Bisthiazolidines: A Substrate-Mimicking Scaffold as an Inhibitor of the NDM-1 Carbapenemase

Abstract

Pathogenic Gram-negative bacteria resistant to almost all β-lactam antibiotics are a major public health threat. Zn(II)-dependent or metallo-β-lactamases (MBLs) produced by these bacteria inactivate most β-lactam antibiotics, including the carbapenems, which are “last line therapies” for life-threatening Gram-negative infections. NDM-1 is a carbapenemase belonging to the MBL family that is rapidly spreading worldwide. Regrettably, inhibitors of MBLs are not yet developed. Here we present the bisthiazolidine (BTZ) scaffold as a structure with some features of β-lactam substrates, which can be modified with metal-binding groups to target the MBL active site. Inspired by known interactions of MBLs with β-lactams, we designed four BTZs that behave as in vitro NDM-1 inhibitors with Ki values in the low micromolar range (from 7 ± 1 to 19 ± 3 μM). NMR spectroscopy demonstrated that they inhibit hydrolysis of imipenem in NDM-1-producing Escherichia coli. In vitro time kill cell-based assays against a variety of bacterial strains harboring blaNDM-1 including Acinetobacter baumannii show that the compounds restore the antibacterial activity of imipenem. A crystal structure of the most potent heterocycle (L-CS319) in complex with NDM-1 at 1.9 Å resolution identified both structural determinants for inhibitor binding and opportunities for further improvements in potency.

Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes

“…However, BTZ binding does not result in global conformational changes for either IMP-1 (IMP:1b compared with As in the IMP-1:2a complex, residues W64 and V67 in the L3 loop are involved in hydrophobic interactions with the inhibitor, with W64 in particular forming π-stacking interactions with the BTZ bicyclic ring. Despite previous observations that the NDM-1 L3 loop may "close" on 1a binding (17), the position of the IMP-1 L3 loop is more stable, with no substantial conformational shifts evident on BTZ binding compared with the native structure. 1b makes very similar interactions (Fig.…”

“…The crystal structures of MBL:BTZ complexes presented here and in previous publications (17,18) identify that BTZs use four distinct modes of binding to the range of MBL targets. L-BTZs adopt similar binding modes to the B1 and B3 binuclear enzymes, with overall BTZ orientation retained across enzymes that use different side chains [K224 (BcII, IMP-1, NDM-1); R228 (VIM-2) and S223 (L1)] in interactions with the BTZ carboxylate group that may or may not involve the intermediacy of bound water molecules.…”

“…An understanding of how BTZs inhibit all three classes of MBL requires structural information for representative BTZ:MBL complexes to complement our previously obtained structures of 1a bound to the B1 MBLs VIM-2 and NDM-1 (17,18). We therefore crystallized the MBLs IMP-1 and BcII (subclass B1), Sfh-I (B2), and L1 (B3) and soaked the crystals in the four BTZs.…”

“…2) (17,18). These βLIs contain both a free thiol, which is a high-affinity zinc-binding group, and two defining properties of β-lactam substrates, a feature hitherto overlooked in MBL inhibitor design.…”

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

“…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.…”