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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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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NONE-No attached Diamond beamline
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Open Access
Abstract: Regulation of the Wnt signaling pathway is critically important for a number of cellular processes in both development and adult mammalian biology. This Perspective will provide a summary of current and emerging therapeutic opportunities in modulating Wnt signaling, especially through inhibition of Notum carboxylesterase activity. Notum was recently shown to act as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group. Inhibition of Notum activity may represent a new approach to treat disease where aberrant Notum activity has been identified as the underlying cause. Reliable screening technologies are available to identify inhibitors of Notum, and structural studies are accelerating the discovery of new inhibitors. A selection of these hits have been optimized to give fit-for-purpose small molecule inhibitors of Notum. Three noteworthy examples are LP-922056 (26), ABC99 (27), and ARUK3001185 (28), which are complementary chemical tools for exploring the role of Notum in Wnt signaling.
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Mar 2021
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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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I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[19946, 14744]
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.
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Jul 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Benjamin N.
Atkinson
,
David
Steadman
,
William
Mahy
,
Yuguang
Zhao
,
James
Sipthorp
,
Elliott D.
Bayle
,
Fredrik
Svensson
,
George
Papageorgiou
,
Fiona
Jeganathan
,
Sarah
Frew
,
Amy
Monaghan
,
Magda
Bictash
,
E.
Yvonne Jones
,
Paul V.
Fish
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20-24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.
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Oct 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Benjamin Nicholas
Atkinson
,
David
Steadman
,
Yuguang
Zhao
,
James
Sipthorp
,
Luca
Vecchia
,
Reinis Reinholds
Ruza
,
Fiona
Jeganathan
,
Georgie
Lines
,
Sarah
Frew
,
Amy
Monaghan
,
Svend
Kjaer
,
Magda
Bictash
,
Yvonne
Jones
,
Paul V.
Fish
Diamond Proposal Number(s):
[16814, 14744]
Open Access
Abstract: NOTUM is a carboxylesterase that has been shown to act by mediating the O-depalmitoleoylation of Wnt proteins resulting in suppression of Wnt signaling. Here, we describe the development of NOTUM inhibitors that restore Wnt signaling for use in in vitro disease models where NOTUM over activity is an underlying cause. A crystallographic fragment screen with NOTUM identified 2-phenoxyacetamide 3 as binding in the palmitoleate pocket with modest inhibition activity (IC50 33 μM). Optimization of hit 3 by SAR studies guided by SBDD identified indazole 38 (IC50 0.032 μM) and isoquinoline 45 (IC50 0.085 μM) as potent inhibitors of NOTUM. The binding of 45 to NOTUM was rationalized through an X-ray cocrystal structure determination which showed a flipped binding orientation compared to 3. However, it was not possible to combine NOTUM inhibition activity with metabolic stability as the majority of the compounds tested were rapidly metabolized in an NADPH-independent manner.
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Apr 2019
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I03-Macromolecular Crystallography
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Niall
Igoe
,
Elliott D.
Bayle
,
Cynthia
Tallant
,
Oleg
Fedorov
,
Julia C.
Meier
,
Pavel
Savitsky
,
Catherine
Rogers
,
Yannick
Morias
,
Sarah
Scholze
,
Helen
Boyd
,
Danen
Cunoosamy
,
David M.
Andrews
,
Anne
Cheasty
,
Paul E.
Brennan
,
Susanne
Müller
,
Stefan
Knapp
,
Paul V.
Fish
Abstract: The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe NI-57 (16) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor 16 preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound 16 has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with 16 was demonstrated in nanoBRET and FRAP assays. The binding of 16 to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show 16 has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for 16 was generated in mouse with single dose administration showing favorable oral bioavailability.
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Jul 2017
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