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

Abstract

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both L-and D-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with K i s of 6-15 μM or 36-84 μM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 μM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the L-BTZ enantiomers exhibit 100-fold lower K i s (0.26-0.36 μM) than D-BTZs (26-29 μM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the L-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. D-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.carbapenemase | antibiotic resistance | inhibitors | bisthiazolidines | metallo-β-lactamase

Structural and biochemical analysis of the metallo‐β‐lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di‐metal scaffold for catalytic activity

“…S. maltophilia L1 is a subclass B3 MBL, one of two chromosome‐encoded β‐lactamases (L2 belongs to a class A) . Several crystal structures of L1 and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis . B3 MBLs share a relatively low sequence identity of 23–35%; however, they are structurally well‐conserved particularly their di‐nuclear zinc active sites.…”

NMR-filtered virtual screening leads to non-metal chelating metallo-β-lactamase inhibitors

“…S1 and S2†). 10–13 Development of new types of MBL-fold enzyme inhibitors that do not work via metal chelation is presently desirable, in part because this may enable improved selectivity than (readily) achievable with zinc ion chelation. Here we report how a virtual screening approach combined with NMR filtering, led to the identification of non-metal chelating inhibitors of the clinically relevant Verona Integron-encoded MBL (VIM)-2.…”

Crystallographic analyses of isoquinoline complexes reveal a new mode of metallo-β-lactamase inhibition

“…S11). 9, 17 Based on these structures we designed and synthesized two compounds ( 3 and 4 , Table 1) combining the tryptophan moiety of 2 and the ( S )-3-mercapto-2-methylpropanal moiety of D-/L-captopril (Supplementary Methods Scheme S1 for 3 / 4 synthesis), with the aim of identifying broad-spectrum MBL inhibitors.…”