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

Abstract

ureas (CEUs) are antimicrotubule agents interacting covalently with ␤-tubulin near the colchicine-binding site (C-BS). Glutamyl 198 residue in ␤-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic residues, Cys239 and Cys354, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). By use of mass spectrometry, we have now identified residues in ␤-tubulin that have become modified irreversibly by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU), and N,NЈ-ethylenebis(iodoacetamide) (EBI). The binding of HP-CEU and 4ZCombCEU to ␤-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs then were used as molecular probes to assess, in mouse B16F0 and human MDA-MB-231 cells, the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU, and 4ZCombCEU on the acetylation of Lys40 on ␣-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation, as observed also with other microtubule depolymerizing agents. Therefore, CEU affects the stability and the dynamics of microtubule, likewise a E198G mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular cross-link between Cys239 and Cys354 of ␤-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/ function and the biological activity of several cellular proteins under physiological conditions.

An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site

“…It is speculated that the active site of the β-tubulin is stereo selective and the R -isomer of the branched chloroethyl group allowed a sterically favored orientation of the alkylating moiety, promoting the approach of the chlorine atom toward the sulfhydryl of Cysβ239 residue. However, subsequent work using mass spectrometry has identified that the Gluβ198, which is adjacent to the colchicine binding site behind the two potent nucleophilic residues, Cysβ239 and Cysβ354, has been shown to covalently react with CEU (129,130). None of the cysteine residues of β-tubulin was linked to the alkylating agent.…”

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

“…This was also confirmed by replacing the TMP moiety of CA-4 by the CEU group resulting in molecules referred to as (Z)-1-(2-chloroethyl)-3-(4-(3-hydroxy-4-methoxystyryl)phenyl)ureas (CEU analogs to CA-4, 5d-g, Fig. 1) exhibiting antiproliferative activity (IC 50 ) at the micromolar level and that are still acylating the C-BS [14,17]. Of note, only the methoxylated phenolic moieties of CA-4 at positions 3 and 4 have been studied so far.…”

“…As mentioned earlier, CEUs such as tBCEU [24] and sBCEU [25] are acylating the glutamic acid residue in position 198 of the C-BS [13,14].…”

“…The general synthesis of SEUs, SCEUs and SIMZs started from triphenylphosphonium bromide salts (14) which were prepared by addition of triphenyphosphine to 4-nitrobenzyl M A N U S C R I P T…”

“…A C C E P T E D ACCEPTED MANUSCRIPT 5 position 198 [13,14]. This CEU fragment was used to prepare several antimicrotubule agents substituted at position 4 of the aromatic ring such as 1-(4-(tert-butyl)phenyl)-3-(2-chloroethyl)urea (tBCEU, 5a) [15] and 1-(4-(sec-butyl)phenyl)-3-(2-chloroethyl)urea (sBCEU, 5b) [15], and substituted at position 3 of the aromatic ring represented by 1-(2-chloroethyl)-3-(4-(5-hydroxypentyl)phenyl)urea (5c) [16].…”