Establishment and application of high throughput screening model for kinase insert domain receptor / 中国药学杂志

ABSTRACT

OBJECTIVE: To establish a KDR high throughput screening model with homogeneous time resolved fluorescence(HTRF) detection technology in order to screen small molecular KDR inhibitors from the compound library. METHODS: A 20 μL assay system in 384-well low-volume white microplate was developed with HTRF based fluorescence detection system to determine the enzyme activity. The optimization steps consisted of the following experiments enzyme concentration and incubation time optimiztion, ATP and substrate Km determination, quality control experiments including signal noise ratio inspection, the inhibitor SU5416 IC50 validation, and Z' value calculation. After a stable assay system was accomplished, a HTS campaign was started with 10560 samples irom the compound library and the IC50 of some compounds were determined. RESULTS: The optimized KDR activity assay conditions were as follows the kinase concentration was 0.10 ng · μL-1; ATP Km was 0.75μmol · L-1; substrate Km was 94.90 nmol · L-1; biotin/ SA ratio was 21; Z' value was 0.85 and the IC50 of SU5416 inhibitor was 1.03 μmol · L-1. Three compounds with the ID of S2-14, S2-16, and S2-38 showed IC50 on KDR of 1.01 × 10-4, 6.04 × 10-5, 7.23 × 10-6 mol · L-1, respectively. CONCLUSION: A KDR high throughput screening model has been successfully established with HTRF methodology and a focused HTS campaign has been accomplished with several leads. This assay system is reliable and the results are stable. The established assay system is suitable for further application in screening natural anti-angiogenesis products.