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61 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Mutate and conjugate: a method to enable rapid in-cell target validation Adam M. Thomas , Marta Serafini , Emma K. Grant , Edward A. J. Coombs , Joseph P. Bluck , Matthias Schiedel , Michael A. Mcdonough , Jessica K. Reynolds , Bernadette Lee , Michael Platt , Vassilena Sharlandjieva , Philip C. Biggin , Fernanda Duarte , Thomas A. Milne , Jacob T. Bush , Stuart J. Conway Acs Chemical Biology DOI: 10.1021/acschembio.3c00437 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.	Oct 2023
I24-Microfocus Macromolecular Crystallography	Structural basis for binding of the renal carcinoma target hypoxia‐inducible factor 2α to prolyl hydroxylase domain 2 William D. Figg , Giorgia Fiorini , Rasheduzzaman Chowdhury , Yu Nakashima , Anthony Tumber , Michael A. Mcdonough , Christopher J. Schofield Proteins: Structure, Function, And Bioinformatics 8 DOI: 10.1002/prot.26541 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.	Jul 2023
I03-Macromolecular Crystallography	In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease Takashi Miura , Tika R. Malla , C. David Owen , Anthony Tumber , Lennart Brewitz , Michael A. Mcdonough , Eidarus Salah , Naohiro Terasaka , Takayuki Katoh , Petra Lukacik , Claire Strain-Damerell , Halina Mikolajek , Martin A. Walsh , Akane Kawamura , Christopher J. Schofield , Hiroaki Suga Nature Chemistry 81 DOI: 10.1038/s41557-023-01205-1 Diamond Proposal Number(s): [27088] Open Access Show Abstract ▼ Abstract: γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.	May 2023
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	An LNA-amide modification that enhances the cell uptake and activity of phosphorothioate exon-skipping oligonucleotides Ysobel R. Baker , Cameron Thorpe , Jinfeng Chen , Laura M. Poller , Lina Cox , Pawan Kumar , Wooi F. Lim , Lillian Lie , Graham Mcclorey , Sven Epple , Daniel Singleton , Michael A. Mcdonough , Jack S. Hardwick , Kirsten E. Christensen , Matthew J. A. Wood , James P. Hall , Afaf H. El-Sagheer , Tom Brown Nature Communications 13 DOI: 10.1038/s41467-022-31636-2 Diamond Proposal Number(s): [18069, 23459] Open Access Show Abstract ▼ Abstract: Oligonucleotides that target mRNA have great promise as therapeutic agents for life-threatening conditions but suffer from poor bioavailability, hence high cost. As currently untreatable diseases come within the reach of oligonucleotide therapies, new analogues are urgently needed to address this. With this in mind we describe reduced-charge oligonucleotides containing artificial LNA-amide linkages with improved gymnotic cell uptake, RNA affinity, stability and potency. To construct such oligonucleotides, five LNA-amide monomers (A, T, C, 5mC and G), where the 3′-OH is replaced by an ethanoic acid group, are synthesised in good yield and used in solid-phase oligonucleotide synthesis to form amide linkages with high efficiency. The artificial backbone causes minimal structural deviation to the DNA:RNA duplex. These studies indicate that splice-switching oligonucleotides containing LNA-amide linkages and phosphorothioates display improved activity relative to oligonucleotides lacking amides, highlighting the therapeutic potential of this technology.	Jul 2022
I04-1-Macromolecular Crystallography (fixed wavelength)	Studies on the reactions of biapenem with VIM metallo β-lactamases and the serine β-lactamase KPC-2 Anka Lucic , Tika R. Malla , Karina Calvopina , Catherine L. Tooke , Jurgen Brem , Michael A. Mcdonough , James Spencer , Christopher J. Schofield Antibiotics 11 DOI: 10.3390/antibiotics11030396 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: Carbapenems are important antibacterials and are both substrates and inhibitors of some β-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-β-lactamases VIM-1 and VIM-2 and the class A serine-β-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the β-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of β-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems.	Mar 2022
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Mechanisms of substrate recognition and N 6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes Simranjeet Kaur , Nok Yin Tam , Michael A. Mcdonough , Christopher J. Schofield , Wei Shen Aik Nucleic Acids Research 28 DOI: 10.1093/nar/gkac195 Diamond Proposal Number(s): [23459] Open Access Show Abstract ▼ Abstract: AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5′-3′ orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.	Mar 2022
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors Jurgen Brem , Tharindi Panduwawala , Jon Ulf Hansen , Joanne Hewitt , Edgars Liepins , Pawel Donets , Laura Espina , Alistair J. M. Farley , Kirill Shubin , Gonzalo Gomez Campillos , Paula Kiuru , Shifali Shishodia , Daniel Krahn , Robert K. Leśniak , Juliane Schmidt , Karina Calvopina , María-Carmen Turrientes , Madeline E. Kavanagh , Dmitrijs Lubriks , Philip Hinchliffe , Gareth W. Langley , Ali F. Aboklaish , Anders Eneroth , Maria Backlund , Andrei G. Baran , Elisabet I. Nielsen , Michael Speake , Janis Kuka , John Robinson , Solveiga Grinberga , Lindsay Robinson , Michael A. Mcdonough , Anna M. Rydzik , Thomas M. Leissing , Juan Carlos Jimenez-Castellanos , Matthew B. Avison , Solange Da Silva Pinto , Andrew D. Pannifer , Marina Martjuga , Emma Widlake , Martins Priede , Iva Hopkins Navratilova , Marek Gniadkowski , Anna Karin Belfrage , Peter Brandt , Jari Yli-Kauhaluoma , Eric Bacque , Malcolm G. P. Page , Fredrik Björkling , Jonathan M. Tyrrell , James Spencer , Pauline A. Lang , Pawel Baranczewski , Rafael Cantón , Stuart P. Mcelroy , Philip S. Jones , Fernando Baquero , Edgars Suna , Angus Morrison , Timothy R. Walsh , Christopher J. Schofield Nature Chemistry 59 DOI: 10.1038/s41557-021-00831-x Open Access Show Abstract ▼ Abstract: Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.	Dec 2021
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structure-based design of selective fat mass and obesity associated protein (FTO) inhibitors Shifali Shishodia , Marina Demetriades , Dong Zhang , Nok Yin Tam , Pratheesh Maheswaran , Caitlin Clunie-O'Connor , Anthony Tumber , Ivanhoe K. H. Leung , Yi Min Ng , Thomas M. Leissing , Afaf H. El-Sagheer , Eidarus Salah , Tom Brown , Wei Shen Aik , Michael A. Mcdonough , Christopher J. Schofield Journal Of Medicinal Chemistry 276 DOI: 10.1021/acs.jmedchem.1c01204 Open Access Show Abstract ▼ Abstract: FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo.	Nov 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis Patrick Rabe , Jos J. A. G. Kamps , Kyle D. Sutherlin , James D. S. Linyard , Pierre Aller , Cindy C. Pham , Mikako Makita , Ian Clifton , Michael A. Mcdonough , Thomas M. Leissing , Denis Shutin , Pauline A. Lang , Agata Butryn , Jurgen Brem , Sheraz Gul , Franklin D. Fuller , In-Sik Kim , Mun Hon Cheah , Thomas Fransson , Asmit Bhowmick , Iris D. Young , Lee O'Riordan , Aaron S. Brewster , Ilaria Pettinati , Margaret Doyle , Yasumasa Joti , Shigeki Owada , Kensuke Tono , Alexander Batyuk , Mark S. Hunter , Roberto Alonso-Mori , Uwe Bergmann , Robin L. Owen , Nicholas K. Sauter , Timothy D. W. Claridge , Carol V. Robinson , Vittal K. Yachandra , Junko Yano , Jan F. Kern , Allen M. Orville , Christopher J. Schofield Science Advances 7 DOI: 10.1126/sciadv.abh0250 Diamond Proposal Number(s): [23459, 19458] Open Access Show Abstract ▼ Abstract: Isopenicillin N synthase (IPNS) catalyzes the unique reaction of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.	Aug 2021
I04-Macromolecular Crystallography	Structural basis of prolyl hydroxylase domain inhibition by molidustat William D. Figg , Michael A. Mcdonough , Rasheduzzaman Chowdhury , Yu Nakashima , Zhihong Zhang , James P. Holt‐martyn , Alen Krajnc , Christopher J. Schofield Chemmedchem 5 DOI: 10.1002/cmdc.202100133 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: 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.	Apr 2021

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