileȱitȱwasȱstillȱconnectedȱtoȱtheȱrabbits.ȱThenȱtheȱcircuitȱwasȱ removed,ȱandȱtheȱdeviceȱwasȱwashedȱwithȱsalineȱcarefullyȱsoȱnoȱclotȱsheddingȱwouldȱ occur.ȱSalineȱwasȱrunȱthroughȱtheȱdeviceȱuntilȱtheȱsalineȱwasȱclear.ȱClotȱvolumeȱwasȱ measuredȱbyȱmeasuringȱtheȱvolumeȱofȱtheȱdeviceȱpriorȱtoȱtheȱexperimentȱandȱagainȱatȱ theȱendȱofȱtheȱexperimentȱafterȱwashing.ȱThisȱwasȱdoneȱbyȱcompletelyȱfillingȱtheȱdeviceȱ withȱsalineȱandȱrecordingȱthisȱamountȱofȱsalineȱasȱtheȱdeviceȱvolume.ȱTheȱdifferenceȱ betweenȱtheȱbeginningȱandȱendingȱvolumeȱwasȱdeterminedȱtoȱbeȱtheȱclotȱvolume.ȱFromȱ theȱclotȱvolume,ȱtheȱpercentȱofȱclotȱwithinȱtheȱdeviceȱwasȱcalculated.ȱ

Dataȱacquisitionȱduringȱextracorporealȱcirculation:ȱPlateletȱandȱwhiteȱbloodȱcellȱcounts,ȱ hematocrit,ȱarterialȱbloodȱgasesȱ(ABG),ȱACT,ȱaPTT,ȱfibrinopeptideȱAȱ(FPA),ȱdeviceȱreȬ sistance,ȱandȱbleedingȱtimeȱwereȱmeasuredȱpriorȱtoȱcircuitȱattachmentȱandȱatȱ10,ȱ30,ȱ120,ȱ andȱ240ȱminutesȱfollowingȱtheȱinitiationȱofȱECMO.ȱForȱtheȱplateletȱcounts,ȱaȱsyringeȱwithȱ 0.05ȱmlȱofȱ3.2%ȱsodiumȱcitrateȱ(w/v)ȱwasȱusedȱtoȱdrawȱ0.45ȱmlȱofȱbloodȱforȱaȱtotalȱvolumeȱ ofȱ0.5ȱml.ȱThisȱwasȱthenȱcentrifugedȱatȱ60ȱgȱforȱ10ȱminutes,ȱandȱ20ȱPlȱofȱtheȱplasmaȱwasȱ placedȱinȱ20ȱmlȱofȱISOTON®ȱdiluentȱandȱcountedȱusingȱaȱCoulterȱCounterȱ(Beckmanȱ Coulter,ȱInc.ȱBrea,ȱCA)ȱwithȱaȱ50ȱPlȱapertureȱtube.ȱForȱcounting,ȱcellsȱwereȱconsideredȱ plateletsȱifȱtheirȱdiametersȱwereȱ1.8–5.6ȱPm.ȱForȱwhiteȱbloodȱcellȱcounts,ȱ40ȱPlȱofȱwholeȱ bloodȱwasȱplacedȱinȱ20ȱmlȱofȱtheȱISOTON®ȱdiluent.ȱSixȱdropsȱofȱZAPȬOGLOBINȱlysingȱ solutionȱwereȱaddedȱtoȱtheȱmixtureȱandȱmixedȱgently.ȱThisȱwasȱallowedȱtoȱsitȱforȱtwoȱ minutes.ȱTheȱwhiteȱbloodȱcellsȱwereȱalsoȱcountedȱusingȱtheȱCoulterȱCounterȱ(Beckmanȱ Coulter)ȱwithȱaȱ50ȱPlȱapertureȱtubeȱwhereȱanyȱparticleȱaboveȱ3.6ȱPmȱinȱdiameterȱwasȱ consideredȱaȱwhiteȱbloodȱcell.ȱArterialȱbloodȱgasesȱwereȱmeasuredȱbyȱdrawingȱ0.4ȱmlȱofȱ bloodȱintoȱaȱheparinizedȱsyringeȱandȱrunȱusingȱanȱarterialȱbloodȱgasȱanalyzerȱ(ABL800ȱ FLEX,ȱ Radiometer).ȱ Theȱ ACTȱ andȱ hematocritȱ wereȱ measuredȱ byȱ collectingȱ 0.5ȱ mlȱ ofȱ blood.ȱTheȱACTȱwasȱmeasuredȱusingȱaȱHemochronȱanalyzerȱwithȱtubesȱcontainingȱglassȱ beadsȱasȱtheȱactivator.ȱTheȱhematocritȱwasȱmeasuredȱviaȱcapillaryȱcentrifugation.ȱForȱ

83 $SHSWLGHPDFURF\FOH);,,LQKLELWRUSURYLGHVVDIHDQWLFRDJXODWLRQ aPTTȱmeasurements,ȱbloodȱsamplesȱ(1.8ȱml)ȱwereȱcollectedȱatȱdifferentȱtimeȱpointsȱintoȱ tubesȱcontainingȱsodiumȱcitrateȱ(0.2ȱml,ȱ3.2%ȱ[w/v]ȱsodiumȱcitrate)ȱandȱimmediatelyȱproȬ cessedȱtoȱplasmaȱbyȱcentrifugationȱatȱ2000ȱgȱforȱ15ȱminutesȱatȱ4°C.ȱTheȱsamplesȱwereȱ thenȱanalyzedȱasȱdescribedȱabove.ȱAllȱplateletȱcounts,ȱWBCȱcounts,ȱFPA,ȱandȱFXIIaȱlevȬ elsȱwereȱcorrectedȱforȱhemodilutionȱbyȱadjustingȱtheȱrawȱvaluesȱbasedȱonȱtheȱhemactoȬ crit.ȱTheȱinletȱandȱoutletȱpressureȱandȱbloodȱflowȱrateȱwereȱmeasuredȱusingȱaȱBiopacȱ systemȱ(AeroȱCaminoȱGoleta,ȱCA)ȱandȱpressureȱtransducersȱatȱtheȱinletȱandȱoutletȱofȱtheȱ deviceȱ(EdwardsȱLifesciences),ȱandȱtheȱresistanceȱwasȱcalculatedȱwithȱtheȱstandardȱRȱ=ȱ (PiȬPo)/QȱwhereȱPiȱisȱtheȱinletȱpressureȱinȱmmHg,ȱPoȱisȱtheȱoutletȱpressureȱinȱmmHg,ȱ andȱQȱisȱtheȱflowȱinȱL/min.ȱTheȱbleedingȱtimeȱwasȱmeasuredȱbyȱcuttingȱsmallȱincisionsȱ ofȱ4–5ȱmmȱatȱdifferentȱsitesȱofȱtheȱrightȱorȱleftȱearȱinȱeachȱanimal.ȱBloodȱfromȱtheȱincisionȱ wasȱremovedȱwithȱgauzeȱeveryȱ30ȱseconds,ȱandȱtheȱtimeȱuntilȱtheȱbleedingȱstoppedȱwasȱ measured.ȱ

Statisticalȱanalysis:ȱForȱallȱexperiments,ȱtheȱmeanȱ±ȱstandardȱdeviationȱisȱindicatedȱunȬ lessȱstatedȱotherwise.ȱThisȱincludesȱallȱvaluesȱandȱdataȱpointsȱinȱgraphs.ȱAllȱstatisticalȱ analysesȱwereȱperformedȱusingȱGraphPadȱPrismȱ5ȱorȱMicrosoftȱExcelȱsoftware.ȱAȱchiȬ squaredȱtestȱwasȱusedȱtoȱdetermineȱwhetherȱmiceȱwereȱprotectedȱagainstȱclotȱformationȱ andȱfullȱocclusionȱinȱtheȱFeCl3ȱthrombosisȱmodel.ȱAȱoneȬtailedȱstudent’sȱtȬtestȱwasȱapȬ pliedȱtoȱdetermineȱtheȱsignificanceȱofȱtheȱdifferenceȱinȱtheȱtimeȱuntilȱclotȱformationȱocȬ curredȱbetweenȱtheȱtwoȱgroups.ȱAȱoneȬtailedȱtestȱisȱappropriateȱsinceȱtheȱeffectȱisȱonlyȱ expectedȱinȱoneȱdirection.ȱTheȱsameȱtestȱwasȱusedȱforȱclotȱformationȱinȱtheȱartificialȱlungȱ devices.ȱForȱallȱtests,ȱpȱ<ȱ0.05ȱwasȱconsideredȱsignificant.ȱ

ȱȱ

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6XSSRUWLQJLQIRUPDWLRQ

TableȱS4.1.ȱKiȱvaluesȱofȱcoumarinȬlabeledȱFXII618ȱmodifiedȱatȱArg1ȱorȱArg8.ȱ

TableȱS4.2.ȱOverviewȱofȱFXII618ȱvariantsȱandȱtheirȱKiȱvalues.ȱ

TableȱS4.3.ȱSpecificityȱofȱFXII900.ȱ

TableȱS4.4.ȱCharacterizationȱofȱFII900ȱwithȱindividuallyȱreversedȱbeneficialȱmutations.ȱ

TableȱS4.5.ȱPharmacokineticȱparametersȱinȱrabbits.ȱ

TableȱS4.6.ȱPharmacokineticȱparametersȱinȱpigs.ȱ

TableȱS4.7.ȱTimeȱtoȱclotȱandȱfullȱocclusionȱinȱferricȱchlorideȬinducedȱthrombosisȱmouseȱ model.ȱ

FigureȱS4.1.ȱStabilityȱofȱFXI618ȱvariantsȱcarryingȱtwoȱDȬaminoȱacidsȱatȱtheȱNȬterminus.ȱ

FigureȱS4.2.ȱFXIIaȱinhibitorsȱFXII850ȱandȱFXII851.ȱ

FigureȱS4.3.ȱSynthesisȱofȱFXII900.ȱ

FigureȱS4.4.ȱReversionȱofȱbeneficialȱaminoȱacidȱsubstitutionsȱinȱFXII900.ȱ

Figureȱ S4.5.ȱ Plasmaȱ stabilityȱ ofȱ FXII900ȱ containingȱ CȬtermȱ Argȱ andȱ variantsȱ withȱ reȬ vertedȱbeneficialȱmutations.ȱ

FigureȱS4.6.ȱNegativeȱcontrolȱpeptideȱFXII901.ȱ

FigureȱS4.7.ȱFerricȱchlorideȬinducedȱthrombosisȱinȱmiceȱtreatedȱwithȱnegativeȱcontrolȱ peptideȱFXII901.ȱ

Figureȱ S4.8.ȱ Ferricȱ chlorideȬinducedȱ thrombosisȱ inȱ miceȱ treatedȱ withȱ FXIIaȱ inhibitorȱ FXII900.ȱ

FigureȱS4.9.ȱInhibitionȱofȱFXIIaȬdrivenȱcoagulationȱinȱartificialȱlungȱrabbitȱmodel:ȱdataȱ forȱindividualȱrabbits.ȱ

FigureȱS4.10.ȱAnalyticalȱdataȱofȱimportantȱpeptidesȱ

ȱȱ

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SupplementaryȱTablesȱ

TableȱS4.1.ȱKiȱvaluesȱofȱcoumarinȬlabeledȱFXII618ȱmodifiedȱatȱArg1ȱorȱArg8.ȱAllȱpeptidesȱwereȱsynthesizedȱ withȱtheȱadditionalȱaminoȱacidȱEȬ(7ȬmethoxyȬcoumarinȬ4Ȭyl)ȬAlaȬOHȱatȱtheȱCȬterminusȱforȱaccurateȱconcenȬ trationȱdetermination.ȱThisȱapproachȱenabledȱtheȱdeterminationȱofȱKiȱvaluesȱforȱtheȱcrudeȱbicyclicȱpeptide.ȱ Forȱcomparison,ȱtheȱcrudeȱbicyclicȱpeptideȱFXII618ȱmodifiedȱwithȱtheȱcoumarinȱaminoȱacidȱinhibitedȱhumanȱ andȱmouseȱFXIIaȱwithȱKiȱvaluesȱofȱ20ȱ±ȱ3ȱnMȱandȱ230ȱ±ȱ30ȱnM,ȱrespectively.ȱTheȱKiȱvaluesȱofȱtheȱcrudeȱ peptidesȱcarryingȱtheȱcoumarinȱaminoȱacidȱareȱaroundȱtwoȬfoldȱhigherȱthanȱthoseȱofȱpurifiedȱpeptidesȱwithȬ outȱcoumarin.ȱStandardȱdeviationsȱareȱindicatedȱforȱKisȱsmallerȱthanȱ300ȱnM.ȱAllȱKisȱwereȱmeasuredȱinȱtripȬ licate.ȱMeanȱ±ȱSDȱareȱindicated.ȱ

ModificationȱinȱArg1ȱ ModificationȱinȱArg8ȱ

Alteredȱ Kiȱhumanȱ Kiȱmouseȱ Alteredȱ Kiȱhumanȱ Kiȱmouseȱ aminoȱacidȱ FXIIaȱ(nM)ȱ FXIIaȱ(nM)ȱ aminoȱacidȱ FXIIaȱ(nM)ȱ FXIIaȱ(nM)ȱ

R1arȱ 32ȱ±ȱ5ȱ 350ȱ R8Aȱ 20.5ȱ±ȱ0.4ȱ 230ȱ±ȱ30ȱ

R1srȱ 59ȱ±ȱ4ȱ 750ȱ R8Sȱ 44.0ȱ±ȱ1.6ȱ 360ȱ

R1frȱ 22ȱ±ȱ5ȱ 190ȱ±ȱ20ȱ R8Fȱ 103ȱ±ȱ3ȱ 900ȱ

R1lrȱ 16ȱ±ȱ5ȱ 131ȱ±ȱ17ȱ R8Lȱ 44.3ȱ±ȱ0.9ȱ 490ȱ

R1qrȱ 32ȱ±ȱ4ȱ 250ȱ±ȱ8ȱ R8Pȱ 88.8ȱ±ȱ1.5ȱ 440ȱ

R1raȱ 12.4ȱ±ȱ0.3ȱ 88ȱ±ȱ4ȱ R8Kȱ 24.9ȱ±ȱ0.9ȱ 250ȱ±ȱ40ȱȱ

R1rsȱ 10.9ȱ±ȱ1.6ȱ 85ȱ±ȱ14ȱ R8Hȱ 13.6ȱ±ȱ0.4ȱ 88ȱ±ȱ13ȱ

R1rfȱ 36.3ȱ±ȱ1.1ȱ 220ȱ±ȱ60ȱ R8Eȱ 41.3ȱ±ȱ0.5ȱ 730ȱ

R1rlȱ 83ȱ±ȱ5ȱ 440ȱ R8Qȱ 38.3ȱ±ȱ1.2ȱ 330ȱ

R1rqȱ 21ȱ±ȱ3ȱ 160ȱ±ȱ30ȱ R8Nȱ 51ȱ±ȱ3ȱ 330ȱ ȱ

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TableȱS4.2.ȱOverviewȱofȱFXII618ȱvariantsȱandȱtheirȱKiȱvalues.ȱKiȱvaluesȱwereȱmeasuredȱatȱleastȱthreeȱtimes.ȱ Meanȱ±ȱSDȱareȱindicated.ȱ

Inhibitorȱ Modificationȱtoȱ Kiȱhumanȱ Kiȱmouseȱ Referenceȱ nameȱ FXII618ȱ FXIIȱ(nM)ȱ FXIIȱ(nM)ȱ

FXII618ȱȬȱ 8.1ȱ±ȱ0.7ȱ 86ȱ±ȱ9ȱ Baeriswyl,ȱV.,ȱetȱal.,ȱ ACSȱChem.ȱBiol.,ȱ 2015ȱ

FXII700ȱ Arg11ÆEhArgȱ 1.5ȱ±ȱ0.1ȱ N.D.ȱ Wilbs,ȱJ.,ȱetȱal.,ȱ ChemBioChem,ȱ 2016ȱ

FXII800ȱ Phe3ÆPhe4Fȱ 0.84ȱ±ȱ0.03ȱ N.D.ȱ Middendorp,ȱS.,ȱetȱ al.,ȱJ.ȱMed.ȱChem.,ȱ 2017ȱ

FXII850ȱȱ Arg1ÆDȬArgȬDȬSerȱ 5ȱ±ȱ2ȱ 35ȱ±ȱ9ȱȱ Ȭȱ

FXII851ȱ Arg8ÆHisȱ 6.5ȱ±ȱ0.3ȱ 36ȱ±ȱ3ȱȬȱ

FXII900ȱ Arg11ÆEhArg,ȱ 0.37ȱ±ȱ0.04ȱ 0.45ȱ±ȱ0.11ȱȬȱ Phe3ÆPhe4F,ȱ Arg1ÆDȬArgȬDȬSer,ȱ Arg8ÆHis,ȱdeletionȱ ofȱArg13ȱ ȱ

ȱȱ

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TableȱS4.3.ȱSpecificityȱofȱFXII900.ȱTheȱindicatedȱvaluesȱareȱaveragesȱofȱatȱleastȱthreeȱmeasurements.ȱMeanȱ ±ȱSDȱareȱindicated.ȱ

Proteaseȱ Kiȱ(nM)ȱ %ȱresidualȱactivityȱ atȱ40ȱΐMȱinhibitorȱ

FXIaȱ >ȱ40,000ȱ 95.1ȱ±ȱ0.6ȱȱ

Plasminȱ >ȱ40,000ȱ 86ȱ±ȱ5ȱȱ

Trypsinȱ 1460ȱ±ȱ120ȱ 9.2ȱ±ȱ1.1ȱ

uPAȱ >ȱ40,000ȱ 98.7ȱ±ȱ1.8ȱ

tPAȱ >ȱ40,000ȱ 97ȱ±ȱ4ȱ

Thrombinȱ >ȱ40,000ȱ 95ȱ±ȱ6ȱ

PlasmaȱKallikreinȱ >ȱ40,000ȱ 72ȱ±ȱ3ȱ

FXaȱ >ȱ40,000ȱ 97.1ȱ±ȱ1.3ȱ

FVIIaȱ >ȱ40,000ȱ 97.1ȱ±ȱ1.3ȱ

ȱȱ

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TableȱS4.4.ȱReversionȱofȱbeneficialȱmutations.ȱTheȱindicatedȱvaluesȱareȱaveragesȱofȱatȱleastȱthreeȱmeasureȬ ments.ȱMeanȱ±ȱSDȱareȱindicated.ȱ

Peptideȱ KiȱhumanȱFXIIȱ(nM)ȱ KiȱmouseȱFXIIȱ(nM)ȱ

FXII618ȱ 8.1ȱ±ȱ0.7ȱ 86ȱ±ȱ9ȱ

FXII900ȱ 0.37ȱ±ȱ0.04ȱ 0.45ȱ±ȱ0.11ȱ

FXII900ȱ(withȱCȬtermȱArg)ȱ 0.34ȱ±ȱ0.03ȱ 0.50ȱ±ȱ0.15ȱ

Phe3ÆPhe4Fȱrevertedȱ 0.70ȱ±ȱ0.06ȱ 1.9ȱ±ȱ0.5ȱ

Arg8ÆHisȱrevertedȱ 0.31ȱ±ȱ0.02ȱ 0.91ȱ±ȱ0.04ȱ

Arg11ÆEhArgȱrevertedȱ 0.51ȱ±ȱ0.10ȱ 0.9ȱ±ȱ0.4ȱ ȱ

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TableȱS4.5.ȱPharmacokineticȱparametersȱinȱrabbits.ȱFXII900ȱwasȱadministratedȱintravenouslyȱ(nȱ=ȱ3,ȱ3.7ȱ mg/kg)ȱandȱtheȱconcentrationȱwasȱquantifiedȱbyȱLCȬMS.ȱ

Parameterȱ Valueȱ

t1/2ȱEȱ(min)ȱ 12ȱ±ȱ2ȱ

CLȱ(ml/kg*min)ȱ 16.1ȱ±ȱ1.0ȱ

VDȱ(ml/kg)ȱ 280ȱ±ȱ50ȱ ȱ

ȱȱ

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TableȱS4.6.ȱPharmacokineticȱparametersȱinȱpigs.ȱFXII900ȱwasȱadministratedȱintravenouslyȱ(nȱ=ȱ3,ȱ4ȱmg/kg)ȱ andȱtheȱconcentrationȱwasȱquantifiedȱbyȱLCȬMS.ȱ

Parameterȱ Valueȱ

t1/2ȱEȱ(min)ȱ 36ȱ±ȱ5ȱ

CLȱ(ml/kg*min)ȱ 11.8ȱ±ȱ1.8ȱ

VDȱ(ml/kg)ȱ 610ȱ±ȱ140ȱ ȱ

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TableȱS4.7.ȱTimeȱtoȱclotȱformationȱandȱfullȱocclusionȱinȱferricȱchlorideȬinducedȱthrombosisȱmouseȱmodel.ȱ Timeȱpointsȱatȱwhichȱclotȱformationȱandȱbloodȱvesselȱocclusionȱwereȱobserved.ȱ

Controlȱ Timeȱtoȱclotȱ Timeȱtoȱfullȱ Inhibitorȱ Timeȱtoȱclotȱ Timeȱtoȱfullȱ (min)ȱ occlusionȱ (min)ȱ occlusionȱ (min)ȱ (min)ȱ

1ȱȱ 14ȱ 18ȱ 1ȱȬȱȬȱ

2ȱ 17ȱȬȱ2ȱȬȱȬȱ

3ȱ 6ȱ 9ȱ 3ȱ 23ȱȬȱ

4ȱȬȱȬȱ4ȱȬȱȬȱ

5ȱ 6ȱ 9ȱ 5ȱȬȱȬȱ

6ȱ 7ȱ 9ȱ 6ȱ 21ȱȬȱ

7ȱ 2ȱ 10ȱ 7ȱ 16ȱȬȱ

8ȱ 15ȱȬȱ8ȱȬȱȬȱ

ȱȱ ȱ9ȱȬȱȬȱ ȱ

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SupplementaryȱFiguresȱ

FigureȱS4.1.ȱStabilityȱofȱFXI618ȱvariantsȱcarryingȱtwoȱDȬaminoȱacidsȱatȱtheȱNȬterminus.ȱTheȱpeptidesȱwereȱ incubatedȱinȱhumanȱplasmaȱasȱindicated.ȱResidualȱinhibitoryȱactivityȱwasȱtestedȱinȱaȱFXIIaȱactivityȱassayȱ usingȱaȱfluorogenicȱsubstrate.ȱResidualȱinhibitionȱinȱ%ȱwasȱcalculatedȱasȱ(IC50,0h/IC50,xh)ȱ×ȱ100,ȱwhereinȱIC50,0hȱ isȱtheȱfunctionalȱstrengthȱofȱtheȱinhibitorȱatȱtimeȱpointȱ0ȱandȱIC50,xhȱisȱtheȱfunctionalȱstrengthȱofȱinhibitorȱafterȱ oneȱofȱtheȱdifferentȱplasmaȱincubationȱperiods.ȱ ȱȱ

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FigureȱS4.2.ȱFXIIaȱinhibitorsȱFXII850ȱandȱFXII851.ȱȱ ȱ

ȱȱ

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FigureȱS4.3.ȱSynthesisȱofȱFXII900.ȱTheȱstructureȱofȱFXII900ȱisȱrepresentedȱandȱtheȱchemicalȱstructuresȱofȱtheȱ unnaturalȱaminoȱacidsȱandȱtheȱchemicalȱlinkerȱareȱshownȱbelow.ȱȱ ȱ

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FigureȱS4.4.ȱReversionȱofȱbeneficialȱaminoȱacidȱsubstitutionsȱinȱFXII900.ȱTheȱthreeȱaminoȱacidsȱPhe4F3,ȱ His8,ȱandȱEhArg11ȱwereȱindividuallyȱrevertedȱinȱFXII900ȱ(containingȱaȱCȬterminalȱArg)ȱtoȱtheȱoriginalȱresiȬ duesȱPhe3,ȱArg8,ȱandȱArg11,ȱinȱorderȱtoȱassessȱtheȱcontributionȱofȱtheseȱmodificationsȱtoȱtheȱstabilityȱofȱtheȱ inhibitorȱasȱwellȱasȱtoȱtheȱinhibitionȱofȱhumanȱandȱmouseȱFXIIa.ȱ(A)ȱTheȱaminoȱacidȱsequencesȱofȱtheȱbicyclicȱ peptidesȱareȱindicated.ȱAllȱpeptidesȱcontainȱaȱCȬterminalȱArg.ȱ(B)ȱTheȱresidualȱactivityȱofȱhumanȱandȱmouseȱ FXIIaȱatȱdifferentȱpeptideȱconcentrationsȱwasȱmeasuredȱinȱtriplicate.ȱMeansȱ±ȱSDȱareȱindicated ȱȱ

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FigureȱS4.5.ȱPlasmaȱstabilityȱofȱFXII900ȱcontainingȱCȬtermȱArgȱandȱvariantsȱwithȱrevertedȱbeneficialȱmuȬ tations.ȱTheȱthreeȱaminoȱacidsȱPhe4F3,ȱHis8,ȱandȱEhArg11ȱwereȱindividuallyȱrevertedȱinȱFXII900ȱcontainingȱ aȱCȬterminalȱArgȱtoȱtheȱoriginalȱresiduesȱPhe3,ȱArg8,ȱandȱArg11,ȱrespectively,ȱandȱtheȱstabilityȱinȱplasmaȱ wasȱassessed.ȱPeptidesȱwereȱincubatedȱatȱaȱconcentrationȱofȱ80ȱΐMȱinȱplasmaȱatȱ37°C.ȱAfterȱprecipitationȱ andȱremovalȱofȱplasmaȱproteins,ȱtheȱpeptidesȱandȱtheirȱdegradationȱproductsȱwereȱanalyzedȱLCȬMS.ȱTotalȱ ionȱcountsȱ(TIC)ȱareȱindicatedȱinȱtheȱtopȱpanels.ȱTheȱlowerȱpanelsȱshowȱtheȱionȱcountȱofȱtheȱintactȱpeptides.ȱ Forȱallȱpeptides,ȱaȱspeciesȱwasȱfoundȱwithȱanȱadditionalȱmassȱofȱoneȱdalton.ȱItȱisȱassumedȱthatȱthisȱmassȱ resultsȱfromȱtheȱhydrolysisȱofȱtheȱCȬterminalȱamideȱgroup ȱ

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FigureȱS4.7.ȱFerricȱchlorideȬinducedȱthrombosisȱinȱmiceȱtreatedȱwithȱnegativeȱcontrolȱpeptideȱFXII901.ȱ IntravitalȱfluorescenceȱmicroscopyȱimagesȱofȱmesentericȱarteriolesȱinȱwhichȱthrombosisȱwasȱinducedȱbyȱtopȬ icalȱapplicationȱofȱFeCl3ȱ(7.5%,ȱ1ȱmin).ȱPlateletsȱwereȱfluorescentlyȱlabeledȱwithȱRhodamineȱ6GȱforȱvisualiȬ zation.ȱVesselȱwallsȱatȱFeCl3ȱapplicationȱsiteȱareȱindicatedȱwithȱyellowȱmarkers ȱ

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FigureȱS4.8.ȱFerricȱchlorideȬinducedȱthrombosisȱinȱmiceȱtreatedȱwithȱFXIIaȱinhibitorȱFXII900.ȱIntravitalȱ fluorescenceȱmicroscopyȱimagesȱofȱmesentericȱarteriolesȱinȱwhichȱthrombosisȱwasȱinducedȱbyȱtopicalȱappliȬ cationȱofȱFeCl3ȱ(7.5%,ȱ1ȱmin).ȱPlateletsȱwereȱfluorescentlyȱlabeledȱwithȱRhodamineȱ6Gȱforȱvisualization.ȱVesȬ selȱwallsȱatȱFeCl3ȱapplicationȱsiteȱareȱindicatedȱwithȱyellowȱmarkers ȱ

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FigureȱS4.9.ȱInhibitionȱofȱFXIIaȬdrivenȱcoagulationȱinȱartificialȱlungȱrabbitȱmodel:ȱdataȱforȱindividualȱ rabbits.ȱ Rabbitsȱ connectedȱ venoȬvenousȱ toȱ anȱ artificialȱ lungȱ systemȱ forȱ fourȱ hoursȱ wereȱ injectedȱ withȱ FXII900ȱasȱaȱbolusȱ(2ȱmg/kg)ȱbeforeȱstartȱofȱtheȱextracorporealȱcirculationȱandȱasȱaȱconstantȱinfusionȱ(0.075ȱ mg/kg/min)ȱoverȱtheȱfullȱtimeȱcourseȱofȱtheȱexperimentȱ(nȱ=ȱ4;ȱdataȱshownȱinȱblue),ȱorȱwereȱnotȱtreatedȱ(nȱ=ȱ 3).ȱDataȱforȱindividualȱrabbitsȱisȱshownȱinȱthisȱfigure.ȱMeanȱvaluesȱ±ȱSDȱforȱtheȱtwoȱgroupsȱareȱshownȱinȱ Figure.ȱ28.ȱValuesȱindicatedȱforȱtheȱtimeȱpointȱ0ȱreferȱtoȱmeasurementsȱmadeȱbeforeȱconnectionȱofȱtheȱartiȬ ficialȱlung.ȱResultsȱofȱFXII900ȱareȱshownȱinȱblueȱandȱthoseȱofȱtheȱuntreatedȱinȱred.ȱ(A)ȱaPTT.ȱCoagulationȱ timesȱofȱ230ȱsȱindicateȱthatȱplasmaȱdidȱnotȱcoagulateȱatȱthisȱtime.ȱ(B)ȱACT.ȱClottingȱtimesȱofȱ1500ȱsȱindicateȱ thatȱbloodȱdidȱnotȱcoagulateȱatȱthisȱtime.ȱ(C)ȱResistanceȱcalculatedȱbasedȱonȱpressureȱatȱtheȱinletȱandȱoutletȱ ofȱtheȱdeviceȱandȱtheȱflowȱrate.ȱ(D)ȱBleedingȱtimeȱmeasuredȱbyȱincisionȬprovokedȱinjuriesȱatȱtheȱears.ȱ(E)ȱ Plateletȱcount.ȱ(F)ȱVolumeȱofȱbloodȱclotsȱindicatedȱinȱ%ȱofȱvolumeȱofȱtheȱartificialȱlungs.ȱForȱrabbitȱ2,ȱtheȱ volumeȱisȱnotȱavailable ȱȱ

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FigureȱS4.9.ȱContinuedȱ ȱȱ

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FigureȱS4.10.ȱȱAnalyticalȱHPLCȱandȱmassȱspectrometricȱdataȱforȱkeyȱpeptidesȱ

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FigureȱS4.10.ȱContinued

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Thisȱchapterȱisȱbasedȱonȱaȱmanuscriptȱforȱsubmissionȱbyȱtheȱfollowingȱauthors:ȱȱ

MaolaȱKhan,ȱJonasȱWilbs,ȱJeremyȱTouatiȱandȱChristianȱHeinisȱ

Authorȱcontributions:ȱM.KȱandȱJ.Wȱcontributedȱequally.ȱM.K.ȱandȱJ.T.ȱexpressedȱMMPȬ 2ȱandȱperformedȱphageȱselections.ȱM.K.ȱintroducedȱtheȱhydroxamicȱacidȱmodification.ȱ J.W.ȱperformedȱsystematicȱaffinityȱandȱstabilityȱimprovementsȱandȱselectivityȱanalysis.ȱ J.W.ȱandȱC.H.ȱwroteȱtheȱmanuscript.ȱAllȱauthorsȱcontributedȱtoȱeditingȱandȱcriticalȱproofȬ readingȱofȱtheȱmanuscript.ȱ

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Matrixȱmetalloproteinasesȱ(MMPs)ȱareȱimportantȱtherapeuticȱtargetsȱbutȱtheȱdevelopȬ mentȱ ofȱ selectiveȱ smallȱ moleculeȱ inhibitorsȱ has,ȱ despiteȱ impressiveȱ efforts,ȱ notȱ beenȱ achievedȱtoȱmostȱmembersȱofȱthisȱclass.ȱInȱthisȱwork,ȱweȱhaveȱaddressedȱthisȱlongȬstandȬ ingȱchallengeȱwithȱaȱcombinatorialȱapproachȱtoȱscreenȱbillionsȱofȱmacrocyclesȱcomposedȱ ofȱnaturalȱaminoȱacidsȱandȱaȱsyntheticȱchemicalȱcore.ȱByȱoptimizationȱofȱtheȱzincȬbindingȱ groupȱandȱsubstitutingȱnaturalȱaminoȱacidsȱtoȱunnaturalȱones,ȱweȱsucceededȱatȱenhancȬ ingȱsubstantiallyȱtheȱbindingȱaffinityȱandȱstabilityȱofȱtheȱphageȬselectedȱbicyclicȱpeptide.ȱ TheȱresultingȱMMPȬ2ȱinhibitorȱhasȱaȱhighȱaffinityȱ(1.9ȱnM)ȱandȱstabilityȱ(4.4ȱh)ȱandȱthusȱ providesȱaȱvaluableȱresearchȱtool.ȱTheȱappliedȱcombinedȱstrategiesȱpromiseȱtoȱdeliverȱ selectiveȱsyntheticȱinhibitorsȱalsoȱtoȱotherȱimportantȱMMPȱtargets.ȱ ,QWURGXFWLRQ

Theȱmatrixȱmetalloproteinasesȱ(MMPs)ȱareȱaȱfamilyȱofȱendopeptidasesȱcomprisedȱofȱ23ȱ membersȱinȱhumansȱ(255).ȱTheȱenzymesȱutilizeȱaȱzincȱionȱinȱtheȱactiveȱsiteȱforȱsubstrateȱ cleavageȱandȱareȱexpressedȱasȱzymogens,ȱinȱwhichȱtheȱzincȱionȱisȱchelatedȱbyȱaȱcysteineȱ residueȱinȱtheȱproȬdomainȱinȱadditionȱtoȱthreeȱhistidineȱresiduesȱinȱtheȱcatalyticȱdomain.ȱ UponȱproteolyticȱcleavageȱofȱtheȱproȬdomain,ȱusuallyȱbyȱanotherȱMMP,ȱtheȱenzymeȱbeȬ comesȱactive.ȱTheȱmainȱphysiologicalȱfunctionȱofȱtheȱMMPsȱisȱdegradationȱofȱtheȱextraȬ cellularȱmatrixȱ(ECM)ȱtoȱfacilitateȱtissueȱremodelingȱandȱangiogenesisȱ(255–257).ȱThisȱisȱ achievedȱbyȱtheȱdegradationȱofȱcollagenȱandȱotherȱECMȱsupportingȱcomponentsȱbutȱalsoȱ byȱtheȱreleaseȱofȱgrowthȱfactorsȱandȱcytokinesȱ(258,ȱ259).ȱEvenȱthoughȱeachȱMMPȱhasȱaȱ largeȱvarietyȱofȱsubstrates,ȱoftenȱoverlappingȱwithȱotherȱMMPs,ȱtheȱenzymesȱwereȱtraȬ ditionallyȱclassifiedȱbasedȱonȱtheirȱsubstrateȱpreference.ȱTheȱMMPsȱhaveȱbeenȱgenerallyȱ dividedȱintoȱtheȱfollowingȱgroups:ȱcollagenasesȱ(MMPȬ1,ȱ8,ȱ13,ȱ18),ȱgelatinasesȱ(MMPȬ2,ȱ 9),ȱ stromelysinsȱ (MMPȬ3,ȱ 10,ȱ 11,ȱ 12),ȱ matrilysinsȱ (MMPȬ7,ȱ 26)ȱ andȱ membraneȱ typeȱ (MMPȬ14,ȱ15,ȱ16,ȱ17,ȱ24,ȱ25)ȱ(260).ȱMMPȱactivityȱisȱregulatedȱinȱvivoȱbyȱtranscription,ȱ zymogenȱactivationȱandȱtissueȱinhibitorsȱofȱmetalloproteinasesȱ(TIMPS)ȱ(256,ȱ261,ȱ262).ȱ Upregulationȱand/orȱincreasedȱexpressionȱofȱMMPsȱhasȱbeenȱobservedȱinȱvariousȱdisȬ eases,ȱpredominantlyȱcancer,ȱwhereȱMMPsȱcontributeȱtoȱtumorȱmetastasisȱandȱneovasȬ cularizationȱ(263–267).ȱMMPȱactivityȱandȱoverexpressionȱhaveȱbeenȱcorrelatedȱtoȱpoorȱ prognosisȱandȱtumorȱaggressivenessȱ(268).ȱAsȱaȱconsequence,ȱlargeȱeffortsȱhaveȱbeenȱ made,ȱbothȱinȱindustryȱandȱacademia,ȱinȱorderȱtoȱdevelopȱMMPȱinhibitorsȱasȱtherapeuȬ tics.ȱSeveralȱcompoundsȱwereȱevaluatedȱinȱclinicalȱtrialsȱasȱcancerȱtherapeutics,ȱhoweverȱ noȱMMPȱinhibitorȱwasȱsuccessfullyȱapprovedȱasȱaȱtherapeuticȱ(269).ȱTheȱearlyȱMMPȱinȬ hibitorsȱwereȱsmallȱmoleculeȱpeptidomimetics,ȱoftenȱutilizingȱaȱhydroxamicȱacidȱmoietyȱ forȱtightȱbindingȱtoȱtheȱactiveȱsiteȱzincȱion.ȱTheȱmostȱprominentȱexamplesȱincludeȱbatiȬ

107 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH mastat,ȱmarimastat,ȱCGSȱ27023A,ȱprinomastatȱandȱtanomastatȱ(270–274).ȱSeveralȱexplaȬ nationsȱforȱtheȱclinicalȱfailuresȱofȱtheȱMMPȱinhibitorsȱhaveȱbeenȱproposed.ȱOneȱisȱpoorȱ designȱofȱtheȱclinicalȱtrials,ȱinȱwhichȱmostlyȱpatientsȱwithȱalreadyȱprogressedȱcancersȱ wereȱenrolledȱevenȱthoughȱinȱpreclinicalȱstudiesȱMMPȱinhibitorsȱwereȱfoundȱtoȱbeȱmostȱ efficientȱinȱtheȱearlyȱstagesȱofȱtheȱdiseaseȱ(275).ȱAȱsecondȱreasonȱisȱtheȱlackȱofȱspecificityȱ ofȱtheȱhydroxamateȱbasedȱinhibitors.ȱWithȱseveralȱMMPsȱbeingȱantitargetsȱinȱcancerȱandȱ extensiveȱoffȱtargetȱbinding,ȱtheȱlackȱofȱspecificityȱcausedȱpoorȱefficacyȱandȱsevereȱsideȱ effects,ȱultimatelyȱleadingȱtoȱtheȱterminationȱofȱtheȱMMPȱinhibitorȱprogramsȱ(276).ȱDeȬ spiteȱtheȱlackȱofȱsuccessȱinȱtheȱclinic,ȱMMPȱinhibitorsȱremainȱpromisingȱasȱpotentialȱtherȬ apeuticsȱorȱasȱresearchȱtoolsȱ(275–277).ȱPresently,ȱtheȱfocusȱliesȱonȱdevelopingȱinhibitorsȱ highlyȱselectiveȱforȱaȱspecificȱMMPȱandȱtoȱthisȱendȱseveralȱapproachesȱhaveȱbeenȱunderȬ takenȱ(278–282).ȱInsteadȱofȱtargetingȱtheȱcatalyticȱdomain,ȱnewȱMMPȱinhibitorsȱcanȱtarȬ getȱ exositesȱ includingȱ theȱ hemopexinȱ domain,ȱ collagenȱ bindingȱ siteȱ andȱ proȱ domainȱ (283).ȱSeveralȱselectiveȱsmallȱmoleculeȱinhibitorsȱhaveȱbeenȱreported,ȱwithȱorȱwithoutȱ zincȱbindingȱmoieties.ȱOtherȱapproachesȱincludeȱinȱsilicoȱscreeningȱandȱnaturalȱproductȱ derivedȱinhibitors.ȱInȱaddition,ȱaȱnumberȱofȱhighlyȱselectiveȱantibodiesȱhaveȱbeenȱgenȬ erated.ȱHowever,ȱdevelopingȱspecificȱMMPȱinhibitorsȱhasȱbeenȱchallengingȱandȱdespiteȱ theȱrecentȱeffortsȱselectiveȱinhibitorsȱhaveȱonlyȱbeenȱfoundȱforȱaȱhandfulȱofȱMMPs.ȱȱ

MMPȬ2ȱisȱaȱtargetȱwhichȱhasȱbeenȱstronglyȱassociatedȱtoȱaggressiveȱcancerȱ(284,ȱ285)ȱandȱ severalȱknockȬoutȱmodelsȱconfirmȱitsȱinvolvementȱinȱtumorȱgrowth,ȱneovascularizationȱ andȱmetastasisȱ(286–290).ȱHowever,ȱtheȱdevelopmentȱofȱaȱspecificȱMMPȬ2ȱinhibitorȱhasȱ beenȱparticularlyȱchallengingȱgivenȱitsȱhighȱsimilarityȱtoȱMMPȬ9.ȱToȱdateȱnumerousȱmoȬ lecularȱformatsȱhaveȱbeenȱutilizedȱinȱorderȱtoȱidentifyȱpotentȱandȱselectiveȱMMPȬ2ȱinȬ hibitors,ȱincludingȱsmallȱmolecules,ȱpeptides,ȱnaturalȱproductsȱandȱantibodies.ȱRoselloȱ andȱcoworkersȱsynthesizedȱsmallȱmoleculesȱwithȱtheȱpurposeȱofȱinhibitingȱMMPȬ2ȱandȱ

14ȱselectively.ȱTheȱmostȱselectiveȱcompoundȱinhibitsȱMMPȬ2ȱwithȱanȱIC50ȱofȱ0.09ȱnM,ȱbutȱ alsoȱinhibitsȱMMPȬ8,ȱ9ȱandȱ14ȱinȱtheȱsingleȱdigitȱnanomolarȱrangeȱ(291,ȱ292).ȱDerivativesȱ ofȱgelatinaseȱinhibitorȱSBȬ3CTȱhaveȱbeenȱreportedȱtoȱinhibitȱMMPȬ2ȱselectivelyȱinȱtheȱ lowȱnanomolarȱrange,ȱbutȱsufferȱfromȱlimitationsȱinȱsolubilityȱandȱmetabolismȱ(293,ȱ294).ȱ RecentlyȱotherȱselectiveȱsmallȱmoleculeȱMMPȬ2ȱinhibitorsȱwereȱdescribedȱwithȱaffinitiesȱ inȱtheȱnanomolarȱrange,ȱhoweverȱtheȱcompoundsȱwereȱnotȱfullyȱselectiveȱorȱwereȱonlyȱ evaluatedȱagainstȱMMPȬ9ȱ(295–297).ȱSeveralȱpeptideȱbasedȱinhibitorsȱandȱtransitionȱstateȱ analoguesȱwereȱreported,ȱbutȱwithȱlackingȱselectivityȱoverȱMMPȬ9ȱ(298,ȱ299).ȱChloroȬ toxin,ȱaȱnaturalȱproduct,ȱinhibitsȱMMPȬ2ȱwithȱaȱselectivityȱoverȱotherȱMMPs,ȱbutȱisȱalsoȱ targetingȱotherȱbiomoleculesȱ(300,ȱ301).ȱInȱaddition,ȱantibodiesȱwereȱdevelopedȱbutȱwithȱ lackȱofȬȱorȱnotȱfullyȱcharacterizedȱselectivityȱ(302,ȱ303).ȱUpȱtoȱnow,ȱtheȱmostȱpromisingȱ MMPȬ2ȱinhibitorȱinȱtermsȱofȱpotencyȱandȱselectivityȱisȱaȱpeptideȱbasedȱinhibitor,ȱAPPȬIPȱ whichȱbindsȱtheȱactiveȱsiteȱofȱMMPȬ2ȱwithȱanȱIC50ȱofȱ30ȱnMȱandȱhasȱaffinityȱinȱtheȱmiȬ cromolarȱrangeȱforȱMMPsȱ3,ȱ7,ȱ9ȱandȱ14ȱ(304–306).ȱWhenȱusedȱasȱaȱ23ȱkDaȱfusionȱproteinȱ

108 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH withȱTIMPȬ2,ȱtherebyȱutilizingȱhemopexinȱdomainȱbinding,ȱtheȱmoleculeȱinhibitsȱMMPȬ

2ȱwithȱaȱKiappȱofȱ0.7ȱpMȱwhereasȱactivitiesȱofȱMMPsȱ1,ȱ3,ȱ7,ȱ8,ȱ9ȱandȱ14ȱremainȱaboveȱ70%ȱ atȱ420ȱnMȱ(307).ȱȱȱȱȱȱȱȱȱ

EvenȱthoughȱAPPȬIPȱisȱpotentȱandȱselective,ȱitȱhasȱaȱpoorȱproteolyticȱstabilityȱwhichȱlimȬ itsȱitsȱuseȱasȱresearchȱtoolȱandȱasȱtherapeutic.ȱRecentlyȱourȱlaboratoryȱhasȱgeneratedȱbiȬ cyclicȱpeptideȱinhibitorsȱtoȱaȱnumberȱofȱproteasesȱwhichȱbindȱtoȱtheirȱtargetsȱwithȱhighȱ affinityȱandȱselectivityȱandȱareȱlessȱproneȱtoȱproteolysisȱ(180,ȱ188,ȱ203).ȱTheȱpeptidesȱareȱ generallyȱdevelopedȱbyȱphageȱdisplayȱandȱsubsequentȱleadȱoptimizationȱ(204,ȱ246).ȱInȱ thisȱworkȱweȱaimedȱatȱutilizingȱthisȱapproachȱtoȱmakeȱaȱpotent,ȱselectiveȱandȱproteolytȬ icallyȱstableȱMMPȬ2ȱinhibitorȱwhichȱisȱreadilyȱavailableȱthroughȱchemicalȱsynthesis.ȱ 5HVXOWV

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PeptidesȱofȱaȱphageȱlibraryȱofȱtheȱformȱACX6CX6CGȬpIIIȱ(diversityȱ>ȱ4ȱbillionȱpeptides;ȱ Figureȱ29A)ȱwereȱcyclizedȱbyȱreactingȱtheȱthreeȱcysteineȱresiduesȱwithȱTBMBȱandȱsubȬ jectedȱtoȱthreeȱroundsȱofȱaffinityȱselectionȱwithȱactivatedȱMMPȬ2.ȱAȱstrongȱenrichmentȱ ofȱphageȱinȱroundȱ2ȱandȱ3ȱofȱtheȱselectionȱandȱfiveȱstrongȱconsensusȱsequencesȱamongȱ isolatedȱpeptidesȱindicatedȱisolationȱofȱbindersȱ(Figureȱ29A).ȱOneȱpeptideȱisolatedȱ20ȱ times,ȱM21ȱinhibitedȱtheȱproteaseȱefficientlyȱwithȱaȱKiȱofȱ4ȱPMȱwhileȱallȱotherȱpeptidesȱ showedȱnoȱinhibitionȱorȱonlyȱatȱhighȱmicromolarȱconcentrations.ȱInȱanȱalanineȱscan,ȱtheȱ criticalȱaminoȱacidsȱwereȱunambiguouslyȱfoundȱtoȱbeȱtheȱfirstȱfourȱinȱtheȱsecondȱringȱofȱ theȱmacrocycleȱ(DAIW)ȱ(Figureȱ29B).ȱTheȱsameȱphageȱpeptideȱlibraryȱwasȱalsoȱoxidizedȱ insteadȱofȱreactedȱwithȱTBMBȱtoȱformȱbicyclicȱpeptidesȱbasedȱonȱtwoȱdisulfideȱbridges,ȱ aȱstrategyȱthatȱweȱhaveȱrecentlyȱreportedȱ(184).ȱInȱthisȱselection,ȱfourȱpeptidesȱcontainingȱ onlyȱtwoȱcysteinesȱwereȱstronglyȱenrichedȱ(Figureȱ29C).ȱOneȱofȱtheȱmonocyclicȱpeptidesȱ inhibitedȱ MMPȬ2ȱ moreȱ efficientlyȱ thanȱ theȱ bestȱ bicyclicȱ peptideȱ inhibitorsȱ describedȱ aboveȱ(Kiȱ=ȱ0.05ȱPM).ȱPeptidesȱwithȱonlyȱtwoȱcysteinesȱareȱveryȱrareȱinȱtheȱappliedȱlibraryȱ andȱindicate,ȱalongȱwithȱtheȱgoodȱinhibitoryȱactivity,ȱaȱstrongȱcompetitiveȱadvantageȱofȱ thisȱpeptideȱformatȱforȱtheȱinhibitionȱofȱtheȱmetalloproteinase.ȱComparisonȱofȱtheseȱseȬ quencesȱwithȱhumanȱproteinsȱrevealedȱaȱstrikingȱsimilarityȱtoȱtheȱ10Ȭaminoȱacidȱlinearȱ peptideȱAPPȬIPȱthatȱwasȱfoundȱbyȱHigashiȱandȱcoȬworkersȱtoȱinhibitȱMMPȬ2ȱ(306)ȱ(FigȬ ureȱ30A).ȱTheȱlinearȱpartȱofȱtheȱisolatedȱmonocyclicȱpeptidesȱisȱlikelyȱformingȱsimilarȱ interactionȱasȱtheȱCȬterminalȱaminoȱacidsȱofȱtheȱlinearȱAPPȬIP.ȱTheseȱlatterȱaminoȱacidȱ segmentȱAspȬAlaȬLeuȬMetȱbindsȱinȱaȱlinearȱandȱstretchedȱconformationȱtoȱtheȱactiveȱsiteȱ ofȱMMPȬ2,ȱpossiblyȱexplainingȱtheȱstrongȱenrichmentȱofȱpeptidesȱwithȱaȱlinearȱmoiety.ȱ

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Figureȱ29.ȱ(A)ȱFiveȱconsensusȱsequencesȱwereȱisolatedȱafterȱthreeȱroundsȱofȱselectionȱasȱillustrated.ȱTheȱmostȱ abundantȱclone,ȱM21,ȱinhibitedȱMMPȬ2ȱwithȱhighestȱaffinity.ȱ(B)ȱAnȱalanineȱscanȱofȱM21ȱshowedȱthatȱresiȬ duesȱD10,ȱA11ȱandȱI12ȱwereȱtheȱmostȱimportantȱforȱbinding.ȱTheȱKiȱvaluesȱofȱsystematicallyȱsubstitutedȱ peptidesȱareȱindicated.ȱ(C)ȱFourȱpeptidesȱcontainingȱonlyȱtwoȱcysteinesȱwereȱenrichedȱinȱtheȱselection.ȱOneȱ ofȱthemȱboundȱtheȱtaretȱwithȱhigherȱaffinityȱthanȱtheȱbestȱbicyclicȱpeptide.ȱAllȱmonocyclicȱpeptidesȱsharedȱ theȱsameȱbindingȱmotifȱasȱidentifiedȱinȱM21.ȱ

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TheȱXȬrayȱstructureȱofȱAPPȬIPȱshowedȱthatȱtheȱtwoȱoxygenȱatomsȱofȱtheȱcarboxylateȱofȱ aspartateȱcoordinateȱinȱaȱbidentateȱmannerȱtoȱtheȱactiveȱsiteȱzincȱionȱofȱMMPȬ2ȱ(PDBȱIDȱ 3AYU)ȱ(Figureȱ30A)ȱ(306).ȱBasedȱonȱsequenceȱcomparisonȱweȱhypothesizedȱthatȱtheȱasȬ partateȱresiduesȱinȱM21ȱandȱtheȱmonocyclicȱpeptideȱformȱtheȱsameȱinteractionȱasȱinȱAPPȬ

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IP.ȱHydroxamateȱisȱknownȱtoȱchelateȱzincȱwithȱaȱhigherȱaffinityȱthanȱcarboxylate,ȱmainlyȱ dueȱtoȱitsȱtendencyȱtoȱformȱbidentateȱratherȱthanȱmonodentateȱinteractionsȱwithȱtheȱionȱ (308).ȱItȱhasȱalsoȱbeenȱfoundȱtoȱformȱadditionalȱhydrogenȱbondingȱinteractionsȱwithȱtheȱ enzymeȱ(309,ȱ310).ȱTherefore,ȱweȱreasonedȱthatȱsubstitutionȱofȱaspartateȱwithȱLȬalanylȬ hydroxialanineȱinȱtheseȱpeptidesȱcouldȱsignificantlyȱincreaseȱtheȱbindingȱstrength.ȱAltȬ houghȱtheȱmonocyclicȱlassoȬtypeȱpeptideȱwasȱaȱbetterȱinhibitorȱthanȱbicyclicȱM21,ȱweȱ choseȱtoȱmodifyȱtheȱlatterȱoneȱasȱtheȱbicyclicȱpeptideȱwasȱfoundȱtoȱbeȱmetabolicallyȱmoreȱ stable.ȱToȱprepareȱbicyclicȱpeptideȱM21hy,ȱaȱFmocȬbasedȱsyntheticȱstrategyȱwasȱdevelȬ opedȱinȱwhichȱtheȱlinearȱpeptideȱM21ȱwasȱsynthesizedȱwithȱaȱ2Ȭphenylisopropylȱesterȱ sideȱchainȱprotectedȱaspartate.ȱThisȱprotectionȱgroupȱwasȱorthogonallyȱremovedȱusingȱ 2%ȱ(v/v)ȱTFAȱinȱDCMȱandȱtheȱhydroxamicȱacidȱmoietyȱwasȱintroducedȱviaȱaȱpeptideȱ couplingȱ ofȱ theȱ asparticȱ acidȱ residueȱ withȱ hydroxylamine.ȱ Allȱ remainingȱ protectingȱ groupsȱwereȱremovedȱunderȱacidicȱconditionsȱandȱtheȱpeptideȱwasȱcyclizedȱwithȱTBMB.ȱ

M21hyȱinhibitedȱMMPȬ2ȱwithȱaȱ35ȬfoldȱimprovedȱKiȱofȱ116ȱ±ȱ8ȱnMȱ(Figureȱ30A).ȱ

Figureȱ30.ȱ(A)ȱTheȱpreviouslyȱidentifiedȱlinearȱpeptideȱMMPȬ2ȱinhibitorȱAPPȬIPȱbindsȱbyȱchelationȱofȱtheȱ activeȱsiteȱZnȬion.ȱTheȱbindingȱofȱM21ȱcouldȱbeȱimprovedȱbyȱintroducingȱaȱZnȱchelatorȱwhichȱisȱstrongerȱ thanȱtheȱcarboxylate.ȱ(B)ȱInȱtheȱaffinityȱmaturationȱstrategyȱofȱM21ȱlibrariesȱofȱdifferentȱsizesȱwereȱused.ȱTheȱ bestȱpeptideȱwasȱidentifiedȱinȱtheȱ4x6ȱlibrary.ȱ(C)ȱHydroxamateȱmodificationȱhadȱaȱstrongȱeffectȱonȱMMPȬ2ȱ binding,ȱbutȱdidȱnotȱinfluenceȱMMPȬ9ȱbindingȱtoȱtheȱsameȱextent.ȱKiȱvaluesȱofȱtheȱdifferentȱpeptideȱforȱtheȱ twoȱenzymesȱareȱindicated.ȱ(D)ȱSDSȬPAGEȱshowingȱthatȱM114hyȱinhibitsȱtheȱautodegradationȱofȱMMPȬ2.ȱ Theȱbandȱatȱ32ȱkDaȱisȱaȱknownȱdegradationȱproductȱformedȱuponȱautodegradationȱofȱMMPȬ2ȱ(E)ȱM114hyȱ inhibitsȱautodegradationȱofȱMMPȬ2ȱwhenȱappliedȱtoȱaȱcellȱcultureȱofȱHT1080ȱfibrosarcomaȱcells.ȱMMPȬ2ȱ speciesȱwereȱdetectedȱinȱtheȱcellȱmediaȱbyȱwesternȱblot.ȱȱ

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Inȱanȱattemptȱtoȱfurtherȱimproveȱtheȱbindingȱaffinityȱofȱtheȱpeptideȱandȱatȱtheȱsameȱtimeȱ toȱoptimizeȱtheȱringȱsizes,ȱweȱperformedȱanȱadditionalȱroundȱofȱphageȱdisplayȱusingȱ affinityȱmaturationȱlibraries.ȱSevenȱdifferentȱlibraryȱformatsȱwereȱconstructedȱinȱwhichȱ theȱsizeȱofȱtheȱtwoȱringsȱwasȱvaried.ȱTheȱbindingȱmotifȱDAIWȱinȱtheȱsecondȱringȱwasȱ keptȱconstant.ȱObviously,ȱtheȱpeptidesȱonȱphageȱdidȱnotȱcontainȱtheȱhydroxamateȱgroup.ȱ

Afterȱsequencingȱofȱtheȱclones,ȱitȱseemedȱthatȱtheȱACX3CX6CGȱlibraryȱwasȱtheȱpreferredȱ formatȱ(Figureȱ30B).ȱWeȱthereforeȱusedȱthisȱformatȱwhenȱoptimizingȱresiduesȱinȱtheȱsecȬ ondȱring.ȱTheȱmotifȱQARȱwasȱchosenȱtoȱbeȱkeptȱconstantȱinȱtheȱfirstȱringȱasȱitȱwasȱtheȱ mostȱcommonȱmotifȱinȱtheȱfirstȱringȱofȱtheȱ3x6ȱlibraryȱandȱmoreoverȱitȱwasȱtheȱonlyȱmotifȱ foundȱinȱtheȱsameȱpositionȱinȱmultipleȱlibraries.ȱTheȱarginineȱresidueȱbeforeȱtheȱmiddleȱ cysteineȱwasȱtheȱmostȱconservedȱresidueȱofȱtheȱfirstȱringȱinȱtheȱ3x6ȱandȱ4x6ȱlibraries,ȱtheȱ twoȱmostȱcommonȱformats.ȱWeȱthenȱsynthesizedȱpeptidesȱhavingȱtheȱfiveȱmostȱabunȬ dantȱresiduesȱinȱpositionȱ5ȱofȱtheȱsecondȱringȱandȱglycineȱinȱpositionȱ6.ȱTheȱresultsȱshowȱ thatȱGluȱisȱtheȱpreferredȱresidueȱinȱpositionȱ5ȱofȱtheȱsecondȱringȱ(FigureȱS5.1).ȱForȱtheȱ firstȱringȱoptimizationȱweȱchooseȱ10ȱdifferentȱmotifsȱpresentȱinȱtheȱisolatedȱpeptidesȱfromȱ theȱ2x6,ȱ3x6ȱandȱ4x6ȱformats.ȱTheȱmotifsȱwereȱchosenȱbasedȱonȱaminoȱacidȱabundance.ȱ Predominantlyȱmotifsȱhavingȱanȱarginineȱinȱtheȱpositionȱbeforeȱtheȱmiddleȱcysteineȱwereȱ selectedȱ andȱ comparedȱ toȱ theȱ QARȱ motif.ȱ Weȱ synthesizedȱ peptidesȱ withoutȱ hydroxȬ amateȱgroupsȱasȱthisȱwasȱtechnicallyȱeasier.ȱSeveralȱofȱtheȱselectedȱmotifsȱhadȱsimilarȱ affinities.ȱWeȱfoundȱthatȱRYQRȱwasȱtheȱpreferredȱmotifȱofȱtheȱfirstȱringȱ(FigureȱS5.1).ȱTheȱ affinityȱmaturedȱpeptideȱM114ȱofȱtheȱformatȱACX4CX6CG,ȱinhibitedȱMMPȬ2ȱwithȱaȱKiȱ valueȱofȱ89ȱ±ȱ4ȱnM.ȱȱȱȱ

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Theȱstrongȱchelationȱofȱtheȱactiveȱsiteȱzincȱionȱhasȱlargelyȱcontributedȱtoȱtheȱoffȱtargetȱ bindingȱ ofȱ previouslyȱ developedȱ MMPȱ inhibitors.ȱ Hereȱ weȱ wantedȱ toȱ determineȱ theȱ specificityȱofȱbicyclicȱpeptideȱMMPȬ2ȱinhibitorsȱcontainingȱaȱhydroxamicȱacidȱmoietyȱforȱ zincȱchelationȱandȱaffinityȱenhancement.ȱWhenȱincorporatedȱinȱtheȱoriginalȱleadȱpeptideȱ

M21ȱ identifiedȱ fromȱ phageȱ displayȱ usingȱ theȱ ACX6CX6CGȱ library,ȱ theȱ hydroxamateȱ modificationȱincreasedȱbindingȱaffinityȱ29ȬfoldȱtoȱMMPȬ2ȱ(Figureȱ30C).ȱTheȱKiȱvalueȱforȱ MMPȬ9ȱwasȱofȱmoreȱthanȱ2ȱordersȱofȱmagnitudeȱlarger.ȱForȱtheȱaffinityȱmaturedȱpeptideȱ M114,ȱwhichȱhadȱaȱ45ȬfoldȱhigherȱaffinityȱforȱMMPȬ2ȱthanȱM21,ȱtheȱhydroxamicȱacidȱ increasedȱtheȱMMPȬ2ȱbindingȱaffinityȱbyȱaȱfactorȱ12.ȱInȱtheȱcaseȱofȱMMPȬ9ȱtheȱbindingȱ wasȱincreasedȱ7ȬfoldȱforȱM114hyȱasȱcomparedȱtoȱtheȱpeptideȱwithoutȱhydroxamicȱacid.ȱ TheȱselectivityȱofȱM114hyȱforȱMMPȬ2ȱoverȱMMPȬ9ȱwasȱ1200Ȭfold.ȱTheseȱresultsȱindicateȱ thatȱbothȱtheȱaffinityȱmaturationȱasȱwellȱasȱhydroxamateȱmodificationȱincreaseȱtheȱaffinȬ ityȱofȱtheȱpeptideȱforȱbothȱenzymes.ȱHowever,ȱtheȱeffectȱwasȱmoreȱpronouncedȱforȱMMPȬ 2,ȱthereforeȱultimatelyȱresultingȱinȱaȱhigherȱselectivity

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WeȱnextȱevaluatedȱtheȱabilityȱofȱM114hyȱtoȱinhibitȱMMPȬ2ȱinȱaȱsituationȱmoreȱphysiologȬ icallyȱrelevantȱthanȱanȱenzymaticȱassayȱusingȱanȱartificialȱsubstrate.ȱPreviousȱstudiesȱ haveȱshownȱthatȱMMPȬ2ȱisȱautoactivatedȱandȱautocatalyticallyȱcleavedȱatȱseveralȱsitesȱ (311,ȱ312).ȱTheseȱprocessesȱareȱlikelyȱofȱimportanceȱinȱvivo.ȱHereȱweȱincubatedȱtheȱactiveȱ 62ȱkDaȱactiveȱenzymeȱtogetherȱwithȱdifferentȱconcentrationsȱofȱtheȱinhibitor.ȱTheȱresultsȱ showȱthatȱM114hyȱdoseȬdependentlyȱinhibitsȱtheȱautoproteolysisȱofȱMMPȬ2ȱ(Figureȱ30D).ȱ Forȱallȱinhibitorȱconcentrations,ȱaȱmajorityȱofȱtheȱactiveȱ62ȱkDaȱenzymeȱremains.ȱWithoutȱ inhibitorȱtheȱenzymeȱisȱdegradedȱtoȱtheȱ32ȱkDaȱfragmentȱobservedȱbyȱHowardȱetȱal.ȱ (311).ȱThisȱbandȱisȱalsoȱdetectedȱforȱtheȱlowerȱinhibitorȱconcentrationsȱwhereasȱforȱtheȱ highestȱconcentrationȱtheȱonlyȱdegradationȱproductȱfoundȱisȱtheȱ42ȱkDaȱfragment,ȱwhichȱ isȱaȱprecursorȱofȱtheȱ32ȱkDaȱspecies.ȱ

WeȱthenȱcomparedȱtheȱdegradationȱofȱMMPȬ2ȱinȱHT1080ȱhumanȱfibrosarcomaȱcellȱculȬ tureȱmediaȱfollowingȱselectiveȱMMPȬ2ȱinhibitionȱbyȱM114hy,ȱbroadȱspectrumȱMMPȱinhiȬ bitionȱbyȱbatimastatȱorȱnoȱinhibition.ȱWithoutȱinhibitorȱorȱwithȱonlyȱM114hyȱaȱbroadȱbandȱ wasȱdetectedȱaroundȱ66ȱkDaȱ(Figureȱ30E).ȱThisȱbandȱisȱlikelyȱconsistingȱofȱtwoȱspecies,ȱ 72ȱkDaȱproȬenzymeȱandȱ62ȱkDaȱactiveȱenzyme,ȱaȱsimilarȱresultȱwasȱdetectedȱbyȱBergȬ mannȱetȱal.ȱ(312)ȱWithȱbroadȱspectrumȱinhibitionȱonlyȱtheȱupperȱpartȱofȱthisȱbandȱisȱ observed,ȱhenceȱnoȱactiveȱMMPȬ2ȱisȱformed.ȱThisȱwouldȱmeanȱthatȱenzymesȱotherȱthanȱ MMPȬ2ȱcontributeȱinȱtheȱactivationȱofȱtheȱenzyme.ȱItȱhasȱbeenȱpreviouslyȱreportedȱthatȱ MMPȬ2ȱisȱactivatedȱbyȱMMPȬ14,ȱwhichȱisȱinhibitedȱbyȱbatimastat,ȱhenceȱtheȱabsenceȱofȱ activeȱ62ȱkDaȱMMPȬ2ȱinȱthisȱsettingȱisȱexpected.ȱNoȱdegradationȱproductsȱareȱpresentȱ forȱtheȱbroadȱspectrumȱinhibition,ȱsuggestingȱtheȱblockageȱofȱallȱMMPȬ2ȱdegradingȱenȬ zymes.ȱAȱdifferenceȱinȱdegradationȱproductsȱisȱobservedȱwhenȱMMPȬ2ȱisȱinhibitedȱasȱ comparedȱtoȱnoȱinhibition.ȱThisȱimpliesȱthatȱotherȱMMPsȱdegradeȱMMPȬ2ȱbutȱaȱcertainȱ degradationȱproductȱ(~26kDa)ȱcanȱonlyȱbeȱformedȱbyȱMMPȬ2ȱautoproteolysis.ȱAnȱuniȬ dentifiedȱbandȱatȱ~46ȱkDaȱisȱlikelyȱaȱdegradationȱproductȱformedȱbyȱaȱspeciesȱnotȱinhibȬ itedȱbyȱbatimastat.

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Asȱdescribedȱinȱtheȱintroduction,ȱaȱpotentȱandȱselectiveȱlinearȱpeptideȱMMPȬ2ȱinhibitor,ȱ APPȬIP,ȱhasȱbeenȱpreviouslyȱreported.ȱHowever,ȱitsȱuseȱasȱaȱtherapeuticȱorȱresearchȱtoolȱ mightȱbeȱlimitedȱdueȱtoȱpoorȱproteolyticȱstability,ȱwhichȱisȱoftenȱtheȱcaseȱforȱlinearȱpepȬ tides.ȱInȱcontrast,ȱbicyclicȱpeptidesȱtypicallyȱshowȱsuperiorȱproteolyticȱstabilityȱasȱcomȬ paredȱtoȱtheirȱlinearȱcounterparts.ȱWeȱcomparedȱtheȱproteolyticȱstabilityȱofȱtheȱbicyclicȱ peptideȱM114hyȱtoȱAPPȬIP.ȱTheȱproteolyticȱstabilityȱwasȱassessedȱbyȱincubatingȱtheȱpepȬ tidesȱinȱmouseȱplasmaȱatȱ37°Cȱandȱquantifyingȱtheȱremainingȱpeptideȱbyȱanalyticalȱȱ

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Figureȱ31.ȱ(A)ȱTheȱpeptidesȱwereȱincubatedȱinȱmouseȱplasmaȱandȱremainingȱpeptideȱquantifiedȱatȱtheȱindiȬ catedȱtimepoints.ȱTheȱstabilityȱanalysisȱshowedȱthatȱtheȱbicyclicȱM114hyȱwasȱmoreȱstableȱthanȱtheȱlinearȱAPPȬ IP.ȱ(B)ȱMassȱspectralȱanalysisȱshowedȱthatȱM114ȱwasȱcleavedȱatȱtheȱNȬterminalȱalanineȱandȱatȱoneȱofȱtheȱ arginines.ȱ(C)ȱAȱseriesȱofȱpeptidesȱwasȱsynthesizedȱinȱwhichȱresiduesȱnonȬcrucialȱforȱbindingȱwereȱsubstiȬ tutedȱwithȱDȬalanine.ȱTheȱbindingȱaffinitesȱareȱindicated.ȱ(D)ȱReplacingȱtheȱArg6ȱofȱtheȱfirstȱloopȱincreasedȱ theȱstabilityȱwithoutȱcompromisingȱaffinity.ȱ(E)ȱAȱseriesȱofȱDȬaminoȱacidȱsubstitutedȱpeptidesȱinȱpositionȱ Gly13ȱwereȱsynthesizedȱsinceȱitȱwasȱfoundȱthatȱDȬAlaȱimprovedȱtheȱbindingȱaffinityȱofȱM114ȱwhenȱinȱthisȱ

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position.ȱ(F)ȱPeptideȱM238hyȱcombinesȱArg6ÆDȬAlaȱandȱGly13ÆDȬTyrȱmodificationsȱandȱhadȱaȱfourȬfoldȱ improvedȱaffinityȱoverȱM114hy.ȱ(G)ȱTheȱstabilityȱofȱM238hyȱwasȱassessedȱbyȱincubatingȱtheȱpeptideȱinȱplasmaȱ andȱquantifyingȱtheȱremainingȱfunctionalȱstrengthȱofȱtheȱinhibitorȱatȱtheȱindicatedȱtimeȱpoints.ȱTheȱimproȬ vedȱinhibitorȱwasȱfoundȱtoȱhaveȱaȱfunctionalȱhalfȬlifeȱofȱ4.4ȱh.ȱInȱcontrast,ȱnoȱremainingȱfunctionalityȱwasȱ detectedȱforȱM114hyȱafterȱ1ȱh.ȱȱ

HPLCȱ areaȱ underȱ curve.ȱ Theȱ experimentȱ showedȱ thatȱ APPȬIPȱ indeedȱ isȱ rapidlyȱ deȬ gradedȱinȱplasma,ȱhavingȱaȱhalfȬlifeȱofȱaroundȱ15ȱminȱ(Figureȱ31A).ȱTheȱbicyclicȱpeptideȱ M114hyȱhadȱaȱsubstantiallyȱlongerȱhalfȬlifeȱofȱ3.00ȱ±ȱ0.04ȱh.ȱThisȱresultsȱdemonstrateȱthatȱ theȱbicyclicȱstructureȱofȱM114hyȱimprovesȱitsȱstabilityȱbyȱmakingȱitȱlessȱaccessibleȱtoȱproȬ teasesȱpresentȱinȱplasma.ȱ

6WDELOLW\LPSURYHPHQWRI003LQKLELWRU

EvenȱthoughȱM114hyȱhadȱaȱrelativelyȱlongȱproteolyticȱhalfȬlifeȱofȱ3ȱh,ȱanȱincreasedȱstabilȬ ityȱwouldȱbeȱdesirableȱforȱitsȱpotentialȱuseȱasȱaȱresearchȱtoolȱorȱtherapeutic.ȱInȱthisȱworkȱ weȱthereforeȱaimedȱtoȱimproveȱtheȱproteolyticȱstabilityȱofȱM114hyȱfurther.ȱWeȱchooseȱanȱ approachȱinȱwhichȱweȱidentifiedȱtheȱproteolyticȱcleavageȱsitesȱinȱtheȱpeptideȱsequenceȱ andȱreplacedȱkeyȱaminoȱacidsȱbyȱDȬalanines.ȱWeȱhypothesizedȱthatȱtheȱincorporationȱofȱ DȬaminoȱacidsȱwouldȱmakeȱtheȱpeptideȱlessȱrecognizableȱbyȱproteases.ȱByȱmassȱspectralȱ analysisȱofȱtheȱearlyȱdegradationȱproducts,ȱmolecularȱmassesȱcorrespondingȱtoȱtheȱpepȬ tideȱwithoutȱNȬterminalȱalanineȱasȱwellȱasȱpeptideȱwithȱoneȱringȱopenedȱandȱmissingȱ oneȱarginineȱresidueȱhadȱbeenȱobserved.ȱItȱwasȱhoweverȱnotȱclearȱwhichȱarginineȱhadȱ beenȱcleavedȱ(Figureȱ31B).ȱȱ

Weȱnextȱwantedȱtoȱseeȱhowȱmodifyingȱtheseȱpotentialȱcleavageȱsitesȱwouldȱaffectȱtheȱ targetȱaffinityȱofȱtheȱpeptide.ȱAȱrequirementȱofȱtheȱstabilityȱimprovingȱpeptideȱmodifiȬ cationsȱwasȱthatȱtheyȱshouldȱhaveȱonlyȱminimalȱorȱnoȱimpactȱonȱtargetȱaffinity.ȱPeptidesȱ containingȱonlyȱsingleȱmutationsȱwereȱcomparedȱtoȱtheȱleadȱpeptideȱM114.ȱInȱtheseȱseȬ ries,ȱallȱpeptidesȱwereȱsynthesizedȱwithoutȱhydroxamateȱmodifications,ȱtoȱallowȱaȱrapidȱ screeningȱapproach.ȱItȱwasȱassumedȱthatȱtheȱpropertiesȱofȱtheseȱpeptidesȱwouldȱlaterȱ translateȱintoȱhydroxamateȱmodifiedȱpeptides.ȱInȱaȱfirstȱseries,ȱtheȱCȬterminalȱandȱNȬ terminalȱresiduesȱwereȱeitherȱremovedȱorȱreplacedȱbyȱDȬAlaȱ(Figureȱ31C).ȱTheseȱmodiȬ ficationsȱhadȱnoȱsubstantialȱeffectȱonȱtheȱaffinityȱofȱtheȱpeptides,ȱandȱwouldȱhenceȱbeȱ allowedȱforȱpotentialȱstabilityȱimprovement.ȱHowever,ȱreplacementȱorȱremovalȱofȱtheȱCȬ terminalȱGlyȱresidueȱslightlyȱdecreasedȱtheȱaffinity.ȱȱ

Inȱtheȱnextȱseriesȱweȱscreenedȱpeptidesȱwhereȱallȱaminoȱacidsȱinsideȱtheȱtwoȱrings,ȱexceptȱ thoseȱofȱtheȱimportantȱAspȬAlaȬIleȱbindingȱmotif,ȱwereȱreplacedȱbyȱDȬAla.ȱTheȱaffinityȱ wasȱsubstantiallyȱdecreasedȱforȱallȱpeptidesȱwithȱtheȱexceptionȱofȱM208,ȱwhereȱArg6ȱinȱ theȱfirstȱringȱwasȱreplacedȱandȱM211ȱwhereȱGly13ȱinȱtheȱsecondȱringȱwasȱreplacedȱ(FigȬ ureȱ31C).ȱBothȱpositionsȱwouldȱthereforeȱallowȱDȬaminoȱacidȱincorporation.ȱInȱorderȱtoȱ identifyȱtheȱpreviouslyȱobservedȱarginineȱcleavageȱsite,ȱweȱincubatedȱinȱmouseȱplasmaȱ

115 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH peptidesȱM205ȱandȱM208ȱwhereȱtheȱArg3ȱandȱArg6ȱrespectivelyȱwereȱindividuallyȱreȬ placedȱbyȱDȬAla,ȱandȱanalysedȱtheȱdegradationȱproductsȱbyȱMS.ȱEndopeptidicȱcleavageȱ wasȱfoundȱonlyȱforȱM205ȱbutȱnotȱforȱM208,ȱindicatingȱthatȱonlyȱArg6ȱwasȱproneȱtoȱproȬ teolysisȱ(FigureȱS5.2).ȱWhenȱfurtherȱanalysingȱtheȱdegradationȱproductsȱofȱM114ȱandȱ M114hy,ȱtheseȱresultsȱwereȱconfirmedȱbyȱtheȱdetectionȱofȱmolecularȱspeciesȱwhichȱcouldȱ onlyȱbeȱformedȱbyȱcleavageȱofȱtheȱArg6ȱ(FigureȱS5.3ȱandȱS5.4).ȱWeȱthenȱsynthesizedȱtheȱ peptideȱM208hy,ȱcontainingȱtheȱArg6ÆDȬAlaȱmodificationȱasȱwellȱasȱtheȱhydroxamicȱacidȱ moietyȱandȱtestedȱitsȱproteolyticȱstability.ȱTheȱreplacementȱofȱArg6ȱbyȱDȬAlaȱinȱtheȱfirstȱ loopȱincreasedȱtheȱproteolyticȱstabilityȱofȱtheȱpeptideȱ2.4Ȭfoldȱtoȱ7.2ȱ±ȱ0.6ȱhȱ(Figureȱ31Aȱ andȱD).ȱNoȱdegradationȱproductsȱexceptȱlossȱofȱhydroxamateȱand/orȱNȬterminalȱargiȬ nineȱwereȱobservedȱ(FigureȱS5.5).ȱȱ

Finally,ȱweȱsynthesizedȱaȱsetȱofȱpeptidesȱwithȱArg3ȱandȱArg6ȱreplacedȱbyȱAlaȱandȱDȬAlaȱ respectivelyȱandȱwithȱandȱwithoutȱCȬȱandȱNȬterminalȱresidues.ȱByȱremovingȱtheȱNȬterȬ minalȱcleavageȱsiteȱasȱwellȱasȱreplacingȱbothȱarginines,ȱwhichȱareȱknownȱtoȱbeȱcleavedȱ byȱtrypsin,ȱwithȱaminoȱacidsȱallowedȱinȱtermsȱofȱaffinityȱweȱhopedȱtoȱfurtherȱimproveȱ theȱstability.ȱHowever,ȱwhenȱcomparingȱtheseȱpeptidesȱtoȱeachȱotherȱandȱtoȱM208ȱandȱ M208hyȱtheȱstabilityȱwasȱfoundȱtoȱdecreaseȱwhenȱendsȱwereȱremovedȱorȱArg3ȱreplacedȱ byȱalanineȱ(FigureȱS5.6).ȱMoreoverȱweȱfoundȱthatȱtheȱhydroxamicȱacidȱmoietyȱdecreasesȱ theȱ halfȬlifeȱ ofȱ theȱ peptides.ȱ Basedȱ onȱ theseȱ resultsȱ weȱ choseȱ toȱ incorporateȱ onlyȱ theȱ Arg6ÆDȬAlaȱmodificationȱforȱstabilityȱimprovement.ȱ

$IILQLW\LPSURYHPHQWRI003LQKLELWRU

Afterȱaffinityȱmaturationȱandȱhydroxamicȱacidȱintroduction,ȱtheȱaffinityȱofȱtheȱbicyclicȱ peptideȱMMPȬ2ȱinhibitorȱincreasedȱtoȱreachȱaȱKiȱvalueȱbelowȱ10ȱnM.ȱHowever,ȱinȱorderȱ toȱbeȱusedȱasȱaȱresearchȱtoolȱorȱtherapeuticȱandȱtoȱfullyȱblockȱMMPȬ2,ȱevenȱstrongerȱ targetȱbindingȱisȱdesirable.ȱInȱparallelȱwithȱtheȱstabilityȱimprovementȱweȱthereforeȱsetȱ outȱtoȱfurtherȱimproveȱtheȱaffinity.ȱToȱidentifyȱwhichȱaminoȱacidsȱofȱtheȱaffinityȱmaturedȱ peptideȱwereȱimportantȱforȱtargetȱbindingȱweȱperformedȱanȱadditionalȱalanineȱscan,ȱbutȱ excludingȱtheȱpreviouslyȱidentifiedȱimportantȱmotifȱofȱtheȱsecondȱringȱ(DAI).ȱTheȱresultsȱ showȱthatȱTrp11ȱbutȱalsoȱTyr4ȱareȱimportantȱforȱtargetȱbindingȱ(FigureȱS5.7A).ȱWithȱtheȱ hypothesisȱthatȱtheȱinteractionsȱformedȱbyȱtheseȱresiduesȱcouldȱbeȱimproved,ȱweȱsubstiȬ tutedȱtheȱTrp11ȱwithȱseveralȱunnaturalȱanaloguesȱ(dataȱnotȱshown)ȱandȱtheȱTyr4ȱwithȱaȱ panelȱofȱnaturalȱaminoȱacidsȱ(FigureȱS5.7B).ȱUnfortunatelyȱnoneȱofȱtheȱsubstitutionsȱinȬ creasedȱtheȱtargetȱaffinity.ȱTyr4ȱcouldȱhoweverȱbeȱsubstitutedȱwithȱPheȱwithoutȱlosingȱ affinity.ȱȱ

InȱtheȱDȬalanineȱscanȱdescribedȱaboveȱitȱwasȱobservedȱthatȱsubstitutingȱGly13ȱinȱtheȱsecȬ ondȱringȱwithȱDȬAlaȱslightlyȱenhancedȱtheȱbindingȱaffinityȱofȱM114ȱ(Figureȱ31C).ȱThisȱ resultȱisȱinȱlineȱwithȱanȱobservationȱinȱaȱpreviousȱstudyȱwhereȱChenȱetȱal.ȱdescribeȱthatȱ glycineȱresiduesȱinȱbicyclicȱpeptideȱligandsȱcanȱsometimesȱbeȱreplacedȱbyȱDȬaminoȱacidsȱ

116 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH forȱimprovedȱtargetȱaffinityȱ(191).ȱIfȱtheȱIdihedralȱangleȱofȱtheȱglycineȱresidueȱisȱpositive,ȱ incorporationȱofȱaȱDȬaminoȱacidȱresidueȱcanȱbeȱfavourableȱatȱthatȱpositionȱsinceȱDȬaminoȱ acidsȱadoptȱpositiveȱIdihedralȱangles.ȱIncludingȱaȱDȬaminoȱacidȱcouldȱpossiblyȱincreaseȱ affinityȱbyȱformingȱnewȱinteractionsȱbetweenȱtheȱsideȱchainȱandȱtheȱtarget.ȱFurthermore,ȱ theȱconformationalȱflexibilityȱofȱtheȱunboundȱpeptideȱcouldȱpotentiallyȱbeȱdecreased,ȱ resultingȱinȱaȱlowerȱentropicȱpenaltyȱuponȱbinding.ȱSinceȱGly13ȱbenefitedȱfromȱtheȱinȬ troductionȱofȱaȱDȬAla,ȱitȱisȱlikelyȱthatȱthisȱresidueȱadoptsȱaȱpositiveȱIdihedralȱangle.ȱWeȱ thereforeȱdecidedȱtoȱscreenȱaȱpanelȱofȱDȬaminoȱacidsȱinȱtheȱGly13ȱposition.ȱSeveralȱofȱtheȱ DȬaminoȱacidsȱimprovedȱtheȱaffinity,ȱwithȱtheȱbestȱonesȱbeingȱDȬTyrȱandȱDȬPhe,ȱwhichȱ improvedȱtheȱaffinityȱ2Ȭfoldȱ(Figureȱ31E).ȱSubsequentlyȱweȱinvestigatedȱseveralȱcombiȬ nationsȱofȱtheȱaboveȱidentifiedȱmodificationsȱinȱaȱseriesȱofȱ4ȱpeptidesȱ(includingȱM208hy,).ȱ TheȱmodificationsȱincludedȱTyr4ÆPhe,ȱArg6ÆDȬAla,ȱandȱGly13ÆDȬTyr/DȬPhe.ȱAllȱpepȬ tidesȱinȱthisȱsetȱcontainedȱtheȱhydroxamicȱacidȱmoiety.ȱByȱanalysingȱthisȱgroupȱofȱpepȬ tidesȱweȱfoundȱthatȱTyrȱwasȱtheȱpreferredȱresidueȱinȱpositionȱ4ȱandȱDȬPheȱwasȱslightlyȱ preferredȱinȱpositionȱ13.ȱTheȱbestȱpeptide,ȱM238hyȱwasȱbindingȱMMPȬ2ȱwithȱaȱKiȱofȱ1.9ȱ±ȱ 0.5ȱnMȱ(Figureȱ31F).ȱȱ

FinallyȱweȱsetȱoutȱtoȱvalidateȱtheȱstabilityȱofȱtheȱpeptideȱM238hy,ȱhavingȱallȱthreeȱmodiȬ ficationsȱinȱplaceȱ(Arg6ÆDȬAla,ȱGly13ÆDȬPheȱandȱhydroaxamicȱacid).ȱInȱorderȱtoȱgetȱ moreȱrelevantȱvalues,ȱweȱdeterminedȱtheȱfunctionalȱhalfȬlifeȱofȱtheȱinhibitorsȱusingȱaȱnewȱ methodȱrecentlyȱdevelopedȱinȱourȱlaboratory.ȱTheȱmethodȱisȱbasedȱonȱenzymaticȱactivityȱ assaysȱfollowingȱtheȱplasmaȱincubationȱratherȱthanȱquantificationȱbyȱanalyticalȱHPLC.ȱ Byȱmeasuringȱtheȱremainingȱfunctionalȱstrengthȱofȱtheȱinhibitorȱbothȱtheȱactivityȱofȱintactȱ inhibitorȱasȱwellȱasȱofȱpotentiallyȱactiveȱmetabolitesȱisȱdetermined.ȱWhenȱanalysedȱinȱ thisȱassayȱM238hyȱshowsȱaȱfunctionalȱhalfȬlifeȱofȱ4.4ȱ±ȱ0.4ȱhȱ(Figureȱ31G).ȱForȱtheȱleadȱ peptideȱM114hyȱnoȱremainingȱfunctionalityȱcouldȱbeȱdetectedȱatȱtheȱearliestȱsampleȱtimeȱ pointȱ(1ȱh).ȱȱȱȱȱ

003VSHFLILFLW\SURILOLQJRI0K\

NextȱweȱsetȱoutȱtoȱdoȱaȱselectivityȱscreenȱagainstȱallȱotherȱMMPsȱwhichȱwouldȱlikelyȱbeȱ inhibitedȱbyȱM238hy.ȱTheȱgoalȱwasȱtoȱcompareȱimportantȱresiduesȱofȱtheȱenzymesȱinȱcloseȱ proximityȱtoȱtheȱbindingȱsiteȱ(Figureȱ32A).ȱToȱidentifyȱtheȱenzymesȱwhichȱcouldȱlikelyȱ beȱinhibitedȱbyȱaȱMMPȬ2ȱinhibitor,ȱweȱfirstȱperformedȱaȱBLASTȱscreenȱofȱtheȱcatalyticȱ domainȱofȱMMPȬ2ȱ(TableȱS5.1).ȱForȱtheȱsimilarityȱsearchȱtheȱaminoȱacidȱsequenceȱofȱtheȱ twoȱpartsȱofȱtheȱMMPȬ2ȱcatalyticȱdomainȱasȱaȱsingleȱsequence,ȱwithȱfibronectinȱdomainsȱ removed,ȱwasȱusedȱasȱreportedȱbyȱHashimotoȱ(306).ȱTheȱBLOSUM62ȱscoringȱmatrixȱwasȱ used.ȱForȱtheȱspecificityȱprofilingȱaȱkitȱofȱ10ȱMMPsȱwasȱanalysed.ȱTheȱkitȱcontainedȱmostȱ ofȱtheȱMMPsȱhavingȱtheȱhighestȱsimilarityȱtoȱtheȱMMPȬ2ȱcatalyticȱdomain.ȱAȱmajorityȱofȱ theȱMMPsȱscreened,ȱwereȱnotȱinhibitedȱbyȱM238hyȱandȱhadȱKiȱvaluesȱexceedingȱ50ȱPMȱ

(Figureȱ32B).ȱWithȱtheȱexceptionȱofȱMMPȬ2,ȱonlyȱtwoȱproteasesȱwereȱinhibitedȱwithȱKiȱ

117 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH

valuesȱbelowȱ1ȱPMȱ(MMPȬ3ȱandȱ12,ȱKiȱvaluesȱofȱ4.1ȱ±ȱ1.1ȱandȱ600ȱ±ȱ100ȱnMȱrespectively).ȱ TheȱstrongȱaffinityȱforȱMMPȬ3ȱwasȱsurprisingȱgivenȱthatȱmostȱpreviouslyȱreportedȱMMPȬ 2ȱinhibitorsȱonlyȱlackedȱselectivityȱoverȱMMPȬ9.ȱThisȱdataȱimplicatesȱthatȱM238hyȱisȱformȬ ingȱ differentȱ interactionȱ withȱ theȱ targetȱ asȱ comparedȱ toȱ mostȱ otherȱ MMPȱ inhibitors.ȱ HenceȱpotentiallyȱalsoȱotherȱMMPsȱcouldȱbeȱinhibited.ȱȱ

Toȱgainȱfurtherȱinsightȱinȱtheȱtargetȱinteractionȱmechanismsȱweȱidentifiedȱkeyȱresiduesȱ withinȱtheȱenzymesȱimportantȱforȱtheȱbindingȱandȱsubsequentlyȱalignedȱtheȱcatalyticȱ domainsȱofȱtheȱMMPsȱandȱcomparedȱtheseȱresidues.ȱTheȱinteractionȱformingȱresiduesȱ wereȱthoughtȱtoȱlieȱwithinȱcloseȱproximityȱtoȱtheȱbindingȱmotifȱDAIWȱofȱtheȱinhibitor.ȱ GivenȱtheȱfactȱthatȱM238hyȱandȱAPPȬIPȱshareȱthisȱconsensusȱmotifȱandȱareȱbothȱchelatingȱ toȱtheȱzincȱionȱthroughȱtheȱAspȱresidueȱtheyȱlikelyȱbindȱtoȱMMPȬ2ȱinȱaȱveryȱsimilarȱway.ȱ Weȱthereforeȱusedȱtheȱpublishedȱcrystalȱstructureȱ(PDBȱIDȱ3AYU)ȱtoȱidentifyȱkeyȱresiȬ duesȱofȱtheȱMMPȬ2ȱcatalyticȱdomain.ȱResiduesȱwithinȱ4Ȭ6ȱÅȱofȱtheȱDALMȱmotifȱinȱAPPȬ IPȱwereȱselectedȱ(Figȱ32AȱandȱB,ȱyellowȱandȱblueȱrespectively).ȱByȱcomparingȱtheseȱresȬ iduesȱofȱallȱMMPsȱinȱtheȱsequenceȱalignmentsȱandȱcrossȬanalysingȱtheȱaffinitiesȱofȱM238hyȱ forȱtheȱMMPsȱinȱtheȱkitȱimportantȱkeyȱresiduesȱforȱselectivityȱcouldȱbeȱidentified.ȱTheȱ mostȱ importantȱ residueȱ wasȱ identifiedȱ asȱ Ala87ȱ (Hashimotoȱ numbering)ȱ inȱ theȱ nonȬ primeȱsideȱofȱtheȱsubstrateȱbindingȱcleft.ȱOnlyȱenzymesȱcontainingȱanȱalanineȱorȱglycineȱ inȱthisȱpositionȱwereȱinhibitedȱbyȱM238hyȱ(MMPȬ2,ȱ3,ȱ7ȱandȱ12,ȱFigureȱ32B).ȱIfȱtheȱenzymeȱ containedȱanyȱlargerȱresidueȱinȱthisȱpositionȱitȱfullyȱabolishedȱbinding.ȱTheseȱresultsȱareȱ wellȱinȱlineȱwithȱaȱpreviouslyȱpublishedȱstudyȱinȱwhichȱtheȱselectivityȱofȱAPPȬIPȱforȱ MMPȬ2ȱoverȱMMPȬ9ȱwasȱstudiedȱ(305).ȱConsequently,ȱweȱtestedȱtheȱaffinityȱofȱM238hyȱ forȱMMPȬ20,ȱwhichȱisȱtheȱonlyȱotherȱMMP,ȱbesidesȱtheȱonesȱalreadyȱtested,ȱhavingȱanȱ alanineȱorȱglycineȱinȱthisȱposition.ȱIndeed,ȱMMPȬ20ȱwasȱalsoȱinhibitedȱwithȱaȱKiȱvalueȱofȱ 410ȱ±ȱ50ȱnM.ȱȱ

WhileȱAla87ȱisȱlikelyȱtheȱmostȱimportantȱresidueȱforȱselectivity,ȱalsoȱotherȱresiduesȱhaveȱ anȱimpact,ȱwhichȱisȱseenȱbyȱtheȱdifferentȱaffinitiesȱofȱM238hyȱforȱMMPsȱ2,ȱ3,ȱ7,ȱ12ȱandȱ20.ȱ SeveralȱstudiesȱindicateȱdifferencesȱinȱtheȱS1’ȱpocketȱasȱsubstrateȱselectivityȱdeterminantsȱ forȱMMPsȱ(313,ȱ314).ȱWeȱhypothesizeȱthatȱM238hyȱformsȱinteractionsȱinȱthisȱpocketȱviaȱ theȱTyr4ȱresidueȱofȱtheȱfirstȱring.ȱItȱisȱknownȱthatȱtheȱS1’ȱpocketȱfavoursȱhydrophobicȱ interactionsȱ(315),ȱasȱutilizedȱbyȱseveralȱsmallȱmoleculeȱinhibitors.ȱIndeedȱalsoȱtheȱTyr3ȱ residueȱofȱAPPȬIPȱisȱformingȱinteractionsȱinȱthisȱpocketȱ(306).ȱMoreoverȱourȱalanineȱscanȱ confirmedȱthatȱTyr4ȱofȱM238hyȱisȱinvolvedȱinȱtargetȱbinding.ȱȱMainlyȱtwoȱimportantȱresȬ iduesȱhaveȱbeenȱidentifiedȱinȱtheȱS1’ȱsiteȱregion,ȱLeu116ȱandȱIle141ȱ(316,ȱ317).ȱBothȱMMPȬ 20ȱandȱMMPȬ12ȱdifferedȱinȱthisȱregionȱasȱcomparedȱtoȱMMPȬ2.ȱMMPȬ20ȱhasȱaȱThrȱresiȬ dueȱinȱbothȱpositionsȱwhereasȱMMPȬ12ȱhasȱaȱThrȱresidueȱinȱ141.ȱItȱisȱlikelyȱthatȱtheseȱ differencesȱcontributeȱtoȱtheȱtwoȱordersȱofȱmagnitudeȱlossȱinȱaffinityȱforȱtheseȱenzymes.ȱ However,ȱalsoȱotherȱdifferencesȱseemȱtoȱhaveȱanȱinfluence,ȱsuchȱasȱLeu81ÆThrȱinȱMMPȬ 20ȱasȱwellȱasȱPhe4ÆMetȱandȱAla87ÆGlyȱinȱMMPȬ12,ȱwhichȱlieȱcloseȱtoȱeachȱotherȱonȱtheȱ

118 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH

nonȬprimeȱside.ȱM238hyȱhasȱevenȱmoreȱthanȱ10ȬfoldȱlowerȱaffinityȱforȱMMPȬ7,ȱwhichȱisȱ likelyȱalsoȱdueȱtoȱdifferencesȱinȱtheȱS1’ȱpocket.ȱMMPȬ7ȱhasȱaȱTyrȱinȱpositionȱ116ȱandȱhasȱ beenȱreportedȱtoȱhaveȱaȱparticularlyȱshallowȱS1’ȱpocketȱ(316).ȱ

ȱ Figureȱ32.ȱ(A)ȱTheȱresiduesȱD10,ȱA11ȱandȱI12ȱareȱmostȱimportantȱforȱbindingȱtoȱMMPȬ2ȱ(left).ȱResiduesȱofȱ MMPȬ2ȱwithinȱ4Ȭ6ȱÅȱofȱDALMȱinȱAPPȬIPȱwereȱselectedȱandȱareȱindicatedȱinȱyellowȱandȱblueȱrespectivelyȱ (right).ȱ(B)ȱInhibitionȱconstantsȱofȱM238hyȱareȱindicatedȱforȱaȱpanelȱofȱMMPsȱ(left).ȱTheȱkeyȱinteractionȱresiȬ duesȱofȱtheseȱenzymesȱwithȱtheȱpeptideȱareȱshownȱ(right).ȱ(C)ȱtheȱsingleȱringsȱofȱM238hyȱandȱtheȱlinearȱ peptideȱwereȱtestedȱforȱMMPȬ2ȱinhibition.ȱTheȱCȬterminalȱringȱwasȱfoundȱtoȱinhibitȱMMPȬ2ȱaroundȱ37Ȭ foldȱlessȱthanȱtheȱbicyclicȱpeptideȱ(left).ȱȱTheȱCȬterminalȱringȱofȱM238hyȱwasȱtestedȱforȱinhibitionȱofȱaȱpanelȱ ofȱMMPs.ȱȱ

119 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH

ThisȱhypothesisȱisȱfurtherȱsupportedȱbyȱHigashiȱetȱal.ȱwhoȱobservedȱthatȱdifferencesȱinȱ theȱS1’ȱpocketȱwereȱtheȱmainȱcauseȱofȱAPPȬIPȱselectivityȱforȱMMPȬ2ȱoverȱMMPȬ7ȱ(305).ȱ HigashiȱandȱcoȬworkersȱfurtherȱclaimȱthatȱtheȱGlnȱinȱpositionȱ93ȱofȱMMPȬ9ȱ(Gly93ȱinȱ MMPȬ2)ȱcontributesȱtoȱselectivity.ȱThisȱobservationȱcouldȱexplainȱwhyȱAPPȬIPȱdoesȱnotȱ inhibitȱMMPȬ3ȱwhereasȱM238hyȱdoes,ȱsinceȱMMPȬ3ȱhasȱanȱAsnȱinȱthisȱposition.ȱFromȱtheȱ crystalȱstructureȱ(PDBȱIDȱ3AYU)ȱitȱisȱevidentȱthatȱPro10ȱofȱAPPȬIPȱwouldȱclashȱwithȱanyȱ residueȱpointingȱtowardsȱtheȱsolventȱinȱpositionȱ93ȱofȱtheȱenzymes,ȱwhichȱindeedȱisȱtheȱ caseȱforȱMMPȬ3ȱandȱ9ȱ(PDBȱIDȱ2D1Oȱandȱ4H3X)ȱ(314,ȱ318).ȱWeȱspeculateȱthatȱM238hyȱ andȱAPPȬIPȱadoptȱsomewhatȱdifferentȱconformationsȱinȱtheȱfarȱpartȱofȱtheȱnonȬprimeȱ sideȱofȱtheȱsubstrateȱbindingȱcleft.ȱȱȱȱȱȱ

003LQKLELWLRQRIVLQJOHULQJVDQGOLQHDU0K\

ToȱunderstandȱwhichȱpartsȱofȱM238hyȱareȱinteractingȱwithȱtheȱMMPs,ȱweȱsynthesizedȱtheȱ peptide’sȱfirstȱandȱsecondȱringȱseparately,ȱcyclizedȱwithȱaȱbivalentȱlinkerȱviaȱtheȱcysteineȱ residues.ȱInȱaddition,ȱaȱlinearȱvariantȱofȱtheȱpeptideȱwasȱmade,ȱhavingȱreplacedȱtheȱcysȬ teinesȱbyȱalanines.ȱTheȱhydroxamicȱacidȱmoietyȱwasȱincludedȱinȱallȱconstructs.ȱAsȱexȬ pected,ȱtheȱNȬterminalȱringȱdidȱnotȱinhibitȱanyȱofȱtheȱenzymesȱ(Figureȱ32C).ȱThisȱemphaȬ sizesȱtheȱfactȱthatȱtheȱinteractionsȱinȱtheȱnonȬprimeȱsideȱofȱtheȱsubstrateȱbindingȱcleftȱareȱ crucialȱforȱinhibitorȱactivity,ȱdespiteȱtheȱpresenceȱofȱtheȱhydroxamateȱandȱS1’ȱbindingȱ residues.ȱȱ

TheȱCȬterminalȱringȱshowsȱinhibitoryȱactivityȱagainstȱMMPsȱ2,ȱ3,ȱ7ȱ(Kiȱaroundȱ35ȱPM),ȱ12ȱ andȱ20.ȱThisȱisȱexpectedȱsinceȱtheseȱproteasesȱhaveȱanȱalanineȱorȱglycineȱresidueȱinȱposiȬ tionȱ87.ȱIfȱcomparedȱtoȱtheȱbicyclicȱpeptide,ȱtheȱaffinityȱisȱalwaysȱimprovedȱbyȱintroducȬ ingȱtheȱfirstȱring,ȱbutȱtoȱvariousȱdegrees.ȱThisȱsuggestsȱthatȱtheȱfirstȱringȱisȱindeedȱmakingȱ interactionsȱwithȱtheȱenzymes,ȱprobablyȱinȱorȱaroundȱtheȱS1’ȱsite.ȱTheȱbestȱinteractionsȱ areȱmadeȱwithȱMMPȬ2ȱandȱ3ȱwhereȱintroductionȱofȱtheȱfirstȱringȱimprovesȱaffinityȱ47Ȭȱ andȱ26Ȭfoldȱrespectively.ȱTheȱeffectȱisȱlessȱpronouncedȱforȱMMPȬ12ȱandȱ20ȱ(11Ȭfoldȱandȱ 8Ȭfoldȱrespectively)ȱandȱevenȱlessȱforȱMMPȬ7ȱ(5Ȭfold).ȱThisȱisȱconsistentȱwithȱourȱpreviȬ ousȱhypothesisȱthatȱS1’ȱinteractionsȱareȱdecreasingȱforȱtheseȱenzymesȱandȱtherebyȱselecȬ tivityȱisȱachieved.ȱȱ

TheȱlinearȱpeptideȱwasȱinhibitingȱMMPȬ2ȱwithȱaȱKiȱofȱ2700ȱ±ȱ300ȱnM,ȱbutȱnoȱotherȱMMPȱ wasȱinhibited.ȱTheȱstrongȱlossȱinȱactivityȱindicatesȱtheȱimportanceȱofȱtheȱbicyclicȱconforȬ mationȱforȱtargetȱbinding.ȱItȱisȱlikelyȱthatȱtheȱpeptideȱisȱlockedȱinȱitsȱbindingȱconforȬ mationȱbyȱtheȱcyclizationȱscaffold.ȱȱ

6HOHFWLYLW\RI0K\

Withȱtheȱpurposeȱofȱmakingȱaȱmoreȱspecificȱinhibitorȱandȱtoȱgainȱfurtherȱinsightȱintoȱ whichȱresiduesȱofȱM238hyȱareȱimportantȱforȱspecificity,ȱweȱsetȱoutȱtoȱscreenȱaȱselectionȱofȱ theȱpreviouslyȱreportedȱpeptidesȱforȱspecificityȱoverȱMMPȬ3.ȱPeptidesȱofȱhighȱdiversityȱ

120 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH butȱwithȱminimalȱlossȱofȱaffinityȱforȱMMPȬ2ȱwereȱchosenȱforȱtheȱscreen,ȱallȱwithoutȱhyȬ droxamate.ȱInitially,ȱallȱpeptidesȱwereȱanalyzedȱforȱMMPȬ3ȱinhibitionȱusingȱtheȱpreviȬ ouslyȱusedȱenzyme,ȱhereȱtermedȱMMPȬ3Ȭaȱ(MMPȬ3ȱcatalyticȱdomain,ȱEnzoȱlifeȱsciences,ȱ kitȱcatȱ#ȱBMLȬAK308Ȭ0001).ȱFromȱtheȱinitialȱscreen,ȱwhichȱwasȱdoneȱonlyȱonceȱandȱwithȱ onlyȱthreeȱdataȱpointsȱforȱeachȱpeptide,ȱaȱsubsetȱofȱpeptidesȱwasȱselected,ȱhavingȱhigherȱ selectivityȱ thanȱ leadȱ peptideȱ M114ȱ(Figureȱ 33A,ȱ indicatedȱbyȱ arrows).ȱTheȱ largestȱinȬ creaseȱinȱselectivityȱwasȱobservedȱforȱpeptideȱM222,ȱwereȱS1’ȱbindingȱTyr4ȱresidueȱwasȱ modifiedȱintoȱGlu.ȱInȱaddition,ȱtheȱintroductionȱofȱDȬAlaȱeitherȱatȱtheȱNȬterminusȱorȱinȱ positionȱ6ȱincreasedȱselectivity.ȱSeveralȱofȱtheȱDȬaminoȱacidsȱinȱpositionȱ13ȱincreasedȱseȬ lectivity.ȱTheȱselectedȱpeptidesȱwereȱthenȱanalysedȱinȱduplicate,ȱbutȱusingȱaȱdifferentȱ enzymeȱconstruct,ȱhereȱtermedȱMMPȬ3Ȭbȱ(MMPȬ3ȱcatalyticȱdomain,ȱEnzoȱlifeȱsciences,ȱ catȱ#ȱALXȬ201Ȭ042ȬC005).ȱWithȱthisȱenzyme,ȱtheȱaffinityȱofȱtheȱpeptidesȱwasȱgenerallyȱ lower,ȱhenceȱselectivityȱincreased.ȱThisȱdifferenceȱinȱbindingȱaffinityȱisȱhardȱtoȱexplain,ȱ sinceȱaccordingȱtoȱtheȱsupplierȱtheȱonlyȱdifferencesȱofȱtheȱenzymesȱareȱthatȱMMPȬ3Ȭaȱ containsȱ residuesȱ Phe100ȬThr272ȱ withȱ aȱ CȬterminalȱ HisȬtagȱ forȱ purificationȱ whereasȱ MMPȬ3ȬbȱinsteadȱcontainsȱresiduesȱGly98ȬPro273.ȱSurprisinglyȱM222ȱshowedȱaȱlowerȱ selectivityȱthanȱM114ȱinȱthisȱassay.ȱInsteadȱM221,ȱ(Tyr4ÆArg)ȱshowedȱhighȱselectivity.ȱ Theȱotherȱselectivityȱimprovingȱmodificationsȱwereȱtheȱsameȱasȱforȱtheȱfirstȱset.ȱȱ

Sinceȱ M238hyȱ containsȱ theȱ selectivityȱ enhancingȱ modificationsȱ Arg6ÆDȬAlaȱ (M212ȱ vsȱ M214)ȱandȱGly13ÆDȬaminoȱacid,ȱweȱwereȱsurprisedȱthatȱitȱwasȱnotȱmoreȱselective.ȱWeȱ hypothesizedȱthatȱeitherȱtheȱhydroxamateȱorȱtheȱGly13ÆDȬPheȱmodificationsȱdecreasedȱ selectivity.ȱPeptidesȱM234ȱandȱM114hyȱwereȱtestedȱagainstȱMMPȬ3Ȭbȱandȱitȱwasȱobservedȱ thatȱwhileȱtheȱspecificityȱwasȱincreasedȱbyȱGly13ÆDȬPheȱindeedȱtheȱhydroxamateȱdeȬ creasedȱspecificityȱ(Figureȱ33B).ȱDespiteȱtheȱdecreaseȱofȱspecificityȱbyȱtheȱhydroxamate,ȱ M114hyȱisȱmoreȱselectiveȱthanȱM238hyȱ(MMPȬ3Ȭbȱassay)ȱevenȱthoughȱtheȱlatterȱcontainsȱ Gly13ÆDȬPhe.ȱWeȱthenȱtestedȱalsoȱM208hyȱwhichȱisȱM238hyȱwithoutȱGly13ÆDȬPhe.ȱUnȬ expectedly,ȱthisȱpeptideȱisȱlessȱselectiveȱthanȱM114hyȱindicatingȱthatȱwhenȱinȱcombinationȱ withȱhydroxamate,ȱtheȱArg6ÆDȬAlaȱmodificationȱdecreasesȱselectivity.ȱFinally,ȱweȱsynȬ thesizedȱfourȱdifferentȱderivativesȱofȱM238hy,ȱeachȱcontainingȱoneȱofȱtheȱidentifiedȱselecȬ tivityȱ increasingȱ modificationsȱ (Figureȱ 33C).ȱ Theseȱ peptidesȱ wereȱ againȱ testedȱ withȱ MMPȬ3Ȭa.ȱOnlyȱoneȱofȱtheseȱpeptidesȱshowedȱanȱimprovedȱselectivityȱasȱcomparedȱtoȱ M238hyȱnamelyȱM240hyȱinȱwhichȱTyr4ȱwasȱreplacedȱbyȱArg.ȱThisȱmodificationȱimprovedȱ theȱselectivityȱ3Ȭfold,ȱbutȱalsoȱdecreasedȱtheȱaffinityȱforȱMMPȬ2,ȱwhichȱhoweverȱstayedȱ wellȱbelowȱ10ȱnM.ȱ

121 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH

ȱ Figureȱ33.ȱ(A)ȱAȱsetȱofȱpreviouslyȱscreenedȱpeptidesȱwereȱselectedȱforȱspecificityȱscreenȱagainstȱMMPȬ3Ȭa.ȱ PeptidesȱwithoutȱhydroxamateȱmodificationsȱwereȱcomparedȱtoȱleadȱpeptideȱM114.ȱTheȱdifferenceȱfromȱ M114ȱisȱindicatedȱinȱgrey.ȱFromȱtheȱinitialȱscreen,ȱpeptidesȱhavingȱaȱhigherȱselectivityȱthanȱM114ȱwereȱseȬ lectedȱasȱindicatedȱbyȱarrows.ȱTheseȱpeptidesȱwereȱthenȱscreenedȱinȱduplicateȱagainstȱaȱdifferentȱenzymeȱ (MMPȬ3Ȭb).ȱTheȱKiȱvaluesȱofȱtheȱpeptideȱforȱenzymesȱMMPȬ2,ȱMMPȬ3ȬaȱandȱMMPȬ3Ȭbȱareȱindicated.ȱ(B)ȱ Hydroxamateȱmodificationȱdecreasesȱspecificity,ȱespeciallyȱwhenȱinȱcombinationȱwithȱArg6ÆDȬAla.ȱModiȬ ficationȱGly13ÆDȬPheȱincreasesȱspecificity.ȱ(C)ȱFourȱpeptidesȱwereȱsynthesizedȱcontainingȱtheȱidentifiedȱ modificationsȱbeneficialȱforȱselectivity.ȱTheȱpeptidesȱadditionallyȱhadȱmodificationsȱArg6ÆDȬAlaȱandȱhyȬ droxamateȱforȱdirectȱcomparisonȱwithȱM238hy.ȱȱȱȱ 'LVFXVVLRQ

DespiteȱtheȱfailureȱofȱMMPȱinhibitorsȱinȱclinicalȱtrials,ȱtheȱenzymeȱclassȱremainsȱimȬ portantȱasȱdrugȱtargets.ȱThisȱisȱhighlightedȱbyȱtheȱrecentȱdevelopmentȱofȱnewȱinhibitorsȱ ofȱdifferentȱformatsȱthanȱtheȱclassicȱsmallȱmoleculeȱhydroxamateȬbasedȱcompounds.ȱTheȱ lackȱofȱselectivityȱhasȱbeenȱstatedȱasȱoneȱofȱtheȱmainȱreasonsȱforȱtheȱfailureȱofȱtheȱearlyȱ MMPȱinhibitors.ȱHowever,ȱforȱtheȱsameȱreason,ȱtheȱbiologicalȱrolesȱandȱinvolvementȱinȱ pathologiesȱofȱtheȱindividualȱMMPsȱisȱnotȱyetȱfullyȱunderstood.ȱToȱgainȱthisȱinsight,ȱ highlyȱspecificȱMMPȱinhibitingȱtoolȱcompoundsȱcouldȱbeȱapplied,ȱforȱexampleȱinȱcellȱ

122 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH basedȱassaysȱorȱdiseaseȱmodels.ȱThisȱknowledgeȱcouldȱthenȱfacilitateȱdrugȱdevelopmentȱ inȱtermsȱofȱtargetȱvalidationȱand/orȱantiȬtargetȱidentificationȱwithinȱtheȱclassȱofȱMMPs.ȱȱ

Hereinȱweȱutilizedȱphageȱdisplayȱofȱbicyclicȱpeptides,ȱwhichȱhaveȱbeenȱdemonstratedȱtoȱ bindȱwithȱhighȱaffinityȱandȱspecificityȱtoȱtheirȱtargets,ȱtoȱdevelopȱaȱselectiveȱMMPȬ2ȱ(/3)ȱ inhibitor.ȱByȱusingȱaffinityȱmaturation,ȱSARȱstudiesȱandȱmedicinalȱchemistry,ȱweȱcouldȱ improveȱtheȱaffinityȱofȱtheȱleadȱpeptideȱisolatedȱinȱtheȱinitialȱphageȱdisplayȱbyȱmoreȱthanȱ threeȱordersȱofȱmagnitudeȱ(M21:ȱKiȱ=ȱ4ȱPM,ȱM238hyȱKiȱ=ȱ1.9ȱnM).ȱThisȱdemonstratesȱtheȱ powerȱofȱtheȱbicyclicȱpeptideȱformatȱwhenȱdevelopedȱbyȱphageȱdisplayȱtogetherȱwithȱ theȱintroductionȱofȱunnaturalȱbuildingȱblocks.ȱȱȱ

ForȱselectiveȱMMPȬ2ȱinhibitors,ȱtheȱmainȱchallengeȱhasȱbeenȱtoȱproveȱselectiveȱoverȱtheȱ otherȱgelatinaseȱMMPȬ9.ȱTheȱtwoȱenzymesȱareȱuniqueȱinȱtheirȱstructure,ȱhavingȱthreeȱ fibronectinȱIIȱtypeȱcollagenȱbindingȱdomainsȱwhichȱareȱrequiredȱforȱtheirȱabilityȱtoȱcleaveȱ gelatine.ȱTheȱpresenceȱofȱtheseȱdomainsȱseparatesȱtheȱgelatinasesȱfromȱotherȱMMPs.ȱInȱ addition,ȱ theȱ twoȱ enzymesȱ shareȱ aȱ 64%ȱ sequenceȱ identityȱ inȱ theȱ catalyticȱ domain.ȱ AchievingȱselectivityȱforȱMMPȱinhibitorsȱcontainingȱaȱhydroxamicȱacidȱmoietyȱhasȱbeenȱ particularlyȱchallenging.ȱOurȱinitialȱleadȱpeptideȱM114hyȱasȱwellȱasȱM238hyȱshowȱstrongȱ selectivityȱforȱMMPȬ2ȱoverȱMMPȬ9.ȱThisȱindicatesȱthatȱtheȱpeptidesȱcontainȱselectivityȱ enhancingȱgroups,ȱnotȱfoundȱforȱmostȱotherȱMMPȬ2ȱinhibitors.ȱHowever,ȱseveralȱotherȱ MMPsȱhaveȱhighȱsimilarityȱtoȱMMPȬ2,ȱthereforeȱitȱisȱimportantȱtoȱscreenȱalsoȱthose.ȱInȱ thisȱscreenȱweȱfoundȱthatȱMMPȬ3ȱwasȱalsoȱinhibitedȱbyȱM238hy.ȱInȱadditionȱweȱidentifiedȱ regionsȱandȱspecificȱresiduesȱofȱtheȱMMPs,ȱcrucialȱforȱselectivityȱofȱtheȱinhibitor.ȱInȱconȬ trastȱtoȱmanyȱotherȱstudiesȱweȱhaveȱhereinȱanalyzedȱtheȱsequencesȱofȱallȱhumanȱMMPs.ȱ InȱparticularȱselectivityȱseemsȱtoȱbeȱdeterminedȱbyȱtheȱS1’ȱpocketȱofȱtheȱenzymeȱasȱwellȱ asȱofȱresiduesȱonȱtheȱnonȬprimeȱsideȱsuchȱasȱAla87.ȱWeȱbelieveȱthatȱourȱfindingsȱcouldȱ beȱofȱvalueȱwhenȱdesigningȱinhibitorsȱspecificȱforȱcertainȱMMPs.ȱHowever,ȱcrystalȱstrucȬ turesȱwouldȱbeȱaȱgreatȱcomplementȱtoȱourȱactivityȱassayȱbasedȱresultsȱinȱorderȱtoȱgainȱ inȬdepthȱknowledgeȱaboutȱwhichȱinteractionsȱcontrolȱMMPȱselectiveȱinhibition.ȱȱȱ

Furthermore,ȱ weȱ haveȱ demonstratedȱ thatȱ byȱ introducingȱ unnaturalȱ aminoȱ acidsȱ weȱ couldȱimproveȱtheȱproteolyticȱstabilityȱandȱaffinity.ȱTheȱstabilityȱwasȱimprovedȱbyȱidenȬ tifyingȱproteolyticȱcleavageȱsitesȱandȱsubsequentlyȱreplacingȱlabileȱresiduesȱbyȱDȬaminoȱ acids.ȱByȱintroducingȱDȬaminoȱacidsȱinȱaȱglycineȱpositionȱnewȱtargetȱinteractionsȱcouldȱ beȱformedȱandȱtherebyȱincreasingȱbindingȱaffinity.ȱTheseȱapproachesȱareȱgeneralȱandȱ canȱbeȱreadilyȱappliedȱtoȱpeptideȱbindersȱofȱanyȱformat.ȱ

Finally,ȱweȱmadeȱanȱeffortȱinȱorderȱtoȱimproveȱtheȱselectivityȱofȱtheȱinhibitorȱforȱMMPȬ 2ȱoverȱMMPȬ3ȱbyȱSARȱstudies.ȱItȱwasȱsomewhatȱunclearȱhowȱselectivityȱwasȱaffected,ȱ butȱ asȱ expectedȱ theȱ hydroxamateȱ decreasedȱ selectivity.ȱ Theȱ selectivityȱ couldȱ beȱ inȬ creasedȱbyȱsubstitutionsȱinȱtheȱTyr4ȱpositionȱasȱwellȱasȱinȱtheȱGly13ȱposition.ȱWeȱbelieveȱ

123 6HOHFWLYHLQKLELWLRQRI003DFKLHYHGZLWKSHSWLGHPDFURF\FOH thatȱitȱisȱlikelyȱthatȱfurtherȱmodificationsȱinȱtheseȱtwoȱpositionsȱcouldȱinfluenceȱaffinityȱ andȱspecificityȱofȱtheȱinhibitorȱbyȱinteractionsȱatȱtheȱS1’ȱsiteȱandȱnonȬprimeȱsideȱofȱtheȱ substrateȱbindingȱpocket.ȱInȱsummary,ȱM238hyȱprovidesȱaȱusefulȱresearchȱtoolȱforȱtheȱ investigationȱofȱtheȱphysiologicalȱandȱpathologicalȱrolesȱofȱMMPȬ2ȱandȱ3.ȱInȱcontrastȱtoȱ otherȱselectiveȱinhibitors,ȱtheȱproteolyticȱstabilityȱofȱM238hyȱallowsȱforȱapplicationȱinȱcellȱ basedȱassaysȱorȱinȱvivo.ȱTheȱinhibitorȱisȱreadilyȱavailableȱthroughȱordinaryȱpeptideȱsynȬ thesisȱfollowedȱbyȱhydroxamateȱmodificationȱandȱcyclization,ȱsimpleȱreactionsȱwhichȱ haveȱbeenȱoptimizedȱinȱourȱlab.ȱFollowingȱtheȱpotentialȱtargetȱvalidation,ȱtheȱbicyclicȱ peptideȱcouldȱpossiblyȱevenȱbeȱdevelopedȱintoȱaȱtherapeutic.ȱȱȱ 0DWHULDOVDQGPHWKRGV

ExpressionȱandȱpurificationȱofȱproȬMMPȬ2:ȱHumanȱproȬMMPȬ2ȱwasȱexpressedȱinȱtranȬ sientlyȱtransfectedȱhumanȱembryonicȱkidneyȱcellsȱasȱfollows.ȱSuspensionȬadaptedȱHEKȬ 293ȱcellsȱwereȱgrownȱinȱ1ȱLȱserumȬfreeȱExCellȱ293ȱmediumȱ(SAFCȱBiosciences)ȱinȱtheȱ presenceȱofȱ4ȱmMȱglutamineȱandȱ3.75ȱmMȱhistoneȱdeacetylaseȱinhibitorȱvalproicȱacidȱinȱ anȱorbitallyȱshakenȱfiveȬliterȱglassȱbottleȱatȱ180ȱrpmȱatȱ37°Cȱinȱtheȱpresenceȱofȱ5%ȱCO2.ȱ Cellsȱatȱaȱdensityȱofȱ20x106ȱcells/mlȱwereȱtransfectedȱwithȱtheȱvectorȱpcDNA3ȬMMP2ȱ(aȱ kindȱgiftȱfromȱProf.ȱDr.ȱEricȱHoward,ȱUniversityȱofȱOklahoma,ȱUSA)ȱusingȱlinearȱpolyȬ ethylenimine.ȱAfterȱ7ȱdays,ȱcellsȱwereȱharvestedȱbyȱcentrifugationȱatȱ2,500ȱrpmȱforȱ15ȱ minȱatȱ4°C.ȱCellȱdebrisȱwasȱremovedȱfromȱtheȱmediumȱbyȱfiltrationȱthroughȱ0.45ȱPmȱPESȱ membranes.ȱTheȱrecombinantȱproteinȱwasȱpurifiedȱfromȱtheȱconditionedȱmediumȱbyȱ sizeȱexclusionȱchromatographyȱonȱaȱHiPrepȱ26/60ȱSephacrylȱSȬ200ȱhighȱresolutionȱcolȬ umnȱ (GEȱ Healthcare)ȱ usingȱ PBSȱ pHȱ 7.4ȱ asȱ buffer.ȱ Theȱ purityȱ ofȱ theȱ proMMPȬ2ȱ wasȱ greaterȱthanȱ95%ȱjudgingȱbyȱSDSȬPAGE.ȱ

BiotinylationȱofȱhumanȱproMMPȬ2:ȱTheȱrecombinantȱproMMPȬ2ȱ(4.5ȱPM)ȱinȱPBSȱ(pHȱ 7.4)ȱwasȱincubatedȱwithȱ5ȬfoldȱexcessȱofȱEZȬlinkȱSulfoȬNHSȬLCȬBiotinȱ(22.5ȱPM,ȱPierce)ȱ forȱ1ȱhȱatȱ25°C.ȱTheȱexcessȱofȱbiotinȱwasȱsubsequentlyȱreducedȱbyȱrepetitiveȱconcentraȬ tionȱandȱdilutionȱstepsȱwithȱTNCȱbufferȱ(50ȱmMȱTrisȬHClȱ(pHȱ7.5),ȱ150ȱmMȱNaCl,ȱ10ȱ mMȱCaCl2,ȱ0.02%ȱ(w/v)ȱNaN3ȱandȱ0.05%ȱ(w/v)ȱBrijȬ35)ȱinȱaȱVivaspinȬ20ȱfilterȱ(MWCOȱofȱ 10’000,ȱSartoriusȬStedimȱBiotechȱGmbH).ȱTheȱabilityȱofȱtheȱbiotinylatedȱMMPȬ2ȱtoȱbindȱ toȱeitherȱstreptavidinȱorȱneutravidinȱwasȱverifiedȱbyȱincubatingȱtheȱproteinȱwithȱmagȬ neticȱstreptavidinȱandȱneutravidinȱbeadsȱrespectivelyȱandȱanalysingȱtheȱboundȱandȱunȬ boundȱproteinȱfractionsȱbyȱSDSȬPAGE.ȱ

ActivationȱofȱhumanȱproȬMMPȬ2:ȱPurifiedȱproȬMMPȬ2ȱ(1ȱtoȱ5ȱPM)ȱinȱTNCȱbufferȱwasȱ incubatedȱ1ȱhȱatȱ37°Cȱwithȱfreshlyȱpreparedȱ4Ȭaminophenylmercuricȱacetateȱ(APMA,ȱ stockȱsolution:ȱ50ȱmMȱdissolvedȱinȱ200ȱmMȱNaOH)ȱatȱaȱfinalȱconcentrationȱofȱ2ȱmM.ȱ Activatedȱ MMPȬ2ȱ wasȱ purifiedȱ byȱ sizeȱ exclusionȱ chromatographyȱ onȱ aȱ Superdexȱ 75ȱ 10/300ȱGLȱhighȱresolutionȱcolumnȱ(GEȱHealthcare)ȱusingȱTNCȱbuffer.ȱȱ

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Phageȱselectionȱofȱbicyclicȱpeptides:ȱBacterialȱcellsȱofȱtheȱlibraryȱglycerolȱstockȱwereȱ inoculatedȱinȱ500ȱmLȱofȱ2YT/chloramphenicolȱ(30ȱPg/ml)ȱmediumȱtoȱobtainȱanȱOD600ȱofȱ 0.1.ȱTheȱcultureȱwasȱshakenȱ(200ȱrpm)ȱforȱ16ȱhȱatȱ30°C.ȱAfterȱ30ȱminȱofȱcentrifugationȱatȱ 8500ȱrpmȱandȱ4°C,ȱtheȱphageȱwereȱpurifiedȱbyȱprecipitationȱwithȱ0.2ȱvolumesȱofȱ20%ȱ (w/v)ȱpolyethyleneȱglycolȱ6000ȱ(PEG6000),ȱ2.5ȱMȱNaClȱonȱiceȱandȱcentrifugationȱatȱ4000ȱ rpmȱforȱ30ȱmin.ȱPEGȱpurifiedȱphage,ȱtypicallyȱ1011Ȭ1012ȱt.u.ȱ(transducingȱunits),ȱwereȱ reducedȱinȱ20ȱmlȱofȱ20ȱmMȱNH4HCO3,ȱ5ȱmMȱEDTA,ȱpHȱ8.0ȱwithȱ1ȱmMȱTCEPȱatȱ42qCȱforȱ 1ȱh.ȱTheȱconcentrationȱofȱTCEPȱwasȱsubsequentlyȱreducedȱbyȱrepetitiveȱconcentrationȱ andȱdilutionȱstepsȱwithȱreactionȱbufferȱ(20ȱmMȱNH4HCO3,ȱ5ȱmMȱEDTA,ȱpHȱ8.0,ȱdeȬ gased)ȱinȱaȱVivaspinȬ20ȱfilterȱ(MWCOȱofȱ10’000,ȱSartoriusȬStedimȱBiotechȱGmbH)ȱasȱdeȬ scribedȱpreviously.ȱTheȱvolumeȱofȱtheȱphageȱsolutionȱwasȱadjustedȱtoȱ32ȱmlȱwithȱreacȬ tionȱbufferȱandȱ8ȱmlȱofȱ50ȱPMȱtrisȬ(bromomethyl)benzeneȱ(TBMB)ȱinȱMeCNȱwereȱaddedȱ toȱobtainȱaȱfinalȱTBMBȱconcentrationȱofȱ10ȱPM.ȱTheȱreactionȱwasȱincubatedȱatȱ30qCȱforȱ1ȱ hȱbeforeȱnonȬreactedȱTBMBȱwasȱremovedȱbyȱprecipitationȱofȱtheȱphageȱwithȱ0.2ȱvolumesȱ ofȱ20%ȱ(w/v)ȱPEG6000,ȱ2.5ȱMȱNaClȱonȱiceȱandȱcentrifugationȱatȱ4000ȱrpmȱforȱ30ȱminutes.ȱ Theȱphageȱpelletȱwasȱthenȱdissolvedȱinȱ3ȱmlȱwashingȱbufferȱ(10ȱmMȱTrisȬCl,ȱpHȱ7.4,ȱ150ȱ mMȱNaCl,ȱ10ȱmMȱMgCl2,ȱ1ȱmMȱCaCl2).ȱBiotinylatedȱactiveȱMMPȬ2ȱ(180ȱPg)ȱwasȱaddedȱ toȱ50ȱPlȱmagneticȱstreptavidinȱbeadsȱ(DynabeadsȱMȬ280ȱfromȱInvitrogenȱDynalȱBiotechȱ AS)ȱinȱwashingȱbufferȱandȱincubatedȱonȱaȱrotatingȱwheelȱforȱ10ȱminȱatȱroomȱRT.ȱTheȱ magneticȱbeadsȱwereȱthenȱwashedȱwithȱ0.5ȱmlȱwashingȱbufferȱandȱincubatedȱforȱ30ȱminȱ atȱRTȱwithȱ0.5ȱmlȱwashingȱbufferȱcontainingȱ1%ȱ(w/v)ȱBSAȱandȱ0.1%ȱ(v/v)ȱTweenȱ20.ȱAtȱ theȱsameȱtimeȱtheȱchemicallyȱmodifiedȱphageȱ(typicallyȱ1010Ȭ1011ȱt.u.ȱdissolvedȱinȱ3ȱmlȱ washingȱbuffer)ȱwereȱblockedȱbyȱadditionȱofȱ1.5ȱmlȱofȱwashingȱbufferȱcontainingȱ3%ȱ (w/v)ȱBSAȱandȱ0.3%ȱ(v/v)ȱTweenȱ20ȱforȱ30ȱminutes.ȱTheȱblockedȱbeads/antigenȱmixtureȱ (0.5ȱml)ȱandȱphageȱ(4.5ȱml)ȱwereȱmixedȱtogetherȱandȱincubatedȱforȱ30ȱminutesȱonȱaȱroȬ tatingȱwheelȱatȱroomȱtemperature.ȱTheȱbeadsȱ(andȱantigen/phageȱboundȱtoȱthem)ȱwereȱ washedȱeightȱtimesȱwithȱwashingȱbufferȱcontainingȱ0.1%ȱ(v/v)ȱTweenȱ20ȱandȱtwiceȱwithȱ washingȱbuffer.ȱTheȱphageȱwereȱelutedȱeitherȱbyȱincubationȱwithȱ100ȱPlȱofȱ50ȱmMȱglyȬ cine,ȱpHȱ2.2ȱforȱ5ȱminutesȱ(pHȬdependentȱelution),ȱandȱthenȱtransferredȱtoȱ50ȱΐlȱofȱ1ȱMȱ TrisȬCl,ȱpHȱ8.0ȱforȱneutralisationȱorȱbyȱincubationȱwithȱ100ȱΐlȱofȱ250ȱΐMȱofȱtheȱcompetȬ itiveȱhydroxamateȱinhibitorȱGM6001ȱ(EnzoȱLifeȱSciences,ȱInc.)ȱinȱwashingȱbufferȱ(pHȱ7.4)ȱ forȱ30ȱminutesȱ(competitiveȱelution).ȱTheȱelutedȱphageȱwereȱincubatedȱwithȱ30ȱmlȱTG1ȱ cellsȱatȱOD600ȱofȱ0.4ȱforȱ90ȱminutesȱatȱ37qCȱandȱtheȱcellsȱwereȱplatedȱonȱlargeȱ2YT/chloȬ ramphenicolȱ (30ȱ Pg/mlȱ chloramphenicol)ȱ plates.ȱ Secondȱ andȱ thirdȱ roundȱ ofȱ panningȱ wereȱ performedȱ followingȱ theȱ sameȱ procedureȱ butȱ usingȱ inȱ theȱ secondȱ roundȱ neuȬ travidinȬcoatedȱmagneticȱbeadsȱinsteadȱofȱstreptavidinȱinȱorderȱtoȱpreventȱtheȱenrichȬ mentȱofȱstreptavidinȬspecificȱpeptideȱbinders.ȱNeutravidinȱbeadsȱwereȱpreparedȱbyȱreȬ actingȱ0.8ȱmgȱneutravidinȱ(Pierce)ȱwithȱ0.5ȱmlȱtosylȬactivatedȱmagneticȱbeadsȱ(2ȱxȱ109ȱ beads/ml;ȱDynabeadsȱMȬ280,ȱInvitrogenȱDynalȱBiotechȱAS)ȱaccordingȱtoȱtheȱsupplierȇsȱ instructions.ȱ

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2.5%ȱthioanisole,ȱ2.5%ȱH2O)ȱforȱ2ȱhȱwithȱshaking.ȱTheȱresinȱwasȱremovedȱbyȱvacuumȱ filtration,ȱandȱtheȱpeptidesȱwereȱprecipitatedȱwithȱiceȬcoldȱdiethylȱetherȱ(50ȱml),ȱincuȬ batedȱforȱ30ȱminȱatȱȬ20°C,ȱandȱpelletedȱbyȱcentrifugationȱ(2700ȱg,ȱ5ȱmin).ȱTheȱdiethylȱ etherȱwasȱdiscarded,ȱtheȱprecipitateȱwashedȱtwiceȱwithȱdiethylȱetherȱandȱtheȱremainingȱ solventȱevaporatedȱatȱRT.ȱȱ

Hydroxamateȱmodification:ȱAllȱpeptidesȱwhichȱwereȱhydroxamateȱmodifiedȱwereȱsynȬ thesizedȱwithȱusingȱFmocȬAspȱ(2Ȭphenylisopropylȱester)ȬOHȱ(Bachem,ȱcatȱ#ȱBȬ2475).ȱTheȱ procedureȱreportedȱaboveȱforȱpeptideȱsynthesisȱwasȱfollowedȱuntilȱtheȱcleavageȱfromȱ theȱ solidȱ support.ȱ Theȱ 2Ȭphenylisopropylȱ esterȱ protectingȱ groupȱ wasȱ selectivelyȱ reȬ movedȱbyȱtheȱadditionȱofȱ5ȱmlȱTFAȱ(2%ȱ(v/v))ȱinȱdichloromethaneȱandȱleftȱshakingȱforȱ2ȱ min.ȱTheȱmixtureȱwasȱremovedȱbyȱfiltrationȱandȱtheȱprocedureȱrepeatedȱfiveȱtimes.ȱTheȱ solidȱsupportȱwasȱthenȱwashedȱfiveȱtimesȱwithȱDMF.ȱForȱintroducingȱtheȱhydroxamicȱ acidȱmoietyȱaȱpeptideȱcouplingȱwasȱperformedȱbyȱaddingȱNH2OH•HClȱ(2ȱequiv,ȱ1ȱml,ȱ 0.06ȱMȱinȱDMFȱaddedȱtoȱequimolarȱamountȱofȱKOH),ȱpyBOPȱ(1.2ȱequiv,ȱ1ȱml,ȱ0.036ȱMȱ inȱDMF)ȱandȱDIPEAȱ(6ȱequiv,ȱ1ȱml,ȱ0.18ȱMȱinȱDMF).ȱTheȱreactionȱwasȱperformedȱtwiceȱ andȱwasȱrunȱatȱRT,ȱshakingȱatȱ400ȱrpmȱforȱ5ȱmin.ȱAfterȱtheȱcouplingȱreactionȱtheȱresinȱ wasȱwashedȱfourȱtimesȱwithȱDMFȱandȱpeptidesȱwereȱsubsequentlyȱcleavedȱfromȱtheȱ solidȱsupportȱasȱreportedȱabove.ȱ

Peptideȱcyclizationȱandȱpurification:ȱCrudeȱpeptideȱatȱaȱconcentrationȱofȱ1ȱmMȱwasȱ reactedȱwithȱ1.4ȱmMȱ1,3,5ȬTris(bromomethyl)benzeneȱ(TBMB)ȱinȱ70%ȱNH4HCO3ȱbufferȱ (60ȱmM,ȱpHȱ8.0)ȱandȱ30%ȱMeCNȱforȱ1ȱhȱatȱ30°C.ȱTheȱpeptideȱandȱTBMBȱwereȱpreparedȱ andȱmixedȱusingȱtheȱsolvents,ȱconcentrationsȱandȱvolumesȱexemplifiedȱforȱtheȱcyclizaȬ tionȱofȱ~ȱ50ȱmgȱcrudeȱpeptide.ȱToȱ6ȱmlȱ4.2ȱmMȱpeptideȱinȱMeCN/H2Oȱ(1:2),ȱ3.5ȱmlȱ10ȱmMȱ

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TBMBȱinȱMeCNȱandȱ2ȱmlȱMeCNȱwereȱaddedȱandȱtheȱreactionȱstartedȱbyȱadditionȱofȱ14ȱ mlȱNH4HCO3ȱbufferȱ(60ȱmM,ȱpHȱ8.0).ȱTheȱcyclizedȱpeptidesȱsynthesizedȱonȱaȱ50ȱmgȱscaleȱ wereȱpurifiedȱbyȱreversedȬphaseȱHPLCȱ(WatersȱPrepȱLCȱ4000ȱsystem)ȱusingȱaȱpreparaȬ tiveȱC18ȱRPȱcolumnȱ(VydacȱC18ȱTP1022ȱcolumn,ȱ250ȱ×ȱ22ȱmm,ȱ10ȱPm,ȱ300ȱÅ)ȱandȱaȱlinearȱ gradientȱofȱ20Ȭ45%ȱsolventȱBȱ(MeCN,ȱ0.1%ȱ(v/v)ȱTFA)ȱinȱsolventȱAȱ(H2O,ȱ0.1%ȱ(v/v)ȱTFA)ȱ inȱ25ȱminȱatȱaȱflowȱofȱ20ȱml/min.ȱAbsorbanceȱwasȱdetectedȱatȱ220ȱnm.ȱTheȱcyclizedȱpepȬ tidesȱwereȱinjectedȱafterȱlyophilizationȱofȱtheȱreactionȱandȱdissolvingȱtheȱpeptideȱinȱ10ȱ mlȱH2Oȱcontainingȱ10%ȱ(v/v)ȱDMSO,ȱ10%ȱ(v/v)ȱMeCNȱandȱ0.1%ȱ(v/)ȱTFA.ȱFractionsȱconȬ tainingȱtheȱdesiredȱpeptideȱwereȱlyophilized.ȱȱ

HPLCȱandȱmassȱspectrometricȱanalysis:ȱTheȱmolecularȱmassȱofȱpurifiedȱpeptidesȱwasȱ determinedȱonȱaȱsingleȱquadrupoleȱmassȱspectrometerȱinȱpositiveȱionȱmodeȱusingȱelecȬ trosprayȱionizationȱ(LCMSȬ2020,ȱShimadzu).ȱTheȱpurityȱofȱtheȱpeptidesȱwasȱdeterminedȱ byȱ analyticalȱ RPȬHPLCȱ (Agilentȱ 1260ȱ HPLCȱ system)ȱ usingȱ anȱ analyticalȱ C18ȱ columnȱ (AgilentȱZorbaxȱ300SBȬC18,ȱ4.6ȱmmȱ×ȱ250ȱmm,ȱ5ȱPm)ȱandȱaȱlinearȱgradientȱofȱ0Ȭ50%ȱ solventȱBȱ(MeCN,ȱ0.1%ȱ(v/v)ȱTFA)ȱinȱsolventȱAȱ(H2O,ȱ5%ȱ(v/v)ȱMeCN,ȱ0.1%ȱ(v/v)ȱTFA)ȱ inȱ15ȱminȱatȱaȱflowȱofȱ1ȱml/min.ȱAbsorbanceȱwasȱdetectedȱatȱ220ȱnm.ȱ

Peptideȱconcentrationȱdetermination:ȱLyophilizedȱpeptideȱwasȱweighedȱandȱdissolvedȱ inȱDMSOȱ(3%ȱ(v/v))ȱinȱwaterȱbyȱsequentialȱadditionȱtoȱobtainȱpeptideȱstocksȱofȱ2ȱmMȱbyȱ weight.ȱTheȱconcentrationsȱofȱtheȱpeptidesȱwereȱdeterminedȱbyȱmeasuringȱtheȱabsorpȬ tionȱatȱ280ȱnm.ȱTheȱstockȱsolutionȱwasȱthenȱdilutedȱbasedȱonȱtheȱabsorbanceȱtoȱaȱfinalȱ concentrationȱofȱ1ȱmM.ȱȱ

MMPȱinhibitionȱassays:ȱTheȱinhibitoryȱactivityȱofȱbicyclicȱpeptidesȱwasȱdeterminedȱbyȱ measuringȱresidualȱproteaseȱactivityȱwithȱaȱfluorogenicȱsubstrate.ȱTheȱassayȱwasȱperȬ formedȱinȱbufferȱcontainingȱ50ȱmMȱTrisȬCl,ȱpHȱ7.5,ȱ150ȱmMȱNaCl,ȱ10ȱmMȱCaCl2ȱandȱ 0.1%ȱw/vȱBSA,ȱinȱaȱvolumeȱofȱ150ȱPl.ȱForȱallȱMMPs,ȱwithȱtheȱexceptionȱofȱMMPȬ19,ȱacȬ tivityȱwasȱmeasuredȱwithȱtheȱFRETȱsubstrateȱMcaȬLysȬProȬLeuȬGlyȬLeuȬDap(DnP)ȬAlaȬ ArgȬNH2ȱ(FSȬ6,ȱGLȱBiochem)ȱatȱfinalȱconcentrationsȱofȱ5Ȭ20ȱPM.ȱForȱMMPȬ19ȱtheȱsubȬ strateȱ OMNIMMP®ȱ RED:ȱ TQ3ȬGABAȬProȬChaȬAbuȬSmcȬHisȬAlaȬDab(6’ȬTAMRA)Ȭ AlaȬLysȬNH2ȱ(Enzoȱlifeȱsciences)ȱwasȱusedȱatȱaȱfinalȱconcentrationȱofȱ0.75ȱPM.ȱTheȱfolȬ lowingȱ enzymesȱ andȱ concentrationsȱ wereȱ used:ȱ MMPȬ1ȱ (Enzoȱ lifeȱ sciencesȱ kit,ȱ catȱ #ȱ BMLȬAK308Ȭ0001)ȱ10ȱmU/Pl,ȱsubstrateȱ20ȱPM,ȱMMPȬ2ȱ(expressedȱinȱhouse)ȱ0.5ȱnM,ȱsubȬ strateȱ10ȱPM,ȱMMPȬ3Ȭaȱ(Enzoȱlifeȱsciencesȱkit)ȱ7.5ȱmU/Pl,ȱsubstrateȱ20ȱ PM,ȱMMPȬ3Ȭbȱ (Enzoȱlifeȱsciences,ȱcatȱ#ȱALXȬ201Ȭ042ȬC005)ȱ3ȱnM,ȱsubstrateȱ20ȱPM,ȱMMPȬ7ȱ(Enzoȱlifeȱ sciencesȱkit)ȱ0.25ȱmU/Pl,ȱsubstrateȱ20ȱPM,ȱMMPȬ8ȱ(Enzoȱlifeȱsciencesȱkit)ȱ3ȱmU/Pl,ȱsubȬ strateȱ20ȱPM,ȱMMPȬ9ȱ(Enzoȱlifeȱsciencesȱkit)ȱ4ȱmU/Pl,ȱsubstrateȱ10ȱPM,ȱMMPȬ12ȱ(Enzoȱ lifeȱ sciencesȱ kit)ȱ 2.5ȱ mU/Pl,ȱ substrateȱ 10ȱ PM,ȱ MMPȬ13ȱ (Enzoȱ lifeȱ sciencesȱ kit)ȱ 0.1875ȱ mU/Pl,ȱ substrateȱ 5ȱ PM,ȱ MMPȬ14ȱ (Enzoȱ lifeȱ sciencesȱ kit)ȱ 2.5ȱ mU/Pl,ȱ substrateȱ 10ȱ PM,ȱ

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MMPȬ19ȱ(Enzoȱlifeȱsciencesȱkit)ȱ0.75ȱmU/Pl,ȱsubstrateȱ0.75ȱPM,ȱMMPȬ20ȱ(EnzoȱlifeȱsciȬ ences,ȱcatȱ#ȱBMLȬSE540Ȭ0010)ȱ7.5ȱmU/Pl,ȱsubstrateȱ5ȱPM.ȱTheȱreactionȱwasȱpipettedȱasȱ follows:ȱ50ȱPlȱbicyclicȱpeptideȱinȱassayȱbufferȱwasȱaddedȱtoȱ50ȱPlȱofȱenzymeȱinȱassayȱ bufferȱandȱincubatedȱforȱ15ȱminutesȱatȱ37°Cȱbeforeȱ50ȱPlȱofȱ15Ȭ60ȱPMȱfluorogenicȱsubȬ strateȱinȱassayȱbufferȱwasȱaddedȱtoȱmeasureȱtheȱresidualȱproteaseȱactivityȱwithȱanȱInfiniteȱ M200Proȱfluorescenceȱplateȱreaderȱ(FSȬ6:ȱexcitationȱatȱ325ȱnm,ȱemissionȱatȱ400ȱnm;ȱOMȬ NIMMP®ȱRED:ȱexcitationȱatȱ545ȱnm,ȱemissionȱatȱ576ȱnm,ȱTecan)ȱforȱaȱperiodȱofȱ30ȱminȱ withȱaȱreadȱeveryȱminuteȱatȱ37°C.ȱTheȱbicyclicȱpeptidesȱwereȱseriallyȱdilutedȱfromȱtheȱ1ȱ mMȱstockȱsolutionsȱ2Ȭfoldȱusingȱassayȱbuffer.ȱSigmoidalȱcurvesȱwereȱfittedȱtoȱtheȱdataȱ usingȱtheȱfollowingȱdoseȱresponseȱequationȱwhereinȱxȱ=ȱpeptideȱconcentration,ȱyȱ=ȱ%ȱ proteaseȱactivity,ȱpȱ=ȱHillȱslope.ȱIC50ȱvaluesȱwereȱderivedȱfromȱtheȱfittedȱcurve.ȱ

ͳͲͲ ݕൌ ȱ ͳ൅ͳͲሺ୪୭୥ ூ஼ହ଴ ି௫ሻ௣

Theȱinhibitionȱconstantsȱ(Ki)ȱwereȱcalculatedȱaccordingȱtoȱtheȱequationȱofȱChengȱandȱ

PrusoffȱKiȱ=ȱIC50/(1+([S]0/Km)ȱwhereinȱIC50ȱisȱtheȱfunctionalȱstrengthȱofȱtheȱinhibitor,ȱ[S]0ȱ isȱtheȱtotalȱsubstrateȱconcentration,ȱandȱKmȱisȱtheȱMichaelisȬMentenȱconstant.ȱKmȱvaluesȱ wereȱdeterminedȱforȱtheȱenzymesȱwhichȱwereȱinhibitedȱwithȱIC50ȱvaluesȱbelowȱ50ȱPMȱ andȱwereȱasȱfollows:ȱMMPȬ2:ȱ30ȱ±ȱ2ȱPM,ȱMMPȬ3:ȱ90ȱ±ȱ7ȱPM,ȱMMPȬ7:ȱ42ȱ±ȱ5ȱPM,ȱMMPȬ 12:ȱ58ȱ±ȱ1ȱPM,ȱMMPȬ14:ȱ10.1ȱ±ȱ0.1ȱPM,ȱMMPȬ20:ȱ37ȱ±ȱ4ȱPM.ȱTheseȱvaluesȱareȱinȱaccordanceȱ ofȱwhatȱhasȱbeenȱpreviouslyȱreportedȱforȱthisȱsubstrateȱ(319).ȱȱȱȱ

InhibitionȱofȱMMPȬ2ȱautoproteolysis:ȱ100ȱnMȱMMPȬ2ȱinȱTNCȱbufferȱwasȱincubatedȱ withȱandȱwithoutȱ0.1,ȱ0.5ȱandȱ1ȱPMȱM114hyȱforȱ1ȱhȱatȱ37°C.ȱ20ȱPlȱofȱtheȱreactionȱwasȱ analyzedȱbyȱSDSȬPAGEȱusingȱaȱ10%ȱacrylamideȱgelȱ(CBSȱScientificȱcatȱ#ȱFK01012Ȭ10)ȱ andȱTrisȬTricineȱClearPAGEȱSDSȱrunningȱbuffer,ȱandȱsilverȱstainingȱ(PierceȱSilverȱStainȱ Kit,ȱThermoȱFisherȱScientific).ȱ

InhibitionȱofȱMMPȬ2ȱinȱcellȱculture:ȱHT1080ȱcellsȱwereȱculturedȱinȱ10ȱmlȱDMEMȱmediaȱ supplementedȱwithȱ10%ȱfetalȱbovineȱserumȱ(FBS)ȱandȱnonȬessentialȱaminoȱacidsȱ(NEAA,ȱ finalȱconc.ȱ0.1ȱmMȱorȱ100ȱPLȱofȱ100Xȱstockȱ(ThermoȱFisherȱScientific)ȱinȱ75ȱcm2ȱflasks.ȱ Cellsȱatȱ80Ȭ90%ȱconfluencyȱwereȱwashedȱwithȱserumȱfreeȱ(SF)ȱmediaȱthriceȱ(DMEMȱwithȱ NEAA),ȱ incubatedȱ inȱ SFȱ mediaȱ forȱ 2ȱ hoursȱ andȱ againȱ washedȱ withȱ SFȱ mediaȱ thrice.ȱ M114hyȱorȱbatimastatȱdissolvedȱinȱ10%ȱDMSOȱ+ȱ90%ȱMilliȬQȱwaterȱwereȱaddedȱ(10ȱPMȱ finalȱconc.)ȱandȱincubatedȱforȱ48ȱhoursȱatȱ37°C,ȱ5%ȱCO2.ȱ10ȱmlȱofȱcellȱcultureȱconditionȱ mediaȱwasȱcollected,ȱfilteredȱthroughȱ0.2ȱPmȱmembraneȱandȱconcentratedȱtoȱ2ȱmlȱusingȱ 5ȱkDaȱcutȱoffȱVivaspinȱ20ȱ(Sartorius)ȱandȱcentrifugationȱatȱ4,000ȱrpm,ȱforȱaroundȱ240ȱminȱ atȱ4°C.ȱAliquotsȱofȱ20ȱPlȱwereȱseparatedȱbyȱSDSȬPAGEȱusingȱonȱaȱ10%ȱacrylamideȱgelȱ (CBSȱScientificȱcatȱ#ȱFK01012Ȭ10)ȱusingȱClearPAGEȱSDSȱrunningȱbufferȱ(TrisȬTricine).ȱ ProteinsȱwereȱtransferredȱtoȱaȱPVDFȱmembraneȱandȱdetectedȱwithȱrabbitȱpolyclonalȱantiȬ

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MMPȬ2ȱantibodyȱ(NovusȱBiologicals;ȱ1000Ȭfoldȱdilution),ȱantiȬrabbitȱIgGȬHRPȱ(Novusȱ Biologicals;ȱ5000ȱdilution)ȱandȱCN/DABȱsubstrateȱ(ThermoȱFisherȱScientific).ȱ

Plasmaȱstabilityȱassaysȱ(HPLC):ȱToȱ720ȱPlȱmouseȱplasmaȱ(Innovativeȱresearch)ȱ80ȱPlȱ(1ȱ mMȱinȱH2O)ȱofȱpeptideȱsolutionȱwasȱaddedȱtoȱobtainȱaȱfinalȱpeptideȱconcentrationȱinȱ plasmaȱofȱ100ȱPM.ȱPeptideȱwasȱincubatedȱinȱmouseȱplasmaȱatȱ37°Cȱinȱaȱwaterȱbath.ȱSamȬ plesȱofȱ90ȱPlȱwereȱcollectedȱatȱdifferentȱtimeȱpointsȱ(0,ȱ0.5,ȱ1,ȱ2,ȱ4,ȱ8,ȱ16ȱandȱ32ȱh)ȱandȱ mixedȱwithȱ60ȱPlȱofȱ6ȱMȱguanidiniumȱhydrochloride.ȱTheȱsamplesȱwereȱthenȱstoredȱatȱȬ 20°C.ȱAfterȱcollectionȱofȱallȱtimeȱpoints,ȱtheȱsamplesȱwereȱcentrifugedȱ(11000ȱg,ȱ5ȱmin)ȱ priorȱtoȱanalysis.ȱTheȱstabilityȱandȱdegradationȱofȱtheȱpeptidesȱwasȱanalysedȱbyȱanalytȬ icalȱ RPȬHPLCȱ (Agilentȱ 1260ȱ HPLCȱ system)ȱ usingȱ anȱ analyticalȱ C8ȱ columnȱ (Waters,ȱ XbridgeȱC8,ȱ4.6ȱmmȱ×ȱ250ȱmm,ȱ5ȱPm,ȱ130ȱA)ȱandȱaȱlinearȱgradientȱofȱ15Ȭ65%ȱsolventȱBȱ

(MeCN,ȱ0.1%ȱ(v/v)ȱTFA)ȱinȱsolventȱAȱ(H2O,ȱ5%ȱ(v/v)ȱMeCN,ȱ0.1%ȱ(v/v)ȱTFA)ȱinȱ30ȱminȱ atȱaȱflowȱofȱ1ȱml/min.ȱAbsorbanceȱwasȱdetectedȱatȱ220ȱandȱ280ȱnm.ȱPeaksȱcorrespondingȱ toȱintactȱpeptideȱorȱdegradationȱproductsȱwereȱidentifiedȱbyȱmassȱspectrometryȱanalysisȱ ofȱtheȱcollectedȱfractionsȱusingȱtheȱLCMSȬ2020ȱsystemȱdescribedȱabove.ȱForȱtheȱquantiȬ ficationȱofȱtheȱintactȱpeptide,ȱareaȱunderȱtheȱcurveȱwasȱcalculatedȱforȱpeaksȱcorrespondȬ ingȱtoȱintactȱpeptideȱorȱwithȱonlyȱtheȱNȬterminalȱalanineȱcleaved.ȱAverageȱvaluesȱofȱ220ȱ andȱ280ȱnmȱchromatogramsȱwereȱused.ȱȱȱ

Plasmaȱstabilityȱassaysȱ(Functional):ȱPeptideȱ(100ȱPlȱofȱ1ȱmMȱinȱH2O)ȱwasȱaddedȱtoȱ900ȱ Plȱmouseȱplasmaȱ(InnovativeȱResearch)ȱtoȱobtainȱaȱfinalȱpeptideȱconcentrationȱofȱ100ȱ PM.ȱTheȱmixtureȱwasȱincubatedȱinȱaȱwaterȱbathȱatȱ37°C.ȱAtȱdifferentȱtimeȱpointsȱ(0,ȱ0.5,ȱ 1,ȱ2,ȱ4,ȱ8,ȱ16,ȱandȱ32ȱh),ȱsamplesȱofȱ30ȱPlȱwereȱremoved,ȱdilutedȱtoȱ200ȱPlȱwithȱaqueousȱ bufferȱ(50ȱmMȱTrisȬHCl,ȱpHȱ7.4,ȱ150ȱmMȱNaCl,ȱ10ȱmMȱCaCl2)ȱandȱincubatedȱforȱ20ȱminȱ atȱ65°Cȱtoȱinactivateȱplasmaȱproteases.ȱTheȱpeptide/plasmaȱsamplesȱwereȱstoredȱatȱȬ20°Cȱ untilȱtheȱresidualȱinhibitoryȱactivityȱofȱtheȱpeptidesȱwasȱmeasuredȱinȱaȱMMPȬ2ȱinhibitionȱ assay.ȱForȱtheȱactivityȱassay,ȱtheȱpeptide/plasmaȱsamplesȱwereȱcentrifugedȱforȱ5ȱminȱatȱ 16,000ȱ g,ȱ serialȱ 2Ȭfoldȱ dilutionsȱ ofȱ theȱ supernatantȱ preparedȱ (peptideȱ concentrationȱ rangesȱfromȱ0.78ȱnMȱtoȱ0.8ȱPM)ȱandȱtheȱresidualȱactivityȱofȱ0.5ȱnMȱMMPȬ2ȱmeasuredȱ usingȱ10ȱPMȱMcaȬLysȬProȬLeuȬGlyȬLeuȬDap(DnP)ȬAlaȬArgȬNH2ȱsubstrate.ȱIC50ȱvaluesȱ wereȱderivedȱfromȱtheȱfittedȱcurveȱusingȱtheȱequationȱindicatedȱabove.ȱResidualȱinhibiȬ tionȱinȱ%ȱwasȱcalculatedȱusingȱtheȱequationȱIC50,0h/IC50,xh*100ȱwhereinȱIC50,0hȱisȱtheȱfuncȬ tionalȱstrengthȱofȱtheȱinhibitorȱatȱtimeȱpointȱ0ȱandȱIC50,xhȱtheȱfunctionalȱstrengthȱofȱinhibȬ itorȱafterȱoneȱofȱtheȱdifferentȱplasmaȱincubationȱperiodsȱmentionedȱabove.ȱ

ȱȱ

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TableȱS5.1.ȱSequenceȱidentityȱandȱsimilarityȱofȱȱcatalyticȱdomainsȱofȱallȱhumanȱMMPȱȱ

FigureȱS5.1.ȱAffinityȱmaturationȱofȱM21ȱ

FigureȱS5.2.ȱDegradationȱproductsȱofȱM205ȱandȱM208ȱ

FigureȱS5.3.ȱPlasmaȱstabilityȱandȱdegradationȱofȱM114ȱȱ

FigureȱS5.4.ȱPlasmaȱstabilityȱandȱdegradationȱofȱM114hyȱ

FigureȱS5.5.ȱPlasmaȱstabilityȱandȱdegradationȱofȱM208hyȱ

FigureȱS5.6.ȱStabilityȱanalysisȱofȱendȱremovalȱandȱArg3ÆAlaȱmodifiedȱpeptidesȱ

FigureȱS5.7.ȱM114ȱalanineȱscanȱandȱTyr4ȱmodificationsȱ

ȱȱ

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TableȱS5.1.ȱSequenceȱidentity,ȱsimilarityȱandȱBLOSUM62ȱscoreȱofȱcatalyticȱdomainsȱofȱallȱ humanȱMMPs.ȱ

Enzymeȱ %ȱIdentityȱ %ȱSimilarityȱ BLOSUM62ȱScoreȱ MMPȬ13ȱ 65ȱ 76ȱ 592ȱ MMPȬ9ȱ 64ȱ 77ȱ 557ȱ MMPȬ3ȱ 58ȱ 70ȱ 521ȱ MMPȬ20ȱ 58ȱ 67ȱ 517ȱ MMPȬ12ȱ 59ȱ 71ȱ 506ȱ MMPȬ10ȱ 56ȱ 68ȱ 493ȱ MMPȬ1ȱ 56ȱ 72ȱ 486ȱ MMPȬ8ȱ 55ȱ 68ȱ 482ȱ MMPȬ7ȱ 53ȱ 64ȱ 469ȱ MMPȬ24ȱ 54ȱ 64ȱ 441ȱ MMPȬ11ȱ 52ȱ 65ȱ 430ȱ MMPȬ27ȱ 54ȱ 64ȱ 427ȱ MMPȬ26ȱ 48ȱ 67ȱ 425ȱ MMPȬ14ȱ 49ȱ 63ȱ 425ȱ MMPȬ16ȱ 54ȱ 64ȱ 424ȱ MMPȬ15ȱ 48ȱ 62ȱ 405ȱ MMPȬ25ȱ 44ȱ 56ȱ 348ȱ MMPȬ17ȱ 40ȱ 43ȱ 331ȱ MMPȬ19ȱ 46ȱ 58ȱ 327ȱ MMPȬ28ȱ 44ȱ 57ȱ 320ȱ MMPȬ23ȱ 39ȱ 50ȱ 278ȱ MMPȬ21ȱ 38ȱ 49ȱ 230ȱ ȱ

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FigureȱS5.1.ȱ(A)ȱConsensusȱsequencesȱofȱaffinityȱmaturationȱlibraries.ȱ(B)ȱSynthesizedȱpeptidesȱwithȱQARȱ motifȱinȱfirstȱloop.ȱ(C)ȱSynthesizedȱpeptideȱforȱfirstȱloopȱoptimizationȱwithȱconstantȱsecondȱloop.ȱ

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FigureȱS5.2.ȱObservedȱdegradationȱproductsȱafterȱincubationȱinȱmouseȱplasma.ȱ

ȱȱ

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FigureȱS5.3.ȱPlasmaȱstabilityȱanalysisȱofȱM114ȱ

ȱȱ

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FigureȱS5.4.ȱPlasmaȱstabilityȱanalysisȱofȱM114hyȱȱ

ȱȱ

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FigureȱS5.5.ȱPlasmaȱstabilityȱanalysisȱofȱM208hyȱ

ȱȱ

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FigureȱS5.6.ȱStabilityȱanalysisȱofȱendȱremovalȱandȱArg3ÆAlaȱmodifiedȱpeptidesȱ

ȱȱ

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FigureȱS5.7.ȱAlanineȱscanȱandȱreplacementȱofȱTyr4ȱ

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Peptidesȱareȱaȱpromisingȱclassȱofȱtherapeuticȱmolecules,ȱwhichȱcombineȱpropertiesȱofȱ smallȱmoleculesȱwithȱthoseȱofȱtheȱlargerȱbiologicals.ȱHowever,ȱdueȱtoȱintrinsicȱlimitaȬ tionsȱofȱtheȱmolecularȱformat,ȱpeptideȱleadȱcompoundsȱoftenȱhaveȱtoȱundergoȱextensiveȱ medicinalȱchemistryȱinȱorderȱtoȱbeȱoptimizedȱintoȱmoreȱdrugȬlikeȱcompounds.ȱWithȱthisȱ PhDȱthesis,ȱmyȱgeneralȱobjectiveȱhasȱbeenȱtoȱdevelopȱbicyclicȱpeptideȱhitȱcompoundsȱ intoȱmoleculesȱthatȱareȱmoreȱlikelyȱtoȱbeȱusedȱasȱtherapeuticsȱorȱresearchȱtools.ȱThisȱworkȱ hasȱaimedȱatȱimprovingȱpropertiesȱsuchȱasȱtargetȱaffinity,ȱspecificityȱandȱstability.ȱWithȱ aȱsuccessfullyȱimprovedȱFXIIȱinhibitorȱinȱhand,ȱmyȱnextȱgoalȱwasȱtoȱcharacterizeȱtheȱ inhibitorȱinȱvariousȱanimalȱmodels,ȱinȱorderȱtoȱevaluateȱitsȱpropertiesȱinȱaȱtherapeuticȱ setting.ȱȱ

Inȱthisȱthesis,ȱIȱhaveȱshownȱthatȱevenȱthoughȱbicyclicȱpeptidesȱisolatedȱdirectlyȱfromȱ phageȱdisplayȱareȱoftenȱnotȱoptimalȱcompoundsȱforȱuseȱasȱtherapeuticsȱorȱresearchȱtools,ȱ theyȱcanȱbeȱoptimizedȱusingȱvariousȱapproaches.ȱStrategiesȱincludingȱDȬaminoȱacidsȱinȬ corporation,ȱ unnaturalȱ aminoȱ acidȱincorporationȱ andȱ macrocycleȱ backboneȱ extensionȱ yieldedȱpeptidesȱhavingȱhighȱaffinityȱandȱstability.ȱTheseȱstrategiesȱareȱnowȱroutinelyȱ usedȱinȱourȱlaboratoryȱforȱtheȱimprovementȱofȱphageȬselectedȱpeptides.ȱMyȱPhDȱworkȱ hasȱbeenȱdividedȱintoȱthreeȱparts,ȱandȱinȱthisȱlastȱchapterȱIȱwillȱsummarizeȱtheȱgeneralȱ conclusionsȱforȱeachȱofȱthem.ȱ %DFNERQHH[WHQVLRQRIDELF\FOLFSHSWLGH

Inȱaȱfirstȱproject,ȱIȱinvestigatedȱifȱinsertionȱofȱoneȱorȱtwoȱcarbonȱatomsȱinȱtheȱmacrocyclicȱ backboneȱofȱaȱbicyclicȱpeptideȱFXIIȱinhibitorȱcanȱimproveȱtheȱbindingȱaffinity.ȱTheȱstratȬ egyȱofȱinsertingȱatomsȱintoȱnaturallyȱderivedȱpeptidesȱhasȱbeenȱappliedȱpreviously,ȱbutȱ hasȱnotȱbeenȱusedȱsystematicallyȱtoȱimproveȱpropertiesȱofȱinȬvitroȬevolvedȱcyclicȱpepȬ tides.ȱWhenȱusingȱlibrariesȱcomposedȱofȱcanonicalȱaminoȱacids,ȱtheȱatomsȱinȱtheȱpeptideȱ backboneȱcanȱonlyȱbeȱvariedȱinȱblocksȱofȱthree.ȱIȱthereforeȱusedȱotherȱstrategiesȱtoȱinsertȱ oneȱorȱtwoȱatomsȱcarbonȱinȱtheȱmacrocyclicȱbackbone.ȱTheȱfirstȱapproachȱwasȱtoȱuseȱEȬ aminoȱacids,ȱwhichȱcontributeȱoneȱextraȱatomȱasȱcomparedȱtoȱnormalȱDȬaminoȱacids.ȱ Theȱsecondȱapproachȱwasȱtoȱreplaceȱtheȱcysteineȱresiduesȱusedȱforȱcyclizationȱbyȱsingleȱ orȱdoubleȱhomologatedȱanalogues,ȱhenceȱaddingȱoneȱorȱtwoȱextraȱcarbonȱatomsȱinȱtheȱ backbone.ȱWeȱhypothesizedȱthatȱextendingȱtheȱbackboneȱbyȱsingleȱorȱdoubleȱatomȱinȬ sertionȱ couldȱ leadȱ toȱ smallȱ conformationalȱ changes,ȱ andȱ thatȱ thisȱ inȱ turnȱ couldȱ strengthenȱexistingȱmolecularȱinteractionsȱorȱallowȱtheȱformationȱofȱnewȱnoncovalentȱ contactsȱwithȱtheȱtarget.ȱ

ToȱidentifyȱpositionsȱthatȱcouldȱbenefitȱfromȱatomȱinsertionȱweȱappliedȱaȱscanningȱstratȬ egyȱinȱwhichȱweȱsynthesizedȱtwoȱseriesȱofȱpeptides,ȱoneȱwhereȱeachȱaminoȱacidȱwasȱ replacedȱbyȱglycineȱandȱaȱsecondȱwhereȱeachȱaminoȱacidȱwasȱreplacedȱbyȱEȬalanine.ȱInȱ thisȱscan,ȱweȱwereȱableȱtoȱidentifyȱtwoȱpositions,ȱoccupiedȱbyȱPro6ȱandȱArg11,ȱinȱwhichȱ

141 ±*HQHUDOFRQFOXVLRQVDQGRXWORRN theȱaffinityȱlossȱwasȱsimilarȱinȱbothȱseriesȱandȱhenceȱwouldȱpotentiallyȱallowȱforȱatomȱ insertion.ȱWeȱthenȱsynthesizedȱpeptidesȱwithȱEȬanaloguesȱinȱtheseȱpositionsȱandȱidentiȬ fiedȱaȱpeptideȱwithȱ4.7ȬfoldȱimprovedȱaffinityȱuponȱchangingȱanȱDȱarginineȱresidueȱintoȱ oneȱofȱtheȱEȱarginineȱresidues.ȱTheȱhomologationȱofȱcysteineȱalsoȱresultedȱinȱaffinityȱ improvementȱinȱcertainȱpositions.ȱ

Thisȱworkȱshowsȱthatȱbyȱintroducingȱsmallȱchangesȱinȱtheȱbackboneȱofȱcyclicȱpeptides,ȱ theȱbindingȱaffinityȱcanȱbeȱincreasedȱsubstantially.ȱSinceȱthisȱchemicalȱspaceȱisȱnotȱsamȬ pledȱinȱlibrariesȱusingȱonlyȱcanonicalȱaminoȱacids,ȱthisȱstrategyȱoffersȱaȱsourceȱforȱimȬ provementȱofȱhitȱcompoundsȱfromȱsuchȱlibraries.ȱTheȱscanningȱapproachȱwithȱreplaceȬ mentȱofȱaminoȱacidsȱfirstȱwithȱglycineȱandȱEȬalanineȱprovidesȱaȱmethodȱforȱtheȱidentifiȬ cationȱofȱpositionsȱlikelyȱtoȱbenefitȱfromȱatomȱinsertion.ȱUsingȱthisȱapproachȱcanȱsaveȱ timeȱandȱmoneyȱsinceȱeveryȱresidueȱdoesȱnotȱhaveȱtoȱbeȱimmediatelyȱreplacedȱwithȱaȱsetȱ ofȱEȬaminoȱacidsȱ(thereȱareȱfourȱEȬaminoȱacidsȱforȱeachȱalphaȱaminoȱacid).ȱInȱaȱsecondȱ step,ȱweȱinsertedȱthenȱEȬaminoȱacidsȱcontainingȱsideȱchainsȱintoȱtheȱpromisingȱpositions.ȱ Itȱhasȱtoȱbeȱnoted,ȱthatȱweȱdidȱnotȱtestȱallȱtheȱpossibleȱfourȱEȬaminoȱacidsȱforȱeachȱofȱtheȱ twoȱ positions,ȱ dueȱ toȱtheȱ lackȱ ofȱ commercialȱ availability.ȱ Itȱ isȱ thereforeȱ possibleȱ thatȱ otherȱEȬanaloguesȱthanȱtheȱhereȱappliedȱ(S)ȬE3Ȭhomoarginineȱcouldȱaffectȱtheȱpropertiesȱ ofȱtheȱpeptideȱdifferently,ȱwithȱpotentiallyȱevenȱfurtherȱincreasedȱaffinityȱand/orȱstabilȬ ity.ȱȱȱȱ

FromȱourȱresultsȱitȱisȱnotȱentirelyȱclearȱhowȱtheȱatomȱinsertionȱinȱpositionȱArg11ȱcontribȬ utesȱtoȱtheȱaffinityȱimprovement.ȱSinceȱtheȱglycineȱandȱEȬalanineȱreplacedȱpeptidesȱhaveȱ similarȱaffinity,ȱweȱhypothesizeȱthatȱtheȱtargetȱinteractionsȱofȱtheȱarginineȱsideȱchainȱinȱ thisȱ positionȱ areȱ moreȱ favoredȱ forȱ theȱ EȬanalogue.ȱ However,ȱ theȱ backboneȱ extensionȱ couldȱpossiblyȱalsoȱintroduceȱmoreȱflexibilityȱtoȱtheȱmacrocycleȱandȱthusȱaffectȱtheȱinȬ teractionsȱofȱotherȱsideȬchains.ȱFinally,ȱweȱlaterȱdiscoveredȱthatȱtheȱsubstitutionȱofȱArg11ȱ toȱaȱEȬaminoȱacidȱlargelyȱcontributedȱtoȱtheȱincreasedȱstabilityȱofȱtheȱbicyclicȱpeptide.ȱ TheȱapproachȱofȱmacrocycleȱbackboneȱextensionȱfacilitatedȱbyȱaȱEȬalanineȱscanȱcanȱlikelyȱ beȱgenerallyȱappliedȱtoȱinȬvitroȬevolvedȱpeptidesȱwithȱtheȱpurposeȱofȱimprovingȱaffinityȱ andȱproteolyticȱstability.ȱ

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FXIIȱrepresentsȱaȱnovelȱtargetȱforȱtheȱdevelopmentȱofȱsafe,ȱbleedingȱfreeȱanticoagulants.ȱ Theȱdevelopmentȱofȱsuchȱanticoagulantsȱhasȱbeenȱchallenging.ȱFXIIȱhasȱbeenȱfoundȱtoȱ beȱinvolvedȱinȱexperimentalȱthrombosisȱbutȱnotȱinȱhemostasisȱandȱcanȱthusȱbeȱtargetedȱ forȱ thisȱ purpose.ȱ Severalȱ proteinȬbasedȱ inhibitorsȱ haveȱ recentlyȱ beenȱ developedȱ andȱ evaluated,ȱbutȱnoȱhighȬaffinityȱsmallȱmoleculeȱhasȱbeenȱreported.ȱInȱtheȱsecondȱprojectȱ ofȱmyȱPhDȱthesis,ȱIȱhaveȱcontinuedȱtheȱworkȱofȱtheȱfirstȱprojectȱwithȱtheȱobjectiveȱtoȱ optimizeȱaȱphageȱdisplayȬselectedȱpeptideȱmacrocycleȱFXIIȱinhibitorȱforȱinȱvivoȱevaluaȬ tionȱofȱselectiveȱFXIIȱinhibitionȱbyȱaȱsyntheticȱmolecule.ȱSubsequentlyȱIȱhaveȱdemonȬ stratedȱinȱvivoȱtheȱpeptide’sȱpotentialȱofȱprovidingȱsafeȱanticoagulation.ȱȱȱȱȱ

Inȱ theȱ phageȱ displayȱ selectionsȱ thatȱ wereȱ performedȱ toȱ identifyȱ theȱ FXIIȱ inhibitorȱ FXII618,ȱonlyȱtheȱsideȱchainsȱofȱtheȱ20ȱcanonicalȱaminoȱacidsȱwereȱsampled.ȱInȱthisȱpartȱ ofȱmyȱwork,ȱIȱhaveȱusedȱunnaturalȱaminoȱacidsȱcontainingȱdifferentȱsideȱchainsȱtoȱimȬ proveȱtheȱFXIIȱinhibitorȱlead.ȱIȱcombinedȱi)ȱaȱsubstitutionȱtoȱanȱunnaturalȱaminoȱacidȱ thatȱwasȱidentifiedȱbyȱaȱcolleague,ȱii)ȱaȱsubstitutionȱtoȱtheȱEȬarginineȱaminoȱacidȱthatȱIȱ identifiedȱinȱtheȱfirstȱproject,ȱandȱiii)ȱtwoȱsubstitutionsȱthatȱIȱfoundȱadditionally.ȱAnȱapȬ proachȱinȱwhichȱpeptidesȱwereȱsynthesizedȱwithȱaȱcoumarinȱtagȱallowedȱmeȱtoȱscreenȱ chemicallyȱcyclizedȱpeptidesȱinȱunpurifiedȱreactionȱmixtures.ȱThisȱmethodȱoffersȱaȱwayȱ toȱfacilitateȱtheȱscreeningȱofȱlargerȱlibrariesȱofȱchemicallyȱmodifiedȱpeptidesȱcontainingȱ unnaturalȱbuildingȱblocks,ȱorȱotherȱsyntheticȱcombinatorialȱlibraries,ȱwithoutȱprecedentȱ purification.ȱTheȱpeptideȱcombiningȱaȱtotalȱofȱfourȱaminoȱacidȱsubstitutionsȱinhibitedȱ theȱ intrinsicȱ coagulationȱ muchȱ moreȱ efficientlyȱ thanȱ theȱ leadȱ compoundȱ FXII618ȱ inȱ plasmaȱfromȱseveralȱspecies,ȱhighlightingȱtheȱneedȱforȱstrongȱaffinitiesȱforȱinhibitorsȱofȱ FXII.ȱ

Theȱpharmacokineticȱstudyȱinȱmice,ȱrabbitsȱandȱpigsȱshowedȱthatȱFXII900ȱcanȱbeȱappliedȱ intravenouslyȱorȱsubȬcutaneouslyȱandȱthatȱitȱinhibitsȱcoagulationȱinȱvivoȱinȱaȱconcentraȬ tionȱdependentȱmanner,ȱwithoutȱanyȱobservedȱsideȱeffectsȱsuchȱasȱgeneralȱtoxicityȱorȱ prolongedȱbleeding.ȱTheȱhalfȬlifeȱofȱtheȱpeptideȱisȱaroundȱthreeȱtimesȱlongerȱinȱpigsȱasȱ comparedȱtoȱrabbits,ȱwhichȱliesȱwithinȱtheȱexpectedȱrangeȱwhenȱtakingȱintoȱconsideraȬ tionȱallometricȱscalingȱofȱpharmacokineticȱparameters.ȱThisȱindicatesȱthatȱanȱevenȱlongerȱ halfȬlifeȱcanȱbeȱexpectedȱinȱhumans.ȱFromȱtheȱstudyȱinȱpigs,ȱaȱtendencyȱwasȱobservedȱ thatȱFXII900ȱaffectedȱaPTTȱstrongerȱthanȱheparin.ȱTheȱFXIIȱinhibitorȱcouldȱthusȱpotenȬ tiallyȱbeȱmoreȱefficientȱthanȱtheȱcurrentlyȱusedȱheparinȱinȱinhibitingȱundesiredȱbloodȱ coagulationȱ initiatedȱ byȱ artificialȱ surfacesȱ presentȱ inȱ variousȱ medicalȱ devices.ȱ Atȱ theȱ sameȱtime,ȱtheȱsideȱeffectsȱofȱbleedingȱcouldȱbeȱreduced.ȱ

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ThrombusȱformationȱwasȱefficientlyȱinhibitedȱinȱaȱFeCl3ȱmodelȱinȱmice,ȱwhichȱclearlyȱ demonstratesȱtheȱpotentȱanticoagulationȱpropertiesȱofȱFXII900.ȱTheȱpeptideȱcouldȱlikelyȱ beȱadministeredȱsubcutaneouslyȱinȱorderȱtoȱprovideȱimmediateȱanticoagulation.ȱTheȱreȬ ducedȱdeviceȱresistanceȱinȱartificialȱlungsȱwhenȱapplyingȱFXII900ȱshowsȱthatȱintrinsicȱ coagulationȱ contributesȱ toȱ theȱ cloggingȱ ofȱ theȱ apparatus.ȱ Theȱ peptideȱ didȱ notȱ affectȱ bleedingȱinȱrabbitsȱwhichȱfurtherȱemphasizesȱitsȱselectivityȱtowardsȱtheȱintrinsicȱcoaguȬ lationȱpathway.ȱSomewhatȱsurprising,ȱtheȱbleedingȱtimesȱareȱinsteadȱincreasedȱinȱtheȱ groupȱreceivingȱnoȱanticoagulants.ȱWeȱspeculateȱthatȱthisȱmayȱbeȱdueȱtoȱtheȱconsumpȬ tionȱofȱcoagulationȱfactorsȱrelatedȱtoȱtheȱclotȱformationȱinȱtheȱdevice.ȱTheseȱresultsȱshowȱ thatȱFXIIȱinhibitionȱbyȱaȱpeptideȱmacrocycleȱprovidesȱsafeȱanticoagulationȱwhenȱusedȱinȱ anȱartificialȱlungȱsystemȱandȱisȱlikelyȱtoȱprolongȱtheȱfunctionalityȱofȱtheȱdeviceȱinȱaȱclinȬ icalȱsituation.ȱ

Theȱfieldȱofȱanticoagulationȱdrugsȱhasȱtraditionallyȱbeenȱrelativelyȱconservative,ȱwithȱ drugsȱsuchȱasȱheparinȱorȱwarfarinȱbeingȱusedȱforȱdecades.ȱHowever,ȱthereȱhasȱbeenȱanȱ unmetȱmedicalȱneedȱforȱanticoagulantsȱwithȱreducedȱbleedingȱrisk.ȱDuringȱtheȱlastȱdecȬ adeȱthisȱhasȱbeenȱtargeted,ȱforȱexample,ȱbyȱtheȱdevelopmentȱofȱtheȱnovelȱoralȱanticoagȬ ulantsȱ(NOAC).ȱTheseȱdrugsȱwereȱintroducedȱwithȱtheȱpurposeȱofȱprovidingȱsaferȱalterȬ nativesȱtoȱtheȱwidelyȱusedȱvitaminȱKȱantagonists,ȱmainlyȱbyȱhavingȱreducedȱintraȬȱandȱ interpatientȱ variability,ȱ requiringȱ lessȱ frequentȱ monitoringȱ andȱ havingȱ insignificantȱ foodȬdrugȱinteractionsȱ(320,ȱ321).ȱTheȱNOACsȱdifferȱfromȱvitaminȱKȱantagonistsȱmechȬ anisticallyȱbyȱdirectlyȱinhibitingȱcoagulationȱfactorsȱ(FXȱorȱthrombin)ȱinsteadȱofȱtheirȱ biosynthesis.ȱWhileȱprovidingȱsomeȱbenefits,ȱtheȱnewȱanticoagulantsȱstillȱinduceȱanȱinȬ creasedȱbleedingȱrisk,ȱwhichȱcanȱhaveȱsevereȱconsequencesȱforȱpatientsȱ(322–324).ȱȱȱ

Severalȱrecentȱstudiesȱhaveȱbeenȱevaluatingȱnewȱstrategiesȱforȱsaferȱanticoagulationȱinȱ variousȱclinicalȱsituations,ȱsuggestingȱaȱnew,ȱopenȬmindedȱwayȱofȱthinkingȱinȱtheȱfield.ȱ Oneȱsuchȱstudyȱanalyzedȱwhetherȱbivalirudin,ȱaȱdirectȱthrombinȱinhibitorȱapprovedȱinȱ 2000,ȱcouldȱreduceȱmajorȱbleedingȱeventsȱundergoingȱpercutaneousȱcoronaryȱintervenȬ tionȱ(PCI)ȱ(325).ȱDespiteȱpreviousȱstudiesȱindicatingȱreducedȱbleedingȱwithȱtheȱuseȱofȱ bivalirudin,ȱnoȱsignificantȱdifferenceȱcouldȱbeȱdetectedȱamongȱtheseȱparametersȱbetweenȱ theȱtwoȱgroups.ȱInȱanotherȱstudyȱtheȱdirectȱthrombinȱinhibitorȱDabigatranȱwasȱfoundȱtoȱ significantlyȱlowerȱtheȱbleedingȱriskȱwhenȱreplacingȱwarfarinȱandȱaspirinȱinȱaȱcombinaȬ tionȱtherapyȱwithȱaȱP2Y12ȱinhibitorȱ(326).ȱTheȱNOACȱrivaroxabanȱwasȱevaluatedȱeitherȱ aloneȱorȱasȱdualȱtherapyȱwithȱaspirinȱvsȱaspirinȱalone.ȱItȱwasȱfoundȱthatȱrivaroxabanȱwasȱ moreȱeffectiveȱthanȱaspirinȱforȱtheȱpreventionȱofȱrecurrentȱvenousȱthromboembolismȱ withȱaȱsimilarȱbleedingȱriskȱ(327).ȱRivaroxabanȱwasȱalsoȱmoreȱeffectiveȱinȱcombinationȱ withȱaspirin,ȱthanȱaspirinȱaloneȱinȱpreventingȱmajorȱadverseȱcardiovascularȱevents,ȱhowȬ everȱ withȱ aȱ significantlyȱ increasedȱbleedingȱ riskȱ (328).ȱ Severalȱ effortsȱhaveȱ alsoȱ beenȱ doneȱtoȱcombatȱtheȱbleedingȱsideȬeffectsȱofȱtheȱNOACs,ȱsuchȱasȱdevelopmentȱofȱFXȱdeȬ coyȱproteinsȱorȱdrugȱtargetingȱantibodiesȱ(322,ȱ329).ȱGivenȱtheȱwillingnessȱofȱtestingȱnewȱ

144 ±*HQHUDOFRQFOXVLRQVDQGRXWORRN anticoagulantȱstrategiesȱandȱtheȱconcernsȱaboutȱbleeding,ȱweȱbelieveȱthatȱaȱselectiveȱFXIIȱ inhibitorȱsuchȱasȱFXII900ȱisȱaȱstrongȱcandidateȱforȱclinicalȱevaluationȱwithȱtheȱpotentialȱ ofȱprovidingȱgreatȱbenefits,ȱespeciallyȱinȱsituationsȱwhereȱrapidȱandȱrelativelyȱshortȱanȬ ticoagulationȱisȱrequiredȱsuchȱasȱinȱPCIȱorȱCPB.ȱ

Anotherȱindicationȱwhereȱtheȱbenefitsȱofȱanticoagulationȱisȱsomewhatȱmoreȱunclearȱisȱ acuteȱischemicȱstrokeȱ(AIS).ȱTheȱgoldȱstandardȱtreatmentȱinvolvesȱthrombolysisȱviaȱtheȱ administrationȱofȱrecombinantȱtissueȱplasminogenȱactivatorȱ(330).ȱInȱaȱsystematicȱreviewȱ aȱcorrelationȱwasȱfoundȱbetweenȱearlyȱanticoagulantȱadministrationȱandȱaȱreducedȱreȬ currenceȱofȱischemicȱstrokeȱ(331).ȱHowever,ȱanticoagulationȱisȱinȱgeneralȱcontraindicatedȱ dueȱtoȱtheȱveryȱhighȱriskȱofȱfatalȱhemorrhage.ȱTakenȱtogetherȱthisȱindicatesȱthatȱbleedingȱ freeȱanticoagulants,ȱsuchȱasȱFXIIȱinhibitorsȱcouldȱprovideȱadvantagesȱinȱtheȱtreatmentȱofȱ AIS.ȱ

Ourȱstudyȱhasȱhoweverȱseveralȱlimitations.ȱEvenȱthoughȱweȱhaveȱfoundȱthatȱFXII900ȱ inhibitsȱthrombosisȱinȱtwoȱdifferentȱanimalȱmodels,ȱtheseȱsituationsȱareȱtoȱvariousȱextentȱ artificial.ȱAlthoughȱtheȱFeCl3ȱmodelȱisȱwidelyȱappliedȱtoȱinvestigateȱtheȱeffectȱofȱanticoȬ agulationȱand/orȱcoagulationȱfactors,ȱitsȱmechanismȱofȱactionȱisȱnotȱwellȱunderstood.ȱ Therefore,ȱitȱisȱdifficultȱtoȱdetermineȱhowȱwellȱthisȱmodelȱrepresentsȱpathologicalȱthromȬ busȱformation.ȱAsȱaȱconsequence,ȱitȱisȱhardȱtoȱestimateȱtheȱthrombusȱpreventingȱpropȬ ertiesȱofȱaȱcompoundȱsolelyȱevaluatedȱwithȱthisȱmodel.ȱTheȱartificialȱlungȱstudyȱrepreȬ sentsȱwellȱtheȱclinicalȱsettingȱofȱthisȱdevice.ȱHowever,ȱinȱorderȱtoȱbetterȱpredictȱtheȱpoȬ tentialȱofȱFXII900ȱaȱlongerȱstudyȱwouldȱhaveȱbeenȱdesired,ȱsinceȱtheȱdevicesȱwillȱbeȱusedȱ forȱlongerȱtimeȱperiods.ȱInȱaddition,ȱitȱisȱnotȱclearȱhowȱwellȱtheȱrabbitsȱandȱsmallȱsizeȱ artificialȱlungsȱresembleȱtheȱlargerȱdeviceȱwhenȱactuallyȱusedȱinȱhumans.ȱȱ

Theȱworkȱinȱthisȱprojectȱhasȱfurtherȱdemonstratedȱtheȱextentȱofȱimprovementsȱthatȱareȱ possibleȱtoȱachieveȱwhenȱunnaturalȱaminoȱacidsȱareȱappliedȱtoȱbicyclicȱpeptideȱisolatedȱ byȱphageȱdisplay.ȱSeveralȱofȱtheȱstrategiesȱappliedȱhereȱareȱnowȱusedȱinȱourȱlaboratoryȱ forȱtheȱimprovementȱofȱpeptides.ȱManyȱofȱtheȱcharacterizationȱassaysȱandȱmodelsȱhaveȱ beenȱestablishedȱbyȱmeȱinȱourȱlaboratoryȱandȱareȱnowȱusedȱalsoȱforȱotherȱprojects.ȱFiȬ nally,ȱFXII900ȱwasȱpatentedȱandȱanȱoptionȱtoȱaȱlicenseȱwasȱrecentlyȱnegotiatedȱwithȱaȱ pharmaceuticalȱcompanyȱwhoȱaimsȱtoȱdevelopȱtheȱinhibitorȱintoȱaȱdrug.ȱȱȱ

ȱȱ

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InȱtheȱthirdȱprojectȱofȱmyȱPhDȱthesis,ȱmyȱobjectiveȱwasȱtoȱimproveȱtheȱaffinityȱandȱstaȬ bilityȱofȱaȱphageȬselectedȱbicyclicȱpeptideȱMMPȬ2ȱinhibitor.ȱWithȱtheȱmainȱobjectiveȱbeȬ ingȱstabilityȱimprovement,ȱIȱhadȱperformedȱaȱDȬalanineȱscanȱinȱorderȱtoȱidentifyȱsitesȱforȱ possibleȱstabilityȱenhancingȱmodifications.ȱAȱproteolyticȱcleavageȱsiteȱcouldȱbeȱidentiȬ fiedȱandȱreplacedȱbyȱaȱ DȬaminoȱacid,ȱresultingȱinȱanȱimprovedȱstability.ȱAtȱtheȱsameȱ time,ȱaȱglycineȱresidueȱcouldȱalsoȱbeȱreplacedȱbyȱaȱDȬaminoȱacidȱforȱaffinityȱimproveȬ ment.ȱReplacingȱglycineȱwithȱaȱDȬaminoȱacidȱwasȱfoundȱtoȱbeforeȱinȱourȱlabȱtoȱbeȱaȱpromȬ isingȱstrategyȱtoȱimproveȱbindingȱaffinityȱ(191).ȱInȱtheȱcaseȱofȱtheȱMMPȬ2ȱinhibitor,ȱnoȱ crystalȱstructureȱwasȱobtained,ȱbutȱitȱisȱtemptingȱtoȱassumeȱthatȱalsoȱtheȱglycineȱinȱthisȱ peptideȱhasȱaȱpositiveȱIȱdihedralȱangleȱandȱthereforeȱbenefitsȱfromȱtheȱintroductionȱofȱaȱ DȬaminoȱacid.ȱ

GivenȱtheȱchallengeȱofȱachievingȱhighȱselectivityȱforȱMMPȱinhibitors,ȱIȱperformedȱanȱ extensiveȱanalysisȱandȱspecificityȱscreeningȱofȱtheȱbicyclicȱpeptideȱMMPȬ2ȱinhibitor.ȱTheȱ experimentsȱshowedȱthatȱinȱadditionȱtoȱMMPȬ2,ȱalsoȱMMPȬ3ȱwasȱinhibitedȱbyȱtheȱpepȬ tide.ȱThisȱisȱsomewhatȱsurprising,ȱsinceȱpreviousȱMMPȬ2ȱinhibitorsȱhaveȱbeenȱspecificȱ forȱ MMPȬ2ȱ overȱ MMPȬ3ȱ butȱ haveȱ alsoȱ beenȱ inhibitingȱ MMPȬ9.ȱ Theȱ specificityȱ couldȱ howeverȱbeȱincreasedȱbyȱaȱlimitedȱnumberȱofȱaminoȱacidȱsubstitutions.ȱItȱisȱthereforeȱ likelyȱthatȱaȱfullyȱspecificȱinhibitorȱcanȱbeȱdevelopedȱbyȱfurtherȱutilizingȱthisȱstrategy.ȱ Anotherȱapproachȱinȱorderȱtoȱyieldȱaȱmoreȱspecificȱinhibitorȱcouldȱbeȱtoȱchangeȱtheȱringȱ sizesȱofȱtheȱbicyclicȱpeptideȱorȱbyȱaddingȱexocyclicȱresiduesȱatȱeitherȱterminusȱofȱtheȱ peptide.ȱ

TheȱuseȱofȱMMPȱinhibitorsȱasȱantiȬcancerȱtherapeuticsȱremainsȱcontroversial.ȱInhibitorsȱ ofȱMMPsȱwereȱextensivelyȱdevelopedȱandȱevaluatedȱinȱclinicalȱstudiesȱinȱtheȱ1990sȱandȱ 2000s,ȱbutȱallȱcompoundsȱfailedȱinȱclinicalȱtrials.ȱTheȱfailuresȱareȱoftenȱexplainedȱbyȱaȱ lackȱofȱspecificityȱofȱtheȱinhibitors,ȱbutȱinȱadditionȱtheȱroleȱofȱMMPsȱinȱvariousȱdiseasesȱ isȱnotȱfullyȱunderstood.ȱItȱhasȱbeenȱpostulatedȱthatȱsomeȱMMPsȱareȱinȱfactȱtargetsȱinȱ someȱdiseasesȱbutȱantiȬtargetsȱinȱothers.ȱTheȱmultifacetedȱrolesȱofȱMMPsȱtogetherȱwithȱ theirȱhighȱsimilarityȱandȱsubstrateȱoverlapsȱmakeȱthemȱveryȱchallengingȱtherapeuticȱtarȬ gets.ȱAȱhighȱaffinity,ȱstableȱandȱspecificȱMMPȱinhibitor,ȱsuchȱasȱtheȱpeptideȱdevelopedȱ inȱthisȱstudy,ȱcouldȱhelpȱtoȱshineȱlightȱonȱtheȱfunctionsȱofȱtheȱdifferentȱMMPsȱinȱvariousȱ diseases.ȱAȱbetterȱunderstandingȱofȱtheȱroleȱofȱindividualȱMMPsȱmayȱtriggerȱnewȱMMPȱ inhibitorȱprogramsȱinȱtheȱfuture.ȱ

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