This proposal is a competing renewal application that concerns the regulation, development of synthetic inhibitors, and catalytic mechanism of serotonin N-acetyltransferase, the melatonin rhythm enzyme. Melatonin is circadian rhythm hormone produced in the pineal gland that is hypothesized to contribute to the regulation of sleep-wake cycle, mood, and the process of aging. Precise details about the role of melatonin in physiology and pathophysiology are still lacking, in part because of a dearth of pharmacologic tools. The biosynthesis of melatonin from the neurotransmitter serotonin involves two enzymatic steps, the first of which is catalyzed by serotonin N-acetyltransferase. The diurnal and light-sensitive fluctuation in serotonin Nacetyltransferase drives the rise and fall of melatonin production, the level of which is highest at night. This proposal has three principal goals. First, we plan to develop and characterize a novel class of potent pantetheinyl-prodrug inhibitors of serotonin N-acetyltransferase that promise to be more potent and less toxic than first generation inhibitors. Second, we will elucidate the impact of phosphorylation of serotonin N-acetyltransferase on the cellular stability of the enzyme. Serotonin N-acetyltransferase is known to be phosphorylated on both its N-and C-termini in response to light, but the complete significance of each of these phosphorylation events is not yet known. By incorporating non-hydrolyzable phosphono amino acid analogs in place of phosphothreonine and phosphoserine into serotonin N-acetyltransferase, the precise role of phosphorylation should be revealed. Third, we will clarify the effects of 14-3-3 protein recruitment on the catalytic properties of serotonin N-acetyltransferase. Since the predominant in vivo form of serotonin Nacetyltransferase is probably complexed with 14-3-3, these studies will permit characterization of the physiologically relevant form of this enzyme. It is expected that these studies will lead to an increase in our understanding about the role of melatonin in health and disease and that newly designed inhibitors may serve as lead agents for the treatment of sleep and mood disorders.
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