AbstractOneofthemainregulatorsinthecellcycleiscyclin-dependentkinase4and6(CDK4/6).FDAhasapprovedCDK4/6inhibitorsforthetreatmentofpatientswithmetastaticbreastcancer.However,thedevelopmentofselectiveagentsremainsproblematicduetotheconservationoftheirATPbindingsites.
细胞周期中的主要调节因子之一是细胞周期蛋白依赖性激酶4和6（CDK4/6）。FDA已批准CDK4/6抑制剂用于治疗转移性乳腺癌患者。然而，由于其ATP结合位点的保守性，选择性药物的开发仍然存在问题。
Inthepreviousinsilicostudy,ZINC585292724,ZINC585292587,ZINC585291674,andZINC585291474havebeenidentifiedaspotentialinhibitors.Therefore,thepresentstudyaimedtoanalyzetheselectivityandinhibitoryactivityofthefourcompoundsagainstCDK4/6invitroaswellasdeterminethepotentialfortheirfurtherdevelopmentinsilico.
。因此，本研究旨在分析四种化合物在体外对CDK4/6的选择性和抑制活性，并确定其在计算机上进一步发展的潜力。
Theinvitroresultsshowedthatthefourcompoundshadgoodselectivitytowardsbothkinases,duetotheirsimilarstructure.Inagreementwiththeinsilicoresults,ZINC585291674producedthebestinhibitoryactivityagainstCDK4andCDK6,withIC50of184.14nMand111.78nM,respectively.TheirADMETprofilewerealsosimilartoreferencecompoundofPalbociclib.
体外结果表明，由于它们的结构相似，这四种化合物对两种激酶都具有良好的选择性。与计算机模拟结果一致，ZINC585291674对CDK4和CDK6产生最佳抑制活性，IC50分别为184.14nM和111.78nM。它们的ADMET谱也类似于Palbociclib的参考化合物。
Basedonthis,ZINC585291674canbeusedasaleadcompoundforfurtherinhibitordevelopment..
基于此，ZINC585291674可用作进一步抑制剂开发的先导化合物。。
IntroductionOneofthefundamentalbiologicalprocessesiscellproliferationwhichoccursinaseriesofstagesconstitutingthecellcycle.Thiscycleincludesinterphase(preparationofDNAreplicationintheG1phase,DNAreplicationintheSphase,andpreparationofmitosisinG2)andmetaphase,characterizedbysisterchromatids’separation.
引言基本的生物学过程之一是细胞增殖，它发生在构成细胞周期的一系列阶段。该周期包括间期（G1期DNA复制的制备，S期DNA复制和G2期有丝分裂的制备）和中期，其特征在于姐妹染色单体的分离。
Besides,thereisaninactiveorG0phase,wheremostofthenon-proliferativecellsarelocated1,2.Thecycleinnormalcellsismonitoredbycheckpointasacontrolsystem,whichinducescellcyclearrestduringabnormaldivision,andenablescellrepair,therebyensuringaproperDNAsynthesisandchromosomalseparation3,4.
此外，还有一个非活动期或G0期，其中大多数非增殖细胞位于1,2。正常细胞的周期由检查点作为控制系统进行监测，该系统在异常分裂过程中诱导细胞周期停滞，并实现细胞修复，从而确保适当的DNA合成和染色体分离3,4。
Cyclin-dependentkinases(CDKs),theserine/threoninefamilyofproteinkinases,areoneofthemostimportantproteinsregulatingcellcycletransitions.CDKformsheterodimerswithcyclin,theregulatorysubunitsforitsactivity5,6.AmongtheCDKfamily,CDK4andCDK6(CDK4/6)areparticularlyimportantastheyplayafundamentalroleintheG1/Stransitionthroughphosphorylationofretinoblastomaprotein(Rb)afteractivationbycyclinDwhencellssensemitogenicsignals6.
细胞周期蛋白依赖性激酶（CDKs）是丝氨酸/苏氨酸蛋白激酶家族，是调节细胞周期转变的最重要蛋白质之一。CDK与细胞周期蛋白形成异二聚体，细胞周期蛋白是其活性的调节亚基5,6。在CDK家族中，CDK4和CDK6（CDK4/6）特别重要，因为它们在细胞周期蛋白D激活后通过视网膜母细胞瘤蛋白（Rb）的磷酸化在G1/S转换中发挥基本作用，当细胞感觉到有丝分裂信号6时。
Thecellcycleisderegulatedinmalignantcells,characterizedbyabnormalanduncontrolledproliferationduetodysregulationandmutationsofvariousregulators,suchasCDK,CAK(CDK-activatingkinase),CKI(CDK-inhibitors),CDKsubstrates,cyclins,andcheckpointproteins5,6,7.Severaloncogenescanbeactivatedincancer,includingthoseintheRAS/RAF/MEK/ERK,PI3K/Akt/mTOR,JAK/STAT,Wnt/β-catenin,andBTK/NF-κBpathways,allofwhichconvergeonCDK4/6-cyclinDcomplex.
恶性细胞的细胞周期失调，其特征是由于CDK，CAK（CDK激活激酶），CKI（CDK抑制剂），CDK底物，细胞周期蛋白和检查点蛋白5,6,7等各种调节因子的失调和突变导致异常和不受控制的增殖。几种癌基因可在癌症中被激活，包括RAS/RAF/MEK/ERK，PI3K/Akt/mTOR，JAK/STAT，Wnt/β-连环蛋白和BTK/NF-κB途径，所有这些都聚集在CDK4/6-细胞周期蛋白D复合物上。
Additionally,mutationsoftumorsuppressorgenese.g.p53,tendtoincreaseCDK4/6activitythroughthereleaseofinhibitoryP21CIP18,9.Therefore,CD.
此外，肿瘤抑制基因（例如p53）的突变倾向于通过释放抑制性P21CIP18,9来增加CDK4/6活性。因此，CD。
Table1Thekinaseactivityof1μMconcentrationofthehitcompounds.FullsizetableCDK4/6inhibitoryactivitywasassessedbydeterminingtheIC50,andtheresultsarepresentedinFig.2andTable2.ZINC585291674hadthelowestIC50valueagainstCDK4/6,followedbyZINC585292724,ZINC585291474,andZINC585292587at185.14nMand111.78nM,286.75nMand196.25nM,225.68nMand360.68nM,and498.31nMand550.13nM,respectively.
表11μM浓度的命中化合物的激酶活性。通过测定IC50来评估全尺寸表CDK4/6的抑制活性，结果如图2和表2所示。ZINC585291674对CDK4/6的IC50值最低，其次是Zinc5852724，ZINC585291474和Zinc5852587，分别为185.14nM和111.78nM，286.75nM和196.25nM，225.68nM和360.68nM，以及498.31nM和550.13nM。
TheinhibitoryactivityshownbythesefourcompoundstoCDK4andCDK6wasinlinewithGbindvaluesobtainedinthepreviousinsilicostudywiththeenergyorderZINC585291674 这四种化合物对CDK4和CDK6的抑制活性与之前的计算机研究中获得的ΔGbind值一致，能量顺序为ZINC585291674 2Thedose–responsecurveofhitcompounds-CDK4(a)andhitcompounds-CDK6(b).FullsizeimageTable2TheIC50ofthehitcompounds.FullsizetableThehitcompounds’IC50indicatedtheorderofinhibitorystrengthonCDK4andCDK6inagreementwiththeinsilicoresults,butthevalueswerestillhighercomparedtopalbociclibasareference. 2命中化合物-CDK4（a）和命中化合物-CDK6（b）的剂量-反应曲线。。全尺寸表命中化合物的IC50表明对CDK4和CDK6的抑制强度顺序与计算机模拟结果一致，但与作为参考的palbociclib相比，该值仍然更高。 Theinsilicostudyshowedthatthefourcompounds’hadlowerGbindthanpalbociclib,hencetheiractivitywaspredictedtobebetter.ThefourhitstructurescontainCchiralasdemonstratedinFig.1,therebyallowingtheirpresenceinamixtureofSandRisomersorenantiomers.Theenantiomersofchiralcompoundscanhavesimilarphysicalandchemicalcharacteristics,buttheenantiomericeffectstendtodiffersignificantlyinachiralenvironment23.InsilicostudiesAccordingtoFig.1,theRisomerwasthechemicalstructurepreviouslyusedinthefourcompounds21. 计算机研究表明，这四种化合物的ΔGbind低于palbociclib，因此它们的活性预计会更好。如图1所示，四击结构含有C手性，从而允许它们存在于S和R异构体或对映体的混合物中。手性化合物的对映体可以具有相似的物理和化学特性，但对映体效应在手性环境中往往存在显着差异23。在计算机研究中，根据图1，R异构体是先前在四种化合物中使用的化学结构21。 ThechiralCatomasthestereocenterisatposition3oftheirpiperidinering.TodeterminetheSisomers’interactionwithCDK4. 手性C原子作为立体中心位于其哌啶环的3位。确定S异构体与CDK4的相互作用。 Table3TheCDK4/6–hitinteractionsrecordedduringdocking.FullsizetableFig.3SuperimposedbindingmodeofhitcompoundsR(blue)andS(magenta)totheATPbindingpocketofCDK4(a)andCDK6(b).FullsizeimageInZINC585291674,SdoesnotformhydrogenbondswiththeLys35residueoftheK/E/D/Dmotifwhichhascatalyticandstructuralimportanceintheactiveproteinkinase25,26. 。全尺寸表图。3命中化合物R（蓝色）和S（洋红色）与CDK4（a）和CDK6（b）的ATP结合口袋的叠加结合模式。全尺寸图像在ZINC585291674中，S不与K/E/D/D基序的Lys35残基形成氢键，其在活性蛋白激酶25,26中具有催化和结构重要性。 ThedifferenceinthebindingmodewasdisplayedbyZINC585292587,whereRformedhydrogenbondswithVal96,His95,andLys35residues,butSbondedwithVal96andAsn145residues.BasedonthevisualizationoftheinteractiondemonstratedinFig.3a,thecompounds’positionisinoppositedirectionsintheATPbindingpocket. ZINC585292587显示了结合模式的差异，其中R与Val96，His95和Lys35残基形成氢键，但S与Val96和Asn145残基结合。基于图3a所示相互作用的可视化，化合物的位置在ATP结合口袋中处于相反的方向。 Inthiscase,theRstructure’spartthatinteractswiththehingeregionistheamino-pyrazine,withtheadeninepocketbeingoccupiedbythepyrazinering.Meanwhile,inS,thesidechainonthepiperidineringandadeninepocketisoccupiedbythepiperidinering.BasedonFig.3b,allRandScompoundsformedthesameinteractionwithCDK6inthehingeregion(Val101residue)throughasecondaryaminegroupconnectingthetwoheterocyclicrings,However,RfurtherreachedtheDFGmotifinZINC585292724,ZINC585291674,andZINC585291474. 在这种情况下，与铰链区相互作用的R结构部分是氨基吡嗪，腺嘌呤口袋被吡嗪环占据。同时，在s中，哌啶环和腺嘌呤口袋上的侧链被哌啶环占据。基于图3b，所有R和s化合物通过连接两个杂环的仲胺基团与铰链区（Val101残基）中的CDK6形成相同的相互作用，然而，R进一步达到ZINC5852724，ZINC585291674和ZINC585291474中的DFG基序。 TheRisomerofZINC585291674formedahydrogenbondwiththeLys43residuethroughtheOatominthepiperidineringsidechain,butthisdidnotoccurinS.AnexampleofligandinteractionwithCDK4andCDK6atthebindingsiteisshowninFig.4throughtheinteractionbetweenZINC585291674withCDK4(a)andCDK6(b)attheirbindingsites.Fig. ZINC585291674的R异构体通过哌啶环侧链中的O原子与Lys43残基形成氢键，但这在S中没有发生。在结合位点与CDK4和CDK6的配体相互作用的例子如图4所示。通过ZINC585291674与CDK4（a）和CDK6（b）在其结合位点之间的相互作用。图。 4ComplexesofCDK4(a)andCDK6(b)withZINC585291674,andtheirbindingmodesatthebindingsites.FullsizeimageBindingstabilityanalysisandbindingfreeenergycalculationThesta. 4CDK4（a）和CDK6（b）与ZINC585291674的复合物，以及它们在结合位点的结合模式。全尺寸图像结合稳定性分析和结合自由能计算sta。 Table4Theoccupancyofhydrogenbondsduring200nssimulations.FullsizetableTable5Thepredictedbindingfreeenergy(kcal/mol)fortheligand–CDK4complexes.FullsizetableTable6Thepredictedbindingfreeenergy(kcal/mol)fortheligandCDK6complexes.FullsizetableTheresultsoftheinsilicostudyonthefourcompoundcomplexesthroughmoleculardockingandmoleculardynamicssimulationsshoweddifferencesbetweenRandS. 表4200ns模拟期间氢键的占据率。全尺寸表5配体-CDK4复合物的预测结合自由能（kcal/mol）。。全尺寸表通过分子对接和分子动力学模拟对四种化合物复合物进行的计算机研究结果表明，R和S之间存在差异。 Overall,RhadbetterresultsthanS,andtheseprovideanoverviewofthepossibledifferencesinthecompounds’affinitytothetargetthatcouldaffecttheiractivity.FurtherseparationofRandSisneededtoconfirmthesecurrentinsilicoresults.However,invitrostudiesshowedthatZINC585291674producedthebestinhibitoryactivityagainstCDK4andCDK6comparedtotheotherthreecompounds. 总体而言，R比S具有更好的结果，并且这些概述了化合物对靶标的亲和力可能影响其活性的可能差异。需要进一步分离R和S以确认这些计算机模拟结果。然而，体外研究表明，与其他三种化合物相比，ZINC585291674对CDK4和CDK6产生最佳的抑制活性。 Thiswasinlinewithpreviousinsilicoresults,therebymakingZINC585291674apotentialCDK4/6inhibitorforfurtherdevelopment.ADMETanalysisForanewmolecularentity(NME)tobecomeadrug,itisnotonlyessentialtohaveahighbiologicalactivity,safeandefficient,butitisalsorequiredtohaveafavorablepharmacokineticprofileincludingtoxicity(ADMET). 这与先前的计算机模拟结果一致，从而使ZINC585291674成为进一步开发的潜在CDK4/6抑制剂。ADMET分析对于新的分子实体（NME）成为药物，不仅必须具有高生物活性，安全有效，而且还需要具有良好的药代动力学特征，包括毒性（ADMET）。 ThepharmacokineticandtoxicitypredictionsforthefourhitcompoundswascarriedoutusingADMETLab2.0webserver.Table7summarizetheresultofADMETpredictionsofthefourcompounds.. 使用ADMETLab2.0web服务器对四种命中化合物进行药代动力学和毒性预测。表7总结了四种化合物的ADMET预测结果。。 Table7TheresultofADMETpredictionsofthefourcompounds.FullsizetableAbsorptionprofilepredictionwascarriedoutusingtheCaco-2permeability,HIA,andMDCKparameters.Thehumancolonadenocarcinomacelllines(Caco-2),asanalternativeapproachforthehumanintestinalepithelium,hasbeencommonlyusedtoestimateinvivodrugpermeabilityduetotheirmorphologicalandfunctionalsimilarities27. 表7四种化合物的ADMET预测结果。使用Caco-2渗透性，HIA和MDCK参数进行全尺寸表吸收曲线预测。人结肠腺癌细胞系（Caco-2）作为人肠上皮的替代方法，由于其形态和功能相似性，通常用于估计体内药物渗透性27。 Humanintestinalabsorption(HIA)oforaldrugisanimportantprerequisiteforitsefficacy,aswellasthereisacloserelationshipbetweenintestinalabsorptionandoralbioavailabilityandHIAcanbeseenasanalternativeindicatorfororalbioavailabilitytosomeextent.Madin–DarbyCanineKidneycells(MDCK)havebeendevelopedasaninvitromodelforpermeabilityscreening. 口服药物的人体肠道吸收（HIA）是其疗效的重要先决条件，肠道吸收与口服生物利用度之间存在密切关系，HIA在一定程度上可以作为口服生物利用度的替代指标。Madin–Darby犬肾细胞（MDCK）已被开发为渗透性筛选的体外模型。 Itsapparentpermeabilitycoefficient,Papp,iswidelyconsideredtobetheinvitrogoldstandardforassessingtheuptakeefficiencyofchemicalsintothebody.AsshowninTable7,thefourcompoundsshowedgoodabsorptionprofilesinallthreeparameters.Distributionprofilepredictionwascarriedoutusingplasmaproteinbinding(PBB),blood–brainbarrier(BBB)penetration,andvolumedistribution(VD)parameters(Table7). 其表观渗透系数Papp被广泛认为是评估化学物质摄入体内效率的体外金标准。如表7所示，四种化合物在所有三个参数中均显示出良好的吸收曲线。使用血浆蛋白结合（PBB），血脑屏障（BBB）穿透和体积分布（VD）参数进行分布曲线预测（表7）。 AllfourcompoundshavehighPPBvalueswhichindicateahighamountofdrugboundtoprotein.ZINC585292724andZINC585291474haveanexcellentabilitytopenetratetheBBBwhileZINC585291674andZINC585292587haveamediumability.However,theVDparametershowsgoodresults,wheretheVDvalueshowsanexcellentvalue. 所有四种化合物都具有高PPB值，这表明大量药物与蛋白质结合。ZINC585292724和ZINC585291474具有出色的穿透血脑屏障的能力，而ZINC585291674和ZINC585292587具有中等能力。然而，VD参数显示出良好的结果，其中VD值显示出优异的值。 Oneofthemainmechanismsofdrugabsorptionanddistributionisthroughplasmaproteinbinding(PPB),wherePPBcandirectlyaffectoralbioavailabilitybecausethefreeconcentrationofthedrugwilldecreasewhenthedrugbindstoseru. 药物吸收和分布的主要机制之一是通过血浆蛋白结合（PPB），其中PPB可以直接影响口服生物利用度，因为当药物与血清结合时，药物的游离浓度会降低。 Dataavailability 数据可用性 Thedatasetsgeneratedduringand/oranalyzedduringthepresentstudyareavailablefromthecorrespondingauthoronreasonablerequest. 本研究期间产生和/或分析的数据集可根据合理要求从通讯作者处获得。 ReferencesZhang,M.etal.Cdkinhibitorsincancertherapy,anoverviewofrecentdevelopment.Am.J.CancerRes.11,1913(2021).ADS PubMed PubMedCentral 公共医学中心 GoogleScholar 谷歌学者 Case,D.A.etal.Amber2016(UniversityofCalifornia,2016). Case，D.A.等人，Amber2016（加利福尼亚大学，2016）。 DownloadreferencesAcknowledgementsTheauthorsacknowledgepartialfundingfromTheMinistryofEducation,Culture,Research,andTechnology,Indonesia(0017/E5/PG.02.00.PT/2022),andBandungInstituteofTechnology(26/IT1.C10/SK-DA/2023).AuthorinformationAuthorsandAffiliationsSchoolofPharmacy,BandungInstituteofTechnology,Bandung,40132,IndonesiaNiMadePitriSusanti,FransiskaKurniawan,SophiDamayanti,RahmanaEmranKartasasmita&DaryonoHadiTjahjonoStudyProgramofPharmacy,FacultyofMathematicsandNaturalSciences,UniversitasUdayana,Badung,80361,IndonesiaNiMadePitriSusantiAuthorsNiMadePitriSusantiViewauthorpublicationsYoucanalsosearchforthisauthorin. 下载参考文献致谢作者感谢印度尼西亚教育，文化，研究和技术部（0017/E5/PG.02.00。PT/2022）和万隆理工学院（26/IT1。C10/SK-DA/2023）的部分资助。作者信息作者和附属机构万隆理工学院药学院，万隆，40132，印尼制造的PitriSusanti，FransiskaKurniawan，SophiDamayanti，RahmanaEmranKartasasmita＆DaryonoHadiTjahjonoStudy药学项目，乌达亚纳大学数学与自然科学学院，巴东，80361，印尼制造的PitriSusantiAuthorsNi制造的PitriSusantiView作者出版物您也可以在中搜索这位作者。 PubMedGoogleScholarFransiskaKurniawanViewauthorpublicationsYoucanalsosearchforthisauthorin PubMedGoogleScholarsiskaKurniawanView作者出版物您也可以在 PubMedGoogleScholarSophiDamayantiViewauthorpublicationsYoucanalsosearchforthisauthorin PubMedGoogleScholarSophiDamayantiView作者出版物您也可以在 PubMedGoogleScholarRahmanaEmranKartasasmitaViewauthorpublicationsYoucanalsosearchforthisauthorin PubMedGoogleScholarahmanaEmranKartasasmitaView作者出版物您也可以在 PubMedGoogleScholarDaryonoHadiTjahjonoViewauthorpublicationsYoucanalsosearchforthisauthorin PubMedGoogleScholarDaryonoHadiTjahjonoView作者出版物您也可以在 PubMedGoogleScholarContributionsD.H.T.conceivedanddesignedtheexperiments(methodology),analyzedthedata,correctedthemanuscript,andparticipatedinthesupervisionaswellasthecontributionofresources.N.M.P.S.addedtothemethodology,performedtheexperiments,collectedthedata,anddraftedthemanuscript. PubMed谷歌学术贡献SD。H、T.构思并设计了实验（方法论），分析了数据，更正了手稿，并参与了监督以及资源的贡献。N、M.P.S.加入了该方法，进行了实验，收集了数据并起草了手稿。 Meanwhile,F.K.,S.D.andR.E.K.analyzedthedataandcorrectedthemanuscript.Allauthorsreviewedthemanuscript.CorrespondingauthorCorrespondenceto. 同时，F.K.，S.D.和R.E.K.分析了数据并更正了手稿。所有作者都审阅了手稿。对应作者对应。 DaryonoHadiTjahjono.Ethicsdeclarations DaryonoHadiTjahjono。道德宣言 Competinginterests 相互竞争的利益 Theauthorsdeclarenocompetinginterests. 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