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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Structural studies of βTrCP reveal plasticity in binding modes of consensus and nonconsensus degrons Gavin W. Collie , Hazel Mak , Marta Acebron-Garcia-De-Eulate , Argyrides Argyrou , Maxime Couturier , Maria Emanuela Cuomo , Daniel H. O Donovan , Isabella C. Russell , Geoffrey Wells , Jon Winter-Holt Acs Chemical Biology DOI: 10.1021/acschembio.5c01007 Diamond Proposal Number(s): [20015] Open Access Show Abstract ▼ Abstract: The E3 ligase βTrCP regulates a significant number of important cytosolic proteins by recognizing and binding to a “DSGXXS” consensus phosphodegron sequence, resulting in the ubiquitination and degradation of target proteins. While many of the substrates of βTrCP have strong disease links, there is high-resolution structural data available for just one of these proteins in complex with βTrCP. Here, we describe the development of a robust crystallographic system for βTrCP and report high-resolution crystal structures for βTrCP in complex with degrons from five new targets, encompassing the important cancer proteins, WEE1, claspin, ATF4, PDCD4, and IκBα. Interestingly, these structures reveal the molecular basis by which βTrCP can recognize and bind both consensus and nonconsensus degron peptides and reveal an overall general plasticity in degron binding mode. We also provide a biochemical assessment of the binding affinities of these peptides for βTrCP, adding further insight into the molecular interactions observed in the crystal structures. Finally, computational analyses of the βTrCP complexes identify opportunities for potential molecular glue approaches.	Mar 2026
I04-Macromolecular Crystallography	Targeting the interaction of NRF2 and β-TrCP with molecular glues Daniel H. O Donovan , Jon Winter-Holt , Gavin W. Collie , Maria E. Cuomo , David J. Mcgarry , Millie Fox , Bo Kyung Alex Seong , Ursula Grazini , Peter Barton , David Longmire , Andrew Lister , Yong Xu , Hazel Mak , Evelyne J. Barrey , Marta Acebrón-García-De-Eulate , Magdalena Richter , Nisha Peter , Maxime Couturier , Gustavo J. Gutierrez , Ryan Guilbert , Wankyu Lee , Pamela A. Lochhead , Argyrides Argyrou Acs Chemical Biology DOI: 10.1021/acschembio.5c01006 Diamond Proposal Number(s): [20015] Show Abstract ▼ Abstract: Inhibiting the oncogenic driver NRF2 in non-small cell lung cancer (NSCLC) represents a promising yet challenging clinical opportunity. Small molecules that enhance the NRF2:β-TrCP interaction (“molecular glues”) could hold therapeutic potential by promoting the ubiquitination and proteasomal degradation of NRF2. NRX-252114 is a molecular glue previously reported to promote the interaction between β-catenin and β-TrCP. We now find that NRX-252114 can also enhance the association between β-TrCP and NRF2 phosphodegron peptides. To leverage this novel interaction for the development of NRF2:β-TrCP molecular glues, we synthesized and evaluated a library of chemical analogues, guided by homology modeling and subsequently by X-ray crystallography. Surprisingly, structural elucidation of the NRF2:β-TrCP complex revealed occlusion of the presumed molecular glue binding pocket. This mechanistic insight explains the limited affinity enhancement for analogues of NRX-252114, and provides a structural rationale for the lack of NRF2 degradation in cells. Our findings broaden the scope of β-TrCP-targeted molecular glues, demonstrate that NRF2 is “glueable” at the peptide level, and provide mechanistic guidance for future efforts to target the pharmacologically elusive NRF2 pathway in cancer.	Mar 2026
I04-Macromolecular Crystallography	Discovery and in vivo efficacy of AZ-PRMT5i-1, a novel PRMT5 inhibitor with high MTA cooperativity James M. Smith , Bernard Barlaam , David Beattie , Lauren Bradshaw , Ho Man Chan , Elisabetta Chiarparin , Olga Collingwood , Sophie L. Cooke , Anna Cronin , Iain Cumming , Emma Dean , Judit É. Debreczeni , Iván Del Barco Barrantes , Coura Diene , Davide Gianni , Carine Guerot , Xiaoxiao Guo , Sinem Guven , Thomas G. Hayhow , Ted Hong , Paul D. Kemmitt , Gillian M. Lamont , Scott Lamont , James T. Lynch , Lisa Mcwilliams , Shaun Moore , Piotr Raubo , Graeme R. Robb , James Robinson , James S. Scott , Bharath Srinivasan , Oliver Steward , Christopher J. Stubbs , Karl Syson , Lixiang Tan , Oliver Turner , Elizabeth Underwood , Jelena Urosevic , Mercedes Vazquez-Chantada , Amy L. Whittaker , David M. Wilson , Jon J. Winter-Holt Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.4c00097 Show Abstract ▼ Abstract: PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel “MTA-cooperative” compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 (“AZ-PRMT5i-1”), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.	Jul 2024
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Fragment-based discovery of novel MUS81 inhibitors Gavin W. Collie , Ulf Börjesson , Yunhua Chen , Zhiqiang Dong , Paolo Di Fruscia , Andrea Gohlke , Anna Hoyle , Thomas A. Hunt , Mehul H. Jesani , Haiou Luo , Jakub Luptak , Alexander G. Milbradt , Priyanka Narasimhan , Martin Packer , Saleha Patel , Jingchuan Qiao , R. Ian Storer , Christopher J. Stubbs , Jonathan Tart , Caroline Truman , Anderson T. Wang , Matthew G. Wheeler , Jon Winter-Holt Acs Medicinal Chemistry Letters DOI: 10.1021/acsmedchemlett.3c00453 Diamond Proposal Number(s): [20015] Open Access Show Abstract ▼ Abstract: MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization. Here we report the fragment-based discovery of novel small molecule MUS81 inhibitors with sub-μM biochemical activity. These inhibitors were used to develop a novel crystal system, providing the first structural insight into the inhibition of MUS81 with small molecules.	Jun 2024
I03-Macromolecular Crystallography	Optimization of an imidazo[1,2-a]pyridine series to afford highly selective type i1/2 dual Mer/Axl kinase inhibitors with in vivo efficacy William Mccoull , Scott Boyd , Martin R. Brown , Muireann Coen , Olga Collingwood , Nichola L. Davies , Ann Doherty , Gary Fairley , Kristin Goldberg , Elizabeth Hardaker , Guang He , Edward J. Hennessy , Philip Hopcroft , George Hodgson , Anne Jackson , Xiefeng Jiang , Ankur Karmokar , Anne-Laure Lainé , Nicola Lindsay , Yumeng Mao , Roshini Markandu , Lindsay Mcmurray , Neville Mclean , Lorraine Mooney , Helen Musgrove , J. Willem M. Nissink , Alexander Pflug , Venkatesh Pilla Reddy , Philip B. Rawlins , Emma Rivers , Marianne Schimpl , Graham F. Smith , Sharon Tentarelli , Jon Travers , Robert I. Troup , Josephine Walton , Cheng Wang , Stephen Wilkinson , Beth Williamson , Jon Winter-Holt , Dejian Yang , Yuting Zheng , Qianxiu Zhu , Paul D. Smith Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.1c00920 Diamond Proposal Number(s): [17180] Show Abstract ▼ Abstract: Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.	Sep 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Generating selective leads for Mer kinase inhibitors - example of a comprehensive lead-generation strategy J. Willem M. Nissink , Sana Bazzaz , Carolyn Blackett , Matthew A. Clark , Olga Collingwood , Jeremy S. Disch , Diana Gikunju , Kristin Goldberg , John P. Guilinger , Elizabeth Hardaker , Edward J. Hennessy , Rachael Jetson , Anthony D. Keefe , William Mccoull , Lindsay Mcmurray , Allison Olszewski , Ross Overman , Alexander Pflug , Marian Preston , Philip B. Rawlins , Emma Rivers , Marianne Schimpl , Paul Smith , Caroline Truman , Elizabeth Underwood , Juli Warwicker , Jon Winter-Holt , Simon Woodcock , Ying Zhang Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.0c01904 Show Abstract ▼ Abstract: Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.	Mar 2021
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	A-loop interactions in Mer tyrosine kinase give rise to inhibitors with two-step mechanism and long residence time of binding Alexander Pflug , Marianne Schimpl , J. Willem M. Nissink , Ross C. Overman , Philip B. Rawlins , Caroline Truman , Elizabeth Underwood , Juli Warwicker , Jon J. Winter-Holt , William Mccoull Biochemical Journal DOI: 10.1042/BCJ20200735 Diamond Proposal Number(s): [14631, 17180, 20015] Open Access Show Abstract ▼ Abstract: The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor ‘example 172’ (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.	Oct 2020
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Discovery of a thiadiazole–pyridazine-based allosteric glutaminase 1 inhibitor series that demonstrates oral bioavailability and activity in tumor xenograft models M. Raymond V. Finlay , Mark Anderton , Andrew Bailey , Scott Boyd , Joanna Brookfield , Ceri Cairnduff , Mark Charles , Anne Cheasty , Susan E. Critchlow , Janet Culshaw , Tennyson Ekwuru , Ian Hollingsworth , Neil Jones , Fred Leroux , Mairi Littleson , Hollie Mccarron , Jennifer Mckelvie , Lorraine Mooney , J. Willem M. Nissink , David Perkins , Steve Powell , Mar Jimenez Quesada , Piotr Raubo , Verity Sabin , James Smith , Peter D. Smith , Andrew Stark , Attilla Ting , Peng Wang , Zena Wilson , Jon J. Winter-Holt , J. Matthew Wood , Gail L. Wrigley , Guoqing Yu , Peng Zhang Journal Of Medicinal Chemistry 62, 6540 - 6560 DOI: 10.1021/acs.jmedchem.9b00260 Show Abstract ▼ Abstract: Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell’s ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.	Jul 2019
I03-Macromolecular Crystallography	A high-throughput screening triage workflow to authenticate a novel series of PFKFB3 inhibitors Stephen A. St-Gallay , Neil Bennett , Susan E. Critchlow , Nicola Curtis , Gareth Davies , Judit Debreczeni , Nicola Evans , Ian Hardern , Geoff Holdgate , Neil P. Jones , Lindsey Leach , Sarita Maman , Sheila Mcloughlin , Marian Preston , Laurent Rigoreau , Andrew Thomas , Andrew P. Turnbull , Graeme Walker , Jarrod Walsh , Richard A. Ward , Ed Wheatley , Jon Winter-Holt Slas Discovery: Advancing Life Sciences R&d 27 DOI: 10.1177/2472555217732289 Diamond Proposal Number(s): [5735] Open Access Show Abstract ▼ Abstract: A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.	Sep 2017
I03-Macromolecular Crystallography	MTH1 substrate recognition - an example of specific promiscuity J. Willem M. Nissink , Michal Bista , Jason Breed , Nikki Carter , Kevin Embrey , Jonathan Read , Jon J. Winter-Holt , Michael Massiah Plos One 11 DOI: 10.1371/journal.pone.0151154 Open Access Show Abstract ▼ Abstract: TH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1.	Mar 2016

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