Labchip system

(1) 5µL of test sample solution, 5 µL of substrate/ATP/metal solution in assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH 7.5), and 10 µL of kinase solution in assay buffer prepared in assay buffer were mixed in the wells of a 384-well polypropylene plate and allowed to react for 5 h at room temperature. (2) The reaction was stopped by adding 70 µL of Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences). (3) Substrate peptides and phosphorylated peptides in the reaction solution were separated and quantified using the LabChip system (Perkin Elmer, Shelton, CT, USA). (4) The product ratio (P/(P+S)) was calculated from the substrate peptide peak height (S) and the phosphorylated peptide peak height (P) of the kinase reaction.

Kinase activity was measured by Off-chip Mobility Shift Assay in Carna Biosciences. The 4× substrate/ATP/metal solution was prepared with kit buffer (20 mM HEPES, 0.01% Triton X-100, 5 mM DTT, pH 7.5), and 2× kinase solution was prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 1 mM DTT, pH 7.5). The 5 μl of 4× compound solution, 5 ml of 4× substrate/ATP/metal solution, and 10 ml of 2× kinase solution were mixed and incubated in a well of a polypropylene 384 well microplate for 1 h at RT. Then, 70 ml of termination buffer (QuickScout Screening Assist MSA; Carna Biosciences) was added to the well. The reaction mixture was applied to LabChip™ system (Perkin Elmer), and the product and substrate peptide peaks were separated and quantitated. The kinase reaction was evaluated by the product ratio calculated from peak heights of product (P) and substrate (S) peptides (P/(P+S)).

Cellular kinase activities were evaluated using nonradioisotopic methods such as the off‐chip mobility shift assay or IMAP (Carna Biosciences, Kobe, Japan).11 A kinase inhibition profiling panel was produced on the basis of the kinase inhibition rates. In off‐chip mobility shift assay, compound solution was prepared in assay buffer (20 mmol/L HEPES, 0.01% Triton X‐100, 2 mmol/L DTT, pH 7.5) and incubated in a 384‐well plate at room temperature. The reaction was stopped by adding Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences, Kobe, Japan). The substrate peptide and the phosphorylated peptide in the reaction solution were separated and quantified by LabChip system (Perkin Elmer, MA, USA). In IMAP assay, compound solution was prepared in assay buffer (20 mmol/L HEPES, 0.01% Tween‐20, 2 mmol/L DTT, pH 7.4) and incubated in a 384‐well black plate at room temperature. IMAP binding reagent (IMAP Screening Express kit; Molecular Devices, CA, USA) was added and incubated for 30 minutes. The kinase reaction was evaluated at the fluorescence polarization.