Mechanism of action of N-phenyl-N'-(2-chloroethyl)ureas in the colchicine-binding site at the interface between alpha- and beta-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 FlexiDock 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-beta198 rather than Cys-beta239. In addition, these studies suggest that Cys-beta239 together with Asn-alpha99, Ser-alpha176, Thr-alpha177, Leu-beta246, Asn-beta247, Ala-beta248, Lys-beta252 and Asn-beta256 are implicated in the stabilization of a C-BS-CEU complex prior to the acylation of Glu-beta198 by CEU. Our molecular models propose the formation of a stabilized C-BS-CEU complex before the completion of the Glu-beta198 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.