I04-Macromolecular Crystallography
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Abstract: β-Lactamases comprise the most important known mode of resistance to β-lactam antibiotics. Boronic acids/boronate esters show promise as a new class of β-lactamase inhibitors with the potential to inhibit both the metallo- and serine-β-lactamases. We report studies employing representative β-lactamases concerning a bicyclic boronate ester with a thioether rather than the more typical β-lactam antibiotic ‘C-6/C-7’ acylamino side chain as present in the penicillin/cephalosporin antibiotics. The results, including a crystal structure of the clinically relevant VIM-2 metallo β-lactamase in complex with the inhibitor, reveal the potential of boronate inhibitors without the canonical acylamino side chain for inhibition of a broader range of serine- and metallo- β-lactamases compared to previous bicyclic boronates, including the metallo-β-lactamase L1. They imply further SAR studies will expand the already broad scope of -lactamase inhibition by bicyclic boronates.
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Dec 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069, 19458]
Open Access
Abstract: The L,D-transpeptidases (Ldts) are promising antibiotic targets for treating tuberculosis. We report screening of cysteine-reactive inhibitors against LdtMt2 from Mycobacterium tuberculosis. Structural studies on LdtMt2 with potent inhibitor ebselen reveal opening of the benzisoselenazolone ring by a nucleophilic cysteine, forming a complex involving extensive hydrophobic interactions with a substrate-binding loop.
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Aug 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Open Access
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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Aug 2019
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I03-Macromolecular Crystallography
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Emma Zsófia Aletta
Nagy
,
Souad Diana
Tork
,
Pauline A.
Lang
,
Alina
Filip
,
Florin Dan
Irimie
,
László
Poppe
,
Monica Ioana
Toşa
,
Christopher J.
Schofield
,
Jurgen
Brem
,
Csaba
Paizs
,
Laszlo Csaba
Bencze
Diamond Proposal Number(s):
[19458]
Abstract: Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity towards phenylalanines and cinnamic acids mono-substituted with both electron donating (-CH3, -OCH3) and electron withdrawing (-CF3, -Br) groups at all positions (o-, m-, p-) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at o-, m-, p-positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of para-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative scale synthesis of (S)-m-bromo-phenylalanine (ee: > 99%, yield: 60%) and (R)-p-methyl-phenylalanine (ee: 97%, yield: 49%), using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.
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Aug 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[17212, 18069]
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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Aug 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Abstract: Bacterial production of β‐lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA‐48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this post‐translational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19F‐NMR spectroscopy to monitor the carbamylation state of 19F‐labelled OXA‐48. This method was used to investigate the interactions of OXA‐48 with clinically used serine β‐ lactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19F‐labelled OXA‐48 provide a structural rationale for the sensitivity of the 19F‐label to active site interactions. The overall results demonstrate the use of 19F‐NMR to monitor reversible covalent post‐translational modifications.
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Jul 2019
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I04-Macromolecular Crystallography
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Samuel T.
Cahill
,
Jonathan M.
Tyrrell
,
Iva
Hopkins Navratilova
,
Karina
Calvopina
,
Sean W.
Robinson
,
Christopher T.
Lohans
,
Michael A.
Mcdonough
,
Ricky
Cain
,
Colin W. G.
Fishwick
,
Matthew B.
Avison
,
Timothy R.
Walsh
,
Christopher J.
Schofield
,
Jurgen
Brem
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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Apr 2019
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I04-Macromolecular Crystallography
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Open Access
Abstract: Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo β-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.
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Dec 2018
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[313]
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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Nov 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Dong
Zhang
,
Marios S.
Markoulides
,
Dmitrijs
Stepanovs
,
Anna M.
Rydzik
,
Ahmed
El-hussein
,
Corentin A. M.
Bon
,
Jos J. A. G.
Kamps
,
Klaus-daniel
Umland
,
Patrick M.
Collins
,
Samuel T.
Cahill
,
David Y.
Wang
,
Timothy D. W.
Claridge
,
Jurgen
Brem
,
Michael
Mcdonough
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[9306, 12346, 16949]
Abstract: Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.
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Feb 2018
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