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

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Discovery and characterization of a cryptic secondary binding site in the molecular chaperone HSP70 Suzanne O'Connor , Yann-Vai Le Bihan , Isaac M. Westwood , Manjuan Liu , Oi Wei Mak , Gabriel Zazeri , Ana P. R. Povinelli , Alan M. Jones , Rob Van Montfort , Jóhannes Reynisson , Ian Collins Molecules 27 DOI: 10.3390/molecules27030817 Diamond Proposal Number(s): [6385] Open Access Show Abstract ▼ Abstract: Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.	Jan 2022
	Target 2035 – update on the quest for a probe for every protein Susanne Müller , Suzanne Ackloo , Arij Al Chawaf , Bissan Al-Lazikani , Albert Antolin , Jonathan B. Baell , Hartmut Beck , Shaunna Beedie , Ulrich A. K. Betz , Gustavo Arruda Bezerra , Paul E. Brennan , David Brown , Peter J. Brown , Alex N. Bullock , Adrian J. Carter , Apirat Chaikuad , Mathilde Chaineau , Alessio Ciulli , Ian Collins , Jan Dreher , David Drewry , Kristina Edfeldt , Aled M. Edwards , Ursula Egner , Stephen V. Frye , Stephen M. Fuchs , Matthew D. Hall , Ingo V. Hartung , Alexander Hillisch , Stephen H. Hitchcock , Evert Homan , Natarajan Kannan , James R. Kiefer , Stefan Knapp , Milka Kostic , Stefan Kubicek , Andrew S. Leach , Sven Lindemann , Brian D. Marsden , Hisanori Matsui , Jordan L. Meier , Daniel Merk , Maurice Michel , Maxwell R. Morgan , Anke Mueller-Fahrnow , Dafydd R. Owen , Benjamin G. Perry , Saul H. Rosenberg , Kumar Singh Saikatendu , Matthieu Schapira , Cora Scholten , Sujata Sharma , Anton Simeonov , Michael Sundström , Giulio Superti-Furga , Matthew H. Todd , Claudia Tredup , Masoud Vedadi , Frank Von Delft , Timothy M. Willson , Georg E. Winter , Paul Workman , Cheryl H. Arrowsmith Rsc Medicinal Chemistry 17 DOI: 10.1039/D1MD00228G Open Access Show Abstract ▼ Abstract: Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.	Dec 2021
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms Alan M. Jones , Isaac M. Westwood , James D. Osborne , Thomas P. Matthews , Matthew D. Cheeseman , Martin G. Rowlands , Fiona Jeganathan , Rosemary Burke , Diane Lee , Nadia Kadi , Manjuan Liu , Meirion Richards , Craig Mcandrew , Norhakim Yahya , Sarah Dobson , Keith Jones , Paul Workman , Ian Collins , Rob L. M. Van Montfort Scientific Reports 6 DOI: 10.1038/srep34701 Open Access Show Abstract ▼ Abstract: The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops.	Oct 2016
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Exploiting protein conformational change to optimize adenosine-derived inhibitors of HSP70 Matthew D. Cheeseman , Isaac Westwood , Olivier Barbeau , Martin Rowlands , Sarah Dobson , Alan M. Jones , Fiona Jeganathan , Rosemary Burke , Nadia Kadi , Paul Workman , Ian Collins , Rob Van Montfort , Keith Jones Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.5b02001 Open Access Show Abstract ▼ Abstract: HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner.	May 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	Molecular mechanisms of human IRE1 activation through dimerization and ligand binding Amar Joshi , Yvette Newbatt , P. Craig Mcandrew , Mark Stubbs , Rosemary Burke , Mark Richards , Chitra Bhatia , John J. Caldwell , Tatiana Mchardy , Ian Collins , Richard Bayliss Oncotarget 6, 13019 - 13035 DOI: 10.18632/oncotarget.3864 Diamond Proposal Number(s): [10369] Open Access Show Abstract ▼ Abstract: IRE1 transduces the unfolded protein response by splicing XBP1 through its C-terminal cytoplasmic kinase-RNase region. IRE1 autophosphorylation is coupled to RNase activity through formation of a back-to-back dimer, although the conservation of the underlying molecular mechanism is not clear from existing structures. We have crystallized human IRE1 in a back-to-back conformation only previously seen for the yeast homologue. In our structure the kinase domain appears primed for catalysis but the RNase domains are disengaged. Structure-function analysis reveals that IRE1 is autoinhibited through a Tyr-down mechanism related to that found in the unrelated Ser/Thr protein kinase Nek7. We have developed a compound that potently inhibits human IRE1 kinase activity while stimulating XBP1 splicing. A crystal structure of the inhibitor bound to IRE1 shows an increased ordering of the kinase activation loop. The structures of hIRE in apo and ligand-bound forms are consistent with a previously proposed model of IRE1 regulation in which formation of a back-to-back dimer coupled to adoption of a kinase-active conformation drive RNase activation. The structures provide opportunities for structure-guided design of IRE1 inhibitors.	Apr 2015
I04-Macromolecular Crystallography	Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases Charlotte E. Allen , Chiau L. Chow , John J. Caldwell , Isaac M. Westwood , Rob L. M. Van Montfort , Ian Collins Bioorganic & Medicinal Chemistry 21 (18), 5707 - 5724 DOI: 10.1016/j.bmc.2013.07.021 PMID: 23920481 Diamond Proposal Number(s): [8015] Open Access Show Abstract ▼ Abstract: With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.	Sep 2013
I02-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Fragment-based screening maps inhibitor interactions in the ATP-binding site of checkpoint kinase 2 Cris Silva-Santisteban , Isaac Westwood , Kathy Boxall , Nathan Brown , Sam Peacock , Craig Mcandrews , Elaine Barrie , Meirion Richards , Amin Mirza , Antony W. Oliver , Rosemary Burke , Swen Hoelder , Keith Jones , G. Wynne Aherne , Julian Blagg , Ian Collins , Michelle D. Garrett , Rob L. M. Van Montfort Plos One 8 (6) DOI: 10.1371/journal.pone.0065689 PMID: 23776527 Open Access Show Abstract ▼ Abstract: Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragmentbased screening campaign using a combination of a high-concentration AlphaScreenTM kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rat	Jun 2013
I03-Macromolecular Crystallography	Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response Maruf M. U. Ali , Tina Bagratuni , Emma L. Davenport , Piotr R. Nowak , M. Cris Silva-Santisteban , Anthea Hardcastle , Craig Mcandrews , Martin G. Rowlands , Gareth J. Morgan , Wynne Aherne , Ian Collins , Faith E. Davies , Laurence H. Pearl The Embo Journal 30 (5), 894 - 905 DOI: 10.1038/emboj.2011.18 PMID: 21317875 Diamond Proposal Number(s): [6385] Open Access Show Abstract ▼ Abstract: Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N?terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C?terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1? bound to ADP, revealing the ‘phosphoryl?transfer’ competent dimeric face?to?face complex, which precedes and is distinct from the back?to?back RNase ‘active’ conformation described for yeast Ire1. We show that the Xbp1?specific ribonuclease activity depends on autophosphorylation, and that ATP?competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1? is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1? as a target for development of ATP?competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies.	Feb 2011
I03-Macromolecular Crystallography	CCT241533 is a potent and selective inhibitor of CHK2 that potentiates the cytotoxicity of PARP inhibitors Victoria E. Anderson , M. I. Walton , P. D. Eve , K. J. Boxall , L. Antoni , J. J. Caldwell , W. Aherne , L. H. Pearl , Antony W. Oliver , I. Collins , M. D. Garrett Cancer Research 71 (2), 463 - 472 DOI: 10.1158/0008-5472.CAN-10-1252 PMID: 21239475 Diamond Proposal Number(s): [6385] Show Abstract ▼ Abstract: CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533. X-ray crystallography confirmed that CCT241533 bound to CHK2 in the ATP pocket. This compound inhibits CHK2 with an IC50 of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 mu mol/L. CCT241533 blocked CHK2 activity in human tumor cell lines in response to DNA damage, as shown by inhibition of CHK2 autophosphorylation at S516, band shift mobility changes, and HDMX degradation. CCT241533 did not potentiate the cytotoxicity of a selection of genotoxic agents in several cell lines. However, this compound significantly potentiates the cytotoxicity of two structurally distinct PARP inhibitors. Clear induction of the pS516 CHK2 signal was seen with a PARP inhibitor alone, and this activation was abolished by CCT241533, implying that the potentiation of PARP inhibitor cell killing by CCT241533 was due to inhibition of CHK2. Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors. Cancer Res; 71(2); 463-72. (C) 2011 AACR.	Jan 2011
I03-Macromolecular Crystallography	Structure-Based Design of Potent and Selective 2-(Quinazolin-2-yl)phenol Inhibitors of Checkpoint Kinase 2 John J. Caldwell , Emma J. Welsh , Cornelis Matijssen , Victoria E. Anderson , Laurent Antoni , Kathy Boxall , Frederique Urban , Angela Hayes , Florence I. Raynaud , Laurent J. M. Rigoreau , Tony Raynham , G. Wynne Aherne , Laurence H. Pearl , Antony W. Oliver , Michelle D. Garrett , Ian Collins Journal Of Medicinal Chemistry 54 (2), 580 - 590 DOI: 10.1021/jm101150b PMID: 21186793 Diamond Proposal Number(s): [6385] Show Abstract ▼ Abstract: Structure-based design was applied to the optimization of a series of 2-(quinazolin-2-yl)phenols to generate potent and selective ATP-competitive inhibitors of the DNA damage response signaling enzyme checkpoint kinase 2 (CHK2). Structure?activity relationships for multiple substituent positions were optimized separately and in combination leading to the 2-(quinazolin-2-yl)phenol 46 (IC50 3 nM) with good selectivity for CHK2 against CHK1 and a wider panel of kinases and with promising in vitro ADMET properties. Off-target activity at hERG ion channels shown by the core scaffold was successfully reduced by the addition of peripheral polar substitution. In addition to showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533).	Jan 2011

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