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Biochem J. 2018 Jan 11;475(1):329-340. doi: 10.1042/BCJ20170579.

Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate.

Author information

1
Crick-GSK Biomedical LinkLabs, GlaxoSmithKline, Stevenage SG1 2NY, U.K.
2
Immune Cell Signalling Laboratory, The Francis Crick Institute, London NW1 1AT, U.K.
3
Bioinformatics and Biostatistics, The Francis Crick Institute, London NW1 1AT, U.K.
4
Structural Biology, Science Technology Platforms, The Francis Crick Institute, London NW1 1AT, U.K.
5
RD Platform Technology & Science, GlaxoSmithKline, Stevenage SG1 2NY, U.K.
6
Immune Cell Signalling Laboratory, The Francis Crick Institute, London NW1 1AT, U.K. steve.ley@crick.ac.uk.

Abstract

The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.

KEYWORDS:

TPL-2; high-throughput assay; inflammation; kinase

PMID:
29229763
PMCID:
PMC5763956
DOI:
10.1042/BCJ20170579
[Indexed for MEDLINE]
Free PMC Article

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