Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of “cross-class” MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.
We thank Diamond Light Source for beamtime (proposals mx12342 and mx17212), and the staff of beamlines I03 and I04-1 for assistance with crystal testing and data collection. This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) to R. A. B. under Award Numbers R01AI063517 and R01AI072219, to R. A. B., G. M., J. S. and A. J. V. under Award Number R01AI100560 and to B. S. under Award Numbers R01 AI130060 and AI117211. This study was also supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, Award Number 1I01BX001974 to R. A. B. from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development, and the Geriatric Research Education and Clinical Center VISN 10. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Department of Veterans Affairs. V. M. and V. V. are recipients of a fellowship from Comisión Académica de Posgrado (CAP-Udelar). This work was supported by grant S2021INIC2019 from Comisión Sectorial de Investigación Científica (CSIC) to V. M. and G. M. and grant PICT-2016-1657 from ANPCyT to A. J. V., C. B. and D. M. M. and A. J. V. are staff members from CONICET. M. A. R. is recipient of a fellowship from CONICET.
© 2021 American Chemical Society.
- antibiotic resistance