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46 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
I04-1-Macromolecular Crystallography (fixed wavelength)	Notum deacylates octanoylated ghrelin Yuguang Zhao , Laura-nadine Schuhmacher , Morgan Roberts , Satoshi Kakugawa , Ganka Bineva-todd , Steve Howell , Nicola O'reilly , Christine Perret , Ambrosius P. Snijders , Jean-paul Vincent , E. Yvonne Jones Molecular Metabolism DOI: 10.1016/j.molmet.2021.101201 Diamond Proposal Number(s): [14744] Open Access Show Abstract ▼ Abstract: Objectives: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. Methods: We used mass-spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. Results: Mass-spectrometry detected the removal of octanoyl from ghrelin by purified active Notum, but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex shows the octanoyl lipid is accommodated in the hydrophobic pocket of Notum. The knock-in allele expressing HA-tagged Notum reveals that Notum is produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed with a high fat diet leads to a small but significant increase in acylated ghrelin in the circulation, while no such increase is seen in wildtype animals on the same diet. Conclusions: Overall our data demonstrate Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high fat diet conditions. Our work therefore adds Notum to the list of enzymes, including butylcholineasterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.	Feb 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Caffeine inhibits Notum activity by binding at the catalytic pocket Yuguang Zhao , Jingshan Ren , James Hillier , Weixian Lu , Edith Yvonne Jones Communications Biology 3 DOI: 10.1038/s42003-020-01286-5 Diamond Proposal Number(s): [14744] Open Access Show Abstract ▼ Abstract: Notum inhibits Wnt signalling via enzymatic delipidation of Wnt ligands. Restoration of Wnt signalling by small molecule inhibition of Notum may be of therapeutic benefit in a number of pathologies including Alzheimer’s disease. Here we report Notum activity can be inhibited by caffeine (IC50 19 µM), but not by demethylated caffeine metabolites: paraxanthine, theobromine and theophylline. Cellular luciferase assays show Notum-suppressed Wnt3a function can be restored by caffeine with an EC50 of 46 µM. The dissociation constant (Kd) between Notum and caffeine is 85 µM as measured by surface plasmon resonance. High-resolution crystal structures of Notum complexes with caffeine and its minor metabolite theophylline show both compounds bind at the centre of the enzymatic pocket, overlapping the position of the natural substrate palmitoleic lipid, but using different binding modes. The structural information reported here may be of relevance for the design of more potent brain-accessible Notum inhibitors.	Oct 2020
I04-1-Macromolecular Crystallography (fixed wavelength)	5-phenyl-1,3,4-oxadiazol-2(3H)-ones are potent inhibitors of notum carboxylesterase activity identified by the optimization of a crystallographic fragment screening hit William Mahy , Nicky J. Willis , Yuguang Zhao , Hannah L. Woodward , Fredrik Svensson , James Sipthorp , Luca Vecchia , Reinis R. Ruza , James Hillier , Svend Kjær , Sarah Frew , Amy Monaghan , Magda Bictash , Patricia C. Salinas , Paul Whiting , Jean-paul Vincent , E. Yvonne Jones , Paul V. Fish Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.0c01391 Diamond Proposal Number(s): [16814] Show Abstract ▼ Abstract: Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-triazole 7 as an attractive starting point for a structure-based drug design hit-to-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and partial blood–brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.	Oct 2020
B21-High Throughput SAXS I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structure of the human cation-independent mannose 6-phosphate/IGF2 receptor domains 7–11 uncovers the mannose 6-phosphate binding site of domain 9 Alice J. Bochel , Christopher Williams , Airlie J. Mccoy , Hans-jürgen Hoppe , Ashley J. Winter , Ryan D. Nicholls , Karl Harlos , E. Yvonne Jones , Imre Berger , A. Bassim Hassan , Matthew P. Crump Structure DOI: 10.1016/j.str.2020.08.002 Diamond Proposal Number(s): [8423] Show Abstract ▼ Abstract: The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7–10 within two multi-domain constructs, D9–10 and D7–11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7–11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7–11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.	Sep 2020
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Glypicans shield the Wnt lipid moiety to enable signalling at a distance Ian J. Mcgough , Luca Vecchia , Benjamin Bishop , Tomas Malinauskas , Karen Beckett , Dhira Joshi , Nicola O’reilly , Christian Siebold , E. Yvonne Jones , Jean-paul Vincent Nature 423 DOI: 10.1038/s41586-020-2498-z Diamond Proposal Number(s): [19946] Show Abstract ▼ Abstract: A relatively small number of proteins have been suggested to act as morphogens—signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1,2,3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4,5,6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.	Jul 2020
I24-Microfocus Macromolecular Crystallography	Screening of a custom-designed acid fragment library identifies 1-phenylpyrroles and 1-phenylpyrrolidines as inhibitors of Notum carboxylesterase activity William Mahy , Mikesh Patel , David Steadman , Hannah L. Woodward , Benjamin N. Atkinson , Fredrik Svensson , Nicky J. Willis , Alister Flint , Dimitra Papatheodorou , Yuguang Zhao , Luca Vecchia , Reinis R. Ruza , James Hillier , Sarah Frew , Amy Monaghan , Artur Costa , Magda Bictash , Magnus Walter , E. Yvonne Jones , Paul V. Fish Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.0c00660 Diamond Proposal Number(s): [19946, 14744] Show Abstract ▼ Abstract: The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions. Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z as the most potent compound from this series. Similarly, optimization of 1-phenylpyrrolidine 8 gave acid 26. This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable in vitro ADME profiles.	Jul 2020
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural basis of semaphorin‐plexin cis interaction Daniel Rozbesky , Marieke G. Verhagen , Dimple Karia , Gergely N. Nagy , Luis Alvarez , Ross A Robinson , Karl Harlos , Sergi Padilla‐parra , R. Jeroen Pasterkamp , Edith Y. Jones The Embo Journal 58 DOI: 10.15252/embj.2019102926 Diamond Proposal Number(s): [10627] Open Access Show Abstract ▼ Abstract: Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell‐attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high‐resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.	Jun 2020
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Drosophila OTK is a glycosaminoglycan-binding protein with high conformational flexibility Daniel Rozbesky , Jim Monistrol , Vitul Jain , James Hillier , Sergi Padilla-parra , E. Yvonne Jones Structure DOI: 10.1016/j.str.2020.02.008 Diamond Proposal Number(s): [14744] Open Access Show Abstract ▼ Abstract: The transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. We have determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. We failed to detect direct binding between Drosophila Plexin A (PlexA) and OTK, which was suggested previously. We found that, instead of PlexA, OTK directly binds semaphorin 1a. Our binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.	Mar 2020
I04-1-Macromolecular Crystallography (fixed wavelength)	The antiepileptic drug carbamazepine binds to a novel pocket on the Wnt receptor Frizzled-8 Yuguang Zhao , Jingshan Ren , James Hillier , Weixian Lu , E. Yvonne Jones Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b02020 Diamond Proposal Number(s): [14744] Show Abstract ▼ Abstract: Misregulation of Wnt signalling is common in human cancer. Development of small molecule inhibitors against the Wnt receptor, Frizzled (FZD), may have potential in cancer therapy. During small molecule screens, we observed binding of carbamazepine (CBZ) to the cysteine-rich domain (CRD) of the Wnt receptor FZD8 using surface plasmon resonance (SPR). Cellular functional assays demonstrated CBZ can suppress FZD8 mediated Wnt/β-catenin signalling. We determined the crystal structure of the complex at 1.7Å resolution, which reveals that CBZ binds at a novel pocket on the FZD8 CRD. The unique residue Tyr52 discriminates FZD8 from the closely-related FZD5 and other FZDs for CBZ binding. The first small molecule-bound FZD structure provides a basis for anti-FZD drug development. Furthermore, the observed CBZ mediated Wnt signalling inhibition may help to explain the phenomenon of bone loss and increased adipogenesis in some patients during long-term CBZ treatment.	Feb 2020
	Hand-foot-and-mouth disease virus receptor KREMEN1 binds the canyon of Coxsackie Virus A10 Yuguang Zhao , Daming Zhou , Tao Ni , Dimple Karia , Abhay Kotecha , Xiangxi Wang , Zihe Rao , E. Yvonne Jones , Elizabeth E. Fry , Jingshan Ren , David I. Stuart Nature Communications 11 DOI: 10.1038/s41467-019-13936-2 Open Access Show Abstract ▼ Abstract: Coxsackievirus A10 (CV-A10) is responsible for an escalating number of severe infections in children, but no prophylactics or therapeutics are currently available. KREMEN1 (KRM1) is the entry receptor for the largest receptor-group of hand-foot-and-mouth disease causing viruses, which includes CV-A10. We report here structures of CV-A10 mature virus alone and in complex with KRM1 as well as of the CV-A10 A-particle. The receptor spans the viral canyon with a large footprint on the virus surface. The footprint has some overlap with that seen for the neonatal Fc receptor complexed with enterovirus E6 but is larger and distinct from that of another enterovirus receptor SCARB2. Reduced occupancy of a particle-stabilising pocket factor in the complexed virus and the presence of both unbound and expanded virus particles suggests receptor binding initiates a cascade of conformational changes that produces expanded particles primed for viral uncoating.	Jan 2020

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