Mechanistic Study of the Reaction of Thiol‐Containing Enzymes with α,β‐Unsaturated Carbonyl Substrates by Computation and Chemoassays

Abstract

We investigated the reactions between substituted alpha,beta-unsaturated carbonyl compounds (Michael systems) and thiols by computations as well as chemoassays. The results give insight into variations in the underlying mechanisms as a function of the substitution pattern. This is of interest for the mechanisms of inhibition of the SARS coronavirus main protease (SARS-CoV M(pro)) by etacrynic acid derivatives as well as for the excess toxicity of substituted alpha,beta-unsaturated carbonyl compounds. This study compares possible reaction courses including 1,4-addition followed by a ketonization step, and underscores the importance of a base-catalyzed step for the reactivity of thiol groups in enzymes. Phenyl and methyl substituents at the Michael system decrease the reactivity of the electrophilic compound, but chlorophenyl substituents partly recover the reactivity. Computations also indicate that electron-pushing substituents lead to a change in the reaction mechanism. The conformation of the Michael system is also found to significantly influence reactivity: the s-cis conformation leads to higher reactivity than the s-trans conformation. The computed data explain the trends in measured inhibition potencies of substituted alpha,beta-unsaturated carbonyl compounds and of reaction rates in chemical assays. They also indicate that the reversibility of inhibition does not stand in contrast to the formation of a new covalent bond between inhibitor and protease.

Mechanism of falcipain-2 inhibition by α,β-unsaturated benzo[1,4]diazepin-2-one methyl ester

“…This is in agreement with the experimental FP-2 kinetic data showing that FP-2 was in a ''cleaving'' active state in a pH range from 4.5 to 8.5, with an optimal value of 7.5 [35]. Additionally, the results obtained for the thiol-containing SARS-CoV protease, an enzyme belonging to the FP-2 family, are in line with our proposals [14]. Furthermore, looking into our complex, the catalytic amino acidic triad (Cys42, His174, Asn204) appeared protected from the contact of the solvent by the presence of the bulky structure of the 1,4-BDZ substrate.…”

“…On the other hand, valuable insights are also achievable from small and simplified models in which the side chains (or some atoms) of the catalytic triad of the enzymes are represented. For example, Paasche et al [14] and Mladenovic et al [18] studying thiol-containing enzymes, investigated the influence of the chemical structure of the inhibitors on the kinetic parameters of papain-like proteases adopting model systems containing a simplified structure of the inhibitors, ammonia and methanethiol [14,18,30].…”

“…A number of publications aiming to the prediction of the inhibition mechanism of cysteine-proteases, and structurally related enzymes, by means of computational approaches, have been published [14][15][16][17][18]. However, to the best of our knowledge, no theoretical studies have been conducted on FP-2.…”

L718Q mutant EGFR escapes covalent inhibition by stabilizing a non-reactive conformation of the lung cancer drug osimertinib

“…Computational studies recently performed by us 30 and others 35 indicate that acrylamide-based inhibitors of EGFR alkylate Cys797 by a direct addition mechanism in which the thiolate group of Cys797 attacks the β carbon of the warhead, while the carboxylic acid group of Asp800 protonates the α carbon, leading to a stable 3-(alkylsulfanyl)propanamide adduct ( Scheme 2 ). Using this reaction scheme, we modelled the Michael-type addition of Cys797 to osimertinib, for both EGFR T790M and EGFR T790M/L718Q mutants.…”

“…No carbanion/enolate species was identified as a stable minimum, consistent with previous DFT calculations on the addition of thiolates to acrylamides. 30 , 35 …”