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17 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
VMXm-Versatile Macromolecular Crystallography microfocus	Alkyne derivatives of SARS-CoV-2 main protease inhibitors including nirmatrelvir inhibit by reacting covalently with the nucleophilic cysteine Lennart Brewitz , Leo Dumjahn , Yilin Zhao , C. David Owen , Stephen M. Laidlaw , Tika R. Malla , Dung Nguyen , Petra Lukacik , Eidarus Salah , Adam D. Crawshaw , Anna J. Warren , Jose Trincao , Claire Strain-Damerell , Miles W. Carroll , Martin A. Walsh , Christopher J. Schofield Journal Of Medicinal Chemistry 374 DOI: 10.1021/acs.jmedchem.2c01627 Diamond Proposal Number(s): [27088] Open Access Show Abstract ▼ Abstract: Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.	Feb 2023
	East Coast fever, a neglected tropical disease with an outdated vaccine approach? Jeannine Kolakowski , Tim Connelley , Petra Lukacik , Mark Pfuhl , Dirk Werling Trends In Parasitology 67 DOI: 10.1016/j.pt.2022.08.006 Open Access Show Abstract ▼ Abstract: Since its discovery, bovine theileriosis has caused major socioeconomic losses in sub-Saharan Africa. Acaricide resistance of the intermediate host, paucity of therapeutics, and lack of sufficiently cross-protective vaccines increase the risk of parasite spread due to global warming. Here, we highlight three important areas that require investigation to develop next-generation vaccines.	Aug 2022
I24-Microfocus Macromolecular Crystallography	xia2.multiplex : a multi-crystal data-analysis pipeline Richard J. Gildea , James Beilsten-Edmands , Danny Axford , Sam Horrell , Pierre Aller , James Sandy , Juan Sanchez-Weatherby , C. David Owen , Petra Lukacik , Claire Strain-Damerell , Robin L. Owen , Martin A. Walsh , Graeme Winter Acta Crystallographica Section D Structural Biology 78 DOI: 10.1107/S2059798322004399 Diamond Proposal Number(s): [26986, 27088] Open Access Show Abstract ▼ Abstract: In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.	Jun 2022
I03-Macromolecular Crystallography	Penicillin derivatives inhibit the SARS-COV-2 main protease by reaction with its nucleophilic cysteine Tika R. Malla , Lennart Brewitz , Dorian-Gabriel Muntean , Hiba Aslam , C. David Owen , Eidarus Salah , Anthony Tumber , Petra Lukacik , Claire Strain-Damerell , Halina Mikolajek , Martin Walsh , Christopher J. Schofield Journal Of Medicinal Chemistry 49 DOI: 10.1021/acs.jmedchem.1c02214 Diamond Proposal Number(s): [27088] Open Access Show Abstract ▼ Abstract: The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.	May 2022
	Mass spectrometric assays reveal discrepancies in inhibition profiles for the SARS‐CoV‐2 papain‐like protease Lennart Brewitz , Jos J. A. G. Kamps , Petra Lukacik , Claire Strain-Damerell , Yilin Zhao , Anthony Tumber , Tika R. Malla , Allen M. Orville , Martin A. Walsh , Christopher Schofield Chemmedchem DOI: 10.1002/cmdc.202200016 Show Abstract ▼ Abstract: The two SARS-CoV-2 proteases, i.e. the main protease (M pro ) and the papain-like protease (PL pro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL pro catalysis in vitro . The assay was applied to investigate the effect of reported small-molecule PL pro inhibitors and selected M pro inhibitors on PL pro catalysis. The results reveal that some, but not all, PL pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL pro . Less selective M pro inhibitors, e.g. auranofin, inhibit PL pro , highlighting the potential for dual PL pro /M pro inhibition. MS-based PL pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.	Jan 2022
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The structure of nontypeable Haemophilus influenzae SapA in a closed conformation reveals a constricted ligand-binding cavity and a novel RNA binding motif Petra Lukacik , C. David Owen , Gemma Harris , Jani Reddy Bolla , Sarah Picaud , Irfan Alibay , Joanne E. Nettleship , Louise E. Bird , Raymond Owens , Philip C. Biggin , Panagis Filippakopoulos , Carol V. Robinson , Martin A. Walsh Plos One 16 DOI: 10.1371/journal.pone.0256070 Diamond Proposal Number(s): [4990, 5073, 4988] Open Access Show Abstract ▼ Abstract: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 μM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 μM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.	Oct 2021
NONE-No attached Diamond beamline	Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding H. T. Henry Chan , Marc A. Moesser , Rebecca K. Walters , Tika R. Malla , Rebecca M. Twidale , Tobias John , Helen M. Deeks , Tristan Johnston-Wood , Victor Mikhailov , Richard B. Sessions , William Dawson , Eidarus Salah , Petra Lukacik , Claire Strain-Damerell , C. David Owen , Takahito Nakajima , Katarzyna Świderek , Alessio Lodola , Vicent Moliner , David R. Glowacki , James Spencer , Martin A. Walsh , Christopher J. Schofield , Luigi Genovese , Deborah K. Shoemark , Adrian J. Mulholland , Fernanda Duarte , Garrett M. Morris Chemical Science 12, 13686 - 13703 DOI: 10.1039/D1SC03628A Open Access Show Abstract ▼ Abstract: The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective ‘peptibitors’ inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.	Oct 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor Daniel Zaidman , Paul Gehrtz , Mihajlo Filep , Daren Fearon , Ronen Gabizon , Alice Douangamath , Jaime Prilusky , Shirly Duberstein , Galit Cohen , C. David Owen , Efrat Resnick , Claire Strain-Damerell , Petra Lukacik , Haim Barr , Martin A. Walsh , Frank Von Delft , Nir London Cell Chemical Biology 63 DOI: 10.1016/j.chembiol.2021.05.018 Diamond Proposal Number(s): [18145, 27963] Show Abstract ▼ Abstract: Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found ∼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 μM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.	Jun 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19 Martin A. Redhead , C. David Owen , Lennart Brewitz , Amelia H. Collette , Petra Lukacik , Claire Strain-Damerell , Sean W. Robinson , Patrick M. Collins , Philipp Schäfer , Mark Swindells , Chris J. Radoux , Iva Navratilova Hopkins , Daren Fearon , Alice Douangamath , Frank Von Delft , Tika R. Malla , Laura Vangeel , Thomas Vercruysse , Jan Thibaut , Pieter Leyssen , Tu-Trinh Nguyen , Mitchell Hull , Anthony Tumber , David J. Hallett , Christopher J. Schofield , David I. Stuart , Andrew L. Hopkins , Martin A. Walsh Scientific Reports 11 DOI: 10.1038/s41598-021-92416-4 Open Access Show Abstract ▼ Abstract: Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.	Jun 2021
	Mass spectrometry reveals potential of β-lactams as SARS-CoV-2 M pro inhibitors Tika R. Malla , Anthony Tumber , Tobias John , Lennart Brewitz , Claire Strain-Damerell , C. David Owen , Petra Lukacik , H. T. Henry Chan , Pratheesh Maheswaran , Eidarus Salah , Fernanda Duarte , Haitao Yang , Zihe Rao , Martin A. Walsh , Christopher J. Schofield Chemical Communications 582 DOI: 10.1039/D0CC06870E Open Access Show Abstract ▼ Abstract: 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.	Jan 2021

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