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23 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength) Krios II-Titan Krios II at Diamond	Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease Andre Schutzer Godoy , Aline Minalli Nakamura , Alice Douangamath , Yun Song , Gabriela Dias Noske , Victor Oliveira Gawriljuk , Rafaela Sachetto Fernandes , Humberto D'Muniz Pereira , Ketllyn irene Zagato Oliveira , Daren Fearon , Alexandre Dias , Tobias Krojer , Michael Fairhead , Alisa Powell , Louise Dunnett , Jose Brandao-Neto , Rachael Skyner , Rod Chalk , Dávid Bajusz , Miklós Bege , Anikó Borbás , György Miklós Keserű , Frank Von Delft , Glaucius Oliva Nucleic Acids Research 579 DOI: 10.1093/nar/gkad314 Diamond Proposal Number(s): [27083, 27023] Open Access Show Abstract ▼ Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.	Apr 2023
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B Tamar Skaist Mehlmam , Justin T. Biel , Syeda Maryam Azeem , Elliot R. Nelson , Sakib Hossain , Louise Dunnett , Neil G. Paterson , Alice Douangamath , Romain Talon , Danny Axford , Helen Orins , Frank Von Delft , Daniel A. Keedy Elife 12 DOI: 10.7554/eLife.84632 Diamond Proposal Number(s): [15751, 18340, 23570] Open Access Show Abstract ▼ Abstract: Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly – but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.	Mar 2023
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Discovery of novel druggable pockets on polyomavirus VP1 through crystallographic fragment-based screening to develop capsid assembly inhibitors Evgenii M. Osipov , Ali H. Munawar , Steven Beelen , Daren Fearon , Alice Douangamath , Conor Wild , Stephen D. Weeks , Arthur Van Aerschot , Frank Von Delft , Sergei V. Strelkov Rsc Chemical Biology 31 DOI: 10.1039/D2CB00052K Diamond Proposal Number(s): [16816, 18582, 19190, 25586, 30588] Open Access Show Abstract ▼ Abstract: Polyomaviruses are a family of ubiquitous double-stranded DNA viruses many of which are human pathogens. These include BK polyomavirus which causes severe urinary tract infection in immunocompromised patients and Merkel cell polyomavirus associated with aggressive cancers. The small genome of polyomaviruses lacks conventional drug targets, and no specific drugs are available at present. Here we focus on the main structural protein VP1 of BK polyomavirus which is responsible for icosahedral capsid formation. To provide a foundation towards rational drug design, we crystallized truncated VP1 pentamers and subjected them to a high-throughput screening for binding drug-like fragments through a direct X-ray analysis. To enable a highly performant screening, rigorous optimization of the crystallographic pipeline and processing with the latest generation PanDDA2 software were necessary. As a result, a total of 144 binding hits were established. Importantly, the hits are well clustered in six surface pockets. Three pockets are located on the outside of the pentamer and map on the regions where the ‘invading’ C-terminal arm of another pentamer is attached upon capsid assembly. Another set of three pockets is situated within the wide pore along the five-fold axis of the VP1 pentamer. These pockets are situated at the interaction interface with the minor capsid protein VP2 which is indispensable for normal functioning of the virus. Here we systematically analyse the three outside pockets which are highly conserved across various polyomaviruses, while point mutations in these pockets are detrimental for viral replication. We show that one of the pockets can accommodate antipsychotic drug trifluoperazine. For each pocket, we derive pharmacophore features which enable the design of small molecules preventing the interaction between VP1 pentamers and therefore inhibiting capsid assembly. Our data lay a foundation towards a rational development of first-in-class drugs targeting polyomavirus capsid.	May 2022
I04-1-Macromolecular Crystallography (fixed wavelength)	Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase Joseph A. Newman , Alice Douangamath , Setayesh Yadzani , Yuliana Yosaatmadja , Anthony Aimon , Jose Brandao-Neto , Louise Dunnett , Tyler Gorrie-Stone , Rachael Skyner , Daren Fearon , Matthieu Schapira , Frank Von Delft , Opher Gileadi Nature Communications 12 DOI: 10.1038/s41467-021-25166-6 Diamond Proposal Number(s): [26998] Open Access Show Abstract ▼ Abstract: There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.	Aug 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	A crystallographic snapshot of SARS-CoV-2 main protease maturation process G. D. Noske , A. M. Nakamura , V. O. Gawriljuk , R. S. Fernandes , G. M. A. Lima , H. V. D. Rosa , H. D. Pereira , A. C. M. Zeri , A. A. F. Z. Nascimento , M. C. L. C. Freire , D. Fearon , A. Douangamath , F. Von Delft , G. Oliva , A. S. Godoy Journal Of Molecular Biology DOI: 10.1016/j.jmb.2021.167118 Diamond Proposal Number(s): [27963] Show Abstract ▼ Abstract: SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.	Jun 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
I04-1-Macromolecular Crystallography (fixed wavelength)	Exploring protein hotspots by optimized fragment pharmacophores Dávid Bajusz , Warren S. Wade , Grzegorz Satała , Andrzej J. Bojarski , Janez Ilaš , Jessica Ebner , Florian Grebien , Henrietta Papp , Ferenc Jakab , Alice Douangamath , Daren Fearon , Frank Von Delft , Marion Schuller , Ivan Ahel , Amanda Wakefield , Sándor Vajda , János Gerencsér , Péter Pallai , György M. Keserű Nature Communications 12 DOI: 10.1038/s41467-021-23443-y Diamond Proposal Number(s): [27001, 18145, 27963] Open Access Show Abstract ▼ Abstract: Fragment-based drug design has introduced a bottom-up process for drug development, with improved sampling of chemical space and increased effectiveness in early drug discovery. Here, we combine the use of pharmacophores, the most general concept of representing drug-target interactions with the theory of protein hotspots, to develop a design protocol for fragment libraries. The SpotXplorer approach compiles small fragment libraries that maximize the coverage of experimentally confirmed binding pharmacophores at the most preferred hotspots. The efficiency of this approach is demonstrated with a pilot library of 96 fragment-sized compounds (SpotXplorer0) that is validated on popular target classes and emerging drug targets. Biochemical screening against a set of GPCRs and proteases retrieves compounds containing an average of 70% of known pharmacophores for these targets. More importantly, SpotXplorer0 screening identifies confirmed hits against recently established challenging targets such as the histone methyltransferase SETD2, the main protease (3CLPro) and the NSP3 macrodomain of SARS-CoV-2.	May 2021
NONE-No attached Diamond beamline	Achieving efficient fragment screening at XChem facility at Diamond Light Source Alice Douangamath , Alisa Powell , Daren Fearon , Patrick M. Collins , Romain Talon , Tobias Krojer , Rachael Skyner , Jose Brandao-Neto , Louise Dunnett , Alexandre Dias , Anthony Aimon , Nicholas M. Pearce , Conor Wild , Tyler J. Gorrie-Stone , Frank Von Delft Journal Of Visualized Experiments DOI: 10.3791/62414 Open Access Show Abstract ▼ Abstract: In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen. Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification. XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.	May 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking Marion Schuller , Galen J. Correy , Stefan Gahbauer , Daren Fearon , Taiasean Wu , Roberto Efraín Díaz , Iris D. Young , Luan Carvalho Martins , Dominique H. Smith , Ursula Schulze-Gahmen , Tristan W. Owens , Ishan Deshpande , Gregory E. Merz , Aye C. Thwin , Justin T. Biel , Jessica K. Peters , Michelle Moritz , Nadia Herrera , Huong T. Kratochvil , Anthony Aimon , James Bennett , Jose Brandao Neto , Aina E. Cohen , Alexandre Dias , Alice Douangamath , Louise Dunnett , Oleg Fedorov , Matteo P. Ferla , Martin R. Fuchs , Tyler J. Gorrie-Stone , James M. Holton , Michael G. Johnson , Tobias Krojer , George Meigs , Alisa J. Powell , Johannes Gregor Matthias Rack , Victor Rangel , Silvia Russi , Rachael E. Skyner , Clyde A. Smith , Alexei S. Soares , Jennifer L. Wierman , Kang Zhu , Peter O’brien , Natalia Jura , Alan Ashworth , John J. Irwin , Michael C. Thompson , Jason E. Gestwicki , Frank Von Delft , Brian K. Shoichet , James S. Fraser , Ivan Ahel Science Advances 7 DOI: 10.1126/sciadv.abf8711 Diamond Proposal Number(s): [27001] Open Access Show Abstract ▼ Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.	Apr 2021

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