I03-Macromolecular Crystallography
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James P.
Holt‐martyn
,
Rasheduzzaman
Chowdhury
,
Anthony
Tumber
,
Tzu‐lan
Yeh
,
Martine I.
Abboud
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Gareth W.
Langley
,
William
Figg
,
Michael A.
Mcdonough
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Christopher J.
Schofield
Abstract: The 2‐oxoglutarate‐dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on a promising class of 4‐hydroxypyrimidine‐containing PHD inhibitors.
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Dec 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Inga
Pfeffer
,
Lennart
Brewitz
,
Tobias
Krojer
,
Sacha A.
Jensen
,
Grazyna T.
Kochan
,
Nadia J.
Kershaw
,
Kirsty S.
Hewitson
,
Luke A.
Mcneill
,
Holger
Kramer
,
Martin
Münzel
,
Richard J.
Hopkinson
,
Udo
Oppermann
,
Penny A.
Handford
,
Michael A.
Mcdonough
,
Christopher J.
Schofield
Open Access
Abstract: AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
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Oct 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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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: JmjC domain containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones, and other proteins including heat shock protein 70 (HSP70), oestrogen receptor α (ERα) and RNA helicase A. Here we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG supported its assignment as a lysyl-hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.
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May 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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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Faraz
Shaikh
,
Yuguang
Zhao
,
Luis
Alvarez
,
Maria
Iliopoulou
,
Christopher Thomas
Lohans
,
Christopher J.
Schofield
,
Sergi
Padilla-parra
,
Shirley W. I.
Siu
,
Elizabeth
Fry
,
Jingshan
Ren
,
David I.
Stuart
Diamond Proposal Number(s):
[10627]
Abstract: Potent Ebolavirus (EBOV) inhibitors will help to curtail outbreaks such as that which occurred in 2014-16 in West Africa. EBOV has on its surface a single glycoprotein (GP) critical for viral entry and membrane fusion. Recent high resolution complexes of EBOV GP with a variety of approved drugs revealed that binding to a common cavity prevented fusion of the virus and endosomal membranes, inhibiting virus infection. We performed docking experiments, screening a database of natural compounds to identify those likely to bind at this site. Using both inhibition assays of HIV-1-derived pseudovirus cell entry and structural analyses of the complexes of the compounds with GP we show here that two of these compounds attach in the common binding cavity, out of eight tested. In both cases two molecules bind in the cavity. The two compounds are chemically similar but the tighter binder has an additional chlorine atom that forms good halogen bonds to the protein and achieves an IC50 of 50 nM, making it the most potent GP-binding EBOV inhibitor yet identified, validating our screening approach for the discovery of novel anti-viral compounds.
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Feb 2019
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