B21-High Throughput SAXS
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Diamond Proposal Number(s):
[20145]
Open Access
Abstract: Ectopic expression in T-cell precursors of LIM only protein 2 (LMO2), a key factor in hematopoietic development, has been linked to the onset of T-cell acute lymphoblastic leukaemia (T-ALL). In the T-ALL context, LMO2 drives oncogenic progression through binding to erythroid-specific transcription factor SCL/TAL1 and sequestration of E-protein transcription factors, normally required for T-cell differentiation. A key requirement for the formation of this oncogenic protein-protein interaction (PPI) is the conformational flexibility of LMO2. Here we identify a small molecule inhibitor of the SCL-LMO2 PPI, which hinders the interaction in vitro through direct binding to LMO2. Biophysical analysis demonstrates that this inhibitor acts through a mechanism of conformational modulation of LMO2. Importantly, this work has led to the identification of a small molecule inhibitor of the SCL-LMO2 PPI, which can provide a starting point for the development of new agents for the treatment of T-ALL. These results suggest that similar approaches, based on the modulation of protein conformation by small molecules, might be used for therapeutic targeting of other oncogenic PPIs.
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May 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12305]
Open Access
Abstract: Zika virus, a flavivirus like Dengue and West Nile viruses, poses a significant risk as a pathogen in the category of emerging infectious diseases. Zika infections typically cause nonspecific, mild symptoms, but can also manifest as a neurological disorder like Guillain-Barré syndrome. Infection in pregnant women is linked to microcephaly in newborn infants. The methyltransferase domain of the non-structural protein 5 is responsible for two sequential methylations of the 5′-RNA cap. This is crucial for genome stability, efficient translation, and escape from the host immune response. Here we present the crystal structures of the Zika methyltransferase domain in complex with the methyl-donor SAM and its by-product SAH. The methyltransferase-SAH binary complex presents a new conformation of a “closed” or “obstructed” state that would restrict the binding of new RNA for capping. The combination and comparison of our new structures with recently published Zika methyltransferase structures provide a first glimpse into the structural mechanism of Zika virus mRNA capping.
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Jan 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Cancer cells reprogram their metabolism and energy production to sustain increased growth, enable metastasis and overcome resistance to cancer treatments. Although primary roles for many metabolic proteins have been identified, some are promiscuous in regards to the reaction they catalyze. To efficiently target these enzymes, a good understanding of their enzymatic function and structure, as well as knowledge regarding any substrate or catalytic promiscuity is required. Here we focus on the characterization of human 3-phosphoglycerate dehydrogenase (PHGDH). PHGDH catalyzes the NAD+-dependent conversion of 3-phosphoglycerate to phosphohydroxypyruvate, which is the first step in the de novo synthesis pathway of serine, a critical amino acid for protein and nucleic acid biosynthesis. We have investigated substrate analogues to assess whether PHGDH might possess other enzymatic roles that could explain its occasional over-expression in cancer, as well as to help with the design of specific inhibitors. We also report the crystal structure of the catalytic subunit of human PHGDH, a dimer, solved with bound cofactor in one monomer and both cofactor and L-tartrate in the second monomer. In vitro enzyme activity measurements show that the catalytic subunit of PHGDH is still active and that PHGDH activity could be significantly inhibited with adenosine 5’-diphosphoribose.
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Nov 2017
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Christopher R.
Coxon
,
Christopher
Wong
,
Richard
Bayliss
,
Kathy
Boxall
,
Katherine H.
Carr
,
Andrew M.
Fry
,
Ian R.
Hardcastle
,
Christopher J.
Matheson
,
David R.
Newell
,
Mangaleswaran
Sivaprakasam
,
Huw
Thomas
,
David
Turner
,
Sharon
Yeoh
,
Lan Z.
Wang
,
Roger J.
Griffin
,
Bernard T.
Golding
,
Céline
Cano
Diamond Proposal Number(s):
[307, 6385]
Open Access
Abstract: Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 μM; Nek2 IC50 = 0.62 μM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 μM; Nek2 IC50 = 0.27 μM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.
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Nov 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: 3-Phosphoglycerate dehydrogenase (PHGDH) has recently been identified as an attractive target in cancer therapy as it links upregulated glycolytic flux to increased biomass production in cancer cells. PHGDH catalyses the first step in the serine synthesis pathway and thus diverts glycolytic flux into serine synthesis. We have used siRNA-mediated suppression of PHGDH expression to show that PHGDH is a potential therapeutic target in PHGDH-amplified breast cancer. Knockdown caused reduced proliferation in the PHGDH-amplified cell line MDA-MB-468, whereas breast cancer cells with low PHGDH expression or with elevated PHGDH expression in the absence of genomic amplification were not affected. As a first step towards design of a chemical probe for PHGDH, we report a fragment-based drug discovery approach for the identification of PHGDH inhibitors. We designed a truncated PHGDH construct that gave crystals which diffracted to high resolution, and could be used for fragment soaking. 15 fragments stabilising PHGDH were identified using a thermal shift assay and validated by X-ray crystallography and ITC competition experiments to exhibit 1.5-26.2 mM affinity for PHGDH. A structure-guided fragment growing approach was applied to the PHGDH binders from the initial screen, yielding greater understanding of the binding site and suggesting routes to achieve higher affinity NAD-competitive inhibitors.
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Aug 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Harshnira
Patani
,
Tom D.
Bunney
,
Nethaji
Thiyagarajan
,
Richard A.
Norman
,
Derek
Ogg
,
Jason
Breed
,
Paul
Ashford
,
Andrew
Potterton
,
Mina
Edwards
,
Sarah V.
Williams
,
Gary S.
Thomson
,
Camilla S. M.
Pang
,
Margaret A.
Knowles
,
Alexander L.
Breeze
,
Christine
Orengo
,
Chris
Phillips
,
Matilda
Katan
Diamond Proposal Number(s):
[9204]
Open Access
Abstract: Frequent genetic alterations discovered in FGFRs and evidence implicating some as drivers in diverse tumors has been accompanied by rapid progress in targeting FGFRs for anticancer treatments. Wider assessment of the impact of genetic changes on the activation state and drug responses is needed to better link the genomic data and treatment options. We here apply a direct comparative and comprehensive analysis of FGFR3 kinase domain variants representing the diversity of point-mutations reported in this domain. We reinforce the importance of N540K and K650E and establish that not all highly activating mutations (for example R669G) occur at high-frequency and conversely, that some “hotspots” may not be linked to activation. Further structural characterization consolidates a mechanistic view of FGFR kinase activation and extends insights into drug binding. Importantly, using several inhibitors of particular clinical interest (AZD4547, BGJ-398, TKI258, JNJ42756493 and AP24534), we find that some activating mutations (including different replacements of the same residue) result in distinct changes in their efficacy. Considering that there is no approved inhibitor for anticancer treatments based on FGFR-targeting, this information will be immediately translatable to ongoing clinical trials.
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Mar 2016
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I03-Macromolecular Crystallography
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Cory A.
Ocasio
,
Mohan B.
Rajasekaran
,
Sarah
Walker
,
Darren
Le Grand
,
John
Spencer
,
Frances M. G.
Pearl
,
Simon E.
Ward
,
Velibor
Savic
,
Laurence H.
Pearl
,
Helfrid
Hochegger
,
Antony
Oliver
Diamond Proposal Number(s):
[8015]
Open Access
Abstract: MASTL (microtubule-associated serine/threonine kinase-like), more commonly known as Greatwall (GWL), has been proposed as a novel cancer therapy target. GWL plays a crucial role in mitotic progression, via its known substrates ENSA/ARPP19, which when phosphorylated inactivate PP2A/B55 phosphatase. When over-expressed in breast cancer, GWL induces oncogenic properties such as transformation and invasiveness. Conversely, down-regulation of GWL selectively sensitises tumour cells to chemotherapy. Here we describe the first structure of the GWL minimal kinase domain and development of a small-molecule inhibitor GKI-1 (Greatwall Kinase Inhibitor-1). In vitro, GKI-1 inhibits full-length human GWL, and shows cellular efficacy. Treatment of HeLa cells with GKI-1 reduces ENSA/ARPP19 phosphorylation levels, such that they are comparable to those obtained by siRNA depletion of GWL; resulting in a decrease in mitotic events, mitotic arrest/cell death and cytokinesis failure. Furthermore, GKI-1 will be a useful starting point for the development of more potent and selective GWL inhibitors.
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Nov 2015
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I02-Macromolecular Crystallography
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Andrew J.
Massey
,
Stephen
Stokes
,
Helen
Browne
,
Nicolas
Foloppe
,
Andreá
Fiumana
,
Simon
Scrace
,
Mandy
Fallowfield
,
Simon
Bedford
,
Paul
Webb
,
Lisa
Baker
,
Mark
Christie
,
Martin J.
Drysdale
,
Mike
Wood
Open Access
Abstract: Chk1 kinase is a critical component of the DNA damage response checkpoint especially in cancer cells and targeting Chk1 is a potential therapeutic opportunity for potentiating the anti-tumor activity of DNA damaging chemotherapy drugs. Fragment elaboration by structure guided design was utilized to identify and develop a novel series of Chk1 inhibitors culminating in the identification of V158411, a potent ATPcompetitive inhibitor of the Chk1 and Chk2 kinases. V158411 abrogated gemcitabine and camptothecin induced cell cycle checkpoints, resulting in the expected modulationof cell cycle proteins and increased cell death in cancer cells. V158411 potentiated the cytotoxicity of gemcitabine, cisplatin, SN38 and camptothecin in a variety of p53 deficient human tumor cell lines in vitro, p53 proficient cells were unaffected. In nude mice, V158411 showed minimal toxicity as a single agent and in combination with irinotecan. In tumor bearing animals, V158411 was detected at high levels in the tumor with a long elim nation half-life; no pharmacologically significant in vivo drug-drug interactions with irinotecan were identified through analysis of the pharmacokinetic profiles. V158411 potentiated the anti-tumor activity of irinotecan in a variety of human colon tumor xenograft models without additional systemic toxicity. These results demonstrate the opportunity for combining V158411 with standard of care chemotherapeutic agents to potentiate the therapeutic efficacy of these agents without increasing their toxicity to normal cells. Thus, V158411 would warrant further clinical evaluation.
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Sep 2015
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B23-Circular Dichroism
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Carrie A.
Duckworth
,
Scott E.
Guimond
,
Paulina
Sindrewicz
,
Ashley J.
Hughes
,
Neil S.
French
,
Lu-Yun
Lian
,
Edwin A.
Yates
,
D. Mark
Pritchard
,
Jonathan M.
Rhodes
,
Jeremy E.
Turnbull
,
Lu-Gang
Yu
Diamond Proposal Number(s):
[9218]
Open Access
Abstract: Concentrations of circulating galectin-3, a metastasis promoter, are greatly
increased in cancer patients. Here we show that 2- or 6-de-O-sulfated, N-acetylated
heparin derivatives are galectin-3 binding inhibitors. These chemically modified heparin
derivatives inhibited galectin-3-ligand binding and abolished galectin-3-mediated
cancer cell-endothelial adhesion and angiogenesis. Unlike standard heparin, these
modified heparin derivatives and their ultra-low molecular weight sub-fractions
had neither anticoagulant activity nor effects on E-, L- or P-selectin binding to their
ligands nor detectable cytotoxicity. Intravenous injection of such heparin derivatives
(with cancer cells pre-treated with galectin-3 followed by 3 subcutaneous injections
of the derivatives) abolished the circulating galectin-3-mediated increase in lung
metastasis of human melanoma and colon cancer cells in nude mice. Structural
analysis using nuclear magnetic resonance and synchrotron radiation circular
dichroism spectroscopies showed that the modified heparin derivatives bind to
the galectin-3 carbohydrate-recognition domain. Thus, these chemically modified,
non-anticoagulant, low-sulfated heparin derivatives are potent galectin-3 binding
inhibitors with substantial potential as anti-metastasis/cancer drugs.
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Jun 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Amar
Joshi
,
Yvette
Newbatt
,
P. Craig
Mcandrew
,
Mark
Stubbs
,
Rosemary
Burke
,
Mark
Richards
,
Chitra
Bhatia
,
John J.
Caldwell
,
Tatiana
Mchardy
,
Ian
Collins
,
Richard
Bayliss
Diamond Proposal Number(s):
[10369]
Open Access
Abstract: IRE1 transduces the unfolded protein response by splicing XBP1 through its C-terminal cytoplasmic kinase-RNase region. IRE1 autophosphorylation is coupled to RNase activity through formation of a back-to-back dimer, although the conservation of the underlying molecular mechanism is not clear from existing structures. We have crystallized human IRE1 in a back-to-back conformation only previously seen for the yeast homologue. In our structure the kinase domain appears primed for catalysis but the RNase domains are disengaged. Structure-function analysis reveals that IRE1 is autoinhibited through a Tyr-down mechanism related to that found in the unrelated Ser/Thr protein kinase Nek7. We have developed a compound that potently inhibits human IRE1 kinase activity while stimulating XBP1 splicing. A crystal structure of the inhibitor bound to IRE1 shows an increased ordering of the kinase activation loop. The structures of hIRE in apo and ligand-bound forms are consistent with a previously proposed model of IRE1 regulation in which formation of a back-to-back dimer coupled to adoption of a kinase-active conformation drive RNase activation. The structures provide opportunities for structure-guided design of IRE1 inhibitors.
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Apr 2015
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