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

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Binding‐site purification of actives (B‐SPA) enables efficient large‐scale progression of fragment hits by combining multi‐step array synthesis with HT crystallography Harold Grosjean , Anthony Aimon , Storm Hassell-Hart , Warren Thompson , Lizbe Koekemoer , James Bennett , Anthony Bradley , Cameron Anderson , Conor Wild , William J. Bradshaw , Edward A. Fitzgerald , Tobias Krojer , Oleg Fedorov , Philip C. Biggin , John Spencer , Frank Von Delft Angewandte Chemie International Edition DOI: 10.1002/anie.202424373 Diamond Proposal Number(s): [19301] Show Abstract ▼ Abstract: Fragment approaches are long-established in target-based ligand discovery yet their full transformative potential lies dormant, because progressing hits to potency remains underserved by methodological work. The only credible progression paradigm is multiple cycles of costly conventional design-make-test-analyse (DMTA) medicinal chemistry, necessitating picking winners early and discarding others. It is effective to cheaply parallelize large numbers of non-uniform multi-step reactions, because, even without compound purification, a high-quality readout of binding is available, viz. crystallography. This can detect low-level binding of slightly active compounds, which the targeted binding site extracts directly from crude reaction mixtures (CRMs). In this proof-of-concept study, we expand a fragment hit from a crystal-based screen of the bromodomain PHIP2, using array synthesis on low-cost robotics to implement 6 independent multi-step reaction routes of up to 5 steps, attempting the synthesis of 1876 diverse expansions, designs entirely driven by synthetic tractability. The expected product was present in 1108 (59%) CRMs, detected by automated mass spectrometry, 22 individual products were resolved in crystal structures of CRMs added to crystals, providing an initial SAR map, pose stability in 19 and instability in 3 products and resolved stereochemical preference. One compound showed biochemical potency (IC50=34 μM) and affinity (Kd=50 μM) after resynthesis.	Feb 2025
	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
I04-1-Macromolecular Crystallography (fixed wavelength)	Novel starting points for human glycolate oxidase inhibitors, revealed by crystallography-based fragment screening Sabrina R. Mackinnon , Gustavo A. Bezerra , Tobias Krojer , Tamas Szommer , Frank Von Delft , Paul E. Brennan , Wyatt W. Yue Frontiers In Chemistry 10 DOI: 10.3389/fchem.2022.844598 Diamond Proposal Number(s): [18145] Open Access Show Abstract ▼ Abstract: Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme’s substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.	May 2022
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Discovery of novel BRD4 ligand scaffolds by automated navigation of the fragment chemical space Serena G. Piticchio , Miriam Martinez-Cartro , Salvatore Scaffidi , Moira Rachman , Sergio Rodriguez-Arevalo , Ainoa Sanchez-Arfelis , Carmen Escolano , Sarah Picaud , Tobias Krojer , Panagis Filippakopoulos , Frank Von Delft , Carles Galdeano , Xavier Barril Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.1c01108 Diamond Proposal Number(s): [15433, 19301] Show Abstract ▼ Abstract: Fragment-based drug discovery (FBDD) is a very effective hit identification method. However, the evolution of fragment hits into suitable leads remains challenging and largely artisanal. Fragment evolution is often scaffold-centric, meaning that its outcome depends crucially on the chemical structure of the starting fragment. Considering that fragment screening libraries cover only a small proportion of the corresponding chemical space, hits should be seen as probes highlighting privileged areas of the chemical space rather than actual starting points. We have developed an automated computational pipeline to mine the chemical space around any specific fragment hit, rapidly finding analogues that share a common interaction motif but are structurally novel and diverse. On a prospective application on the bromodomain-containing protein 4 (BRD4), starting from a known fragment, the platform yields active molecules with nonobvious scaffold changes. The procedure is fast and inexpensive and has the potential to uncover many hidden opportunities in FBDD.	Dec 2021
I03-Macromolecular Crystallography	Structure and activation mechanism of the human liver-type glutaminase GLS2 Igor M. Ferreira , José Edwin N. Quesñay , Alliny C. S. Bastos , Camila T. Rodrigues , Melanie Vollmar , Tobias Krojer , Claire Strain-Damerell , Nicola A. Burgess-Brown , Frank Von Delft , Wyatt W. Yue , Sandra M G. Dias , Andre L. B. Ambrosio Biochimie 185, 96 - 104 DOI: 10.1016/j.biochi.2021.03.009 Diamond Proposal Number(s): [8421] Open Access Show Abstract ▼ Abstract: Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called “lid loop” is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS “activation” loop, formerly known as the “gating” loop) renders a highly active protein in stable tetrameric form. We conclude that the “activation” loop, a known target for GLS inhibition, may also be a drug target for GLS2.	Jun 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	Fragment screening reveals starting points for rational design of galactokinase 1 inhibitors to treat classic galactosemia Sabrina R. Mackinnon , Tobias Krojer , William R. Foster , Laura Diaz-Saez , Manshu Tang , Kilian V. M. Huber , Frank Von Delft , Kent Lai , Paul Brennan , Gustavo Arruda Bezerra , Wyatt W. Yue Acs Chemical Biology 152 DOI: 10.1021/acschembio.0c00498 Diamond Proposal Number(s): [18145] Open Access Show Abstract ▼ Abstract: Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP- competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.	Mar 2021
	The low-cost Shifter microscope stage transforms the speed and robustness of protein crystal harvesting Nathan David Wright , Patrick Collins , Lizbe Koekemoer , Tobias Krojer , Romain Talon , Elliot Nelson , Mingda Ye , Radoslaw Nowak , Joseph Newman , Jia Tsing Ng , Nick Mitrovic , Helton Wiggers , Frank Von Delft Acta Crystallographica Section D Structural Biology 77, 62 - 74 DOI: 10.1107/S2059798320014114 Open Access Show Abstract ▼ Abstract: Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100–240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.	Jan 2021

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