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The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.

Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β‑lactamase from Escherichia coli , by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β‑lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β‑lactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.

The bicyclic boronate VNRX-5133 (taniborbactam) 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 MBL NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp 3 ) 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 binding modes for bicyclic boronates for SBLs and MBLs imply scope for side-chain optimization. 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.

The β-lactams remain the most important antibacterials, but their use is increasingly compromised by resistance, importantly by β-lactamases. Although β-lactam and non-β-lactam inhibitors forming stable acyl-enzyme complexes with nucleophilic serine β-lactamases (SBLs) are widely used, these are increasingly susceptible to evolved SBLs and do not inhibit metallo-β-lactamases (MBLs). Boronic acids and boronate esters, especially cyclic ones, can potently inhibit both SBLs and MBLs. Vaborbactam, a monocyclic boronate, is approved for clinical use, but its β-lactamase coverage is limited. Bicyclic boronates rapidly react with SBLs and MBLs forming stable enzyme-inhibitor complexes that mimic the common anionic high-energy tetrahedral intermediates in SBL/MBL catalysis, as revealed by crystallography. The ability of boronic acids to 'morph' between sp 2 and sp 3 hybridisation states may help enable potent inhibition. There is limited structure-activity relationship information on the (bi)cyclic boronate inhibitors compared to β-lactams, hence scope for creativity towards new boron-based β-lactamase inhibitors/antibacterials., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)