title: Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay.
abstract: 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 Gӧ6983, bisindolylmaleimide II, staurosporine, and rottlerin in the assay resulted in IC50values 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.
authors: Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea.|Department of Biological Chemistry, University of Science and Technology, Daejeon, Korea.
authors: Binh Thanh
authors: BT
authors: Nguyen
authors: Deparment of Materials Science and Engineering, Hallym University, Seoul, Korea.
authors: Min
authors: M
authors: Park
authors: Department of Materials and Sciences, Yonsei University, Seoul, Korea.
authors: Jae-Chul
authors: JC
authors: Pyun
authors: Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea.
authors: Young Sook
authors: YS
authors: Yoo
authors: Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea.|Department of Biological Chemistry, University of Science and Technology, Daejeon, Korea.
authors: Min-Jung
authors: MJ
authors: Kang
mesh_terms: D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors / Q000032:analysis*; D015203:Reproducibility of Results
chemical_list: D047428:Protein Kinase Inhibitors; D011493:Protein Kinase C; D016650:Fluorescein-5-isothiocyanate
keywords: CE-LIF; Kinase; Protein Kinase C; Screening of kinase inhibitor