Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay
- Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay
- 유영숙; 강민정; Hafeza Akter; 박민; 변재철
- CE-LIF; Kinase; Protein Kinase C; Screening of kinase inhibitor
- Issue Date
- VOL 37-3153
- 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 Gӧ 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.
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