Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).
SLAS Discov
; 22(10): 1193-1202, 2017 12.
Article
de En
| MEDLINE
| ID: mdl-28692323
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.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Protein-Serine-Threonine Kinases
/
Inhibiteurs de protéines kinases
/
Tests de criblage à haut débit
/
Inflammation
Type d'étude:
Diagnostic_studies
/
Screening_studies
Limites:
Humans
Langue:
En
Journal:
SLAS Discov
Année:
2017
Type de document:
Article
Pays d'affiliation:
Royaume-Uni