I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: N-Acyl indolines 4 are potent, non-covalent Notum inhibitors developed from a covalent virtual screening hit 2a. The lead compounds were simple to synthesise, achieved excellent potency in a biochemical Notum-OPTS assay and restored Wnt signalling in a cell-based TCF/LEF reporter assay. Multiple high resolution X-ray structures established a common binding mode of these inhibitors with the indoline bound centred in the palmiteolate pocket with key interactions being aromatic stacking and a water mediated hydrogen bond to the oxyanion hole. These N-acyl indolines 4 will be useful tools for use in vitro studies to investigate the role of Notum in disease models, especially when paired with a structurally related covalent inhibitor (e.g. 4w and 2a). Overall, this study highlights the designed switch from covalent to non-covalent Notum inhibitors and so illustrates a complementary approach for hit generation and target inhibition.
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Jan 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Lucy C.
Walters
,
Daniel
Rozbesky
,
Karl
Harlos
,
Max
Quastel
,
Hong
Sun
,
Sebastian
Springer
,
Robert P.
Rambo
,
Fiyaz
Mohammed
,
E. Yvonne
Jones
,
Andrew J.
Mcmichael
,
Geraldine M.
Gillespie
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.
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Jun 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuguang
Zhao
,
William
Mahy
,
Nicky J.
Willis
,
Hannah L.
Woodward
,
David
Steadman
,
Elliott D.
Bayle
,
Benjamin N.
Atkinson
,
James
Sipthorp
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Karl
Harlos
,
Fiona
Jeganathan
,
Stefan
Constantinou
,
Artur
Costa
,
Svend
Kjær
,
Magda
Bictash
,
Patricia C.
Salinas
,
Paul
Whiting
,
Jean-Paul
Vincent
,
Paul V.
Fish
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[16814]
Open Access
Abstract: The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer’s disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments’ diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.
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Jun 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Nicky J.
Willis
,
William
Mahy
,
James
Sipthorp
,
Yuguang
Zhao
,
Hannah L.
Woodward
,
Benjamin N.
Atkinson
,
Elliott D.
Bayle
,
Fredrik
Svensson
,
Sarah
Frew
,
Fiona
Jeganathan
,
Amy
Monaghan
,
Stefano
Benvegnù
,
Sarah
Jolly
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Svend
Kjær
,
Steven
Howell
,
Ambrosius P.
Snijders
,
Magda
Bictash
,
Patricia C.
Salinas
,
Jean-Paul
Vincent
,
E. Yvonne
Jones
,
Paul
Whiting
,
Paul V.
Fish
Diamond Proposal Number(s):
[16814, 19446]
Open Access
Abstract: Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer’s disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.
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May 2022
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I03-Macromolecular Crystallography
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Dapeng
Li
,
Simon
Brackenridge
,
Lucy C.
Walters
,
Olivia
Swanson
,
Karl
Harlos
,
Daniel
Rozbesky
,
Derek W.
Cain
,
Kevin
Wiehe
,
Richard M.
Scearce
,
Maggie
Barr
,
Zekun
Mu
,
Robert
Parks
,
Max
Quastel
,
Robert J.
Edwards
,
Yunfei
Wang
,
Wes
Rountree
,
Kevin O.
Saunders
,
Guido
Ferrari
,
Persephone
Borrow
,
E. Yvonne
Jones
,
S. Munir
Alam
,
Mihai L.
Azoitei
,
Geraldine M.
Gillespie
,
Andrew J.
Mcmichael
,
Barton F.
Haynes
Open Access
Abstract: The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.
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Mar 2022
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I03-Macromolecular Crystallography
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David
Steadman
,
Benjamin N.
Atkinson
,
Yuguang
Zhao
,
Nicky J.
Willis
,
Sarah
Frew
,
Amy
Monaghan
,
Chandni
Patel
,
Emma
Armstrong
,
Kathryn
Costelloe
,
Lorenza
Magno
,
Magda
Bictash
,
E. Yvonne
Jones
,
Paul V.
Fish
,
Fredrik
Svensson
Diamond Proposal Number(s):
[19946]
Abstract: Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC50 < 500 nM. A critical selection process was then applied with two clusters and two singletons (1–4d) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/β-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.
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Jan 2022
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I03-Macromolecular Crystallography
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Yuguang
Zhao
,
Fredrik
Svensson
,
David
Steadman
,
Sarah
Frew
,
Amy
Monaghan
,
Magda
Bictash
,
Tiago
Moreira
,
Rod
Chalk
,
Weixian
Lu
,
Paul V.
Fish
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: The carboxylesterase Notum hydrolyzes a palmitoleate moiety from Wingless/Integrated(Wnt) ligands and deactivates Wnt signaling. Notum inhibitors can restore Wnt signaling which may be of therapeutic benefit for pathologies such as osteoporosis and Alzheimer’s disease. We report the identification of a novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures of the Notum inhibitor complexes reveal a common covalent adduct formed between the nucleophile serine-232 and hydrolyzed butyric esters. The covalent interaction in solution was confirmed by mass spectrometry analysis. Inhibitory potencies vary depending on the warheads used. Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom is positioned at an unfavorable angle for the approach of the active site water, which, combined with strong hydrophobic interactions with the enzyme pocket residues, hinders the intermediate from being further processed and results in covalent inhibition. These insights into Notum catalytic inhibition may guide development of more potent Notum inhibitors.
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Jul 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Diamond Proposal Number(s):
[14744]
Open Access
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.
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Feb 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Open Access
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.
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Oct 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Diamond Proposal Number(s):
[16814]
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.
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Oct 2020
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