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RelationshipsbetweenInhibitionConstants,TypesofInhibitionandIC50CalculationandComparisononIC50ofTwoModelTyrosinaseInhibitorsHairongMao1,JiangangXie1,QimengZhang2,XinyuLü3,ShubaiLi2,1DepartmentofChemistry,ZhengzhouTeachersCollege,Zhengzhou,China2DepartmentofChemicalEngineeringTechnology,ChangzhouInstituteofEngineeringTechnology,Changzhou,China3InstituteofFineChemicals,JiangsuPolytechnicUniversity,Changzhou,ChinaCorrespondingauthor.Emailsbliemail.czie.netAbstractCalculatingmethodsforinhibitorconcentrationIC50leadingto50activitylostfordiphenolaseactivityofanovelparaboliccompetitiveinhibitorthetrifluoromethylcontaining1,2,3triazole,TFTAandanovelmixedinhibitorE41imidazoylmethylcinnamicacidozagrelwerederivedonthebasisofkineticsoftyrosinaseinhibition.ThesemethodswereappliedtocalculateIC50valuesofthetwotyrosinaseinhibitingcompounds.TheexperimentalIC50valuesofTFTAandozagrelwere41.9μMand3.45mM,respectiviely.Moreover,thecalculatedIC50valueofTFTAandozagrelwere82.8μMand3.15mM,respectively.Thecalculationresultofozagrelisclosertoexperimentaldata.KeywordstyrosinaseIC50trifluoromethylcontaining1,2,3triazoleTFTAE41imidazoylmethylcinnamicacidozagrelI.INTRODUCTIONTheregulationofenzymeactivityisofgreatinterestforbiochemicalresearchpurposesandforanumberofapplicationsinmedicine,physiology,andpharmacology.Inhibitionofenzymesfromanybiologicalsourcecanbeachievedbyseveralagents,suchasselfinactivatingsubstratesandreversibleorirreversibleinhibitors.TyrosinaseEC1.14.18.1isacoppercontainingenzymewithresponsibilitiesforskin,hair,melanizationandenzymaticbrowninginfruitsandvegetables13.Itcatalyzesthehydroxylationofmonophenolsmonophenolaseactivityandtheoxidationofodiphenolstooquinonesdiphenolaseactivity,bothdependingonmolecularoxygen4,5.Thebrowningisresponsibleforlossinnutritionalquality,andthereforebecomesamajorprobleminthefoodindustry.Therefore,thecontrolofthetyrosinaseactivityisofimportanceinpreventingthesynthesisofmelanininthebrowningofmushroomsandothervegetablesandfruits.Manyeffortshavebeenputinthesearchforfeasibleandeffectivetyrosinaseinhibitors69.Inoursearchfortyrosinasenovelinhibitors,trifluoromethylcontaining1,2,3triazoles10,andozagrelanalogues11,werefoundtohaveobviousinhibitoryeffectsonthemonophenolaseactivityandthediphenolaseactivityofmushroomtyrosinase.TheIC50inhibitorconcentrationleadingto50activitylostisameasureoftheeffectivenessofacompoundininhibitingbiologicalorbiochemicalfunction.Often,thecompoundinquestionisadrugcandidate.Thisquantitativemeasureindicateshowmuchofaparticulardrugorotherinhibitorsneededtoinhibitagivenbiologicalprocessbyhalf.Itiscommonlyusedasameasureofantagonistdrugpotencyinpharmacologicalresearch.However,itappearsnottohavebeennoticedpreviouslythatthereisaverysimplerelationshipbetweenIC50withinhibitionconstantsandtypesofinhibitiontype.Therefore,theaimofthepresentworkwastodevelopamethodtocalculateIC50oftwonoveltyrosinaseinhibitors.II.CALCULATIONANDCOMPARISONONIC50A.CalculationofIC50fortheParaboliccompetitiveInhibitorThetrifluoromethylcontaining1,2,3triazolesexhibitantimicrobial,antiviralandantitumoractivitiesandhavearangeofimportantapplicationsinpharmaceuticalandagrochemicalindustries12.NNNF3COHFig.1.Structureofthetrifluoromethylcontaining1,2,3triazoleTFTA.Wefoundanoveltrifluoromethylcontaining1,2,3triazoleTFTA,seestructureinFig.1caninhibitmushroomtyrosinaseactivityIC5041.9μMwithitsincreasingconcentrations.Theinhibitionkinetics,analyzedbyLineweaverBurkplots,indicatedTFTAtobeaparaboliccompetitiveinhibitorofdiphenolasewhenLDOPAwasusedassubstrate.ItissuggestedthattwoTFTAmoleculescancombinewithfreetyrosinasetoformadeadendcomplexatenzymeactivesite.TheinhibitionconstantsKi1forinhibitorenzymecomplexandKi2forinhibitorenzymeinhibitor9781424447138/10/25.00©2010IEEEcomplexwereestimatedtobe92.9mMand1.36mM,respectively.ThemechanismrepresentingparaboliccompetitiveinhibitionforthetyrosinasecatalyzedreactionisasfollowsESESEPk1k2k1k3k3k4IIEIk4EI2IIE,S,I,Pdenoteenzymemushroomtyrosinase,substrateLDOPA,inhibitorTFTA,andproductdopachrome,respectivelyES,EI,andEI2aretherespectivecompounds.AsitisusuallythecasethatS»ETandI»ET.Inthesteadystate,therateofformationanddisappearanceofthecomplexesareidenticalESES211kkk−1.133IEEIkk−1.2EIIEI244−kk1.3TheexpressionsforthetotalenzymeconcentrationandthereactionrateEIEIESEE2T1.4ES2kV1.5InsertionoftheexpressionsforE,EI,andEI2intermofES,intotheexpressionforthetotalenzymeconcentrationgivesESI1IS1SE4433121121T⎥⎦⎤⎢⎣⎡⋅−−−−kkkkkkkkkk1.6I1ISSE4433121121T2−−−−⋅kkkkkkkkkkkV1.7Becausek1k2/k1istheMichaelisconstantKmforthereactionintheabsenceofinhibitor,k3/k3istheequilibriumconstantKi1forthedissociationofthecomplexEIintoEandI,andk4/k4istheequilibriumconstantKi2forthedissociationofthecomplexEI2intoEIandI,therateequationmaybewrittenintheformII1SSi2i12i1mmaxKKKKVV1.8whichinreciprocalforbecomesmaxi2i12i1maxm1II1S1VKKKVKV⋅1.9ItisevidentfromEq.1.9thattheslopeofLineweaverBurklinesisinfluencedbythechangingIandI2.AccordingtoEq.1.8,intheabsenceofTFTZ,thekineticfunctionisSSmmax0KVV1.10Theinhibitionratio01100ViV−SSII1SS1mmaxi2i12i1mmax−KVKKKKV1.11Whenireaches50,IIC50,so,II1SS21i2i12i1mmKKKKK1.12TheIC50valuethenegativevalueshouldberemovedismm2mmi1i1i1i250mi1i24SIC2KKKKKKKKKKK1.13AccordingtotheexperimentconditionsandEq.1.13Km0.117mM,Ki192.9mM,Ki21.36mM,andS1.0mM,sothecalculatedIC50valueofistobe82.8μM.Then,accordingtoFig.5,theexperimentalIC50valueis41.9μM.Inthisinvestigation,thecalculatedIC50valueisabouttwotimesthatoftheexperimentalone.B.CalculationofIC50fortheMixedInhibitorOzagrelE41imidazoylmethylcinnamicacid,seestructureinFig.2,asaselectiveinhibitorofthromboxanesynthaseTXASinhumanplateletsespecially,isusedinthetreatmentandpreventionofvariousthromboticdiseases13.Wefoundthatozagrelinhibitedmushroomtyrosinaseactivity.TheIC50valuewas3.45mM.OzagrelwasestimatedtobeareversiblemixedtypeinhibitorofdiphenolaseactivitywhenLDOPAwasusedassubstratewiththeconstantsKS1,KS2,Ki1,andKi2determinedtobe2.21,3.89,0.454,0.799mM,repectively.Theinhibitionkinetics,analyzedbyLineweaverBurkplots,indicatedozagreltobeamixedinhibitorofdiphenolasewhenLDOPAwasusedassubstrate.Itdemonstratedthatozagrelboundtheenzymeatasitedistinctedfromthesubstrateactivesite,butitboundtoeitherEorES.Fig.2.StructureofE41imidazoylmethylcinnamicacidozagrel.Inthistypeofreversibleinhibition,ozagrelcaninteractwithboththefreeenzymetyrosinaseandtheenzymesubstratecomplexatasiteotherthantheactivesitewithaclassicalMichaelisMentenreactionmechanismESESEPkcatEISIIESIKi1KS1KS2Ki2Here,E,S,I,Pdenoteenzymemushroomtyrosinase,substrateLDOPA,inhibitorozagrel,andproductdopachrome,respectivelyES,EI,andESIaretherespectivecompounds.AsitisusuallythecasethatS»E0andI»E0.ItisassumedthattheeffectisnotonlyonaffinitybutalsoontherateofthebreakdownoftheEScomplexthusozagrelwouldinfluencetheenzymeactivityintwoways,affectingVmaswellasKm.ItisformallyconvenienttoregardKS1,KS2,Ki1andKi2asthedissociationconstantsoftherespectivecomplexesESintoEandS,ESIintoEIandS,EIintoEandI,andESIintoESandI,respectively.IntheMichaelisMentenstate,thesubstratebindingstepandformationoftheEScomplexarefastrelativetothebreakdownrate.Asistheusuallythecase,theequilibriumequationsoftheenzyme,substrate,inhibitor,product,andtheirrespectivecompoundsES,EI,ESIaregivenS1ESESK2.1i1EIEIK2.2S2EISESIK2.3i2ESIESIK2.4AccordingtoEq.2.1toEq.2.4,therelationshipwithKS1,KS2,Ki1andKi2isgivenS1i1S2i2KKKK2.5TheexpressionsforthetotalenzymeconcentrationandthereactionrateTEEESEIESI2.6TheoverallvelocitywillbegivenasfollowscatESVk2.7InsertionoftheexpressionsforE,EI,andESIintermofES,intotheexpressionforthetotalenzymeconcentrationgivesS1S1Ti1i2IIE1ESSSKKKK2.8catTS1i1i2ESII1S1kVKKK2.9TherateequationmaybewrittenintheformmaxS1i1i2SII1S1VVKKK2.10whichinreciprocalformbecomesS1i1i2maxmaxII1111SKKKVVV⋅2.11ItisevidentfromEquation11thatthemixedtypeinhibitionaffectsbothKmandVmaxitsspecialcaseisnoncompetitiveinhibitionwhichwouldaffecttheVmaxbutnottheKm.So,theslopeandinterceptofEquation11aregivenasthefollowingformS1i1maxI1SlopeKKV2.12i2maxI1InterceptKV2.13Forthesteadystatemodel,substratebindingoccursfasterthanthebreakdownoftheEScomplex,sothekineticfunctionintheabsenceofinhibitorismax0S1SSVVK2.14Theinhibitionratio01100ViV−maxS1i1i2maxS1SII1S11SSVKKKVK−2.15Whenireaches50,IIC50,so,S15050S1i1i2S11ICIC21S1KKKK−2.16TheIC50valueisS150S1i2i1SICSKKKK2.17AccordingtoEq.2.17andKS1,KS2,Ki1,andKi2,thecalculatedIC50is3.15mM,itisveryclosetotheexperimentaldataofIC503.45mM.III.CONCLUSIONTheIC50valuesoftwonoveltyrosinaseinhibitorswerecalculatedandcompared.ThecalculatedIC50valueofozagreliscloserthanitsexperimentaldata.Theresultssofarobtainedindicatedthatthefurtherassayisneeded,fromnotonlyoneaspect,butfromawholeperspective.REFERENCES1L.Yu,InhibitoryeffectsofSandR6hydroxy2,5,7,8tetramethylchroman2carboxylicacidontyrosinaseactivity,J.Agr.FoodChem.vol51,pp.23442347,2003.2Y.J.Kim,J.E.Chung,M.Kurisawa,H.Uyama,andS.Kobayashi,Newtyrosinaseinhibitors,catechinaldehydepolycondensates,Biomacromolecules.vol.5,pp.474479,2004.3M.Jiménez,S.Chazarra,J.Escribano,J.Cabanes,andF.GarcíaCarmona,Competitiveinhibitionoftyrosinaseby4substitutedbenzaldehydes,J.Agr.FoodChem.vol.49,pp.40604063,2001.4F.G.Molina,J.L.Muňoz,R.Varón,J.N.RodríguezLópez,F.GarcíaCánovas,andJ.Tudela,Anapproximateanalyticalsolutiontothelagperiodofmonophenolaseactivityoftyrosinase,Int.J.Biochem.CellBiol.vol.39,pp.238252,2007.5L.G.Fenoll,M.J.Peňalver,J.N.RodríguezLópez,R.Varón,F.GarcíaCánovas,J.Tudela,Tyrosinasekineticsdiscriminationbetweentwomodelstoexplaintheoxidationmechanismofmonophenolanddiphenolsubstrates,Int.J.Biochem.CellBiol.vol.36,pp.235246,2004.6J.C.EspínandH.J.Wichers,KineticsofactivationoflatentmushroomAgaricusbisporustyrosinasebybenzylalcohol,J.Agr.FoodChem.vol.47,pp.35033508,1999.7I.Kubo,I.KinstHori,Y.Kubo,Y.Yamagiwa,T.Kamikawa,andH.Haraguchi,Moleculardesignofantibrowningagents,J.Agr.FoodChem.vol.48,pp.13931399,2000.8L.G.Fenoll,P.A.GarcíRuiz,R.Varón,andF.GarcíaCánovas,Kineticsstudyofoxidationofquercetinbymushroomtyrosinase,J.Agr.FoodChem.vol.51,pp.77817787,2003.9J.J.Xie,K.K.Song,L.Qiu,Q.He,H.Huang,andQ.X.Chen,Inhibitoryeffectsofsubstrateanalogu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