I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
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.
|
May 2019
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
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.
|
Sep 2018
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14891, 6385, 8015, 10088]
Abstract: TLE1 is an oncogenic transcriptional co-repressor that exerts its repressive effects through binding of transcription factors. Inhibition of this protein-protein interaction represents a putative cancer target but no small molecule inhibitors have been published for this challenging interface. In this manuscript, we report the structure enabled design and synthesis of a constrained peptide inhibitor of TLE1. Our design featured introduction of a four carbon atom linker into the peptide epitope found in many TLE1 binding partners. We developed a concise synthetic route to a proof of concept peptide cycFWRPW. Biophysical testing by ITC and thermal shift assays showed that whilst the constrained peptide bound potently, it had an approximately five fold higher Kd than the unconstrained peptide. Our co-crystal structure suggested that the reduced affinity is likely due to a small shift of one side-chain compared to the otherwise well conserved conformation of the acyclic peptide. Our work describes a constrained peptide inhibitor that may serve as the basis for improved inhibitors.
|
Mar 2017
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Nov 2016
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
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.
|
Oct 2016
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
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
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.
|
May 2016
|
|
I02-Macromolecular Crystallography
|
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.
|
Oct 2013
|
|
I04-Macromolecular Crystallography
|
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.
|
Sep 2013
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
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.
|
Sep 2012
|
|
I03-Macromolecular Crystallography
|
Abstract: The Nek family of serine/threonine kinases regulates centrosome and cilia function; in addition, several of its members are potential targets for drug discovery. Nek2 is dimeric, is cell cycle regulated and functions in the separation of centrosomes at G2/M. Here, we report the crystal structures of wild-type human Nek2 kinase domain bound to ADP at 1.55-Å resolution and T175A mutant in apo form as well as that bound to a non-hydrolyzable ATP analog. These show that regions of the Nek2 structure around the nucleotide-binding site can adopt several different but well-defined conformations. None of the conformations was the same as that observed for the previously reported inhibitor-bound structure, and the two nucleotides stabilized two conformations. The structures suggest mechanisms for the auto-inhibition of Nek2 that we have tested by mutagenesis. Comparison of the structures with Aurora-A and Cdk2 gives insight into the structural mechanism of Nek2 activation. The production of specific inhibitors that target individual kinases of the human genome is an urgent challenge in drug discovery, and Nek2 is especially promising as a cancer target. We not only identify potential challenges to the task of producing Nek2 inhibitors but also propose that the conformational variability provides an opportunity for the design of Nek2 selective inhibitors because one of the conformations may provide a unique target.
|
Feb 2009
|
|
