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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Molecular basis of Class A β-lactamase inhibition by relebactam Catherine L. Tooke , Philip Hinchliffe , Pauline A. Lang , Adrian J. Mulholland , Jurgen Brem , Christopher J. Schofield , James Spencer Antimicrobial Agents And Chemotherapy DOI: 10.1128/AAC.00564-19 Diamond Proposal Number(s): [17212] Open Access Show Abstract ▼ 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.	Aug 2019
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Bicyclic boronate VNRX-5133 inhibits metallo- and serine-β-lactamases Alen Krajnc , Jurgen Brem , Philip Hinchliffe , Karina Calvopina , Tharindi Panduwawala , Pauline A. Lang , Jos J. A. G. Kamps , Jonathan M. Tyrell , Emma Widlake , Benjamin G. Saward , Timothy R. Walsh , James Spencer , Christopher J. Schofield Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b00911 Diamond Proposal Number(s): [17212, 18069] Show Abstract ▼ 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.	Aug 2019
I02-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Crystal structures of VIM-1 complexes explain active site heterogeneity in VIM-class metallo-β-lactamases Ramya Salimraj , Philip Hinchliffe , Magda Kosmopoulou , Jonathan M. Tyrrell , Jurgen Brem , Sander S. Van Berkel , Anil Verma , Raymond J. Owens , Michael A. Mcdonough , Timothy R. Walsh , Christopher J. Schofield , James Spencer The Febs Journal DOI: 10.1111/febs.14695 Diamond Proposal Number(s): [313] Show Abstract ▼ 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.	Nov 2018
I02-Macromolecular Crystallography	Structural and Kinetic Studies on the Potent Inhibition of Metallo-β-Lactamases by 6-Phosphonomethylpyridine-2-Carboxylates Philip Hinchliffe , Carol A. Tanner , Anthony P. Krismanich , Geneviève Labbé , Valerie J. Goodfellow , Laura Marrone , Ahmed Desoky , Karina Calvopina , Emily E. Whittle , Fanxing Zeng , Matthew Avison , Niels C. Bols , Stefan Siemann , James Spencer , Gary I. Dmitrienko Biochemistry DOI: 10.1021/acs.biochem.7b01299 Diamond Proposal Number(s): [12342] Show Abstract ▼ Abstract: There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs), enzymes which hydrolyze β-lactam antibiotics and confer resistance on Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values 0.3 – 7.2 µM; Ki 0.03 – 1.5 µM). Minimum inhibitory concentration assays demonstrated potentiation of β-lactam (meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations where eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of inhibitor binding to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions, but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221/223, respectively (3 Å distance). The potency, low toxicity, cellular activity and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.	Feb 2018
I03-Macromolecular Crystallography	Cyclobutanone Mimics of Intermediates in Metallo-β-Lactamase Catalysis Martine I. Abboud , Magda Kosmopoulou , Anthony Krismanich , Jarrod W. Johnson , Philip Hinchliffe , Jurgen Brem , Timothy D. W. Claridge , James Spencer , Christopher J. Schofield , Gary I. Dmitrienko Chemistry - A European Journal DOI: 10.1002/chem.201705886 Diamond Proposal Number(s): [313] Show Abstract ▼ 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.	Dec 2017
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structural/mechanistic insights into the efficacy of non-classical β-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates Karina Calvopina , Philip Hinchliffe , Jurgen Brem , Kate J. Heesom , Samar Johnson , Ricky Cain , Christopher T. Lohans , Colin W. G. Fishwick , Christopher J. Schofield , James Spencer , Matthew B. Avison Molecular Microbiology DOI: 10.1111/mmi.13831 Diamond Proposal Number(s): [12342] Show Abstract ▼ Abstract: Clavulanic acid and avibactam are clinically deployed serine β-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo β-lactamases. Here we show that avibactam and a bicyclic boronate inhibit L2 (serine β-lactamase) but not L1 (metallo β-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyper-produce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like β-lactams plus non-classical β-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.	Sep 2017
I04-Macromolecular Crystallography	1.12 Å resolution crystal structure of the catalytic domain of the plasmid-mediated colistin resistance determinant MCR-2 Katie Coates , Timothy R. Walsh , James Spencer , Philip Hinchliffe Acta Crystallographica Section F Structural Biology Communications 73, 443 - 449 DOI: 10.1107/S2053230X17009669 Diamond Proposal Number(s): [12342] Open Access Show Abstract ▼ Abstract: MCR-2 confers resistance to colistin, a `last-line' antibiotic against extensively resistant Gram-negative pathogens. It is a plasmid-encoded phosphoethanolamine transferase that is closely related to MCR-1. To understand the diversity in the MCR family, the 1.12 Å resolution crystal structure of the catalytic domain of MCR-2 was determined. Variable amino acids are located distant from both the di-zinc active site and the membrane-proximal face. The exceptionally high resolution will provide an accurate starting model for further mechanistic studies.	Aug 2017
I04-Macromolecular Crystallography	13 C-Carbamylation as a mechanistic probe for the inhibition of class D β-lactamases by avibactam and halide ions Christopher T. Lohans , David Y. Wang , Christian Jorgensen , Samuel T. Cahill , Ian J. Clifton , Michael A. Mcdonough , Henry P. Oswin , James Spencer , Carmen Domene , Timothy D. W. Claridge , Jurgen Brem , Christopher J. Schofield Org. Biomol. Chem. 6 DOI: 10.1039/C7OB01514C Diamond Proposal Number(s): [12346] Show Abstract ▼ Abstract: The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the ‘hydrolytic water’ molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.	Jun 2017
I04-Macromolecular Crystallography	Crystallographic analyses of isoquinoline complexes reveal a new mode of metallo-β-lactamase inhibition Guo-bo Li , Jurgen Brem , Robert Lesniak , Martine I. Abboud , Christopher T. Lohans , Ian J. Clifton , Sheng-yong Yang , Juan-carlos Jiménez-castellanos , Matthew B. Avison , James Spencer , Michael A. Mcdonough , Christopher J. Schofield Chem. Commun. 54 DOI: 10.1039/C7CC02394D Diamond Proposal Number(s): [12346] Show Abstract ▼ Abstract: Crystallographic analyses of the VIM-5 metallo-β-lactamase (MBL) with isoquinoline inhibitors reveal non zinc ion binding modes. Comparison with other MBL–inhibitor structures directed addition of a zinc-binding thiol enabling identification of potent B1 MBL inhibitors. The inhibitors potentiate meropenem activity against clinical isolates harboring MBLs.	Apr 2017

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