I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Grace Q.
Gong
,
Benoit
Bilanges
,
Ben
Allsop
,
Glenn
Masson
,
Victoria
Roberton
,
Trevor
Askwith
,
Sally
Oxenford
,
Ralitsa R.
Madsen
,
Sarah E.
Conduit
,
Dom
Bellini
,
Martina
Fitzek
,
Matt
Collier
,
Osman
Najam
,
Zhenhe
He
,
Ben
Wahab
,
Stephen H.
Mclaughlin
,
A. W. Edith
Chan
,
Isabella
Feierberg
,
Andrew
Madin
,
Daniele
Morelli
,
Amandeep
Bhamra
,
Vanesa
Vinciauskaite
,
Karen E.
Anderson
,
Silvia
Surinova
,
Nikos
Pinotsis
,
Elena
Lopez-Guadamillas
,
Matthew
Wilcox
,
Alice
Hooper
,
Chandni
Patel
,
Maria A.
Whitehead
,
Tom D.
Bunney
,
Len R.
Stephens
,
Phillip T.
Hawkins
,
Matilda
Katan
,
Derek M.
Yellon
,
Sean M.
Davidson
,
David M.
Smith
,
James B.
Phillips
,
Richard
Angell
,
Roger L.
Williams
,
Bart
Vanhaesebroeck
Diamond Proposal Number(s):
[28677]
Abstract: Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
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May 2023
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I03-Macromolecular Crystallography
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Alice C.
Harnden
,
Owen A.
Davis
,
Gary M.
Box
,
Angela
Hayes
,
Louise D.
Johnson
,
Alan T.
Henley
,
Alexis K.
De Haven Brandon
,
Melanie
Valenti
,
Kwai-Ming J.
Cheung
,
Alfie
Brennan
,
Rosemary
Huckvale
,
Olivier A.
Pierrat
,
Rachel
Talbot
,
Michael D.
Bright
,
Hafize Aysin
Akpinar
,
Daniel S. J.
Miller
,
Dalia
Tarantino
,
Sharon
Gowan
,
Selby
De Klerk
,
Peter C.
Mcandrew
,
Yann-Vai
Le Bihan
,
Mirco
Meniconi
,
Rosemary
Burke
,
Vladimir
Kirkin
,
Rob
Van Montfort
,
Florence I.
Raynaud
,
Olivia W.
Rossanese
,
Benjamin R.
Bellenie
,
Swen
Hoelder
Diamond Proposal Number(s):
[24828]
Open Access
Abstract: B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.
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Apr 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signalling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. Whilst inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterised a pair of nanobodies that are specific for mouse Plexin-B1, and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signalling, and provides a potential innovative route for therapeutic modulation of Plexin-B1.
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Apr 2023
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B21-High Throughput SAXS
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Tobias
Schmidt
,
Adrianna
Dabrowska
,
Joseph A.
Waldron
,
Kelly
Hodge
,
Grigorios
Koulouras
,
Mads
Gabrielsen
,
June
Munro
,
David C.
Tack
,
Gemma
Harris
,
Ewan
Mcghee
,
David
Scott
,
Leo m.
Carlin
,
Danny
Huang
,
John
Le quesne
,
Sara
Zanivan
,
Ania
Wilczynska
,
Martin
Bushell
Diamond Proposal Number(s):
[21657]
Open Access
Abstract: Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5’UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5’UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.
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Feb 2023
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I03-Macromolecular Crystallography
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Abstract: The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (wtB-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization. Here we present a new B-Raf kinase domain inhibitor, based on a phenyl(1H-pyrrolo [2,3-b]pyridin-3-yl)methanone template, that represents a hybrid between 4 and 3. This novel inhibitor borrows the hinge binding region from 4 and the back pocket binding moiety from 3. We determined its binding mode, performed activity/selectivity studies, and molecular dynamics simulations in order to study the conformational effects induced by this inhibitor on wt and V600E mutant B-Raf kinase. We discovered that the inhibitor was active and selective for B-Raf, binds in a DFG-out/αC-helix-in conformation, and did not induce the aforementioned paradoxical hyperactivation in the MAPK pathway. We propose that this merging approach can be used to design a novel class of B-Raf inhibitors for translational studies.
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Feb 2023
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I04-Macromolecular Crystallography
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James S.
Scott
,
Darren
Stead
,
Bernard
Barlaam
,
Jason
Breed
,
Rodrigo J.
Carbajo
,
Elisabetta
Chiarparin
,
Natalie
Cureton
,
Paul R. J.
Davey
,
David I.
Fisher
,
Eric T.
Gangl
,
Tyler
Grebe
,
Ryan D.
Greenwood
,
Sudhir
Hande
,
Holia
Hatoum-Mokdad
,
Samantha J.
Hughes
,
Thomas A.
Hunt
,
Tony
Johnson
,
Stefan L.
Kavanagh
,
Teresa C. M.
Klinowska
,
Carrie J. B.
Larner
,
Mandy
Lawson
,
Andrew S.
Lister
,
David
Longmire
,
Stacey
Marden
,
Thomas M.
Mcguire
,
Caroline
Mcmillan
,
Lindsay
Mcmurray
,
Christopher J.
Morrow
,
J. Willem M.
Nissink
,
Thomas A.
Moss
,
Daniel H.
O’donovan
,
Radoslaw
Polanski
,
Stephen
Stokes
,
Kumar
Thakur
,
Dawn
Trueman
,
Caroline
Truman
,
Michael J.
Tucker
,
Haixia
Wang
,
Nicky
Whalley
,
Dedong
Wu
,
Ye
Wu
,
Bin
Yang
,
Wenzhan
Yang
Diamond Proposal Number(s):
[20015]
Abstract: Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.
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Feb 2023
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I03-Macromolecular Crystallography
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Xiao
Liu
,
Raphael
Reinbold
,
Shuang
Liu
,
Ryan A.
Herold
,
Patrick
Rabe
,
Stéphanie
Duclos
,
Rahul B.
Yadav
,
Martine I.
Abboud
,
Sandrine
Thieffine
,
Fraser A.
Armstrong
,
Lennart
Brewitz
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutate (2OG) to (2R)-hydroxyglutarate (2HG). However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3 and C4 alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of wildtype IDH1/2. Absorbance-based, NMR and electrochemical assays were employed to monitor wildtype IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of wildtype IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates compared to 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.
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Jan 2023
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[24509, 20755, 21202]
Open Access
Abstract: Background: Galectin-9 is a member of the family of lectin proteins and crucially regulates human immune responses, particularly because of its ability to suppress the anticancer activities of T lymphocytes and natural killer cells. Recent evidence demonstrated that galectin-9 is highly expressed in a wide range of human malignancies including the most aggressive tumors, such as high-grade glioblastomas and pancreatic ductal adenocarcinomas, as well as common malignancies such as breast, lung and colorectal cancers. However, solid tumor cells at rest are known to secrete either very low amounts of galectin-9 or, in most of the cases, do not secrete it at all. Our aims were to elucidate whether T cells can induce galectin-9 secretion in human cancer cells derived from solid malignant tumors and whether this soluble form displays higher systemic immunosuppressive activity compared with the cell surface-based protein. Methods: A wide range of human cancer cell lines derived from solid tumours, keratinocytes and primary embryonic cells were employed, together with helper and cytotoxic T cell lines and human as well as mouse primary T cells. Western blot analysis, ELISA, quantitative reverse transcriptase-PCR, on-cell Western and other measurement techniques were used to conduct the study. Results were validated using in vivo mouse model. Results: We discovered that T lymphocytes induce galectin-9 secretion in various types of human cancer cells derived from solid malignant tumors. This was demonstrated to occur via two differential mechanisms: first by translocation of galectin-9 onto the cell surface followed by its proteolytic shedding and second due to autophagy followed by lysosomal secretion. For both mechanisms a protein carrier/trafficker was required, since galectin-9 lacks a secretion sequence. Secreted galectin-9 pre-opsonised T cells and, following interaction with other immune checkpoint proteins, their activity was completely attenuated. As an example, we studied the cooperation of galectin-9 and V-domain Ig-containing suppressor of T cell activation (VISTA) proteins in human cancer cells. Conclusion: Our results underline a crucial role of galectin-9 in anticancer immune evasion. As such, galectin-9 and regulatory pathways controlling its production should be considered as key targets for immunotherapy in a large number of cancers.
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Jan 2023
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Open Access
Abstract: Newcastle disease virus (NDV) is an oncolytic agent against various types of mammalian cancers. As with all cancer therapies, the development of cancer resistance, both innate and acquired, is becoming a challenge. In this study, we investigated persistently NDV-infected Caco-2 colon cancer cells, designated as virus-resistant (VR) Caco-2 cells, which were then able to resist NDV-mediated oncolysis. We applied single-cell Raman spectroscopy, combined with deuterium isotope probing (Raman-DIP) techniques, to investigate the metabolic adaptations and dynamics in VR Caco-2 cells. A linear discriminant analysis (LDA) model demonstrated excellent performance in differentiating VR Caco-2 from Caco-2 cells at single-cell level. By comparing the metabolic profiles in a time-resolved manner, the de novo synthesis of proteins and lipids was found upregulated, along with decreased DNA synthesis in VR Caco-2. The results suggest that VR Caco-2 cells might reprogram their metabolism and divert energy from proliferation to protein synthesis and lipidic modulation. The ability to identify and characterise single resistant cells among a population of cancer cells would help develop a deeper understanding of the resistance mechanisms and better tactics for developing effective cancer treatment.
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Jan 2023
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I22-Small angle scattering & Diffraction
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Miguel
Paez-Perez
,
Aurimas
Vyšniauskas
,
Ismael
López-Duarte
,
Eulalie J.
Lafarge
,
Raquel
López-Ríos De Castro
,
Carlos M.
Marques
,
André P.
Schroder
,
Pierre
Muller
,
Christian D.
Lorenz
,
Nicholas J.
Brooks
,
Marina K.
Kuimova
Open Access
Abstract: Lipid peroxidation is a process which is key in cell signaling and disease, it is exploited in cancer therapy in the form of photodynamic therapy. The appearance of hydrophilic moieties within the bilayer’s hydrocarbon core will dramatically alter the structure and mechanical behavior of membranes. Here, we combine viscosity sensitive fluorophores, advanced microscopy, and X-ray diffraction and molecular simulations to directly and quantitatively measure the bilayer’s structural and viscoelastic properties, and correlate these with atomistic molecular modelling. Our results indicate an increase in microviscosity and a decrease in the bending rigidity upon peroxidation of the membranes, contrary to the trend observed with non-oxidized lipids. Fluorescence lifetime imaging microscopy and MD simulations give evidence for the presence of membrane regions of different local order in the oxidized membranes. We hypothesize that oxidation promotes stronger lipid-lipid interactions, which lead to an increase in the lateral heterogeneity within the bilayer and the creation of lipid clusters of higher order.
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Jan 2023
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