I04-Macromolecular Crystallography
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Irene
Georgiou
,
Colin
Robinson
,
Sean N.
O'Byrne
,
Alex
Matsuda
,
Przemysław
Grygier
,
Craig D.
Smith
,
Sandra
O'Neill
,
Shamshad A.
Ahmad
,
Suzanne
Norval
,
John M.
Post
,
Mirjam
Groenewold
,
Nadya
Urakova
,
Patrick
Wanningen
,
Leanid
Kresik
,
Jacek
Plewka
,
Adrien
Delpal
,
Kexin
See
,
Thomas
Eadsforth
,
Kinga
Wierzbicka
,
Etienne
Decroly
,
Kumar Singh
Saikatendu
,
Edcon
Chang
,
Eric J.
Snijder
,
Krzysztof
Pyrć
,
Anna
Czarna
,
Duncan E.
Scott
,
Ian H.
Gilbert
Diamond Proposal Number(s):
[31671]
Abstract: SARS-CoV-2 non-structural protein 14 (nsp14) is essential for viral mRNA cap guanine-N7 methylation and represents a promising but underexplored antiviral target. Herein we describe a structure-guided campaign based on a hit from a focussed SAM mimetic library. Systematic SAR exploration guided by six X-ray co-crystal structures in complex with SARS-CoV-2 led to compound 26, a bi-substrate inhibitor that bridges the SAM and RNA cap binding sites. Compound 26 achieved nanomolar potency against nsp14 from SARS-CoV-2 (IC50 = 53 nM), SARS-CoV-1, and two alphacoronaviruses, with excellent selectivity over human RNMT and flaviviral MTase. In general, the compounds demonstrated favourable metabolic stability, passive permeability, and no HepG2 cytotoxicity. However, cellular antiviral activity was limited, revealing disconnects between enzyme inhibition and phenotypic response. These findings provide a structural framework for optimizing bi-substrate methyltransferase inhibitors against coronaviruses with a view for pan-coronaviral activity.
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Jan 2026
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Simon R.
Stockwell
,
Duncan E.
Scott
,
Gerhard
Fischer
,
Estrella
Guarino
,
Timothy P. C.
Rooney
,
Tzu-Shean
Feng
,
Tommaso
Moschetti
,
Rajavel
Srinivasan
,
Esther
Alza
,
Alice
Asteian
,
Claudio
Dagostin
,
Anna
Alcaide
,
Mathieu
Rocaboy
,
Beata
Blaszczyk
,
Alicia
Higueruelo
,
Xuelu
Wang
,
Maxim
Rossmann
,
Trevor R.
Perrior
,
Tom L.
Blundell
,
David R.
Spring
,
Grahame
Mckenzie
,
Chris
Abell
,
John
Skidmore
,
Ashok R.
Venkitaraman
,
Marko
Hyvonen
Diamond Proposal Number(s):
[9537, 14043]
Open Access
Abstract: Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein–protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.
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Aug 2024
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I03-Macromolecular Crystallography
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Timothy P. C.
Rooney
,
Gregory G.
Aldred
,
Helen K.
Boffey
,
Henriëtte M. G.
Willems
,
Simon
Edwards
,
Stephen J.
Chawner
,
Duncan E.
Scott
,
Christopher
Green
,
David
Winpenny
,
John
Skidmore
,
Jonathan H.
Clarke
,
Stephen P.
Andrews
Diamond Proposal Number(s):
[20026]
Open Access
Abstract: Owing to their central role in regulating cell signaling pathways, the phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive therapeutic targets in diseases such as cancer, neurodegeneration, and immunological disorders. Until now, tool molecules for these kinases have been either limited in potency or isoform selectivity, which has hampered further investigation of biology and drug development. Herein we describe the virtual screening workflow which identified a series of thienylpyrimidines as PI5P4Kγ-selective inhibitors, as well as the medicinal chemistry optimization of this chemotype, to provide potent and selective tool molecules for further use. In vivo pharmacokinetics data are presented for exemplar tool molecules, along with an X-ray structure for ARUK2001607 (15) in complex with PI5P4Kγ, along with its selectivity data against >150 kinases and a Cerep safety panel.
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Dec 2022
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Helen K.
Boffey
,
Timothy P. C.
Rooney
,
Henriette M. G.
Willems
,
Simon
Edwards
,
Christopher
Green
,
Tina
Howard
,
Derek
Ogg
,
Tamara
Romero
,
Duncan E.
Scott
,
David
Winpenny
,
James
Duce
,
John
Skidmore
,
Jonathan H.
Clarke
,
Stephen P.
Andrews
Diamond Proposal Number(s):
[29347, 20026]
Open Access
Abstract: Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 (1). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kβ. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.
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Feb 2022
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Duncan E.
Scott
,
Nicola J.
Francis-Newton
,
May E.
Marsh
,
Anthony G.
Coyne
,
Gerhard
Fischer
,
Tommaso
Moschetti
,
Andrew R.
Bayly
,
Timothy D.
Sharpe
,
Kalina T.
Haas
,
Lorraine
Barber
,
Chiara R.
Valenzano
,
Rajavel
Srinivasan
,
David J.
Huggins
,
Miyoung
Lee
,
Amy
Emery
,
Bryn
Hardwick
,
Matthias
Ehebauer
,
Claudio
Dagostin
,
Alessandro
Esposito
,
Luca
Pellegrini
,
Trevor
Perrior
,
Grahame
Mckenzie
,
Tom L.
Blundell
,
Marko
Hyvonen
,
John
Skidmore
,
Ashok R.
Venkitaraman
,
Chris
Abell
Diamond Proposal Number(s):
[315, 7141]
Open Access
Abstract: BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
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Mar 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Tommaso
Moschetti
,
Timothy
Sharpe
,
Gerhard
Fischer
,
May E.
Marsh
,
Hong Kin
Ng
,
Matthew
Morgan
,
Duncan E.
Scott
,
Tom L.
Blundell
,
Ashok R.
Venkitaraman
,
John
Skidmore
,
Chris
Abell
,
Marko
Hyvonen
Diamond Proposal Number(s):
[315, 6889, 7141, 9007]
Open Access
Abstract: Protein–protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to “humanise” thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.
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Nov 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[315]
Open Access
Abstract: RAD51 is a recombinase involved in the homologous recombination of double strand breaks in DNA. RAD51 forms oligomers by binding to another molecule of RAD51 via an “FxxA” motif, and the same recognition sequence is similarly utilized to bind BRCA2. We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic. We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X-ray crystallography. Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed, and the importance of secondary structure in interpreting alanine-scanning and mutational data of this nature is considered.
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Mar 2016
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[315]
Open Access
Abstract: Homologous recombination is essential for repair of DNA double–strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single–stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single–stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric P. furiosus RadA and monomerised Human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that whilst phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.
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Mar 2016
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[315]
Open Access
Abstract: The ability to identify inhibitors of protein–protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment-based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment-based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X-ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein–protein interaction site.
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Jan 2013
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I03-Macromolecular Crystallography
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Abstract: A new strategy that combines the concepts of fragment-based drug design and dynamic combinatorial chemistry (DCC) for targeting adenosine recognition sites on enzymes is reported. We demonstrate the use of 5?-deoxy-5?-thioadenosine as a noncovalent anchor fragment in dynamic combinatorial libraries templated by Mycobacterium tuberculosis pantothenate synthetase. A benzyl disulfide derivative was identified upon library analysis by HPLC. Structural and binding studies of protein–ligand complexes by X-ray crystallography and isothermal titration calorimetry informed the subsequent optimisation of the DCC hit into a disulfide containing the novel meta-nitrobenzyl fragment that targets the pantoate binding site of pantothenate synthetase. Given the prevalence of adenosine-recognition motifs in enzymes, our results provide a proof-of-concept for using this strategy to probe adjacent pockets for a range of adenosine binding enzymes, including other related adenylate-forming ligases, kinases, and ATPases, as well as NAD(P)(H), CoA and FAD(H2) binding proteins.
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Oct 2009
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