Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs)
| Autor: | Lesley-Anne Pearson, David W. Gray, Rachel E. Heap, Anthony G. Hope, Kathleen M. S. E. Reyskens, C. James Hastie, J. Simon C. Arthur, Matthias Trost, Stuart P. McElroy, David W. Porter |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
kinase interleukin-10 Inflammation Peptide macrophage Biology Protein Serine-Threonine Kinases Mass spectrometry 01 natural sciences Biochemistry Analytical Chemistry drug discovery 03 medical and health sciences salt inducible kinases medicine Humans high-throughput screen IC50 Protein Kinase Inhibitors Original Research mass spectrometry chemistry.chemical_classification Cell Nucleus Drug discovery Kinase Cellular Assay Molecular biology In vitro 0104 chemical sciences 3. Good health High-Throughput Screening Assays 010404 medicinal & biomolecular chemistry Protein Transport 030104 developmental biology chemistry MALDI TOF Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Molecular Medicine medicine.symptom Biotechnology |
| Zdroj: | Slas Discovery |
| ISSN: | 2472-5560 |
| Popis: | Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays. |
| Databáze: | OpenAIRE |
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