Publications by authors named "Christopher J Schofield"

437 Publications

Discovery of neuroprotective agents that inhibit human prolyl hydroxylase PHD2.

Bioorg Med Chem 2021 May 24;38:116115. Epub 2021 Mar 24.

School of Chemistry, University of New South Wales (UNSW), Sydney, Australia. Electronic address:

Prolyl hydroxylase (PHD) enzymes play a critical role in the cellular responses to hypoxia through their regulation of the hypoxia inducible factor α (HIF-α) transcription factors. PHD inhibitors show promise for the treatment of diseases including anaemia, cardiovascular disease and stroke. In this work, a pharmacophore-based virtual high throughput screen was used to identify novel potential inhibitors of human PHD2. Two moderately potent new inhibitors were discovered, with IC values of 4 μM and 23 μM respectively. Cell-based studies demonstrate that these compounds exhibit protective activity in neuroblastoma cells, suggesting that they have the potential to be developed into clinically useful neuroprotective agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2021.116115DOI Listing
May 2021

JMJD6 Is a Druggable Oxygenase That Regulates AR-V7 Expression in Prostate Cancer.

Cancer Res 2021 02;81(4):1087-1100

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.

Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased ( < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival ( = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. SIGNIFICANCE: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-20-1807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025710PMC
February 2021

Structural Basis of Prolyl Hydroxylase Domain Inhibition by Molidustat.

ChemMedChem 2021 Apr 1. Epub 2021 Apr 1.

Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.

Human prolyl-hydroxylases (PHDs) are hypoxia-sensing 2-oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia-inducible factor target genes. PHD inhibition enables the treatment of anaemia/ischaemia-related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late-stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain-penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active-site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π-π-stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking β2-β3, which are involved in dynamic substrate binding/product release.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.202100133DOI Listing
April 2021

Detection, discrimination and quantification of amphetamine, cathinone and nor-ephedrine regioisomers using benchtop H and F nuclear magnetic resonance spectroscopy.

Magn Reson Chem 2021 Mar 30. Epub 2021 Mar 30.

Department of Natural Sciences, Faculty of Science and Engineering, Manchester Metropolitan University, Manchester, UK.

Amphetamine and cathinone derivatives are abused recreationally due to the sense of euphoria they provide to the user. Methodologies for the rapid detection of the drug derivative present in a seized sample, or an indication of the drug class, are beneficial to law enforcement and healthcare providers. Identifying the drug class is prudent because derivatisation of these drugs, to produce regioisomers, for example, occurs frequently to circumvent global and local drug laws. Thus, newly encountered derivatives might not be present in a spectral library. Employment of benchtop nuclear magnetic resonance (NMR) could be used to provide rapid analysis of seized samples as well as identifying the class of drug present. Discrimination of individual amphetamine-, methcathinone-, N-ethylcathinone and nor-ephedrine-derived fluorinated and methylated regioisomers is achieved herein using qualitative automated H NMR analysis and compared to gas chromatography-mass spectrometry (GC-MS) data. Two seized drug samples, SS1 and SS2, were identified to contain 4-fluoroamphetamine by H NMR (match score median = 0.9933) and GC-MS (RR  = 5.42-5.43 min). The amount of 4-fluoroamphetamine present was 42.8%-43.4% w/w and 48.7%-49.2% w/w for SS1 and SS2, respectively, from quantitative F NMR analysis, which is in agreement with the amount determined by GC-MS (39.9%-41.4% w/w and 49.0%-49.3% w/w). The total time for the qualitative H NMR and quantitative F NMR analysis is ~10 min. This contrasts to ~40 min for the GC-MS method. The NMR method also benefits from minimal sample preparation. Thus, benchtop NMR affords rapid, and discriminatory, analysis of the drug present in a seized sample.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mrc.5156DOI Listing
March 2021

Faropenem reacts with serine and metallo-β-lactamases to give multiple products.

Eur J Med Chem 2021 Apr 9;215:113257. Epub 2021 Feb 9.

Chemistry Research Laboratory, The Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom. Electronic address:

Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2021.113257DOI Listing
April 2021

The methyltransferase METTL9 mediates pervasive 1-methylhistidine modification in mammalian proteomes.

Nat Commun 2021 02 9;12(1):891. Epub 2021 Feb 9.

Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316, Oslo, Norway.

Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20670-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873184PMC
February 2021

JMJD6 promotes self-renewal and regenerative capacity of hematopoietic stem cells.

Blood Adv 2021 Feb;5(3):889-899

Laboratory of Haematopoietic Stem Cell and Leukaemia Biology, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom.

Lifelong multilineage hematopoiesis critically depends on rare hematopoietic stem cells (HSCs) that reside in the hypoxic bone marrow microenvironment. Although the role of the canonical oxygen sensor hypoxia-inducible factor prolyl hydroxylase has been investigated extensively in hematopoiesis, the functional significance of other members of the 2-oxoglutarate (2-OG)-dependent protein hydroxylase family of enzymes remains poorly defined in HSC biology and multilineage hematopoiesis. Here, by using hematopoietic-specific conditional gene deletion, we reveal that the 2-OG-dependent protein hydroxylase JMJD6 is essential for short- and long-term maintenance of the HSC pool and multilineage hematopoiesis. Additionally, upon hematopoietic injury, Jmjd6-deficient HSCs display a striking failure to expand and regenerate the hematopoietic system. Moreover, HSCs lacking Jmjd6 lose multilineage reconstitution potential and self-renewal capacity upon serial transplantation. At the molecular level, we found that JMJD6 functions to repress multiple processes whose downregulation is essential for HSC integrity, including mitochondrial oxidative phosphorylation (OXPHOS), protein synthesis, p53 stabilization, cell cycle checkpoint progression, and mTORC1 signaling. Indeed, Jmjd6-deficient primitive hematopoietic cells display elevated basal and maximal mitochondrial respiration rates and increased reactive oxygen species (ROS), prerequisites for HSC failure. Notably, an antioxidant, N-acetyl-l-cysteine, rescued HSC and lymphoid progenitor cell depletion, indicating a causal impact of OXPHOS-mediated ROS generation upon Jmjd6 deletion. Thus, JMJD6 promotes HSC maintenance and multilineage differentiation potential by suppressing fundamental pathways whose activation is detrimental for HSC function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2020002702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876897PMC
February 2021

Cyclic boronates as versatile scaffolds for KPC-2 β-lactamase inhibition.

RSC Med Chem 2020 Apr 10;11(4):491-496. Epub 2020 Jan 10.

School of Cellular and Molecular Medicine , Biomedical Sciences Building , University of Bristol , Bristol , BS8 1TD , UK . Email:

carbapenemase-2 (KPC-2) is a serine-β-lactamase (SBL) capable of hydrolysing almost all β-lactam antibiotics. We compare KPC-2 inhibition by vaborbactam, a clinically-approved monocyclic boronate, and VNRX-5133 (taniborbactam), a bicyclic boronate in late-stage clinical development. Vaborbactam inhibition is slowly reversible, whereas taniborbactam has an off-rate indicating essentially irreversible complex formation and a 15-fold higher on-rate, although both potentiate β-lactam activity against KPC-2-expressing . High resolution X-ray crystal structures reveal closely related binding modes for both inhibitors to KPC-2, with differences apparent only in positioning of the endocyclic boronate ester oxygen. The results indicate the bicyclic boronate scaffold as both an efficient, long-lasting, KPC-2 inhibitor and capable of supporting further iterations that may improve potency against specific enzyme targets and pre-empt the emergence of inhibitor resistant KPC-2 variants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9md00557aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536818PMC
April 2020

Monitoring protein-metal binding by F NMR - a case study with the New Delhi metallo-β-lactamase 1.

RSC Med Chem 2020 Mar 21;11(3):387-391. Epub 2020 Feb 21.

The Department of Chemistry , University of Oxford , 12 Mansfield Road , Oxford , OX1 3TA , UK . Email:

F NMR protein observed spectroscopy is evaluated as a method for analysing protein metal binding using the New Delhi metallo-β-lactamase 1. The results imply F NMR is useful for analysis of different metallated protein states and investigations on equilibrium states in the presence of inhibitors. One limitation is that F labelling may affect metal ion binding. The sensitive readout of changes in protein behaviour observed by F NMR spectra coupled with the broad scope of tolerated conditions ( buffer variations) means F NMR should be further investigated for studying metal ion interactions and the inhibition of metallo-enzymes during drug discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9md00416eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484990PMC
March 2020

Mass spectrometry reveals potential of β-lactams as SARS-CoV-2 M inhibitors.

Chem Commun (Camb) 2021 Feb;57(12):1430-1433

Chemistry Research Laboratory, Department of Chemistry, 12 Mansfield Road, Oxford, OX1 3TA, UK.

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some β-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0cc06870eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006714PMC
February 2021

Improved Synthesis of Phosphoramidite-Protected -Methyladenosine via BOP-Mediated SAr Reaction.

Molecules 2020 Dec 31;26(1). Epub 2020 Dec 31.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.

-methyladenosine(mA) is the most abundant modification in mRNA. Studies on proteins that introduce and bind mA require the efficient synthesis of oligonucleotides containing mA. We report an improved five-step synthesis of the mA phosphoramidite starting from inosine, utilising a 1--benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP)mediated SAr reaction in the key step. The route manifests a substantial increase in overall yield compared to reported routes, and is useful for the synthesis of phosphoramidites of other adenosine derivatives, such as ethanoadenosine, an RNA analogue of the DNA adduct formed by the important anticancer drug Carmustine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules26010147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796277PMC
December 2020

Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2.

Sci Rep 2020 12 15;10(1):21964. Epub 2020 Dec 15.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK.

Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein-protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-76307-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738489PMC
December 2020

Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance.

J Biol Chem 2020 Nov 30. Epub 2020 Nov 30.

School of Cellular and Molecular Medicine, University of Bristol, United Kingdom.

Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), e.g. cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than binding (KM). High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω-, 240- and 270-loops, that border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme, and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω-loop, adopts two distinct conformations in KPC-2, either pointing 'in' or 'out' of the active site; with only the 'in' form compatible with deacylation. The 'out' conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω-loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.016461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949053PMC
November 2020

Structural Investigations of the Inhibition of Escherichia coli AmpC β-Lactamase by Diazabicyclooctanes.

Antimicrob Agents Chemother 2021 01 20;65(2). Epub 2021 Jan 20.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom

β-Lactam antibiotics are presently the most important treatments for infections by pathogenic , but their use is increasingly compromised by β-lactamases, including the chromosomally encoded class C AmpC serine-β-lactamases (SBLs). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimize the DBO core. We report kinetic and structural studies, including four high-resolution crystal structures, concerning inhibition of the AmpC serine-β-lactamase from (AmpC ) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (apparent inhibition constant [], 0.69 μM) against AmpC compared to that of the other DBOs ( = 5.0 to 7.4 μM) due to an ∼10-fold accelerated carbamoylation rate. However, zidebactam also has an accelerated off-rate, and with sufficient preincubation time, all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpC -zidebactam carbamoyl complex compared to those for the other DBOs. The results suggest the carbamoyl complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.02073-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849013PMC
January 2021

A small-molecule probe for monitoring binding to prolyl hydroxylase domain 2 by fluorescence polarisation.

Chem Commun (Camb) 2020 Nov 28;56(91):14199-14202. Epub 2020 Oct 28.

Laboratory of Drug Design and Discovery, Department of Chemistry, China Pharmaceutical University, Nanjing 211198, China.

Inhibition of the dioxygen sensing hypoxia-inducible factor prolyl hydroxylases has potential therapeutic benefit for treatment of diseases, including anaemia. We describe the discovery of a small-molecule probe useful for monitoring binding to human prolyl hydroxylase domain 2 (PHD2) via fluorescence polarisation. The assay is suitable for high-throughput screening of PHD inhibitors with both weak and strong affinities, as shown by work with clinically used inhibitors and naturally occurring PHD inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0cc06353cDOI Listing
November 2020

Metampicillin is a cyclic aminal produced by reaction of ampicillin with formaldehyde.

Sci Rep 2020 10 21;10(1):17955. Epub 2020 Oct 21.

Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.

Metampicillin is a β-lactam antibiotic that is prepared by the reaction of ampicillin with formaldehyde. Although metampicillin has been studied for treatment of infections in animals and humans, its structure has been unclear. We report NMR studies revealing that metampicillin contains a formaldehyde-derived cyclic aminal. NMR time-course experiments with excess formaldehyde in solution show formation of another product with an additional exocyclic hemiaminal group formed by reaction with the cyclic aminal nitrogen. The exocyclic hemiaminal group is readily removed by reaction with the formaldehyde scavenger 1,3-cyclohexanedione, whereas the cyclic aminal methylene exhibits greater stability. The overall results assign the structure of metampicillin as containing a cyclic aminal and further reveal the potential for complexity in the reaction of formaldehyde with biomedicinally relevant molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-74990-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577985PMC
October 2020

Small-molecule active pharmaceutical ingredients of approved cancer therapeutics inhibit human aspartate/asparagine-β-hydroxylase.

Bioorg Med Chem 2020 10 6;28(20):115675. Epub 2020 Aug 6.

Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, OX1 3TA Oxford, United Kingdom. Electronic address:

Human aspartate/asparagine-β-hydroxylase (AspH) is a 2-oxoglutarate (2OG) dependent oxygenase that catalyses the hydroxylation of Asp/Asn-residues of epidermal growth factor-like domains (EGFDs). AspH is reported to be upregulated on the cell surface of invasive cancer cells in a manner distinguishing healthy from cancer cells. We report studies on the effect of small-molecule active pharmaceutical ingredients (APIs) of human cancer therapeutics on the catalytic activity of AspH using a high-throughput mass spectrometry (MS)-based inhibition assay. Human B-cell lymphoma-2 (Bcl-2)-protein inhibitors, including the (R)-enantiomer of the natural product gossypol, were observed to efficiently inhibit AspH, as does the antitumor antibiotic bleomycin A. The results may help in the design of AspH inhibitors with the potential of increased selectivity compared to the previously identified Fe(II)-chelating or 2OG-competitive inhibitors. With regard to the clinical use of bleomycin A and of the Bcl-2 inhibitor venetoclax, the results suggest that possible side-effects mediated through the inhibition of AspH and other 2OG oxygenases should be considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2020.115675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588595PMC
October 2020

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism.

J Biol Chem 2020 12 22;295(49):16604-16613. Epub 2020 Sep 22.

Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom. Electronic address:

An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase-catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining β-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs ( Trp, Val, and Leu, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, the OXA-48 V120L and OXA-23 V128L variants, catalyze increased β-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.014607DOI Listing
December 2020

Anaerobic fixed-target serial crystallography.

IUCrJ 2020 Sep 21;7(Pt 5):901-912. Epub 2020 Aug 21.

Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, United Kingdom.

Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitro-gen cryo-stream at 100 K) enable, is data collection of di-oxy-gen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for di-oxy-gen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the 'sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent l-arginine hy-droxy-lase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2052252520010374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467159PMC
September 2020

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from and .

J Biol Chem 2020 12 15;295(49):16545-16561. Epub 2020 Sep 15.

Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom. Electronic address:

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, and the protozoan parasite, , both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, , and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013998DOI Listing
December 2020

The SNM1A DNA repair nuclease.

DNA Repair (Amst) 2020 11 31;95:102941. Epub 2020 Jul 31.

Department of Oncology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS, UK. Electronic address:

Unrepaired, or misrepaired, DNA damage can contribute to the pathogenesis of a number of conditions, or disease states; thus, DNA damage repair pathways, and the proteins within them, are required for the safeguarding of the genome. Human SNM1A is a 5'-to-3' exonuclease that plays a role in multiple DNA damage repair processes. To date, most data suggest a role of SNM1A in primarily ICL repair: SNM1A deficient cells exhibit hypersensitivity to ICL-inducing agents (e.g. mitomycin C and cisplatin); and both in vivo and in vitro experiments demonstrate SNM1A and XPF-ERCC1 can function together in the 'unhooking' step of ICL repair. SNM1A further interacts with a number of other proteins that contribute to genome integrity outside canonical ICL repair (e.g. PCNA and CSB), and these may play a role in regulating SNM1As function, subcellular localisation, and post-translational modification state. These data also provide further insight into other DNA repair pathways to which SNM1A may contribute. This review aims to discuss all aspects of the exonuclease, SNM1A, and its contribution to DNA damage tolerance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2020.102941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607226PMC
November 2020

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

Angew Chem Int Ed Engl 2020 12 15;59(52):23544-23548. Epub 2020 Oct 15.

Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Rd., OX1 3QZ, Oxford, UK.

The SARS-CoV-2 main protease (M ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. M is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of M . Analysis of the monomer/dimer equilibria reveals a dissociation constant of K =0.14±0.03 μM, indicating M has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate M activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202010316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461284PMC
December 2020

Isocitrate dehydrogenase variants in cancer - Cellular consequences and therapeutic opportunities.

Curr Opin Chem Biol 2020 08 8;57:122-134. Epub 2020 Aug 8.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK. Electronic address:

Abnormal metabolism is common in cancer cells and often correlates with mutations in genes encoding for enzymes involved in small-molecule metabolism. Isocitrate dehydrogenase 1 (IDH1) is the most frequently mutated metabolic gene in cancer. Cancer-associated substitutions in IDH1 and IDH2 impair wild-type production of 2-oxoglutarate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) from isocitrate and oxidised nicotinamide adenine dinucleotide phosphate (NADP ), and substantially promote the IDH variant catalysed conversion of 2-oxoglutarate to d-2-hydroxyglutarate (d-2HG). Elevated d-2HG is a biomarker for some cancers, and inhibition of IDH1 and IDH2 variants is being pursued as a medicinal chemistry target. We provide an overview of the types of cancer-associated IDH variants, discuss some of the proposed consequences of altered metabolism as a result of elevated d-2HG, summarise therapeutic efforts targeting IDH variants and identify areas for future research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbpa.2020.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487778PMC
August 2020

Hypoxia and hypoxia mimetics differentially modulate histone post-translational modifications.

Epigenetics 2021 Jan 1;16(1):14-27. Epub 2020 Jul 1.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford , Oxford, UK.

Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. Hypoxia affects histone modifications by altering the activities of histone-modifying enzymes and the levels of hypoxia-inducible factor (HIF) isoforms. Synthetic hypoxia mimetics promote a similar response, but how accurately the hypoxia mimetics replicate the effects of limited oxygen availability on the levels of histone PTMs is uncertain. Here we report studies on the profiling of the global changes to PTMs on intact histones in response to hypoxia/hypoxia-related stresses using liquid chromatography-mass spectrometry (LC-MS). We demonstrate that intact protein LC-MS profiling is a relatively simple and robust method for investigating potential effects of drugs on histone modifications. The results provide insights into the profiles of PTMs associated with hypoxia and inform on the extent to which hypoxia and hypoxia mimetics cause similar changes to histones. These findings imply chemically-induced hypoxia does not completely replicate the substantial effects of physiological hypoxia on histone PTMs, highlighting that caution should be used in interpreting data from their use.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15592294.2020.1786305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889154PMC
January 2021

Bicyclic Boronates as Potent Inhibitors of AmpC, the Class C β-Lactamase from .

Biomolecules 2020 06 12;10(6). Epub 2020 Jun 12.

Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, UK.

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 , 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10060899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356297PMC
June 2020

Role of Structural Dynamics in Selectivity and Mechanism of Non-heme Fe(II) and 2-Oxoglutarate-Dependent Oxygenases Involved in DNA Repair.

ACS Cent Sci 2020 May 8;6(5):795-814. Epub 2020 May 8.

Department of Chemistry, Michigan Technological University, Houghton, Michigan 49931, United States.

AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (mC) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic β-hairpin, are involved in substrate binding in AlkBH2-dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)=O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2-dsDNA and AlkB-dsDNA; in AlkB-ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein-DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscentsci.0c00312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256942PMC
May 2020

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

Chembiochem 2020 10 18;21(20):2898-2902. Epub 2020 Aug 18.

Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.

l-Ascorbate (l-Asc) is often added to assays with isolated Fe - and 2-oxoglutarate (2OG)-dependent oxygenases to enhance activity. l-Asc is proposed to be important in catalysis by some 2OG oxygenases in vivo. We report observations on the nonenzymatic conversion of 2OG to succinate, which is mediated by hydrogen peroxide generated by the reaction of l-Asc and dioxygen. Slow nonenzymatic oxidation of 2OG to succinate occurs with some, but not all, other reducing agents commonly used in 2OG oxygenase assays. We intend these observations will help in the robust assignment of substrates and inhibitors for 2OG oxygenases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.202000185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693218PMC
October 2020

Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors.

Sci Rep 2020 05 26;10(1):8650. Epub 2020 May 26.

Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, OX1 3TA, Oxford, United Kingdom.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-65123-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251097PMC
May 2020