I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Matthew J.
Beech
,
Edmond C.
Toma
,
Helen G.
Smith
,
Maria M.
Trush
,
Jit H. J.
Ang
,
Mei Y.
Wong
,
Chung H. J.
Wong
,
Hafiz S.
Ali
,
Zakia
Butt
,
Viha
Goel
,
Fernanda
Duarte
,
Alistair J. M.
Farley
,
Timothy R.
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[31353]
Open Access
Abstract: The Tet(X) flavin-dependent monooxygenases enable tetracycline antibiotic resistance by catalysing inactivating hydroxylation, so preventing inhibition of bacterial ribosomes. Tet(X) resistance is growing rapidly, threatening the efficacy of important last-resort tetracyclines such as tigecycline. Tet(X) inhibitors have potential to protect tetracyclines in combination therapies, but their discovery has been hampered by lack of high-throughput assays. We report the development of an efficient fluorescence polarisation Tet(X) binding assay employing a tetramethylrhodamine-glycyl-minocycline conjugate that enables inhibitor discovery. The assay was applied to tetracycline substrates and reported inhibitors, providing insight into their binding modes. Screening of a bioactive molecule library identified novel Tet(X) inhibitors, including psychoactive phenothiazine derivatives and the 5-HT4 agonist tegaserod, the activities of which were validated by turnover assays. Crystallographic studies of Tet(X4)-inhibitor complexes reveal two new inhibitor binding modes, importantly providing evidence for active site binding of Tet(X) inhibitors that do not share structural similarity with tetracycline substrates. In some cases, potentiation of tigecycline activity was observed in bacteria expressing Tet(X4). The combined results provide non-tetracycline scaffolds for development of potent Tet(X) inhibitors and highlight the need to evaluate the impact of non-antibiotics on antimicrobial resistance.
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May 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Zihao
Wang
,
Guodong
Cao
,
Miranda P.
Collier
,
Xingyu
Qiu
,
Sophie
Broadway-Stringer
,
Dominik
Šaman
,
Jediael Z. Y.
Ng
,
Navoneel
Sen
,
Amar J.
Azad
,
Charlotte
Hooper
,
Johannes
Zimmermann
,
Michael A.
Mcdonough
,
Jurgen
Brem
,
Patrick
Rabe
,
Haigang
Song
,
T. Reid
Alderson
,
Christopher J.
Schofield
,
Jani R.
Bolla
,
Kristina
Djinovic-Carugo
,
Dieter O.
Fürst
,
Bettina
Warscheid
,
Matteo T.
Degiacomi
,
Timothy M.
Allison
,
Georg K. A.
Hochberg
,
Carol V.
Robinson
,
Katja
Gehmlich
,
Justin L. P.
Benesch
Open Access
Abstract: The biomechanical properties and responses of tissues underpin a variety important of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. FLNC is a multi-functional protein that interacts with a variety of partners, however, how it is regulated at the molecular level is not well understood. Here we investigate its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We find that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we solve by X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work therefore shows, structurally, how HSPB7 acts as a specific molecular chaperone that regulates FLNC dimerisation.
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May 2025
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I03-Macromolecular Crystallography
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Yue
Wu
,
Zhihong
Li
,
Samanpreet
Kaur
,
Zewei
Zhang
,
Jie
Yue
,
Anthony
Tumber
,
Haoshu
Zhang
,
Zhe
Song
,
Peiyao
Yang
,
Ying
Dong
,
Fulai
Yang
,
Xiang
Li
,
Christopher J.
Schofield
,
Xiaojin
Zhang
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Factor inhibiting hypoxia-inducible factor (FIH) is a JmjC domain 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase that catalyzes protein hydroxylations, including of specific asparagines in the C-terminal transcriptional activation domains of hypoxia-inducible factor alpha (HIF-α) isoforms. FIH is of medicinal interest due to its ability to alter metabolism and modulate the course of the HIF-mediated hypoxic response. We report the development of a light-induced, lysine (Lys106)-targeting irreversible covalent inhibitor of FIH. The approach is complementary to optogenetic methods for regulation of transcription. The covalently reacting inhibitor NBA-ZG-2291 was the result of structure-guided modification of the reported active site binding FIH inhibitor ZG-2291 with an appropriately positioned o-nitrobenzyl alcohol (o-NBA) group. The results demonstrate that NBA-ZG-2291 forms a stable covalent bond in a light-dependent process with Lys106 of FIH, inactivating its hydroxylation activity and resulting in sustained upregulation of FIH-dependent HIF target genes. The light-controlled inhibitors targeting a lysine residue enable light and spatiotemporal control of FIH activity in a manner useful for dissecting the context-dependent physiological roles of FIH.
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May 2025
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I03-Macromolecular Crystallography
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Dóra
Laczi
,
Sofia Schönbauer
Huamán
,
Taylah
Andrews-Clark
,
Stephen M.
Laidlaw
,
Eidarus
Salah
,
Leo
Dumjahn
,
Petra
Lukacik
,
Hani
Choudhry
,
Martin A.
Walsh
,
Miles W.
Carroll
,
Christopher J.
Schofield
,
Lennart
Brewitz
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Nirmatrelvir is a substrate-related inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro) that is clinically used in combination with ritonavir to treat COVID-19. Derivatives of nirmatrelvir, modified at the substrate P2-equivalent position, have been developed to fine-tune inhibitor properties and are now in clinical use. We report the synthesis of nirmatrelvir derivatives with a (R)-4,4-dimethyl-4-silaproline (silaproline) group at the P2-equivalent position. Mass spectrometry (MS)-based assays demonstrate that silaproline-bearing nirmatrelvir derivatives efficiently inhibit isolated recombinant Mpro, albeit with reduced potency compared to nirmatrelvir. Investigations with SARS-CoV-2 infected VeroE6 cells reveal that the silaproline-bearing inhibitors with a CF3 group at the P4-equivalent position inhibit viral progression, implying that incorporating silicon atoms into Mpro inhibitors can yield in vivo active inhibitors with appropriate optimization. MS and crystallographic studies show that the nucleophilic active site cysteine residue of Mpro (Cys145) reacts with the nitrile group of the silaproline-bearing inhibitors. Substituting the electrophilic nitrile group for a non-activated terminal alkyne shifts the inhibition mode from reversible covalent inhibition to irreversible covalent inhibition. One of the two prochiral silaproline methyl groups occupies space in the S2 pocket that is unoccupied in Mpro:nirmatrelvir complex structures, highlighting the value of sila-derivatives in structure-activity-relationship (SAR) studies. The combined results highlight the potential of silicon-containing molecules for inhibition of Mpro and, by implication, other nucleophilic cysteine enzymes.
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Apr 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31353]
Open Access
Abstract: The 2-oxoglutarate (2OG)/Fe(II)-dependent γ-butyrobetaine hydroxylase (BBOX) catalyzes the final step in l-carnitine biosynthesis, i.e., stereoselective C-3 oxidation of γ-butyrobetaine (GBB). BBOX inhibition is a validated clinical strategy to modulate l-carnitine levels and to enhance cardiovascular efficiency. Reported BBOX inhibitors, including the clinically used cardioprotective agent Mildronate, manifest moderate inhibitory activity in vitro, limited selectivity, and/or unfavorable physicochemical properties, indicating a need for improved BBOX inhibitors. We report that the clinically used hypoxia-inducible factor-α prolyl residue hydroxylase (PHD) inhibitors Desidustat, Enarodustat, and Vadadustat efficiently inhibit isolated recombinant BBOX, suggesting that BBOX inhibition by clinically used PHD inhibitors should be considered as a possible off-target effect. Structure–activity relationship studies on the Desidustat scaffold enabled development of potent BBOX inhibitors that manifest high levels of selectivity for BBOX inhibition over representative human 2OG oxygenases, including PHD2. The Desidustat derivatives will help to enable investigations into the biological roles of l-carnitine and the therapeutic potential of BBOX inhibition.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Hypoxia inducible transcription factors (HIFs) mediate the hypoxic response in metazoans. When sufficient O2 is present, Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases (human PHD1-3) promote HIFα degradation via prolyl-hydroxylation. We report crystallographic, spectroscopic, and biochemical characterization of stable and inactive PHD2.Fe(III).2OG complexes. Aerobic incubation of PHD2 with Fe(II) and 2OG enables formation of PHD2.Fe(III).2OG complexes which bind HIF1-2α to give inactive PHD2.Fe(III).2OG.HIF1-2α complexes. The Fe(III) oxidation state in the inactive complexes was shown by EPR spectroscopy. L-Ascorbate hinders formation of the PHD2.Fe(III).2OG.(+/-HIFα) complexes and slowly regenerates them to give the catalytically active PHD2.Fe(II).2OG complex. Crystallographic comparison of the PHD2.Fe(III).2OG.HIF2α complex with the analogous anaerobic Fe(II) complex reveals near identical structures. Exposure of the anaerobic PHD2.Fe(II).2OG.HIF2α crystals to O2 enables in crystallo hydroxylation. The resulting PHD2.product structure, manifests conformational changes compared to the substrate structures. The results have implications for the role of the PHDs in hypoxia sensing and open new opportunities for inhibition of the PHDs and other 2OG dependent oxygenases by promoting formation of stable Fe(III) complexes.
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Oct 2024
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I03-Macromolecular Crystallography
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Open Access
Abstract: The essential L,D-transpeptidase of Mycobacterium tuberculosis (LdtMt2) catalyses the formation of 33 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit LdtMt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on LdtMt2. We determined a crystal structure of wild-type LdtMt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the LdtMt2 active site. The results will be useful in design of LdtMt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.
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Sep 2024
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I03-Macromolecular Crystallography
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Abstract: Prolyl hydroxylase domain-containing proteins 1-3 (PHD1-3) are 2-oxoglutarate (2OG)-dependent oxygenases catalysing C-4 hydroxylation of prolyl residues in α-subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), modifications that promote HIF-α degradation via the ubiquitin-proteasome pathway. Pharmacological inhibition of the PHDs induces HIF-α stabilisation, so promoting HIF target gene transcription. PHD inhibitors are used to treat anaemia caused by chronic kidney disease (CKD) due to their ability to stimulate erythropoietin (EPO) production. We report studies on the effects of the approved PHD inhibitors Desidustat and Enarodustat, and the clinical candidate TP0463518, on activities of a representative set of isolated recombinant human 2OG oxygenases. The three molecules manifest selectivity for PHD inhibition over that of the other 2OG oxygenases evaluated. We obtained crystal structures of Desidustat and Enarodustat in complex with the human 2OG oxygenase factor inhibiting hypoxia-inducible factor-α (FIH), which, together with modelling studies, inform on the binding modes of Desidustat and Enarodustat to active site Fe(II) in 2OG oxygenases, including PHD1-3. The results will help in the design of selective inhibitors of both the PHDs and other 2OG oxygenases, which are of medicinal interest due to their involvement inter alia in metabolic regulation, epigenetic signalling, DNA-damage repair, and agrochemical resistance.
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Sep 2024
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I03-Macromolecular Crystallography
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Open Access
Abstract: The breakthrough cephalosporin cefiderocol, approved for clinical use in 2019, has activity against many Gram-negative bacteria. The catechol group of cefiderocol enables it to efficiently enter bacterial cells via the iron/siderophore transport system thereby reducing resistance due to porin channel mutations and efflux pump upregulation. Limited information is reported regarding the binding of cefiderocol to its key proposed target, the transpeptidase penicillin binding protein 3 (PBP3). We report studies on the reaction of cefiderocol and the related cephalosporins ceftazidime and cefepime with Pseudomonas aeruginosa PBP3, including inhibition measurements, protein observed mass spectrometry, and X-ray crystallography. The three cephalosporins form analogous 3-exomethylene products with P. aeruginosa PBP3 following elimination of the C3′ side chain. pIC50 and kinact/Ki measurements with isolated PBP3 imply ceftazidime and cefiderocol react less efficiently than cefepime and, in particular, meropenem with P. aeruginosa PBP3. Crystal structures inform on conserved and different interactions involved in binding of the three cephalosporins and meropenem to P. aeruginosa PBP3. The results will aid development of cephalosporins with improved PBP3 inhibition properties.
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Sep 2024
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I03-Macromolecular Crystallography
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Yue
Wu
,
Yafen
Chen
,
Thomas
Corner
,
Yu
Nakashima
,
Eidarus
Salah
,
Zhihong
Li
,
Linjian
Zhang
,
Le
Yang
,
Anthony
Tumber
,
Zhuoli
Sun
,
Yukang
Wen
,
Ailin
Zhong
,
Fulai
Yang
,
Xiang
Li
,
Zhihong
Zhang
,
Christopher
Schofield
,
Xiaojin
Zhang
Diamond Proposal Number(s):
[23459]
Abstract: In animals limiting oxygen upregulates hypoxia-inducible factor (HIF) promoting a metabolic shift towards glycolysis. Factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that regulates HIF function by reducing its interaction with histone acetyl transferases. HIF levels are negatively regulated by the HIF prolyl hydroxylases (PHDs), which like FIH, are 2-oxoglutarate(2OG) oxygenases. Genetic loss of FIH promotes both glycolysis and aerobic metabolism. FIH has multiple non-HIF substrates making it challenging to connect its biochemistry with physiology. A structure-mechanism guided approach identified a highly potent in vivo active FIH inhibitor, ZG-2291, binding of which promotes a conformational flip of a catalytically important tyrosine, enabling selective inhibition of FIH over other JmjC subfamily 2OG oxygenases. Consistent with genetic studies, ZG-2291 promotes thermogenesis and ameliorates symptoms of obesity and metabolic dysfunction in ob/ob mice. The results reveal ZG-2291 as a useful probe for the physiological functions of FIH and identify FIH inhibition as a promising strategy for obesity treatment.
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Jun 2024
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