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Abstract: β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI is in phase-3 clinical trials in combination with imipenem, for treatment of infections by multi-drug resistant Enterobacteriaceae. We show that relebactam inhibits five clinically-important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e. the extended spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM); and the carbapenemases, KPC-2, -3 and -4 (1 - 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared to the clinically approved DBO avibactam, (9 to 120-fold differences in IC50). Minimum inhibitory concentration assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3 and KPC-4 reveal its C2 linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity compared to avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3 and -4. This comprehensive comparison of relebactam binding across five clinically-important class A SBLs will inform the design of future DBOs with the aim of improving clinical efficacy of BLI:β-lactam combinations.

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Abstract: The bicyclic boronate VNRX-5133 is a new type of β-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-β-lactamases (SBLs) and some clinically important metallo-β-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1 and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds to the class D SBL OXA-10 and NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography we observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen onto the boron of the bicyclic core. Different side-chain-dependent binding modes for bicyclic boronates imply scope for optimisation. The results further support the ‘high energy intermediate’ analogue approach for broad-spectrum β-lactamase inhibitor development and highlight the ability of boron-inhibitors to interchange between different hybridization states / binding modes.

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Abstract: Background: The β-lactam antibiotics represent the most successful drug class for treatment of bacterial infections. Resistance to them, importantly via production of β-lactamases, which collectively are able to hydrolyse all classes of β-lactams, threatens their continued widespread use. Bicyclic boronates show potential as broad spectrum inhibitors of the mechanistically distinct serine- (SBL) and metallo- (MBL) β-lactamase families. Methods: Using biophysical methods, including crystallographic analysis, we have investigated the binding mode of bicyclic boronates to clinically important β-lactamases. Induction experiments and agar-based MIC screening against MDR-Enterobacteriaceae (n = 132) were used to evaluate induction properties and the in vitro efficacy of a bicyclic boronate in combination with meropenem. Results: Crystallographic analysis of a bicyclic boronate in complex with AmpC from Pseudomonas aeruginosa reveals it binds to form a tetrahedral boronate species. Microbiological studies on the clinical coverage (in combination with meropenem) and induction of β-lactamases by bicyclic boronates further support the promise of such compounds as broad spectrum β-lactamase inhibitors. Conclusions: Together with reported studies on the structural basis of their inhibition of class A, B and D β-lactamases, biophysical studies, including crystallographic analysis, support the proposal that bicyclic boronates mimic tetrahedral intermediates common to SBL and MBL catalysis. General significance: Bicyclic boronates are a new generation of broad spectrum inhibitors of both SBLs and MBLs.

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Abstract: Metallo‐β‐Lactamases (MBLs) protect bacteria from almost all β‐lactam antibiotics. VIM enzymes are among the most clinically important MBLs, with VIM‐1 increasing in carbapenem‐resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are amongst the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here we present crystal structures of VIM‐1 and its complexes with a substrate‐mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM‐1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water‐mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221‐bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs.

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Abstract: The most important resistance mechanism to β-lactam antibiotics involves hydrolysis by two β-lactamase categories: the nucleophilic serine (SBL) and the metallo- (MBL) β-lactamases. Cyclobutanones are hydrolytically stable β-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using 19F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone via interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a 13C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition state and/or intermediate analogues as inhibitors of all β-lactamase classes.