Anthony Shillings

GlaxoSmithKline (United Kingdom)

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Andrew Fosberry

GlaxoSmithKline (United Kingdom)

B.D. Bax

Cardiff University

Pan F. Chan

GlaxoSmithKline (United States)

Peter D. Craggs

GlaxoSmithKline (United Kingdom)

Alexandre Wohlkönig

VIB-VUB Center for Structural Biology

Neil D. Pearson

South College

Sue Hutchinson

GlaxoSmithKline (United Kingdom)

Rab K. Prinjha

GlaxoSmithKline (United Kingdom)

Ryan P. Bingham

GlaxoSmithKline (United Kingdom)

David F. Tough

GlaxoSmithKline (United Kingdom)

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GlaxoSmithKline (United Kingdom)

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GlaxoSmithKline (United States)

Molecular Discovery (United Kingdom)

Age UK

South College

Research Triangle Park Foundation

GlaxoSmithKline (Spain)

GlaxoSmithKline (Netherlands)

University College London

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Additional file 13 of Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

Figshare (2022)

Mohammed Ghiboub Jan Köster Peter D. Craggs Andrew Y. F. Li Yim Anthony Shillings

Additional file 13: Table S13. The normalized read count for ChIP-seq (GSK761-pretreated “M1” macrophages stimulated with 4h LPS (100 ng/mL)).

Modulation (music)

10.6084/m9.figshare.20521105

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The 2.98A crystal structure of the catalytic core (B'A' region) of Staphylococcus aureus DNA Gyrase

B.D. Bax Pan F. Chan Drake S. Eggleston Andrew Fosberry Daniel R. Gentry

10.2210/pdb2xco/pdb

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CRYSTAL STRUCTURE OF MOXIFLOXACIN, DNA, and A. BAUMANNII TOPO IV (PARE-PARC FUSION TRUNCATE)

Alexandre Wohlkönig Pan F. Chan Andrew Fosberry Paul Homes Ji Huang

10.2210/pdb2xkk/pdb

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Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Molecular Therapy — Methods & Clinical Development (2020)

Yu Hua Chen Celeste Pallant Christopher Sampson Alessia Boiti Sabine Johnson

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

HEK 293 cells

Transduction (biophysics)

10.1016/j.omtm.2020.08.011

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Citations (33)

Crystal structures of ASK1‐inhibtor complexes provide a platform for structure‐based drug design

Protein Science (2013)

Onkar Singh Anthony Shillings Peter D. Craggs Ian D. Wall Paul Rowland

ASK1, a member of the MAPK Kinase Kinase family of proteins has been shown to play a key role in cancer, neurodegeneration and cardiovascular diseases and is emerging as a possible drug target. Here we describe a 'replacement-soaking' method that has enabled the high-throughput X-ray structure determination of ASK1/ligand complexes. Comparison of the X-ray structures of five ASK1/ligand complexes from 3 different chemotypes illustrates that the ASK1 ATP binding site is able to accommodate a range of chemical diversity and different binding modes. The replacement-soaking system is also able to tolerate some protein flexibility. This crystal system provides a robust platform for ASK1/ligand structure determination and future structure based drug design.

ASK1

10.1002/pro.2298

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Citations (25)

Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry

SLAS DISCOVERY (2011)

Sue Hutchinson Melanie Leveridge Michelle L. Heathcote Peter Francis Laura Williams

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

High-Throughput Screening

10.1177/1087057111416660

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Citations (94)

Additional file 11 of Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

Figshare (2022)

Mohammed Ghiboub Jan Köster Peter D. Craggs Andrew Y. F. Li Yim Anthony Shillings

Additional file 11: Table 11. The normalized read count for ChIP-seq (GSK761-pretreated “M1” macrophages).

Modulation (music)

10.6084/m9.figshare.20521099

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Identification of KasA as the cellular target of an anti-tubercular scaffold

Nature Communications (2016)

Katherine A. Abrahams Chun-wa Chung Sonja Ghidelli-Disse Joaquín Rullás María José Rebollo-López

Abstract Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase ( kas ) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.

10.1038/ncomms12581

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Citations (83)

Lead Discovery for Microsomal Prostaglandin E Synthase Using a Combination of High-Throughput Fluorescent-Based Assays and RapidFire Mass Spectrometry

SLAS DISCOVERY (2012)

Melanie Leveridge Ana I. Bardera William A. LaMarr Andrew Billinton Benjamin R. Bellenie

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.

High-Throughput Screening

10.1177/1087057111435700

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Citations (23)

Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance

Nature Structural & Molecular Biology (2010)

Alexandre Wohlkönig Pan F. Chan Andrew Fosberry Paul Homes Jianzhong Huang

Quinolone

Topoisomerase IV

Cleavage (geology)

10.1038/nsmb.1892

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Citations (278)