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Instruments / Technical Area	Publication Details	Published
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] 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
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Demonstration of the utility of DOS-derived fragment libraries for rapid hit derivatisation in a multidirectional fashion Sarah L. Kidd , Elaine Fowler , Till Reinhardt , Thomas Compton , Natalia Mateu , Hector Newman , Dom Bellini , Romain Talon , Joseph Mcloughlin , Tobias Krojer , Anthony Aimon , Anthony Bradley , Michael Fairhead , Paul Brear , Laura Diaz-Saez , Katherine Mcauley , Hannah F. Sore , Andrew Madin , Daniel H. O'Donovan , Kilian Huber , Marko Hyvonen , Frank Von Delft , Christopher G. Dowson , David R. Spring Chemical Science DOI: 10.1039/D0SC01232G Diamond Proposal Number(s): [18145, 15649, 14303, 14493] Open Access Show Abstract ▼ Abstract: Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.	May 2020
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A chemical probe for tudor domain protein spindlin1 to investigate chromatin function Vincent Fagan , Catrine Johansson , Carina Gileadi , Octovia Monteiro , James Edward Dunford , Reshma Nibhani , Martin Philpott , Jessica Malzahn , Graham Wells , Ruth Farham , Adam Cribbs , Nadia Halidi , Fengling Li , Irene Chau , Holger Greschik , Srikannathasan Velupillai , Abdellah Allali-Hassani , James M. Bennett , Thomas Christott , Charline Giroud , Andrew M. Lewis , Kilian V. M. Huber , Nick Athanasou , Chas Bountra , Manfred Jung , Roland Schüle , Masoud Vedadi , Cheryl H. Arrowsmith , Yan Xiong , Jian Jin , Oleg Fedorov , Gillian Farnie , Paul E. Brennan , Udo C. T. Oppermann Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b00562 Diamond Proposal Number(s): [10619, 15433] Show Abstract ▼ Abstract: Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.	Sep 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Rapid covalent-probe discovery by electrophile-fragment screening Efrat Resnick , Anthony Bradley , Jinrui Gan , Alice Douangamath , Tobias Krojer , Ritika Sethi , Paul P. Geurink , Anthony Aimon , Gabriel Amitai , Dom Bellini , James Bennett , Michael Fairhead , Oleg Fedorov , Ronen Gabizon , Jin Gan , Jingxu Guo , Alexander Plotnikov , Nava Reznik , Gian Filippo Ruda , Laura Diaz-Saez , Verena M. Straub , Tamas Szommer , Srikannathasan Velupillai , Daniel Zaidman , Yanling Zhang , Alun R. Coker , Christopher G. Dowson , Haim Barr , Chu Wang , Kilian V. M. Huber , Paul E. Brennan , Huib Ovaa , Frank Von Delft , Nir London Journal Of The American Chemical Society DOI: 10.1021/jacs.9b02822 Show Abstract ▼ Abstract: Covalent probes can display unmatched potency, selectivity and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.	May 2019
I04-Macromolecular Crystallography	Design, synthesis and characterization of covalent kdm5 inhibitors Saleta Vazquez-Rodriguez , Miranda Wright , Catherine M. Rogers , Adam P. Cribbs , Srikannathasan Velupillai , Martin Philpott , Henry Lee , James E. Dunford , Kilian V. M. Huber , Matthew B. Robers , James D. Vasta , Marie-Laetitia Thezenas , Sarah Bonham , Benedikt Kessler , James Bennett , Oleg Fedorov , Florence Raynaud , Adam Donovan , Julian Blagg , Vassilios Bavetsias , Udo Oppermann , Chas Bountra , Akane Kawamura , Paul E. Brennan Angewandte Chemie International Edition 58, 515 - 519 DOI: 10.1002/anie.201810179 Diamond Proposal Number(s): [15433] Open Access Show Abstract ▼ Abstract: Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub‐family. The covalent binding to the targeted proteins was confirmed by MS and time‐dependent inhibition. Additional competition assays show that compounds were non 2‐OG competitive. Target engagement and ChIP‐seq analysis showed that the compounds inhibited the KDM5 members in cells at nano‐ to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.	Jan 2019
I04-Macromolecular Crystallography	Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling Matous Hrdinka , Lisa Schlicher , Bing Dai , Daniel M. Pinkas , Joshua C. Bufton , Sarah Picaud , Jennifer A. Ward , Catherine Rogers , Chalada Suebsuwong , Sameer Nikhar , Gregory D. Cuny , Kilian V. M. Huber , Panagis Filippakopoulos , Alex N. Bullock , Alexei Degterev , Mads Gyrd‐hansen The Embo Journal DOI: 10.15252/embj.201899372 Diamond Proposal Number(s): [15433] Open Access Show Abstract ▼ Abstract: RIPK2 mediates inflammatory signaling by the bacteria‐sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD‐mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP‐binding and XIAP‐mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N‐lobe of the kinase, which is in close proximity to the ATP‐binding pocket. Through characterization of a new series of ATP pocket‐binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2‐XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP‐binding pocket in RIPK2 can be exploited to interfere with the RIPK2‐XIAP interaction for modulation of NOD signaling.	Jul 2018
I02-Macromolecular Crystallography	Benzoisoquinolinediones as potent and selective inhibitors of BRPF2 and TAF1/TAF1L bromodomains Léa Bouché , Clara D. Christ , Stephan Siegel , Amaury E. Fernández-Montalván , Simon J. Holton , Oleg Fedorov , Antonius Ter Laak , Tatsuo Sugawara , Detlef Stöckigt , Cynthia Tallant , James Bennett , Octovia Monteiro , Laura Díaz-Sáez , Paulina Siejka , Julia Meier , Vera Pütter , Jörg Weiske , Susanne Müller , Kilian V. M. Huber , Ingo V. Hartung , Bernard Haendler Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.7b00306 Diamond Proposal Number(s): [15558, 10619] Show Abstract ▼ Abstract: Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure–activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2– or TAF1–histone H3.3 or H4 interaction assay.	May 2017
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy Kilian V. M. Huber , Eidarus Salah , Branka Radic , Manuela Gridling , Jonathan M. Elkins , Alexey Stukalov , Ann-Sofie Jemth , Camilla Göktürk , Kumar Sanjiv , Kia Strömberg , Therese Pham , Ulrika Warpman Berglund , Jacques Colinge , Keiryn L. Bennett , Joanna I. Loizou , Thomas Helleday , Stefan Knapp , Giulio Superti-Furga Nature 508 (7495), 222 - 227 DOI: 10.1038/nature13194 PMID: 24695225 Diamond Proposal Number(s): [8421] Show Abstract ▼ Abstract: Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.	Apr 2014
I02-Macromolecular Crystallography	Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing Oleg Fedorov , Kilian Huber , Andreas Eisenreich , Panagis Filippakopoulos , Oliver King , Alex N. Bullock , Damian Szklarczyk , Lars J. Jensen , Doriano Fabbro , Jörg Trappe , Ursula Rauch , Franz Bracher , Stefan Knapp Chemistry & Biology 18 (1), 67 - 76 DOI: 10.1016/j.chembiol.2010.11.009 Open Access Show Abstract ▼ Abstract: There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).	Jan 2011

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