I04-1-Macromolecular Crystallography (fixed wavelength)
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Benoit
Carbain
,
David J.
Paterson
,
Elizabeth
Anscombe
,
Allyson J.
Campbell
,
Celine
Cano
,
Aude
Echalier
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Karen
Haggerty
,
Ian R.
Hardcastle
,
Philip J.
Jewsbury
,
David R.
Newell
,
Martin E. M.
Noble
,
Celine
Roche
,
Lan Z.
Wang
,
Roger J.
Griffin
Abstract: Evaluation of the effects of purine C-8 substitution within a series of CDK1/2-selective O6-cyclohexylmethylguanine derivatives revealed that potency decreases initially with increasing size of the alkyl substituent. Structural analysis showed that C-8 substitution is poorly tolerated, and to avoid unacceptable steric interactions, these compounds adopt novel binding modes. Thus, 2-amino-6-cyclohexylmethoxy-8-isopropyl-9H-purine adopts a “reverse” binding mode where the purine backbone has flipped 180°. This provided a novel lead chemotype from which we have designed more potent CDK2 inhibitors using, in the first instance, quantum mechanical energy calculations. Introduction of an ortho-tolyl or ortho-chlorophenyl group at the purine C-8 position restored the potency of these “reverse” binding mode inhibitors to that of the parent 2-amino-6-cyclohexylmethoxy-9H-purine. By contrast, the corresponding 8-(2-methyl-3-sulfamoylphenyl)-purine derivative exhibited submicromolar CDK2-inhibitory activity by virtue of engineered additional interactions with Asp86 and Lys89 in the reversed binding mode, as confirmed by X-ray crystallography.
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Dec 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yaw Sing
Tan
,
Judith
Reeks
,
Christopher J.
Brown
,
Dawn
Thean
,
Fernando Jose
Ferrer Gago
,
Tsz Ying
Yuen
,
Eunice Tze Leng
Goh
,
Xue Er Cheryl
Lee
,
Claire E.
Jennings
,
Thomas L.
Joseph
,
Rajamani
Lakshminarayanan
,
David P.
Lane
,
Martin E. M.
Noble
,
Chandra S.
Verma
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design.
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Sep 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Mélanie
Uguen
,
Gemma
Davison
,
Lukas J.
Sprenger
,
James H.
Hunter
,
Mathew P.
Martin
,
Shannon
Turberville
,
Jessica
Watt
,
Bernard T.
Golding
,
Martin E. N.
Noble
,
Hannah L.
Stewart
,
Michael J.
Waring
Open Access
Abstract: High-throughput screening provides one of the most common ways of finding hit compounds. Lead-like libraries, in particular, provide hits with compatible functional groups and vectors for structural elaboration and physical properties suitable for optimization. Library synthesis approaches can lead to a lack of chemical diversity because they employ parallel derivatization of common building blocks using single reaction types. We address this problem through a “build–couple–transform” paradigm for the generation of lead-like libraries with scaffold diversity. Nineteen transformations of a 4-oxo-2-butenamide scaffold template were optimized, including 1,4-cyclizations, 3,4-cyclizations, reductions, and 1,4-additions. A pool-transformation approach efficiently explored the scope of these transformations for nine different building blocks and synthesized a >170-member library with enhanced chemical space coverage and favorable drug-like properties. Screening revealed hits against CDK2. This work establishes the build–couple–transform concept for the synthesis of lead-like libraries and provides a differentiated approach to libraries with significantly enhanced scaffold diversity.
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Aug 2022
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Liz
Potterton
,
Jon
Agirre
,
Charles
Ballard
,
Kevin
Cowtan
,
Eleanor
Dodson
,
Phil R.
Evans
,
Huw T.
Jenkins
,
Ronan
Keegan
,
Eugene
Krissinel
,
Kyle
Stevenson
,
Andrey
Lebedev
,
Stuart J.
Mcnicholas
,
Robert A.
Nicholls
,
Martin
Noble
,
Navraj S.
Pannu
,
Christian
Roth
,
George
Sheldrick
,
Pavol
Skubak
,
Johan
Turkenburg
,
Ville
Uski
,
Frank
Von Delft
,
David
Waterman
,
Keith
Wilson
,
Martyn
Winn
,
Marcin
Wojdyr
Open Access
Abstract: The CCP4 (Collaborative Computational Project, Number 4) software suite for macromolecular structure determination by X-ray crystallography groups brings together many programs and libraries that, by means of well established conventions, interoperate effectively without adhering to strict design guidelines. Because of this inherent flexibility, users are often presented with diverse, even divergent, choices for solving every type of problem. Recently, CCP4 introduced CCP4i2, a modern graphical interface designed to help structural biologists to navigate the process of structure determination, with an emphasis on pipelining and the streamlined presentation of results. In addition, CCP4i2 provides a framework for writing structure-solution scripts that can be built up incrementally to create increasingly automatic procedures.
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Feb 2018
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I02-Macromolecular Crystallography
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Open Access
Abstract: CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1–Cks1 and CDK1–cyclin B–Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2–cyclin A, CDK1–cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.
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Nov 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
Abstract: Cyclin-dependent kinase 9/cyclin T, the protein kinase heterodimer that constitutes positive transcription elongation factor b, is a well-validated target for treatment of several diseases, including cancer and cardiac hypertrophy. In order to aid inhibitor design and rationalize the basis for CDK9 selectivity, we have studied the CDK-binding properties of six different members of a 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile series that bind to both CDK9/cyclin T and CDK2/cyclin A. We find that for a given CDK, the melting temperature of a CDK/cyclin/inhibitor complex correlates well with inhibitor potency, suggesting that differential scanning fluorimetry (DSF) is a useful orthogonal measure of inhibitory activity for this series. We have used DSF to demonstrate that the binding of these compounds is independent of the presence or absence of the C-terminal tail region of CDK9, unlike the binding of the CDK9-selective inhibitor 5,6-dichlorobenzimidazone-1-?-d-ribofuranoside (DRB). Finally, on the basis of 11 cocrystal structures bound to CDK9/cyclin T or CDK2/cyclin A, we conclude that selective inhibition of CDK9/cyclin T by members of the 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile series results from the relative malleability of the CDK9 active site rather than from the formation of specific polar contacts.
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Dec 2012
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Alice
Douangamath
,
Daren
Fearon
,
Paul
Gehrtz
,
Tobias
Krojer
,
Petra
Lukacik
,
C. David
Owen
,
Efrat
Resnick
,
Claire
Strain-Damerell
,
Anthony
Aimon
,
Péter
Ábrányi-Balogh
,
Jose
Brandao-Neto
,
Anna
Carbery
,
Gemma
Davison
,
Alexandre
Dias
,
Thomas D.
Downes
,
Louise
Dunnett
,
Michael
Fairhead
,
James D.
Firth
,
S. Paul
Jones
,
Aaron
Keeley
,
György M.
Keserü
,
Hanna F.
Klein
,
Mathew P.
Martin
,
Martin M.
Noble
,
Peter
O’brien
,
Ailsa
Powell
,
Rambabu N.
Reddi
,
Rachael
Skyner
,
Matthew
Snee
,
Michael J.
Waring
,
Conor
Wild
,
Nir
London
,
Frank
Von Delft
,
Martin A.
Walsh
Open Access
Abstract: COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
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Oct 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Christopher R
Coxon
,
Elizabeth
Anscombe
,
Suzannah Jane
Harnor
,
Mathew P.
Martin
,
Benoit Jean-Pierre
Carbain
,
Bernard Thomas
Golding
,
Ian Robert
Hardcastle
,
Lisa K
Harlow
,
Svitlana
Korolchuk
,
Christopher J.
Matheson
,
David R.
Newell
,
Martin E. M.
Noble
,
Mangaleswaran
Sivaprakasam
,
Susan J.
Tudhope
,
David M.
Turner
,
Lan-Zhen
Wang
,
Stephen R
Wedge
,
Christopher
Wong
,
Roger John
Griffin
,
Jane A.
Endicott
,
Celine
Cano
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Purines and related heterocycles substituted at C-2 with 4’-sulfamoylanilino and at C-6 with a variety of groups have been synthesized with the aim of achieving selectivity of binding to CDK2 over CDK1. 6-Substituents that favour competitive inhibition at the ATP binding site of CDK2 were identified and typically exhibited 10-80-fold greater inhibition of CDK2 compared to CDK1. Most impressive was 4-((6-([1,1'-biphenyl]-3-yl)-9H-purin-2-yl)amino) benzenesulfonamide (73) that exhibited high potency towards CDK2 (IC50 0.044 μM), but was ~ 2000-fold less active towards CDK1 (IC50 86 μM). This compound is therefore a useful tool for studies of cell cycle regulation. Crystal structures of inhibitor-kinase complexes showed that the inhibitor stabilizes a glycine-rich loop conformation that shapes the ATP ribose binding pocket, and that is preferred in CDK2, but has not been observed in CDK1. This aspect of the active site may be exploited for the design of inhibitors that distinguish between CDK1 and CDK2.
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Dec 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598, 13587]
Open Access
Abstract: Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.
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Nov 2018
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Marco
Salamina
,
Bailey C.
Montefiore
,
Mengxi
Liu
,
Daniel J.
Wood
,
Richard
Heath
,
James R.
Ault
,
Lan-Zhen
Wang
,
Svitlana
Korolchuk
,
Arnaud
Basle
,
Martyna
Pastok
,
Judith
Reeks
,
Natalie J.
Tatum
,
Frank
Sobott
,
Stefan T.
Arold
,
Michele
Pagano
,
Martin E. M.
Noble
,
Jane A.
Endicott
Diamond Proposal Number(s):
[13587, 16970]
Open Access
Abstract: The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.
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Mar 2021
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