Metallo-β-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding

Abstract

Bacterial resistance is compromising the use of β-lactam antibiotics including carbapenems. The main resistance mechanism against β-lactams is hydrolysis of the β-lactam ring mediated by serine- or metallo-β-lactamases (MBLs). Although several inhibitors of MBLs have been reported, none has been developed into a clinically useful inhibitor. Mercaptocarboxylic acids are among the most prominent scaffolds reported as MBL inhibitors. In this study, the carboxylate group of mercaptocarboxylic acids was replaced with bioisosteric groups like phosphonate esters, phosphonic acids and NH-tetrazoles. The influence of the replacement on the bioactivity and inhibitor binding was evaluated. A series of bioisosteres of previously reported inhibitors was synthesized and evaluated against the MBLs VIM-2, NDM-1 and GIM-1. The most active inhibitors combined a mercapto group and a phosphonate ester or acid, with two/three carbon chains connecting a phenyl group. Surprisingly, also compounds containing thioacetate groups instead of thiols showed low IC values. High-resolution crystal structures of three inhibitors in complex with VIM-2 revealed hydrophobic interactions for the diethyl groups in the phosphonate ester (inhibitor 2b), the mercapto bridging the two active site zinc ions, and tight stacking of the benzene ring to the inhibitor between Phe62, Tyr67, Arg228 and His263. The inhibitors show reduced enzyme activity in Escherichia coli cells harboring MBL. The obtained results will be useful for further structural guided design of MBL inhibitors.

Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding

“…By separating the two enantiomers, the use of the more efficient enantiomer may increase the data quality and may in addition result in better binding affinity data for both the IC 50 and SPR measurements. This idea is supported by the data determined for the 1.55-Å structure of VIM-2_2b, where only the (R)-enantiomer fits in the observed election density maps; thus, the enzyme had been selective (40).…”

“…In order to investigate the interaction between TMB-1 and inhibitor 2b, the inhibitor was modeled into the active site through superposition with the previously solved 1.55-Å crystal structure of VIM2_2b (40). The IC 50 value of inhibitor 2b toward VIM-2 was 0.38 M (40), which is within the same range as the IC 50 value for TMB-1 of 0.62 M.…”

“…In our recent study, we described the organic synthesis details and inhibitor properties of many new thiol-based inhibitors against VIM-2, NDM-1, and GIM-1 (40). Here, the halfmaximal (50%) inhibitory concentration (IC 50 ) values were determined for L-captopril and eight of the new thiol-based inhibitors (40) with TMB-1 at 25°C in 96-well plates using a SpectraMax M2 e spectrophotometer.…”

“…Thiolbased inhibitors have previously been tested for their inhibition potential against the MBLs VIM-2 and NDM-1 (8)(9)(10)39) and have been shown in several studies to be good MBL inhibitors (see, e.g., references 7 and 31). In this study, eight new thiol-based inhibitors synthetized by us (40) were included and compared to L-captopril. The binding affinity (K m ) value for nitrocefin in TMB-1 was determined to be 25 M compared to 34 M for VIM-2, 37 M for GIM-1, and 5 M for NDM-1 (data not shown) and thus was determined to be a suitable reporter substrate for TMB-1.…”

Kinetic, Thermodynamic, and Crystallographic Studies of 2-Triazolylthioacetamides as Verona Integron-Encoded Metallo-β-Lactamase 2 (VIM-2) Inhibitor

“…The SβLs inhibitors, such as clavulanic acid, tazobactam, sulbactam, vaborbactam, and avibactam have been used clinically [4,5]. Although a large number of MβL inhibitors have been reported, including thiols [6][7][8][9], carboxylic acids [10,11], rhodanine [12][13][14], cyclic boronates [15,16], and ebselen [17,18], there are no MβL inhibitors for available clinical purposes to date. Consequently, it is an urgent need to develop novel MβL inhibitors and to investigate their action mechanism.…”