Cyclic Boronates Inhibit All Classes of -Lactamases. Antimicrobial

(cid:2) -Lactamase-mediated resistance is a growing threat to the continued use of (cid:2) -lactam antibiotics. The use of the (cid:2) -lactam-based serine- (cid:2) -lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non- (cid:2) lactam inhibitor avibactam has extended the utility of (cid:2) -lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo- (cid:2) -lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo- (cid:2) -lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difﬁcult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo- (cid:2) -lactamases. Here we report that cyclic boronates are able to inhibit all four classes of (cid:2) -lactamase, including the class A extended spectrum (cid:2) -lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa , and class D OXA enzymes with carbapenem-hydrolyzing capa-bilities. We demonstrate that cyclic boronates can potentiate the use of (cid:2) -lactams against Gram-negative clinical isolates expressing a variety of (cid:2) -lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other (cid:2) -lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo- (cid:2) -lactamase catalysis. Ultra mM mM containing pure His-tagged at 4°C with His-tagged 3C protease wt/wt) to remove N-terminal His tag. 3C together protein digestion mM mM mM enzyme identiﬁed were buffer into mM mM The inhibitory effects of cyclic boronate 2 on susceptibility to 19 diverse (cid:2) -lactam compounds was also assessed in Kirby-Bauer disc diffusion tests. Combination discs (Oxoid) were prepared with a ﬁxed ratio of 2:1 between the (cid:2) -lactam ( (cid:3) g) and cyclic boronate 2. Zones of inhibition around combined and unsupplemented discs were compared following overnight incubation on MH II plates. NMR spectroscopy. 13 C NMR experiments used a Bruker AVIII 600 MHz spectrometer equipped with a Prodigy broadband cryoprobe. Spectra were acquired at 298 K using a standard Bruker 13 C pulse sequence. The experimental parameters used were as follows: 2,048 scans, 36,058-Hz spectral width, 2.0-s relaxation delay, and 65,536 data points. A line broadening of 10 Hz was applied to all spectra. NMR samples contained 560 (cid:3) M OXA-10 and 10 mM NaH 13 CO 3 and were supplemented with 10% D 2 O. The impact of cyclic boronate 1 was tested at a concentration of 5 mM. Crystallization, X-ray data collection, and processing. Crystallization experiments were set up using a 13 mg/ml solution of CTX-M-15 in 50 mM HEPES (pH 7.5)–100 mM NaCl supplemented with 10 mM cyclic boronate 1. Crystallization was performed at room temperature using sitting-drop vapor diffusion methods. Crystals were obtained after 2 days using 100 (cid:3) l of 100 mM HEPES (pH 7.5)–70% 2,4-methylpentanediol in the reservoir and a 1:2 mixture (1 (cid:3) l:2 (cid:3) l) of protein to reservoir solution in the crystallization drop. Crystals were cryoprotected using 25% glycerol in reservoir solution before harvest-ing with nylon loops and ﬂash-cooling in liquid nitrogen. Diffraction data were collected at 100 K using a Rigaku FRE (cid:6) Superbright diffractometer. Diffraction data were integrated and scaled using HKL3000 (62). The structure was solved by molecular replacement with Phaser (63) using a published structure (PDB accession code 4HBT [35]) as a search model. The structure was then ﬁt and reﬁned iteratively using PHENIX and Coot (64, 65). Structural energy minimization. Energy minimization of small-molecule structures was performed using the MM2 energy minimization function in ChemBio3D Ultra (66). Accession number(s). Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 5T66.