I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Olivier A.
Pierrat
,
Manjuan
Liu
,
Gavin W.
Collie
,
Kartika N.
Shetty
,
Matthew J.
Rodrigues
,
Yann-Vai
Le Bihan
,
Emma A.
Gunnell
,
P. Craig
Mcandrew
,
Mark
Stubbs
,
Martin G.
Rowlands
,
Norhakim
Yahya
,
Erald
Shehu
,
Rachel
Talbot
,
Lisa
Pickard
,
Benjamin R.
Bellenie
,
Kwai-Ming J.
Cheung
,
Ludovic
Drouin
,
Paolo
Innocenti
,
Hannah
Woodward
,
Owen A.
Davis
,
Matthew G.
Lloyd
,
Ana
Varela
,
Rosemary
Huckvale
,
Fabio
Broccatelli
,
Michael
Carter
,
David
Galiwango
,
Angela
Hayes
,
Florence I.
Raynaud
,
Christopher
Bryant
,
Steven
Whittaker
,
Olivia W.
Rossanese
,
Swen
Hoelder
,
Rosemary
Burke
,
Rob L. M.
Van Montfort
Open Access
Abstract: By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein–protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.
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Nov 2022
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I03-Macromolecular Crystallography
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Rosemary
Huckvale
,
Alice C.
Harnden
,
Kwai-Ming J.
Cheung
,
Olivier A.
Pierrat
,
Rachel
Talbot
,
Gary M.
Box
,
Alan T.
Henley
,
Alexis K.
De Haven Brandon
,
Albert E.
Hallsworth
,
Michael D.
Bright
,
Hafize Aysin
Akpinar
,
Daniel S. J.
Miller
,
Dalia
Tarantino
,
Sharon
Gowan
,
Angela
Hayes
,
Emma A.
Gunnell
,
Alfie
Brennan
,
Owen A.
Davis
,
Louise D.
Johnson
,
Selby
De Klerk
,
Craig
Mcandrew
,
Yann-Vai
Le Bihan
,
Mirco
Meniconi
,
Rosemary
Burke
,
Vladimir
Kirkin
,
Rob L. M.
Van Montfort
,
Florence I.
Raynaud
,
Olivia W.
Rossanese
,
Benjamin R.
Bellenie
,
Swen
Hoelder
Open Access
Abstract: The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.
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Jun 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Owen A.
Davis
,
Kwai-Ming J.
Cheung
,
Alfie
Brennan
,
Matthew G.
Lloyd
,
Matthew J.
Rodrigues
,
Olivier A.
Pierrat
,
Gavin W.
Collie
,
Yann-Vai
Le Bihan
,
Rosemary
Huckvale
,
Alice C.
Harnden
,
Ana
Varela
,
Michael D.
Bright
,
Paul
Eve
,
Angela
Hayes
,
Alan T.
Henley
,
Michael D.
Carter
,
P. Craig
Mcandrew
,
Rachel
Talbot
,
Rosemary
Burke
,
Rob
Van Montfort
,
Florence I.
Raynaud
,
Olivia W.
Rossanese
,
Mirco
Meniconi
,
Benjamin R.
Bellenie
,
Swen
Hoelder
Open Access
Abstract: To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.
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Jun 2022
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I24-Microfocus Macromolecular Crystallography
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Suzanne
O'Connor
,
Yann-Vai
Le Bihan
,
Isaac M.
Westwood
,
Manjuan
Liu
,
Oi Wei
Mak
,
Gabriel
Zazeri
,
Ana P. R.
Povinelli
,
Alan M.
Jones
,
Rob
Van Montfort
,
Jóhannes
Reynisson
,
Ian
Collins
Diamond Proposal Number(s):
[6385]
Open Access
Abstract: Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.
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Jan 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Matthew G.
Lloyd
,
Rosemary
Huckvale
,
Kwai-Ming J.
Cheung
,
Matthew J.
Rodrigues
,
Gavin W.
Collie
,
Olivier A.
Pierrat
,
Mahad
Gatti Iou
,
Michael
Carter
,
Owen A.
Davis
,
P. Craig
Mcandrew
,
Emma
Gunnell
,
Yann-Vai
Le Bihan
,
Rachel
Talbot
,
Alan T.
Henley
,
Louise D.
Johnson
,
Angela T.
Hayes
,
Michael D.
Bright
,
Florence I.
Raynaud
,
Mirco
Meniconi
,
Rosemary
Burke
,
Rob L. M.
Van Montfort
,
Olivia W.
Rossanese
,
Benjamin R.
Bellenie
,
Swen
Hoelder
Diamond Proposal Number(s):
[14891, 20145]
Open Access
Abstract: We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.
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Nov 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Benjamin R.
Bellenie
,
Kwai-Ming J.
Cheung
,
Ana
Varela
,
Olivier A.
Pierrat
,
Gavin W.
Collie
,
Gary M.
Box
,
Michael D.
Bright
,
Sharon
Gowan
,
Angela
Hayes
,
Matthew J.
Rodrigues
,
Kartika N.
Shetty
,
Michael
Carter
,
Owen A.
Davis
,
Alan T.
Henley
,
Paolo
Innocenti
,
Louise D.
Johnson
,
Manjuan
Liu
,
Selby
De Klerk
,
Yann-Vai
Le Bihan
,
Matthew G.
Lloyd
,
P. Craig
Mcandrew
,
Erald
Shehu
,
Rachel
Talbot
,
Hannah L.
Woodward
,
Rosemary
Burke
,
Vladimir
Kirkin
,
Rob L. M.
Van Montfort
,
Florence I.
Raynaud
,
Olivia W.
Rossanese
,
Swen
Hoelder
Diamond Proposal Number(s):
[14891, 20145]
Open Access
Abstract: Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.
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Apr 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Yann-Vai
Le Bihan
,
Rachel M.
Lanigan
,
Butrus
Atrash
,
Mark G.
Mclaughlin
,
Srikannathasan
Velupillai
,
Andrew G.
Malcolm
,
Katherine S.
England
,
Gian Filippo
Ruda
,
N. Yi
Mok
,
Anthony
Tumber
,
Kathy
Tomlin
,
Harry
Saville
,
Erald
Shehu
,
Craig
Mcandrew
,
Leanne
Carmichael
,
James M.
Bennett
,
Fiona
Jeganathan
,
Paul
Eve
,
Adam
Donovan
,
Angela
Hayes
,
Francesca
Wood
,
Florence I.
Raynaud
,
Oleg
Fedorov
,
Paul
Brennan
,
Rosemary
Burke
,
Rob
Van Montfort
,
Olivia W.
Rossanese
,
Julian
Blagg
,
Vassilios
Bavetsias
Diamond Proposal Number(s):
[20145]
Open Access
Abstract: Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, the conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggest that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.
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May 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Vassilios
Bavetsias
,
Rachel M.
Lanigan
,
Gian Filippo
Ruda
,
Butrus
Atrash
,
Mark G.
Mclaughlin
,
Anthony
Tumber
,
N. Yi
Mok
,
Yann-Vaï
Le Bihan
,
Sally
Dempster
,
Katherine J.
Boxall
,
Fiona
Jeganathan
,
Stephanie B.
Hatch
,
Pavel
Savitsky
,
Velupillai
Srikannathasan
,
Tobias
Krojer
,
Katherine S.
England
,
Jimmy
Sejberg
,
Ching
Thai
,
Adam
Donovan
,
Akos
Pal
Open Access
Abstract: We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.
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Jan 2016
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