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Instruments / Technical Area	Publication Details	Published
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
	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
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Relative binding energies predict crystallographic binding modes of ethionamide booster lead compounds Natalie J. Tatum , Fernanda Duarte , Shina C. L. Kamerlin , Ehmke Pohl The Journal Of Physical Chemistry Letters DOI: 10.1021/acs.jpclett.9b00741 Diamond Proposal Number(s): [18598] Show Abstract ▼ Abstract: Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilised for filtering of the large dataset. We obtained biophysically validated hits, some which yielded complex crystal structures. In some cases, the crystallised binding mode and top scoring mode agree, while for others the alternate ligand binding orientation was found. In this contribution we combine rigid docking, MD simulations and the LIE method to calculate free energies of binding and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favourable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.	Apr 2019
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset Charlotte M. Miton , Stefanie Jonas , Gerhard Fischer , Fernanda Duarte , Mark F. Mohamed , Bert Van Loo , Bálint Kintses , Shina C. L. Kamerlin , Nobuhiko Tokuriki , Marko Hyvonen , Florian Hollfelder Proceedings Of The National Academy Of Sciences 47 DOI: 10.1073/pnas.1607817115 Show Abstract ▼ Abstract: The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme–substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (β leaving group from −1.08 to −0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.	Jul 2018

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