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

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


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.

MeSH terms

  • Adaptor Proteins, Signal Transducing / antagonists & inhibitors*
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • Anti-Inflammatory Agents / chemical synthesis
  • Anti-Inflammatory Agents / pharmacology*
  • Gene Expression
  • HEK293 Cells
  • High-Throughput Screening Assays
  • Humans
  • MAP Kinase Kinase Kinases / antagonists & inhibitors*
  • MAP Kinase Kinase Kinases / genetics
  • MAP Kinase Kinase Kinases / metabolism
  • NF-kappa B p50 Subunit / antagonists & inhibitors*
  • NF-kappa B p50 Subunit / genetics
  • NF-kappa B p50 Subunit / metabolism
  • Peptide Library
  • Peptides / chemical synthesis
  • Peptides / pharmacology*
  • Protein Binding
  • Protein Kinase Inhibitors / chemical synthesis
  • Protein Kinase Inhibitors / pharmacology*
  • Proto-Oncogene Proteins / antagonists & inhibitors*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity


  • Adaptor Proteins, Signal Transducing
  • Anti-Inflammatory Agents
  • NF-kappa B p50 Subunit
  • Peptide Library
  • Peptides
  • Protein Kinase Inhibitors
  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • TNIP2 protein, human
  • MAP Kinase Kinase Kinases
  • MAP3K8 protein, human