一氧化氮合酶抑制剂和增强剂的高通量筛选

HIGH THROUGHPUT SCREENING METHOD OF NITRIC OXIDE SYNTHASE INHIBITORS AND ENHANCERS

目的 建立一氧化氮合酶 (NOS)活性的高通量检测方法 ,筛选调节NOS活性的药物。方法 通过NADPH荧光值的变化 ,间接反映NOS活性。通过对反应体系的优化 ,调整各反应底物 (NADPH ,L Arg ,NOS)及抑制剂 (L NNA)的浓度 ,建立高通量筛选模型并对 5 6 0 0个样品进行筛选。结果 方法简便、容易操作、灵敏度高、结果稳定 ,发现了一批对NOS具有抑制或增强作用的化合物。结论 此法适用于高通量药物筛选。

AIM In order to discover new inhibitors and enhancers of nitric oxide synthase (NOS), an in vitro assay to determine NOS activity was established for high throughput screening. METHODS The activity of NOS was detected based on the change of nicotinamide-adenine dinucleotide phosphate (NADPH) concentration in the reaction system by the fluorescence density. The enzyme was prepared from bovine brain by gradient centrifugation. The reaction performed in black 96 well micro-plate with a final volume of 90 μL. Every factor which would affect the results such as the concentration of NADPH, L -arginine ( L -Arg, used as substrate) and enzyme protein was optimized in different conditions. At last, 5 600 samples (compounds and extracts) were screened by the method. RESULTS The test signal (fluorescence density) in the reaction system was influenced by many different factors such as temperature and concentration of substrates. The ideal system contains protein 1 50 mg·mL -1 , L -Arg 1 mmol·L -1 , NADPH 0 1 mmol·L -1 at 37℃. In this method, there were about 2% samples which emit fluorescence, and about 0 5% samples which quench the fluorescence. So these samples were deleted from the sample library. The effects of these samples on activity of NOS were distributed in a normal manner. About 2% samples had potential effects on the NOS activity (including inhibitors and enhancers). CONCLUSION The method can be performed by high throughput screening and gives the stable data, not only for inhibitors, but also for enhancers of NOS activity.