%0 Journal Article
%T Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site
%A S¨¦bastien Fortin
%A Jacques Lacroix
%A Marie-France C£¿t¨¦
%A Emmanuel Moreau
%A ¨¦ric Petitclerc
%A Ren¨¦ C-Gaudreault
%J Biological Procedures Online
%D 2010
%I BioMed Central
%R 10.1007/s12575-010-9029-5
%X Antimicrotubule agents are important in the management of several cancers treatments notably breast, ovarian, and lung. Unfortunately, their effect is often hampered by chemoresistance, and they exhibit biopharmaceutical properties suitable for the treatment of only a limited number of cancers [1]. New antimicrotubule agents are therefore highly desirable, and several laboratories are eager to develop new drugs exhibiting improved antitumor efficacy and better biopharmaceutical properties [2]. One of the key end-points used in the screening of new molecules, beside the antiproliferative activity, is the binding affinity of the drug to one or another of the specific binding sites present on tubulin. Although binding sites for pironetin [3], tryprostatin [4], epothilone [5], Vinca [6], Taxus [7], and Colchicum [8] alkaloids have been identified so far, the colchicine-binding site remains the main target of numerous research programs. The determination and the characterization of the binding site(s) for each and every newly prepared drug to the latter binding sites are costly, time-consuming, and require specialized equipment, highly purified tubulin, and expensive radiolabeled ligands and are therefore applied only to the most promising molecules.To circumvent these limitations, we have developed an inexpensive and simple detection technique to assess the binding of antimicrotubule agents acting on the colchicine-binding site. This method is based on the property of N,N'-ethylene-bis(iodoacetamide) (EBI), a homobifunctional thioalkylating agent, to crosslink the Cys-239 and the Cys-354 residues of ¦Â-tubulin involved in the colchicine-binding site [9]. The covalent binding of EBI to ¦Â-tubulin forms an adduct that is easily detected by Western blot as a second immunoreacting band of ¦Â-tubulin that migrates faster than the native ¦Â-tubulin band on SDS-PAGE (sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis) [10]. Luduena has used that property to character
%K colchicine-binding site inhibitor
%K taxol-binding site inhibitor
%K N
%K N'-ethylene-bis(iodoacetamide)
%K EBI
%K tubulin affinity assay
%K antimicrotubule agent
%U http://www.biologicalproceduresonline.com/content/12/1/113