Efficient PKC inhibitor screening achieved using a quantitative CE‐LIF assay

An assay for protein kinase C delta (PKCδ) activity based on the quantification of a synthetic substrate using capillary electrophoresis with laser‐induced fluorescence detection was developed. The peptides labeled with fluorescein isothiocyanate F‐ERK (where ERK is extracellular signal‐regulated kinase) and the phosphorylated form, P‐F‐ERK, were utilized for the method development and validation. The migration time of F‐ERK and P‐F‐ERK were 6.3 ± 0.1 and 8.7 ± 0.2 min, respectively. LOD and LOQ values of F‐ERK were 2 and 6 ng/mL and those of P‐F‐ERK were 4 and 12 ng/mL. The correlation coefficients obtained from two standard curves were approximately 0.99. The reproducibility and accuracy of the method for F‐ERK ranged 1.5–4.7 and 86–109%, respectively, and those for P‐F‐ERK were 1.6–6.1 and 93–109%, respectively. The activity of PKCδ was studied in vitro using the human gastric cancer cell line MKN‐1. The use of PKCδ inhibitor candidates, including G?6983, bisindolylmaleimide II, staurosporine, and rottlerin in the assay resulted in IC₅₀ values of 50 nM, 15 nM, 795 nM, and 4 μM, respectively. Comparison of our assay with a commercial PKC kit revealed that our assay is more adaptable to differing enzyme isoforms. This method has potential for high throughput screening for kinase inhibitors as part of a drug discovery program.