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[High throughput screening method of nitric oxide synthase inhibitors and enhancers]
Mian-en Sun1, Yong-hong Chen, Guan-hua Du
1National Center of Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100050, China.
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 microL. 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 degrees C. 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.