I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Cellular retinoic acid binding protein 2 (CRABP2) transports retinoic acid from the cytoplasm to the nucleus where it then transfers its cargo to retinoic acid receptor-containing complexes leading to activation of gene transcription. We demonstrate using purified proteins that CRABP2 is also a cyclin D3-specific binding protein and that the CRABP2 cyclin D3 binding site and the proposed CRABP2 nuclear localization sequence overlap. Both sequences are within the helix-loop-helix motif that forms a lid to the retinoic acid binding pocket. Mutations within this sequence that block both cyclin D3 and retinoic acid binding promote formation of a CRABP2 structure in which the retinoic acid binding pocket is occupied by an alternative lid conformation. Structural and functional analysis of CRABP2 and cyclin D3 mutants combined with AlphaFold models of the ternary CDK4/6-cyclin D3-CRABP2 complex supports the identification of an α-helical protein binding site on the cyclin D3 C-terminal cyclin box fold.
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Oct 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[28576]
Open Access
Abstract: The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.
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Aug 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Gemma
Davison
,
Mathew P.
Martin
,
Shannon
Turberville
,
Selma
Dormen
,
Richard
Heath
,
Amy B.
Heptinstall
,
Marie
Lawson
,
Duncan C.
Miller
,
Yi Min
Ng
,
James N.
Sanderson
,
Ian
Hope
,
Daniel
Wood
,
Céline
Cano
,
Jane A.
Endicott
,
Ian R.
Hardcastle
,
Martin E. M.
Noble
,
Michael J.
Waring
Open Access
Abstract: The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.
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Nov 2022
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Duncan C.
Miller
,
Tristan
Reuillon
,
Lauren
Molyneux
,
Timothy
Blackburn
,
Simon J.
Cook
,
Noel
Edwards
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Roger J.
Griffin
,
Ian
Hardcastle
,
Suzannah J.
Harnor
,
Amy
Heptinstall
,
Pamela
Lochhead
,
Mathew P.
Martin
,
Nick C.
Martin
,
Stephanie
Myers
,
David R.
Newell
,
Richard A.
Noble
,
Nicole
Phillips
,
Laurent
Rigoreau
,
Huw
Thomas
,
Julie A.
Tucker
,
Lan-Zhen
Wang
,
Michael J.
Waring
,
Ai-Ching
Wong
,
Stephen R.
Wedge
,
Martin E. M.
Noble
,
Celine
Cano
Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.
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Apr 2022
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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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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Gianni
Chessari
,
Ian R.
Hardcastle
,
Jong Sook
Ahn
,
Burcu
Anil
,
Elizabeth
Anscombe
,
Ruth H.
Bawn
,
Luke D.
Bevan
,
Timothy J.
Blackburn
,
Ildiko
Buck
,
Celine
Cano
,
Benoit
Carbain
,
Juan
Castro
,
Ben
Cons
,
Sarah J.
Cully
,
Jane A.
Endicott
,
Lynsey
Fazal
,
Bernard T.
Golding
,
Roger J.
Griffin
,
Karen
Haggerty
,
Suzannah J.
Harnor
,
Keisha
Hearn
,
Stephen
Hobson
,
Rhian S.
Holvey
,
Steven
Howard
,
Claire E.
Jennings
,
Christopher N.
Johnson
,
John
Lunec
,
Duncan C.
Miller
,
David R.
Newell
,
Martin E. M.
Noble
,
Judith
Reeks
,
Charlotte H.
Revill
,
Christiane
Riedinger
,
Jeffrey D.
St. Denis
,
Emiliano
Tamanini
,
Huw
Thomas
,
Neil T.
Thompson
,
Mladen
Vinković
,
Stephen R.
Wedge
,
Pamela A.
Williams
,
Nicola E.
Wilsher
,
Bian
Zhang
,
Yan
Zhao
Abstract: Inhibition of murine double minute 2 (MDM2)-p53 protein–protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.
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Mar 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Stephanie
Myers
,
Duncan C.
Miller
,
Lauren
Molyneux
,
Mercedes
Arasta
,
Ruth H.
Bawn
,
Timothy
Blackburn
,
Simon J.
Cook
,
Noel
Edwards
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Roger J.
Griffin
,
Tim
Hammonds
,
Ian R.
Hardcastle
,
Suzannah J.
Harnor
,
Amy
Heptinstall
,
Pamela
Lochhead
,
Mathew P.
Martin
,
Nick C.
Martin
,
David R.
Newell
,
Paul J.
Owen
,
Leon C.
Pang
,
Tristan
Reuillon
,
Laurent J. M.
Rigoreau
,
Huw
Thomas
,
Julie A.
Tucker
,
Lan-Zhen
Wang
,
Ai-Ching
Wong
,
Martin E. M.
Noble
,
Stephen R.
Wedge
,
Celine
Cano
Diamond Proposal Number(s):
[9948, 13587]
Abstract: Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 μM for ERK5; IC50 > 120 μM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.
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May 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Daniel J.
Wood
,
José Daniel
Lopez-Fernandez
,
Leanne E.
Knight
,
Islam
Al-Khawaldeh
,
Conghao
Gai
,
Shengyin
Lin
,
Mathew P.
Martin
,
Duncan C.
Miller
,
Celine
Cano
,
Jane A.
Endicott
,
Ian R.
Hardcastle
,
Martin E. M.
Noble
,
Michael J.
Waring
Abstract: Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource intensive screening of large libraries of lead-like or fragment molecules. Here we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.
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Mar 2019
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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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Duncan C.
Miller
,
Mathew P.
Martin
,
Santosh
Adhikari
,
Alfie
Brennan
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Ian R.
Hardcastle
,
Amy
Heptinstall
,
Stephen
Hobson
,
Claire
Jennings
,
Lauren
Molyneux
,
Yvonne
Ng
,
Stephen R.
Wedge
,
Martin
Noble
,
Celine
Cano
Open Access
Abstract: ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure–activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.
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Feb 2018
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