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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Yanchun
Peng
,
Alexander J.
Mentzer
,
Guihai
Liu
,
Xuan
Yao
,
Zixi
Yin
,
Danning
Dong
,
Wanwisa
Dejnirattisai
,
Timothy
Rostron
,
Piyada
Supasa
,
Chang
Liu
,
César
López-camacho
,
Jose
Slon-campos
,
Yuguang
Zhao
,
David I.
Stuart
,
Guido C.
Paesen
,
Jonathan M.
Grimes
,
Alfred A.
Antson
,
Oliver W.
Bayfield
,
Dorothy E. D. P.
Hawkins
,
De-sheng
Ker
,
Beibei
Wang
,
Lance
Turtle
,
Krishanthi
Subramaniam
,
Paul
Thomson
,
Ping
Zhang
,
Christina
Dold
,
Jeremy
Ratcliff
,
Peter
Simmonds
,
Thushan
De Silva
,
Paul
Sopp
,
Dannielle
Wellington
,
Ushani
Rajapaksa
,
Yi-ling
Chen
,
Mariolina
Salio
,
Giorgio
Napolitani
,
Wayne
Paes
,
Persephone
Borrow
,
Benedikt M.
Kessler
,
Jeremy W.
Fry
,
Nikolai F.
Schwabe
,
Malcolm G.
Semple
,
J. Kenneth
Baillie
,
Shona C.
Moore
,
Peter J. M.
Openshaw
,
M. Azim
Ansari
,
Susanna
Dunachie
,
Eleanor
Barnes
,
John
Frater
,
Georgina
Kerr
,
Oliver
Gould
,
Teresa
Lockett
,
Robert
Levin
,
Yonghong
Zhang
,
Ronghua
Jing
,
Ling-pei
Ho
,
Richard J.
Cornall
,
Christopher P.
Conlon
,
Paul
Klenerman
,
Gavin R.
Screaton
,
Juthathip
Mongkolsapaya
,
Andrew
Mcmichael
,
Julian C.
Knight
,
Graham
Ogg
,
Tao
Dong
Open Access
Abstract: The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
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Sep 2020
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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Jiangdong
Huo
,
Audrey
Le Bas
,
Reinis R.
Ruza
,
Helen M. E.
Duyvesteyn
,
Halina
Mikolajek
,
Tomas
Malinauskas
,
Tiong Kit
Tan
,
Pramila
Rijal
,
Maud
Dumoux
,
Philip N.
Ward
,
Jingshan
Ren
,
Daming
Zhou
,
Peter J.
Harrison
,
Miriam
Weckener
,
Daniel K.
Clare
,
Vinod K.
Vogirala
,
Julika
Radecke
,
Lucile
Moynie
,
Yuguang
Zhao
,
Javier
Gilbert-jaramillo
,
Michael L.
Knight
,
Julia A.
Tree
,
Karen R.
Buttigieg
,
Naomi
Coombes
,
Michael J.
Elmore
,
Miles W.
Carroll
,
Loic
Carrique
,
Pranav N. M.
Shah
,
William
James
,
Alain R.
Townsend
,
David I.
Stuart
,
Raymond J.
Owens
,
James H.
Naismith
Diamond Proposal Number(s):
[27031, 27051]
Open Access
Abstract: The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody–RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD–ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4–6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
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Jul 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
Krios I-Titan Krios I at Diamond
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Daming
Zhou
,
Helen M. E.
Duyvesteyn
,
Cheng-pin
Chen
,
Chung-guei
Huang
,
Ting-hua
Chen
,
Shin-ru
Shih
,
Yi-chun
Lin
,
Chien-yu
Cheng
,
Shu-hsing
Cheng
,
Yhu-chering
Huang
,
Tzou-yien
Lin
,
Che
Ma
,
Jiandong
Huo
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Pranav
Shah
,
Tiong Kit
Tan
,
Pramila
Rijal
,
Robert F.
Donat
,
Kerry
Godwin
,
Karen R.
Buttigieg
,
Julia A.
Tree
,
Julika
Radecke
,
Neil
Paterson
,
Piyada
Supasa
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Miles W.
Carroll
,
Javier
Gilbert-jaramillo
,
Michael L.
Knight
,
William
James
,
Raymond J.
Owens
,
James H.
Naismith
,
Alain R.
Townsend
,
Elizabeth E.
Fry
,
Yuguang
Zhao
,
Jingshan
Ren
,
David I.
Stuart
,
Kuan-ying A.
Huang
Diamond Proposal Number(s):
[19946, 26983]
Abstract: The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD–EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.
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Jul 2020
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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Jiandong
Huo
,
Yuguang
Zhao
,
Jingshan
Ren
,
Daming
Zhou
,
Helen M. E.
Duyvesteyn
,
Helen M.
Ginn
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Pranav N. M.
Shah
,
Tiong Kit
Tan
,
Pramila
Rijal
,
Naomi
Coombes
,
Kevin R.
Bewley
,
Julia A.
Tree
,
Julika
Radecke
,
Neil
Paterson
,
Piyasa
Supasa
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Miles
Carroll
,
Alain
Townsend
,
Elizabeth E.
Fry
,
Raymond J.
Owens
,
David I.
Stuart
Diamond Proposal Number(s):
[19946, 26983]
Open Access
Abstract: There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognises angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.
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Jun 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[14744]
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.
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Feb 2020
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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
Open Access
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.
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Jan 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[16814, 14744]
Abstract: The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin, came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high resolution structures (≤ 1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid; and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µM), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which up-regulation of Wnt signalling may be beneficial.
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Dec 2019
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