Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site 英文参考文献.docVIP

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Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site 英文参考文献.doc

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Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site 英文参考文献

BiolProcedOnline(2010)12:113–117 DOI10.1007/s12575-010-9029-5 QuickandSimpleDetectionTechniquetoAssesstheBinding ofAntimicrotubuleAgentstotheColchicine-BindingSite SébastienFortinJacquesLacroix Marie-FranceC?téEmmanuelMoreau éricPetitclercRenéC.-Gaudreault Received:29January2010/Accepted:8March2010/Publishedonline:8April2010 #TheAuthor(s)2010.ThisarticleispublishedwithopenaccessatS Abstract Development of antimitotic binding to the colchicine-bindingsiteforthetreatmentofcancerisrapidly expanding. Numerous antimicrotubule agents are prepared everyyear,andthedeterminationoftheirbindingaffinityto tubulinrequirestheuseofpurifiedtubulinsandradiolabeled ligands.Suchaprocedureiscostlyandtime-consumingand thereforeislimitedtothemostpromisingcandidates.Here, wereportaquickandinexpensivemethodthatrequiresonly usuallaboratoryresourcestoassessthebindingofantimicro- tubulestocolchicine-bindingsite.Themethodisbasedonthe ability of N,N-ethylene-bis(iodoacetamide) (EBI) to cross- linkinlivingcellsthecysteineresiduesatposition239and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreactingbandofβ-tubulinthatmigratesfasterthan β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β- tubulinadduct,resultinginanassaythatallowsthescreening ofnewmoleculestargetingthisbindingsite. Keywords colchicine-bindingsiteinhibitor.taxol-binding siteinhibitor.N,N-ethylene-bis(iodoacetamide).EBI. tubulinaffinityassay.antimicrotubuleagent 1Introduction Antimicrotubuleagentsareimportantinthemanagementof several cancers treatments notably breast, ovarian, and lung. Unfortunately, their effect is often hampered by chemoresistance,andtheyexhibitbiopharmaceuticalprop- ertiessuitableforthetreatmentofonlyalimitednumberof cancers [1]. New antimicrotubule agents are therefore highly desirable, and several laboratories are eager to