Mechanism of action of N-phenyl-N′-(2-chloroethyl)ureas in the colchicine-binding site at the interface between α-and β-tubulin

Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure-activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N'-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on beta(II)-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The Flexi-Dock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-beta 198 rather than Cys-beta 239. In addition, these studies suggest that Cys-beta 239 together with Asn-alpha 99, Ser-alpha 176, Thr-alpha 177, Leu-beta 246, Asn-beta 247, Ala-beta 248, Lys-beta 252 and Asn-beta 256 are implicated in the stabilization of a C-BS-CEU complex prior to the acylation of Glu-b198 by CEU. Our molecular models propose the formation of a stabilized C-BS-CEU complex before the completion of the Glu-beta 198 acylation; acylation triggering conformational changes of beta-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin. (C) 2009 Elsevier Ltd. All rights reserved.