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Instruments / Technical Area	Publication Details	Published
	Accelerating drug discovery with high-throughput crystallographic fragment screening and structural enablement Daren Fearon , Ailsa Powell , Alice Douangamath , Alexandre Dias , Charles W. E. Tomlinson , Blake H. Balcomb , Jasmin C. Aschenbrenner , Anthony Aimon , Isabel A. Barker , Jose Brandao-Neto , Patrick Collins , Louise E. Dunnett , Michael Fairhead , Richard J. Gildea , Mathew Golding , Tyler Gorrie-Stone , Paul V. Hathaway , Lizbe Koekemoer , Tobias Krojer , Ryan Lithgo , Elizabeth M. Maclean , Peter G. Marples , Xiaomin Ni , Rachael Skyner , Romain Talon , Warren Thompson , Conor F. Wild , Max Winokan , Nathan D. Wright , Graeme Winter , Elizabeth J. Shotton , Frank Von Delft Applied Research DOI: 10.1002/appl.202400192 Open Access Show Abstract ▼ Abstract: Fragment-based drug discovery is a well-established method for the identification of chemical starting points for development into clinical candidates. Historically, crystallographic fragment screening was perceived to be low-throughput and time consuming. However, thanks to advances in synchrotron capabilities and the introduction of dedicated facilities, such as the XChem platform at Diamond Light Source, there have been substantial improvements in throughput and integration between sample preparation, data collection and hit identification. Herein we share our experiences of establishing a crystallographic fragment screening facility, our learnings from operating a user programme for ten years and our perspective on applying structural enablement to rapidly progress initial fragment hits to lead-like molecules.	Nov 2024
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)	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
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
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease Alice Douangamath , Daren Fearon , Paul Gehrtz , Tobias Krojer , Petra Lukacik , C. David Owen , Efrat Resnick , Claire Strain-Damerell , Anthony Aimon , Péter Ábrányi-Balogh , Jose Brandao-Neto , Anna Carbery , Gemma Davison , Alexandre Dias , Thomas D. Downes , Louise Dunnett , Michael Fairhead , James D. Firth , S. Paul Jones , Aaron Keeley , György M. Keserü , Hanna F. Klein , Mathew P. Martin , Martin M. Noble , Peter O’brien , Ailsa Powell , Rambabu N. Reddi , Rachael Skyner , Matthew Snee , Michael J. Waring , Conor Wild , Nir London , Frank Von Delft , Martin A. Walsh Nature Communications 11 DOI: 10.1038/s41467-020-18709-w Open Access Show Abstract ▼ Abstract: COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.	Oct 2020

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