流感病毒药物

egAvailableonlineatwww.sciencedirect.comir(HA)andneuraminidase(NA).InfluenzaBviruseshavedivergedintotwomajorantigeniclineagesinthe1980s(Victoria-likeandYamagata-like).ViralentryrequiresattachmentoftheHAproteintoterminalsialicacidresidueslinkedtogalactoseviaa2,3-linkagesora2,6-linkages[1]followedbyclathrin-dependentendocytosisandmacropinocytosis[2].UnderacidicpH,theHAproteinundergoesconformationalchangestoexposethefusionpeptide,whichmediatesfusionbetweentheM2ionchannelblockersAdamantanederivatives,amantadineandrimantadine(Figure1)inhibittheprotonconductivityoftheM2ionchannelofinfluenzaAvirusesbutpossessnoinhibi-toryeffectfortheionchannelsofinfluenzaBviruses,astheAM2andBM2arefunctionalhomologswithoutapparentsequencehomology[8].InhibitionoftheM2ionchannelactivitypreventsuncoatingoftheinfluenzaAvirusesattheearlyphaseoftheviralreplicationandmayCurrentOpinioninVirology2016,18:126–134www.sciencedirect.comCurrentandnovelantiviralstratinfectionHui-LingYenInfluenzaAandBvirusesaremajorcausesforrespiratoryinfectionsinchildrenandadults.Viralandhostfactorsdetermineclinicalmanifestationswhichrangefromself-resolvinguncomplicatedinfections,severeviralorbacterialsecondarypneumonia,todeath.Emergenceoftransmissibleresistantvariantsandtime-dependenteffectivenessarethemajorchallengesforthecurrentlyapprovedantivirals,M2ionchannelblockersandneuraminidase(NA)inhibitors.FavipiravirthatinhibitstheRNA-dependentRNApolymeraseofmultipleRNAvirusesisapprovedinJapanagainstinfluenzastrainsresistanttoavailableantivirals.Withexpandedknowledgeonviralnucleoprotein(NP)andpolymerasestructures,novelsmallmoleculeinhibitorstargetingNPoligomerformation,PAendonucleasedomain,andthePB2cap-bindingdomainarebeingdeveloped.Combinationtherapywithdifferentantiviralcompoundsorwithhostimmuneresponsemodulatorsmayfurtherbenefitclinicaloutcomes.AddressSchoolofPublicHealth,LiKaShingFacultyofMedicine,TheUniversityofHongKong,HongKongSpecialAdministrativeRegionCorrespondingauthor:Yen,Hui-Ling(hyen@hku.hku)CurrentOpinioninVirology2016,18:126–134ThisreviewcomesfromathemedissueonAntiviralstrategiesEditedbyRaymondFSchinaziandErikdeClercqForacompleteoverviewseetheIssueandtheEditorialAvailableonline23rdJune2016http://dx.doi.org/10.1016/j.coviro.2016.05.0041879-6257/PublishedbyElsevierB.V.IntroductionInfluenzaAandBvirusesaremembersoftheOrthomyx-oviridaewitheightsegmentedsingle-strandednegative-senseRNAgenomes.InfluenzaAvirusesaregroupedintosubtypesbythesurfaceglycoproteinshemagglutininScienceDiesforinfluenzaviralenvelopeandendosomemembranetoreleaseviralribonucleoproteins(RNPs)[3].TheM2ionchannelsmediateprotonflowfromtheendosomeintotheinterioroftheviriontodissociateRNPsfromtheM1matrixproteins.TheRNPs,consistingofhetero-trimericinflu-enzapolymeraseproteins(PB2,PB1,andPA)andtheviralRNAgenomeencapsidatedbythenucleoprotein(NP),aretransportedintothenucleusforviralmRNAsynthesisandgenomereplication[4]mediatedbytheRNA-dependentRNApolymerasePB1protein.Thecap-bindingactivityofthePB2proteinandtheendonucleaseactivityofthePAproteinarecriticalforcapsnatchingandmRNAsynthesis.ThemRNAsaretransportedtothecytoplasmforproteinsynthesis.TheHA,NAandM2proteinsaresynthesizedattheendoplasmicreticulumandaretransportedviathetrans-Golginetworktotheapicalcellmembrane.TheM1matrixandthenucleusexportprotein(NEP)mediatenucleusexportationoftheRNPstothecytoplasmfollowedbyfinalassemblyandbuddingfromtheapicalcellmembrane[5].ViralreleaseandspreadintherespiratorytractarefacilitatedbythesialidaseactivityoftheNAproteins.Clinicalmanifestationsofinfluenzainfectionsmayrangefromself-resolvinguncomplicatedinfectionstocompli-cationsinthehigh-riskpopulations.Amongpatientswithsevereinfections,highviralloadsandelevatedpro-in-flammatorycytokinesaccompaniedbyincreasedimmunecellinfiltrationscanbeobserved,suggestingthattheclinicaloutcomeisdeterminedbycomplexviralandhostdeterminants.Overall,theannualepidemicsandperiodicglobalpandemicscausedbyinfluenzavirusesmayleadto250000–500000to30–50millionglobalmortality,re-spectively[6��,7].Effectivepharmaceuticalinterventionsarethereforeindispensable.Todate,threedifferentclassesofantiviralcompoundsthateachtargettheM2ionchannel,NAenzymesite,andtheRNA-dependentRNApolymerasehavebeenapprovedforclinicaluse.EfficacyandresistanceofcurrentlyapprovedantiviralcompoundsectAntiviralstrategiesforinfluenzaYen127allowthelowpHtotriggerconformationalchangeofthenewlysynthesizedHAatthetrans-Golginetwork.InadditiontothepotentialCNSsideeffects,fullytransmis-sibleresistancevariantsemergerapidlyfrompatientsaftertreatment[9].SurveillancestudiesreportedasteadyincreaseoftheresistantvariantsamongtheseasonalH3N2virusessince2000whichreached90.6%amongglobalH3N2isolatesin2005–2006[10,11],althoughamantadineandrimantadinewerestilleffectiveagainstthemajorityoftheseasonalH1N1virus.Since2009,amantadineandrimantadinearenotrecommendedforclinicaluseduetotheemergenceofthenewpandemicH1N1(pdmH1N1)virus,whichnaturallycarriestheM2-S31Nmutationthatconfersresistancetobothdrugs[12].Systematicreviewsuggestslimitedbenefitofamatandineandrimantadineinpreventing,treating,andshorteningthedurationofinfluenzaAinfectioninchildrenandtheelderly[13].Figure1NH2NH2AmantadineRimantadineCurrentOpinioninVirologyChemicalstructuresofamantadineandrimantadine.AmantadineandrimantadineblockprotonflowthroughM2ionchannelsofinfluenzaAviruses.NAinhibitorsTheNAofinfluenzaAandBvirusesshare30%aminoacidsequencehomology[14]andarehomo-tetramersof240kDawithsialidaseactivityatacidicpH(5.5–6.5)[15].PhylogeneticanalysisfurtherseparatestheNAofinflu-enzaAvirusesintoGroup1(N1,N4,N5,N8)andGroup2(N2,N3,N6,N7,N9)[16].Theenzymesitessharesimilarstructureandconservedresiduesasfoundinotherviralandbacterialsialidases(R118,D151,R152,R224,E276,R292,R371,andY406,N2numbering).Addition-ally,influenzaAandBvirusesshareconservedframeworkresidues(E119,R156,W178,S179,D/N198,I222,E227,H274,E277,N294,E425)[17].ThedevelopmentofNAinhibitorswasinitiatedinthe1970swiththenaturalinhibitor2-deoxy-2,3-dehydro-N-acetylneuraminicacid(Neu5Ac2en),atransitionstateanalogofinfluenzaNA[18,19].ThedetailstructureoftheNAconservedenzymesiteinteractingwithsialicacidorNeu5Ac2enfacilitatedthestructural-baseddesignofwww.sciencedirect.comthecurrentlyapprovedNAinhibitors[20–22].Since1999,fourcompetitiveNAinhibitors(zanamivir,oseltamivircarboxylate,peramivir,andlaninamivir)havebeenap-provedfortreatmentandprophylaxisforinfluenzaAandBinfections.Theseinhibitorspossessdifferentside-chainmodificationstoimprovebioavailabilityandbind-ingtotheNAactivesite(Figure2).Zanamivirreplacedthe4-hydroxylgroupofNeu5Ac2enwiththebasicgua-nidinogrouptoimprovebindingthroughtheterminalnitrogenswiththeNAresiduesE119andE227[23,24];zanamivirisadministeredbyoralinhalation.Tofurtherimproveoralbioavailability,oseltamivircarboxylate[25]andperamivir[26]weredesignedbasedonnon-carbohy-dratetemplates,withcyclohexenesandcyclopentanes,respectively.Oseltamivircarboxylatefurtherdiffersfromzanamivirbythebulkyhydrophobic6-pentylethersidechainthatreplacedtheglycerolsidechainandthe4-aminogroup[25].Toaccommodatethehydrophobicsubstitutionatthe6-position,NAresidueE276needtore-orientandformasaltbridgewithR224andresultsintheformationofahydrophobicpocket[27].Peramivirdiffersfromzanamivirandoseltamivircarboxylatebythecyclopentanering;inaddition,itpossessesa4-guanidinogroupthatresembleszanamivir,andahydrophobicsidechainthatresemblesoseltamivircarboxylate[28�];thedesignleadstomultiplebindinginteractionsofperamivirattheNAenzymesite.Inhumans,theoralbioavailabilitywaspoorandparenteralformulationsviaintra-muscularorintravenous(IV)injectionswerepursued.Currently,itisapprovedinJapan,Korea,andUSAfortreatmentofacuteuncomplicatedinfluenzainadultsbyIVinjection[29].Laninamivirisalong-actingNAinhibitorcontaininga4-guanidinogroupanda7-methoxygroup.Theprodruglaninamiviroctanoateisprocessedintolaninamivirinthelungswithahighretentiontime;thisprovidesthelonglastinganti-influenzaactivityafterasingleintranasaladministration[30,31].Laninamiviriscurrentlyonlyap-provedforclinicaluseinJapan.Inaddition,thenewlydeveloped2,3-difluorosialicacid(DFSA)-basedNAinhi-bitorsthatformstabilizedcovalentintermediateatNAenzymesiteshowedsignificantlyimprovedinhibitoryeffectagainstinfluenzaAandBvariantsresistanttocurrentlyapprovedNAinhibitors[32�].ThesensitivityandresistanceprofileofinfluenzaAandBvirusesNAproteinsaredeterminedbytheinteractionsbetweentheNAconservedresidueswiththeNAinhi-bitorsofdifferentside-chainmodifications.InfluenzaAvirusesaremoresusceptibletothefourapprovedNAinhibitorsthaninfluenzaBviruseswithlowerIC50valuesbytheenzyme-basedNAinhibitionassays[33].ThepdmH1N1virusismarginallymoresusceptibletozana-mivir,peramivir,andlaninamivirwiththe4-guanidinogroupthantheH3N2virus[33],possiblyduetothe150-loopcavitypossessedbytheGroup-1NA[34].Similarly,theresistanceprofileofN1,N2,andinfluenzaBNAproteinscanalsobedifferentiated.ThemutationsthatCurrentOpinioninVirology2016,18:126–134128AntiviralstrategiesHOvirFigure2OHOHOHOHOHOOHOOOOOOOONHNHNHNHNHNH2HHHHHHHNeu5Ac5enZanamiPeramivirconfertodrugresistanceareoftenlocatedatthecon-servedenzymeorframeworkresidues[28�];however,mutationatthemonomer–monomerinterfacehasbeenalsoreportedtoconferresistance[35].Furthermore,somemutationsconfercross-resistancetomultipleNAinhibi-torswhilesomemutationsonlyleadtoreducedsuscepti-bilityofoneNAinhibitoralone.ForN1,theH274Ymutationismostcommonlyobservedmutationclinically[28�];itleadstohighlyreducedsusceptibility(>100-folddifferenceinIC50)tooseltamivirandperamivirbutremainsusceptibleforzanamivirandlaninamivir.Inaddition,aminoacidchangesinresidue222(fromItoK/T/R)werereportedinpdmH1N1virustoconferre-ducedsusceptibility(10–100-folddifferenceinIC50)tooseltamivir;thecombinationofI222RandH274Ymuta-tionsleadtocross-resistancetoallfourNAinhibitors[36].ForN2,theR292KandE119Vmutationsarethemostcommonlyreportedmutationsclinically[28�,33,36].TheR292Kmutationleadstohighlyreducedsusceptibilitytooseltamivirandperamivir,reducedsusceptibilityforzanamivir,and<10-foldincreaseinIC50(normalinhibi-tion)forlaninamivir;theE119Vmutationleadstohighlyreducedsusceptibilityforoseltamivirbutremainsuscep-tibletootherdrugs[33,36].TheN294SmutationhasbeenidentifiedinbothN1(H5N1andpdmH1N1clinicalOHNHNHNH2ChemicalstructuresofNeu5Ac2enandthecurrentlyapprovedNAinhibitorsNeu5Ac2en,atransitionstateanalogofsialicacidwithdifferentmodificatiocarboxylateandperamivirweredesignedbasedonnon-carbohydratetempCurrentOpinioninVirology2016,18:126–134OHOHOHOHOHOHHOOOOONHNHNHNH2NH2LaninamivirOseltamivircarboxylateisolates)andN2(H3N2clinicalisolates)thatconferresistancetooseltamivir[33,37,38].ForinfluenzaB,R152K,D198NorI222Lmutationshavebeendetectedfromimmunocompromisedpatientsafterreceivingpro-longedNAinhibitortreatment[39–41].ThemutationsfoundinclinicalspecimenwithreducedsusceptibilitytoNAinhibitorshavebeensummarizedbyWorldHealthOrganization(WHO)[42]andarereportedannuallybytheWHOcollaboratingcenters[33,36].Ingeneral,thestructure-baseddesignlimitstheemer-genceofresistantvariantswithuncompromisedNAen-zymefunction,asmutationsattheconservedNAresiduesthatalterbindingtotheNAinhibitorswillsimilarlyreducebindingtosialicacids.AlthoughresistantvariantscanbeidentifiedfrompatientsafterNAinhibitortreatment,especiallyamongchildrenwithhigherviralloadthanadults[38,43–46]orimmunocompromisedpatientswithprolongedviralshedding[39–41,47–52],theresistanceratewerelowatthecommunitylevelbefore2007duetothelackoftransmissionofdrugresistantvariants.SeasonalH1N1influenzavariantwithanH274YNAmutation(N1numbering)thatconfersresistancetooseltamivircarbox-ylatewasreportedatincreasingpercentagefrompatientswithoutpriortreatmentin2007–2008influenzaseasonandOONHCurrentOpinioninVirology.ThestructuralbasedesignedNAinhibitorsweredevelopedbasedonnsatC4andC6.Tofurtherimproveoralbioavailability,oseltamivirlatesofcyclohexenesandcyclopentanes.www.sciencedirect.comspreadgloballyasthedominantH1N1strainbytheendof2008.StudiesshownthatNAmutationsemergedinchronologicalorderbefore(V233M,R221Q,K328E,D343N)andpostto(D353G)theacquisitionoftheH274YmutationfacilitatedtherestorationoftheNAfunction[53�,54–56].BecauseofthemechanismofactionoftheNAinhibitorsduringviralreplication,clinicaleffectivenessisassociatedwithtimelyadministration,preferablywithin48hoursofsymptomonset[57,58��,59],althoughtreatmentstartedwithin3–5daysalsoimprovedsurvivalinadultpatientshospitalizedforseasonalinfluenza[58��].Recentsystem-AntiviralstrategiesforinfluenzaYen129aticreviewsonrandomizedcontroltrialsreportedthatoseltamivirtreatmentreducedthetimetosymptomalle-viationinadultsandhealthychildren[60�,61�],decreasedtheriskforlowerrespiratorytractcomplicationsandhospitaladmissioninadults[60�],whilezanamivirre-ducedthetimetosymptomalleviationinadults[62].NucleosideanalogsfortheRNA-dependentRNApolymeraseFavipiravir(T-705,6-fluoro-3-hydroxy-2-pyrazinecarboxa-mide)isanucleosideanalogthatinhibitstheRNA-dependentRNApolymeraseactivityforinfluenzaandotherRNAviruses[63](Figure3).TheRTPformofT-705wasshowntocompetitivelyinhibitATPandGTPincorporationintotheRNAtranscriptsandpreventRNAelongation[64].Inaddition,lethalmutagenesiscanbeanalternativeantiviralmechanismofactionforRNAvirusesasT-705showedmutagenesiseffectforinfluenzaAvirusinvitro[65]andfornorovirusinvivo[66].Inspiteofextensivesearch,favipiravirresistantvarianthasonlybeenselectedforthechikungunyavirustodatewithasinglemutationataconservedresidueoftheRNA-dependentRNApolymerase[67].Despiteitspotentialtetratogenicandembryotoxicpotential,favipiravirwasapprovedin2014inJapanagainstnovelorre-emerginginfluenzavirusinfectionsunderconditionsthatotheranti-influenzavirusdrugsareineffective;inaddition,itwasalsoapprovedinJapanforthetreatmentofEbola[68].Figure3OHOHOHOHOOONH2NH2NFNNNNFavipiravirRibavirinCurrentOpinioninVirologyNucleosideanalogsthatinhibitRNA-dependentRNApolymerase.Favipiravirandribavirinarenucleosideanalogsthatshownbroad-spectruminhibitoryactivityformultipleRNAviruses.www.sciencedirect.comSimilartofavipiravir,ribavirinisaguanosineanalogwithbroadrangeantiviralactivityagainstRNAandDNAviruses[69](Figure3).Therearetwomajorpotentialantiviralactivities,oneisforthemonophosphateformtocompetewiththecellularinosinemonophosphatefortheIMPdehydrogenaseleadingtodepletionofthecellularGTPpool;theotherviathetriphosphateformthatisincorporatedintotheRNAtranscript,whichmayblockRNAelongationandmayalsoleadtothemutagenesiseffect.AlthoughribavirinexhibitedantiviralactivityforinfluenzaAandBvirusesinvitroandinanimalmodels,clinicalusageofribavirinforinfluenzatreatmenthasbeenlimited.Dependingonthedoseandviralstrain,combi-nationtherapywithribavirinandNAinhibitorsmayleadtosynergisticoradditiveeffectsinanimalmodels[6].NovelinhibitorsthatblockinfluenzaRNPfunctionInthepastfewyears,thedetailstructuresofinfluenzaNPandpolymeraseproteins[70–72]havefacilitatedthestructural-baseddesignorrefinementofinhibitorsthatblockinfluenzaRNPfunction.NPinhibitorsThetransportationofnewlysynthesizedNPproteinsfromthecytoplasmtothenucleus,theformationsofNPoligomerstoencapsidatethenewlysynthesizedviralRNA,andnucleusexportofviralRNPareessentialduringinfluenzareplication.CurrentantiviralstrategiesfocusedoninterferingtheNP–NPinteraction,NP–RNAinterac-tion,andNPnucleusexportsignal(NES)fornucleusexportationofinfluenzaAvirus.Nucleozin(Figure4)wasidentifiedthroughhighthroughputscreenings[73–75];itinducesNPhighorderoligomerformationthatinhibitsNPtransportationfromthecytoplasmintothenucleus.Asanalternativestrategy,disruptingNPoligomerformationbytargetingNPresiduesE339-R416saltbridgeformationhasbeenproposedandvalidatedinvitrousingpeptideorsmallmoleculesidentifiedthroughmoleculardocking[76].Naproxen(Figure4),anon-steroidalanti-inflamma-torydrugthatinhibitscyclooxygenaseIandII,wasidentifiedthroughvirtualscreeningtodisrupttheNP–RNAinteractionwithitsantiviralactivityvalidatedinvitroandinthemousemodel[77].Inaddition,RK424(Figure4)wasidentifiedfromhighthroughputscreeningthatbindstoaNPfunctionaldomaincriticalforNP–NPandNP–RNAinteractionsaswellastheNES3–CRM1interactionfornucleusexportation[78].SmallmoleculesthatinhibitsPB2cap-bindingandPAendonucleaseactivitiesInfluenzamRNAsynthesisrelyonthe‘cap-snatching’mechanismwhichrequiresbindingofviralPB2proteintothehostpre-mRNA50-capstructurefollowedbythePAendonucleaseactivitytogeneratethe50-cappedoligonu-cleotidesthatwouldbeusedasprimersforviralmRNAtranscription.Thecap-snatchingmechanismissharedCurrentOpinioninVirology2016,18:126–134130AntiviralstrategiesFigure4OOOONOHOOHOO–N+NCINNNucleozinNaproxenRK424CurrentOpinioninVirologyInhibitorsthatinterfereNP–NPorNP–RNAinteractions.NucleozinandRK424wereidentifiedviahigh-throughputscreening;naproxenthatinhibitscyclooxygenaseIandIIwereidentifiedthroughvirtualscreening.amongsegmentednegative-strandedRNAvirusesintheOrthomyxoviridae,BunyaviridaeandArenaviridae[79�].Anoral-bioavailableazaindole-basedinhibitor(VX-787)wasidentifiedthroughscreeningtocompetitivelybindtothe7-methylGTPbindingsiteofPB2proteintoinhibitcap-bindingactivity[80](Figure5).Inthemousemodel,VX-787waseffectiveunderprophylaxisorpost-exposuretreatmentregimens,with100%survivalachievedupto96hourspost-infection[81].AnM431Imutationwasidentifiedin4outof72patientsfromahumanchallengestudyfollowedbyVX-787treatment;theresistantvirusshowedcompromisedgrowthinvitro[82].Inaddition,resistantvariantwasdetectedaftertwopassagesinvitrowithanF323SmutationinPB2[82].InfluenzaBviruseslackingtheF323inPB2alsoshowedpoorbindingtoVX-787[82],suggestingtheconformationaldifferencesbe-tweeninfluenzaAandBvirusesatthecap-bindingdomaincouldbeachallengeforthedrugdesign.Currently,VX-787isunderphase2btrial.TheconservedresiduesatthePAN-terminalendonu-cleasedomainposeanothertargetfordrugdesign.BeforethedetailPAN-terminalstructurewasavailable,multipleFigure5VX-787FFONNNNHHNOHSmallmoleculesthatinhibitsPB2cap-bindingandPAendonucleaseactivitCurrentOpinioninVirology2016,18:126–134chemicalcompoundshavebeenreportedtoinhibittheendonucleaseactivityforcapsnatchingsince1994[83,84].DetailedstructuralinsightsshowedthattheN-hydroxyimideandthe4-substituted2,4-dioxobu-tanoicacidinhibitorschelateswithtwomanganeseionsattheendonucleaseactivesite[83,85].Althoughresistantvariantswerenotidentifiedafter10serialpassagesunderthecompoundL-742,001(Figure5),onemostpotent4-substituted2,4-dioxobutanoicacidinhibitorinvitro,mutagenesisstudyidentifiedpotentialresiduesinPAproteinsthatmayconferresistance[86].CombinationtherapyThebenefitofcombinationtherapyincludesreducingtheemergenceofresistantvariantsandprovidingpoten-tialadditiveorsynergisticeffectoftheantiviralcom-pounds.However,clinicalapplicationsofcombinationtherapyagainstinfluenzainfectionshavebeenlimited,possiblyduetothefewclassesofantiviralsapprovedforclinicaltreatment.CombinationtherapyofoseltamivirandzanamivirtreatmentintworandomizedcontroltrialsamongpatientsinfectedwithhumanH3N2orpdmH1N1virusesfailedtodemonstratesuperiorefficacythantheL-742001OONOHHO•HCICICurrentOpinioninVirologyy.www.sciencedirect.comAntiviralstrategiesforinfluenzaYen131monotherapyalone[87,88].Favipiravirandoseltamivirshowedsynergisticeffectwhilegivenatlowdosesinmice[89];favipiravirandperamivirshowedsynergyagainstapdmH1N1variantwithanH274Ymutationinmice[90].Combinationofamantadine,ribavirin,andoseltamivirhasshownadditiveorsynergisticeffectinanimalmodels[91–93];clinically,arandomizedcontrolledtrialcompar-ingthetriplecombinationantiviraldrugversusoseltami-viraloneiscurrentlyinprogress[6].Asthenovelclassesofantiviralcompoundsagainstinfluenzabecameavail-able,thebenefitofcombinationtherapycanbefurtherevaluatedclinically.Combinationsbetweenantiviralagentswithimmunemodulatorycompoundshavebeenevaluatedinanimalmodelsandamongpatientswithsevereinfluenzainfec-tions.ThecombinationofoseltamivirwithN-acetylcyc-teineorthesphingosineanalogAAL-R,zanamivirwiththeCOX-2inhibitorandmesalazine,orlaninamivirwiththeartificialsurfactanthavebeenshowntoincreasesur-vivalinanimalmodels[6].Clinically,systemicglucocor-ticoidsmaybeusedtoincr