Publications by authors named "Christopher J Schofield"

2-Oxoglutarate (2OG) dependent N-methyl lysine demethylases (JmjC-KDMs) regulate eukaryotic transcription. We report studies showing that isolated forms of all human KDM4 and KDM5 JmjC enzymes catalyse demethylation of N-methylated Arg-3 of histone H2a. Unexpectedly, the results reveal that KDM4E and, less efficiently, KDM4D catalyse C-4 hydroxylation of Arg-20 of H2a on peptides, recombinant H2a, and calf histone extracts, including when the Arg-20 guanidino group is N-methylated.

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Histone lysine demethylase 4 A (KDM4A), a non-heme Fe(II)/2-oxoglutarate (2OG) dependent oxygenase that catalyzes the demethylation of tri-methylated lysine residues at the 9, 27, and 36 positions of histone H3 (H3 K9me3, H3 K27me3, and H3 K36me3). These methylated residues show contrasting transcriptional roles; therefore, understanding KDM4A's catalytic mechanisms with these substrates is essential to explain the factors that control the different sequence-dependent demethylations. In this study, we use molecular dynamics (MD)-based combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate determinants of KDM4A catalysis with H3 K9me3, H3 K27me3 and H3 K36me3 substrates.

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Hypoxia inducible transcription factors (HIFs) mediate the hypoxic response in metazoans. When sufficient O 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.

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Genetic loss of the 2-oxoglutarate oxygenase factor inhibiting hypoxia-inducible factor (FIH) enhances both glycolysis and aerobic metabolism. FIH is thus a potential target for adiposity control and improving hepatic steatosis. We describe development of a series of novel, potent, and selective FIH inhibitors that occupy both the FIH catalytic site and a recently defined tyrosine conformational-flip pocket.

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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 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).

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Aspartate/asparagine-β-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer.

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The essential L,D-transpeptidase of Mycobacterium tuberculosis (Ldt) catalyses the formation of 3 3 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit Ldt 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.

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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.

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Article Synopsis
  • - The SNM1A exonuclease is crucial for repairing interstrand crosslinks (ICLs) in DNA, and it interacts with the Cockayne Syndrome B protein (CSB) during transcription-coupled DNA repair.
  • - The research shows that certain regions in CSB, particularly the C-terminal region, are essential for binding and enhancing the activity of SNM1A, especially in its ability to process ICLs.
  • - The findings suggest that targeting the specific protein interactions that boost SNM1A's function may offer a new therapeutic strategy for treating conditions related to DNA damage, rather than just inhibiting the nuclease's active site.
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  • JMJD7 is a human enzyme that specifically modifies lysine residues in certain proteins, which is important for their function.
  • Research indicates that JMJD7 has a limited range of substrates compared to other similar enzymes, but can efficiently use altered lysines for its reactions.
  • The study identifies ways to inhibit JMJD7 by using variants of its substrates, which could aid in developing targeted therapies against diseases related to JMJD7 activity.
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Enhancing thermogenic brown adipose tissue (BAT) function is a promising therapeutic strategy for metabolic disease. However, predominantly thermoneutral modern human living conditions deactivate BAT. We demonstrate that selective adipocyte deficiency of the oxygen-sensor HIF-prolyl hydroxylase (PHD2) gene overcomes BAT dormancy at thermoneutrality.

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Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.

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Rhodanines have been characterised as 'difficult to progress' compounds for medicinal use, though one rhodanine is used for diabetes mellitus treatment and others are in clinical development. Rhodanines can undergo hydrolysis to enethiols which are inhibitors of metallo-enzymes, such as metallo β-lactamases. We report that in DMSO, rhodanine derived enethiols undergo dimerisations to give 1,3-dithiolanes and mixed disulfides.

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In seeding plants, biosynthesis of the phytohormone ethylene, which regulates processes including fruit ripening and senescence, is catalyzed by 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The plant pathogen Pseudomonas savastanoi (previously classified as: Pseudomonas syringae) employs a different type of ethylene-forming enzyme (psEFE), though from the same structural superfamily as ACC oxidase, to catalyze ethylene formation from 2-oxoglutarate (2OG) in an arginine dependent manner. psEFE also catalyzes the more typical oxidation of arginine to give L-Δ-pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO.

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Nirmatrelvir (PF-07321332), a first-in-class inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (M), was developed by Pfizer under intense pressure during the pandemic to treat COVID-19. A weakness of nirmatrelvir is its limited metabolic stability, which led to the development of a combination therapy (paxlovid), involving coadministration of nirmatrelvir with the cytochrome P450 inhibitor ritonavir. However, limitations in tolerability of the ritonavir component reduce the scope of paxlovid.

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Article Synopsis
  • Scientists found that a specific marker in melanoma (a type of skin cancer), called ALDH1A3, works with another protein called ACSS2 to help cancer cells use sugar (glucose) better and change their genes.
  • They discovered that acetaldehyde, which is a toxic substance, plays a role in this process and can help change gene activity in cancer cells.
  • In experiments with zebrafish that have melanoma, they showed that blocking ALDH1A3 can stop cancer cells from becoming resistant to treatment and may help doctors find new ways to fight the disease.
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In animals, limiting oxygen upregulates the hypoxia-inducible factor (HIF) and promotes 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.

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DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited.

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Aspartyl/asparaginyl hydroxylase (AspH) catalyzes the post-translational hydroxylations of vital human proteins, playing an essential role in maintaining their biological functions. Single-point mutations in the Second Coordination Sphere (SCS) and long-range (LR) residues of AspH have been linked to pathological conditions such as the ophthalmologic condition Traboulsi syndrome and chronic kidney disease (CKD). Although the clinical impacts of these mutations are established, there is a critical knowledge gap regarding their specific atomistic effects on the catalytic mechanism of AspH.

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Article Synopsis
  • Cyclic β-amino acids (cβAAs) are important for creating compact and stable peptide structures, enhancing their resistance to breakdown and improving their ability to bind to target proteins.
  • An engineered tRNA allowed for the efficient incorporation of cβAAs into a macrocyclic peptide library, which was then tested against the SARS-CoV-2 main protease.
  • The peptides BM3 and BM7 demonstrated strong inhibitory effects and exceptional stability in serum, indicating that cβAAs play a crucial role in enhancing the functionality and longevity of peptides in a biological context.
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The three human SNM1 metallo-β-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site.

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Article Synopsis
  • The SARS-CoV-2 main protease is an important target for COVID-19 drugs, with existing inhibitors like nirmatrelvir and simnotrelvir reacting with a specific cysteine residue.
  • New research indicates that γ-lactams might effectively inhibit the main protease through a similar reaction with the cysteine, suggesting a potential pathway for creating new inhibitors for this and other cysteine enzymes.
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Nonheme Fe(II) and 2-oxoglutarate (2OG)-dependent histone lysine demethylases 2A (KDM2A) catalyze the demethylation of the mono- or dimethylated lysine 36 residue in the histone H3 peptide (H3K36me1/me2), which plays a crucial role in epigenetic regulation and can be involved in many cancers. Although the overall catalytic mechanism of KDMs has been studied, how KDM2 catalysis takes place in contrast to other KDMs remains unknown. Understanding such differences is vital for enzyme redesign and can help in enzyme-selective drug design.

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