Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes.
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 Kis 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 Kis (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.
School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol, Bristol BS8 1TD, United Kingdom;
Philip
P
Hinchliffe
Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Nacional de Rosario, 2000 Rosario, Argentina;
Mariano M
MM
González
Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106; Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106;
Maria F
MF
Mojica
Instituto de Bionanotecnología, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Nacional de Santiago del Estero, G4206XCP Santiago del Estero, Argentina;
Javier M
JM
González
Laboratorio de Química Farmacéutica, Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay;
Valerie
V
Castillo
Laboratorio de Química Farmacéutica, Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay;
Cecilia
C
Saiz
School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol, Bristol BS8 1TD, United Kingdom;
Magda
M
Kosmopoulou
School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol, Bristol BS8 1TD, United Kingdom;
Catherine L
CL
Tooke
Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Nacional de Rosario, 2000 Rosario, Argentina;
Leticia I
LI
Llarrull
Laboratorio de Química Farmacéutica, Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay;
Graciela
G
Mahler
Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106; Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106; Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106; Department of Microbiology, Case Western Reserve University, Cleveland, OH 44106; Department of Molecular Biology, Case Western Reserve University, Cleveland, OH 44106.
Robert A
RA
Bonomo
Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Nacional de Rosario, 2000 Rosario, Argentina; vila@ibr-conicet.gov.ar jim.spencer@bristol.ac.uk.
Alejandro J
AJ
Vila
School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol, Bristol BS8 1TD, United Kingdom; vila@ibr-conicet.gov.ar jim.spencer@bristol.ac.uk.
James
J
Spencer
D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D015195:Drug Design; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D053778:Thiazolidines / Q000737:chemistry*; D065093:beta-Lactamase Inhibitors / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D053778:Thiazolidines; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
antibiotic resistance; bisthiazolidines; carbapenemase; inhibitors; metallo-β-lactamase