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

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	Crystallographic fragment screening of CDK2-cyclin A: FragLites map sites of protein-protein interaction Ian Hope , Mathew P. Martin , Ziwei Jiang , Michael J. Waring , Martin E. M. Noble , Jane A. Endicott , Natalie J. Tatum Structure 15 DOI: 10.1016/j.str.2025.07.016 Diamond Proposal Number(s): [18598] Open Access Show Abstract ▼ Abstract: Sites of protein-protein interaction (PPI) are potentially more selective binding sites for therapeutics than protein substrate-binding sites. PPIs include distinct regions frequently called “hotspots,” sites of key amino acid interactions. Prospective identification of these hotspots through X-ray crystallographic screening could assist in the identification of separation of function mutants for experimental validation, enhance confidence in AI-generated multiprotein complex predictions, and accelerate development of selective chemical probes. To explore these applications, we utilize the FragLite library to examine the binding surfaces of CDK2-cyclin A. The many protein- and peptide-CDK2-cyclin A complexes that have been structurally characterized make this complex an appropriate test case. We show that FragLites comprehensively map both known sites of protein-protein interaction on CDK2-cyclin A and identify a possible uncharacterized site, providing a structural method toward directing mechanistic studies and starting points for chemical probe design.	Aug 2025
I03-Macromolecular Crystallography	Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a Owen Burbidge , Martyna W. Pastok , Diana Papini , Samantha L. Hodder , Grasilda Zenkevičiūtė , Kenneth Z. J. Tan , Shannon Turberville , Martin E. M. Noble , Jane A. Endicott , Laura Itzhaki Structure 366 DOI: 10.1016/j.str.2025.07.017 Diamond Proposal Number(s): [13587] Open Access Show Abstract ▼ Abstract: We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 is a cell cycle regulator that inhibits cyclin-dependent kinases CDK4 and CDK6 and is inactivated in sporadic and familial cancers. The majority of p16 missense mutations cause loss of function by destabilizing the protein’s structure. We identify nanobodies that bind p16 with nanomolar affinities and restore the stability of many different cancer-associated p16 mutations located at sites throughout the protein. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. We confirm that nanobodies bind to p16 in a cellular setting and do not preclude p16 binding to CDK6 and its ability to induce cell-cycle arrest. These findings indicate that nanobodies merit testing as pharmacological chaperones for p16 reactivation in the cell.	Aug 2025
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural requirements for the specific binding of CRABP2 to cyclin D3 Martyna W. Pastok , Charles W. E. Tomlinson , Shannon Turberville , Abbey M. Butler , Arnaud Basle , Martin E. M. Noble , Jane A. Endicott , Ehmke Pohl , Natalie J. Tatum Structure 173 DOI: 10.1016/j.str.2024.09.020 Diamond Proposal Number(s): [18598] Open Access Show Abstract ▼ Abstract: Cellular retinoic acid binding protein 2 (CRABP2) transports retinoic acid from the cytoplasm to the nucleus where it then transfers its cargo to retinoic acid receptor-containing complexes leading to activation of gene transcription. We demonstrate using purified proteins that CRABP2 is also a cyclin D3-specific binding protein and that the CRABP2 cyclin D3 binding site and the proposed CRABP2 nuclear localization sequence overlap. Both sequences are within the helix-loop-helix motif that forms a lid to the retinoic acid binding pocket. Mutations within this sequence that block both cyclin D3 and retinoic acid binding promote formation of a CRABP2 structure in which the retinoic acid binding pocket is occupied by an alternative lid conformation. Structural and functional analysis of CRABP2 and cyclin D3 mutants combined with AlphaFold models of the ternary CDK4/6-cyclin D3-CRABP2 complex supports the identification of an α-helical protein binding site on the cyclin D3 C-terminal cyclin box fold.	Oct 2024
Krios I-Titan Krios I at Diamond	Cryo-EM structure of the CDK2-cyclin A-CDC25A complex Rhianna J. Rowland , Svitlana Korolchuk , Marco Salamina , Natalie J. Tatum , James R. Ault , Sam Hart , Johan P. Turkenburg , James N. Blaza , Martin E. M. Noble , Jane A. Endicott Nature Communications 15 DOI: 10.1038/s41467-024-51135-w Diamond Proposal Number(s): [28576] Open Access Show Abstract ▼ Abstract: The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.	Aug 2024
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Mapping ligand interactions of bromodomains BRD4 and ATAD2 with FragLites and PepLites - halogenated probes of druglike and peptide-like molecular interactions Gemma Davison , Mathew P. Martin , Shannon Turberville , Selma Dormen , Richard Heath , Amy B. Heptinstall , Marie Lawson , Duncan C. Miller , Yi Min Ng , James N. Sanderson , Ian Hope , Daniel Wood , Céline Cano , Jane A. Endicott , Ian R. Hardcastle , Martin E. M. Noble , Michael J. Waring Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.2c01357 Open Access Show Abstract ▼ Abstract: The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.	Nov 2022
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Parallel optimization of potency and pharmacokinetics leading to the discovery of a pyrrole carboxamide ERK5 kinase domain inhibitor Duncan C. Miller , Tristan Reuillon , Lauren Molyneux , Timothy Blackburn , Simon J. Cook , Noel Edwards , Jane A. Endicott , Bernard T. Golding , Roger J. Griffin , Ian Hardcastle , Suzannah J. Harnor , Amy Heptinstall , Pamela Lochhead , Mathew P. Martin , Nick C. Martin , Stephanie Myers , David R. Newell , Richard A. Noble , Nicole Phillips , Laurent Rigoreau , Huw Thomas , Julie A. Tucker , Lan-Zhen Wang , Michael J. Waring , Ai-Ching Wong , Stephen R. Wedge , Martin E. M. Noble , Celine Cano Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.1c01756 Diamond Proposal Number(s): [9948, 13587] Open Access Show Abstract ▼ Abstract: The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.	Apr 2022
B21-High Throughput SAXS I04-1-Macromolecular Crystallography (fixed wavelength)	Discriminative SKP2 interactions with CDK-cyclin complexes support a cyclin a-specific role in p27KIP1 degradation Marco Salamina , Bailey C. Montefiore , Mengxi Liu , Daniel J. Wood , Richard Heath , James R. Ault , Lan-Zhen Wang , Svitlana Korolchuk , Arnaud Basle , Martyna Pastok , Judith Reeks , Natalie J. Tatum , Frank Sobott , Stefan T. Arold , Michele Pagano , Martin E. M. Noble , Jane A. Endicott Journal Of Molecular Biology 433 DOI: 10.1016/j.jmb.2020.166795 Diamond Proposal Number(s): [13587, 16970] Open Access Show Abstract ▼ Abstract: The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.	Mar 2021
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Structure-based design of potent and orally active isoindolinone inhibitors of MDM2-p53 protein–protein interaction Gianni Chessari , Ian R. Hardcastle , Jong Sook Ahn , Burcu Anil , Elizabeth Anscombe , Ruth H. Bawn , Luke D. Bevan , Timothy J. Blackburn , Ildiko Buck , Celine Cano , Benoit Carbain , Juan Castro , Ben Cons , Sarah J. Cully , Jane A. Endicott , Lynsey Fazal , Bernard T. Golding , Roger J. Griffin , Karen Haggerty , Suzannah J. Harnor , Keisha Hearn , Stephen Hobson , Rhian S. Holvey , Steven Howard , Claire E. Jennings , Christopher N. Johnson , John Lunec , Duncan C. Miller , David R. Newell , Martin E. M. Noble , Judith Reeks , Charlotte H. Revill , Christiane Riedinger , Jeffrey D. St. Denis , Emiliano Tamanini , Huw Thomas , Neil T. Thompson , Mladen Vinković , Stephen R. Wedge , Pamela A. Williams , Nicola E. Wilsher , Bian Zhang , Yan Zhao Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.0c02188 Show Abstract ▼ Abstract: Inhibition of murine double minute 2 (MDM2)-p53 protein–protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.	Mar 2021
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4 Stephanie Myers , Duncan C. Miller , Lauren Molyneux , Mercedes Arasta , Ruth H. Bawn , Timothy Blackburn , Simon J. Cook , Noel Edwards , Jane A. Endicott , Bernard T. Golding , Roger J. Griffin , Tim Hammonds , Ian R. Hardcastle , Suzannah J. Harnor , Amy Heptinstall , Pamela Lochhead , Mathew P. Martin , Nick C. Martin , David R. Newell , Paul J. Owen , Leon C. Pang , Tristan Reuillon , Laurent J. M. Rigoreau , Huw Thomas , Julie A. Tucker , Lan-Zhen Wang , Ai-Ching Wong , Martin E. M. Noble , Stephen R. Wedge , Celine Cano European Journal Of Medicinal Chemistry DOI: 10.1016/j.ejmech.2019.05.057 Diamond Proposal Number(s): [9948, 13587] Show Abstract ▼ Abstract: Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 μM for ERK5; IC50 > 120 μM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.	May 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	FragLites - minimal, halogenated fragments displaying pharmacophore doublets. An efficient approach to druggability assessment and hit generation Daniel J. Wood , José Daniel Lopez-Fernandez , Leanne E. Knight , Islam Al-Khawaldeh , Conghao Gai , Shengyin Lin , Mathew P. Martin , Duncan C. Miller , Celine Cano , Jane A. Endicott , Ian R. Hardcastle , Martin E. M. Noble , Michael J. Waring Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b00304 Show Abstract ▼ Abstract: Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource intensive screening of large libraries of lead-like or fragment molecules. Here we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.	Mar 2019

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