Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate
journal contributionposted on 2020-08-27, 15:19 authored by Sandra Kümper, Thorsten Gantke, Chao-Sheng Chen, Yasmina Soneji, Michael J Pattison, Probir Chakravarty, Svend Kjær, Daniel Thomas, Carl Haslam, Bill J Leavens, David House, David J Powell, Steven C Ley
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.
TPL-2high-throughput assayinflammationkinaseAdaptor Proteins, Signal TransducingAmino Acid SequenceAnti-Inflammatory AgentsGene ExpressionHEK293 CellsHigh-Throughput Screening AssaysHumansMAP Kinase Kinase KinasesNF-kappa B p50 SubunitPeptide LibraryPeptidesProtein BindingProtein Kinase InhibitorsProto-Oncogene ProteinsRecombinant ProteinsStructure-Activity RelationshipSubstrate SpecificityLey FC001103CBSBGSK06 Biological Sciences11 Medical and Health Sciences03 Chemical SciencesBiochemistry & Molecular Biology