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Updated: Feb 17, 2026
Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
Published on: October 23, 2019
Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate
Sandra Kümper1, Thorsten Gantke2, Chao-Sheng Chen2
1Crick-GSK Biomedical LinkLabs, GlaxoSmithKline, Stevenage SG1 2NY, U.K.
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.
Insights
Tumour progression locus 2 (TPL-2) regulates TNFα production. Studying the TPL-2 complex, not just the kinase domain, reveals altered inhibitor sensitivities, paving the way for new anti-inflammatory drugs.
Area of Science:
- Biochemistry
- Immunology
- Pharmacology
Background:
- Tumour progression locus 2 (TPL-2) is a kinase crucial for tumour necrosis factor alpha (TNFα) production in innate immunity.
- TPL-2 is a potential anti-inflammatory drug target, but previous inhibitors targeting its isolated kinase domain failed clinical development.
- TPL-2 activity regulates macrophage TNF production via association with NF-κB1 p105 and ABIN-2, independent of MKK1/2 phosphorylation.
Purpose of the Study:
- To determine the substrate specificity of the TPL-2/NF-κB1 p105/ABIN-2 complex.
- To compare the inhibitor sensitivities of the TPL-2 complex versus the isolated TPL-2 kinase domain.
- To explore novel therapeutic strategies for anti-inflammatory drug development targeting TPL-2.
Main Methods:
- Utilized a positional scanning peptide library to define optimal substrate specificity for the TPL-2 complex.
- Employed a high-throughput mass spectrometry assay to monitor kinase activity.
- Screened the TPL-2 complex against existing ATP-competitive TPL-2 inhibitors.
Main Results:
- The TPL-2 complex exhibits significantly altered sensitivities to existing ATP-competitive inhibitors compared to the isolated TPL-2 kinase domain.
- The study identified an optimal peptide substrate for the TPL-2 complex.
- These findings suggest a more physiologically relevant approach to TPL-2 inhibitor screening.
Conclusions:
- Screening the TPL-2/NF-κB1 p105/ABIN-2 complex can identify novel chemical series for TPL-2 inhibitors.
- Both ATP-competitive and allosteric inhibitors targeting the complex show promise for anti-inflammatory drug development.
- This approach may overcome limitations of previous TPL-2 inhibitor development efforts.
