title: A Dual Readout Assay Based on Fluorescence Polarization and Time-Resolved Fluorescence Resonance Energy Transfer to Screen for RSK1 Inhibitors.
abstract: A dual readout assay based on fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) exhibits many advantages over single assay technology in terms of screening quality and efficiency. In this study, we developed a dual readout assay combining FP and TR-FRET to identify ribosomal S6 kinase 1 (RSK1) inhibitors. This dual readout assay can monitor both FP and TR-FRET signals from a single RSK1 kinase reaction by using the immobilized metal affinity for phosphochemical (IMAP)-based assay. The Z' value and signal to background (S/B) ratio were 0.85 and 4.0 using FP, and 0.79 and 10.6 using TR-FRET, which led to performance of a pilot library screening against the drug repositioning set consisting of 2320 compounds with a reasonable reproducibility. From this screening, we identified 16 compounds showing greater than 50% inhibition against RSK1 for both FP and TR-FRET; 6 compounds with greater than 50% inhibition only for FP; and 4 compounds with greater than 50% inhibition only for TR-FRET. In a cell-based functional assay to validate the hit compounds, 10 compounds identified only in a single assay had little effect on the RSK-mediated phosphorylation of liver kinase B1, whereas 5 compounds showing greater than 80% inhibition for both FP and TR-FRET reduced the phosphorylation of liver kinase B1. These results demonstrate that the dual readout assay can be used to identify hit compounds by subsequently monitoring both FP and TR-FRET signals from one RSK1 reaction.
authors.affiliation: Bio-Organic Science Division, Korea Research Institute of Chemical Technology.
authors.forename: Eun-mi
authors.initials: EM
authors.lastname: Jeong
authors.forename: Mi Young
authors.initials: MY
authors.lastname: Lee
authors.forename: Jeong Hyun
authors.initials: JH
authors.lastname: Lee
authors.forename: Byung Ho
authors.initials: BH
authors.lastname: Lee
authors.forename: Kwang-Seok
authors.initials: KS
authors.lastname: Oh
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001681:Biological Assay; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa