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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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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Hannah L.
Woodward
,
Paolo
Innocenti
,
Kwai-Ming J.
Cheung
,
Angela
Hayes
,
Jennie
Roberts
,
Alan T.
Henley
,
Amir
Faisal
,
Grace Wing-Yan
Mak
,
Gary
Box
,
Isaac M.
Westwood
,
Nora
Cronin
,
Michael
Carter
,
Melanie
Valenti
,
Alexis
De Haven Brandon
,
Lisa
O’fee
,
Harry
Saville
,
Jessica
Schmitt
,
Rosemary
Burke
,
Fabio
Broccatelli
,
Rob L. M.
Van Montfort
,
Florence I.
Raynaud
,
Suzanne A.
Eccles
,
Spiros
Linardopoulos
,
Julian
Blagg
,
Swen
Hoelder
Diamond Proposal Number(s):
[10088]
Open Access
Abstract: Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4-d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4-d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.
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Sep 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Dominic
Tisi
,
Elisabetta
Chiarparin
,
Emiliano
Tamanini
,
Puja
Pathuri
,
Joseph E.
Coyle
,
Adam
Hold
,
Finn P.
Holding
,
Nader
Amin
,
Agnes C. L.
Martin
,
Sharna J.
Rich
,
Valerio
Berdini
,
Jeff
Yon
,
Paul
Acklam
,
Rosemary
Burke
,
Ludovic
Drouin
,
Jenny E.
Harmer
,
Fiona
Jeganathan
,
Rob
Van Montfort
,
Yvette
Newbatt
,
Marcello
Tortorici
,
Maura
Westlake
,
Amy
Wood
,
Swen
Hoelder
,
Tom D.
Heightman
Abstract: The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.
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Nov 2016
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Alan M.
Jones
,
Isaac M.
Westwood
,
James D.
Osborne
,
Thomas P.
Matthews
,
Matthew D.
Cheeseman
,
Martin G.
Rowlands
,
Fiona
Jeganathan
,
Rosemary
Burke
,
Diane
Lee
,
Nadia
Kadi
,
Manjuan
Liu
,
Meirion
Richards
,
Craig
Mcandrew
,
Norhakim
Yahya
,
Sarah
Dobson
,
Keith
Jones
,
Paul
Workman
,
Ian
Collins
,
Rob L. M.
Van Montfort
Open Access
Abstract: The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops.
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Oct 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Matthew D.
Cheeseman
,
Isaac
Westwood
,
Olivier
Barbeau
,
Martin
Rowlands
,
Sarah
Dobson
,
Alan M.
Jones
,
Fiona
Jeganathan
,
Rosemary
Burke
,
Nadia
Kadi
,
Paul
Workman
,
Ian
Collins
,
Rob
Van Montfort
,
Keith
Jones
Open Access
Abstract: HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner.
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May 2016
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I02-Macromolecular Crystallography
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Sylvain
Couty
,
Isaac
Westwood
,
Andrew
Kalusa
,
Celine
Cano
,
Jon
Travers
,
Kathy
Boxall
,
Chiau Ling
Chow
,
Sam
Burns
,
Jessica
Schmitt
,
Lisa
Pickard
,
Caterina
Barillari
,
P. Craig
Mcandrew
,
Paul A.
Clarke
,
Spiros
Linardopoulos
,
Roger J.
Griffin
,
G. Wynne
Aherne
,
Florence I.
Raynaud
,
Paul
Workman
,
Keith
Jones
,
Rob
Van Montfort
Open Access
Abstract: The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases.
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Oct 2013
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8015]
Open Access
Abstract: With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.
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Sep 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[6385]
Open Access
Abstract: Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.
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Sep 2012
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