Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs)

Rachel E. Heap, Anthony G. Hope, Lesley-Anne Pearson, Kathleen M. S. E. Reyskens, Stuart P. McElroy, C. James Hastie, David W. Porter, J. Simon C. Arthur, David W. Gray, Matthias Trost (Lead / Corresponding author)

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Abstract

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.

Original languageEnglish
Pages (from-to)1193-1202
Number of pages10
JournalSLAS Discovery
Volume22
Issue number10
Early online date10 Jul 2017
DOIs
Publication statusPublished - 1 Dec 2017

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Keywords

  • Journal article
  • MALDI TOF
  • Mass spectrometry
  • Salt inducible kinases
  • Kinase
  • High-throughput screen
  • Inflammation
  • Drug discovery
  • Macrophage
  • Interleukin-10

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