I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Pauline
Lang
,
Anete
Parkova
,
Thomas
Leissing
,
Karina
Calvopina
,
Ricky
Cain
,
Alen
Krajnc
,
Tharindi
Panduwawala
,
Jules
Philippe
,
Colin W. G.
Fishwick
,
Peteris
Trapencieris
,
Malcolm
Page
,
Christopher J.
Schofield
,
Jurgen
Brem
Open Access
Abstract: 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.
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Jun 2020
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I04-Macromolecular Crystallography
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Open Access
Abstract: The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z’-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.
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May 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Hwanho
Choi
,
Adam P.
Hardy
,
Thomas M.
Leissing
,
Rasheduzzaman
Chowdhury
,
Yu
Nakashima
,
Wei
Ge
,
Marios
Markoulides
,
John S.
Scotti
,
Philip A.
Gerken
,
Helen
Thorbjornsrud
,
Dahye
Kang
,
Sungwoo
Hong
,
Joongoo
Lee
,
Michael A.
Mcdonough
,
Hwangseo
Park
,
Christopher J.
Schofield
Open Access
Abstract: Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.
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May 2020
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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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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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