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

Metallo-beta-lactamases (MBLs) hydrolyze almost all beta-lactam antibiotics and are unaffected by clinically available beta-lactamase inhibitors (beta LIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of beta LIs 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 beta LIs of all MBL classes in vitro, with K(i)s of 6-15 mu M or 36-84 mu M for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 mu M 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 mu M) than D-BTZs (26-29 mu M). Importantly, cell-based time-kill assays show BTZs restore beta-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 beta-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 beta-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.