Design and Evaluation of Mechanism-Based Inhibitors of D-Alanyl-D-alanine Dipeptidase VanX

Abstract

VanX protein is a D‐alanyl‐D‐alanine dipeptidase essential for vancomycin resistance in Enterococcus. It is also a key drug target in circumventing clinical glycopeptide antibiotic resistance. The dipeptide‐like compound D‐Ala‐D‐Gly(SC6H4‐p‐CHF2) (1) was recently reported as the first mechanism‐based inhibitor with high affinity for the enzyme but poor inhibitory efficiency (kinact/Kirr = 9320 M−1 s−1) and a high partition ratio (7600). In order to assess the effects of variations in aromatic substituents on the in vitro inhibition properties of 1, we have prepared a few derivatives and analyzed them using the alternative substrate D‐Ala‐D‐Gly(SPh)‐OH (15) designed according to a similar strategy. Whereas moving the electrophilic difluoromethyl group to the ortho position slightly improved the inhibition parameters (kinact/Kirr = 1140 M−1 s−1) with a small decrease in the partition ratio (7200), introduction of a methoxy group in D‐Ala‐D‐ortho/para‐(difluoromethyl)phenylthioglycines resulted in a marked decrease in inhibitory efficiency. These results demonstrate the difficulties in improving the leading compound 1 given the restricted space available at the VanX active site. (© Wiley‐VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Design, Synthesis, and Biological Evaluation of Conformationally Restricted Rivastigmine Analogues

“…Compounds were named following IUPAC rules as applied by Beilstein-Institut AutoNom (version 2.1), a PC integrated software package for systematic names in organic chemistry. Miotine 30 (2) and dimethylthiocarbamic acid S-(2-formyl-6-methoxyphenyl) ester (15) 19,20 were prepared according to literature methods.…”

“…In contrast, the synthesis of 5 (Scheme ) could be performed using the strategy previously described for 3 , through reaction of the key intermediate 2-allylsulfanyl-3-methoxybenzaldehyde ( 16 ) and sarcosine trimethylsilyl ester. The preparation of 16 was readily accomplished in one pot by sequential treatment of 15 , with sodium hydroxide and allyl bromide. After formation of the tricyclic derivative 17 , the synthesis proceeded as depicted for 4 in Scheme and, in agreement with the parent system, the cis fusion ring was assigned through the coupling constant of the benzylic methine proton.…”

A Five-coordinate Metal Center in Co(II)-substituted VanX

“…The lack of a direct activity assay complicates mechanistic studies, results from which could drive rational inhibitor design or redesign efforts. A ninhydrin-based assay, a capillary electrophoresis procedure, and several coupled assays have been reported (17,(25)(26)(27); however, all of these have experimental problems that limit their use in mechanistic studies. As an alternative, we propose to follow the mechanism by preparing the Co(II)-substituted form of VanX and probing the spectroscopic properties of the metal center during the catalytic cycle with use of stopped-flow and rapid freeze quench coupled with spectroscopic studies.…”

Targeting Metalloenzymes for Therapeutic Intervention

“…While the exact nucleophilic residue is presently unknown, it is understood that this covalent binding effectively blocks the active site and prevents further action . Moving the difluoromethyl group from the para position in compound VanXi-8 to the ortho position in VanXi-9 was found to improve the stability of the leaving electrophile and improved the K irr from 22 to 10.7 μM. , Unfortunately, additional attempts to induce selectivity for the unknown nucleophilic residue(s) were unsuccessful, and this approach of covalent inhibitors against VanX has not progressed further.…”