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

Abstract

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.

Styryl-N-phenyl-N′-(2-chloroethyl)ureas and styrylphenylimidazolidin-2-ones as new potent microtubule-disrupting agents using combretastatin A-4 as model

“…This adduct is easily detectable by Western blot as a second immunoreacting band of β-tubulin [16,21,[26][27][28][29]. As seen in Figure These results also show that that the TMP moiety is not essential for the affinity and the acylation of the C-BS.…”

“…This might be due to the steric hindrance that may prevent the binding of the IMZ moiety to the adjacent pocket behind CYS239 and 354 residues of β-tubulin [20,21].…”

Characterization of the Covalent Binding ofN-Phenyl-N′-(2-chloroethyl)ureas to β-Tubulin: Importance of Glu198 in Microtubule Stability

“…We speculate that the acylation of Glu198 occurs instead of the alkylation of Cys239 due to the structure of CEUs, which is different from the other electrophilic agents studied, and because the 2-chloroethyl moiety of CEU is a much weaker electrophile than that of any other alkylating agent tested so far. According to computational simulations, the acylation of Glu198 by CEUs is favored over the alkylation of Cys239 because it requires less energy of stabilization between the electrophilic moiety of CEUs (i.e., C1 in the 2-chloroethyl moiety of CEUs) and the nucleophilic Glu198 (Fortin et al, 2009).…”

Synthesis, Biological Evaluation, and Structure–Activity Relationships of Novel Substituted N-Phenyl Ureidobenzenesulfonate Derivatives Blocking Cell Cycle Progression in S-Phase and Inducing DNA Double-Strand Breaks

“…13 Of interest, Gluβ198, which is located in a small pocket adjacent to the colchicine-binding site, is involved in microtubule stability and dynamics and is also associated with a mechanism of resistance to Taxotere (docetaxel). 14,15 …”