Development of a novel fluorescent nitric oxide synthase assay

Abstract

Many therapeutic indications exist for nitric oxide synthase (NOS) making it a target of interest for drug discovery. The NOS family of enzymes, which include inducible NOS, endothelial NOS, and neuronal NOS, regulate functions such as cell communication, immune defense, and vasodilation. NOS enzymes convert L‐arginine and NADPH to L‐citrulline and nitric oxide (NO). NO is the agent responsible for the downstream physiological effects of NOS. In the absence of any downstream targets, NO is rapidly converted to nitrite in in vitro assays.Several NOS assays exist, but lack the sensitivity required for HTS‐compatible formats. This poster describes a new NOS assay using a fluorescent detection system. The detection system utilizes 4,5 Diaminofluorescein (DAF2) in which the probe reacts with nitrite under acidic conditions. Initial concentrations of various assay components were selected based on each component's individual effect on the detection system. The assay was further optimized based on the overall effect of all components on both the enzyme reaction and the detection system. Several known NOS inhibitors were evaluated under the final assay conditions resulting in a validated, robust HTS‐compatible assay.