Selective Inhibition of Neuronal Nitric Oxide Synthase

DESCRIPTION (provided by applicant): Nitric oxide synthase (NOS) is a family of enzymes that produces nitric oxide for use in regulating blood pressure, in immune system functioning, and in brain development, memory, and learning. Under pathological conditions, excess nitric oxide is produced, which can result in damage to tissues, leading to neurodegenerative diseases. The broad objective of inhibition of neuronal nitric oxide synthase is the development of a treatment for neurodegeneration in Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke, and cerebral palsy. Selective inhibition of the neuronal isozyme of NOS is important to prevent hypertension and interference with the immune defense system. The specific aims for the next budget period have been streamlined from the seven proposed in the last version of this proposal to five, which emphasize the design, synthesis, potency, and bioavailability of enhanced nNOS-selective inhibitors. Approaches will be presented to increase potency, selectivity, and their ability to cross the blood-brain barrier. The first specific aim, however, focuses on a mechanistic question related to a class of NOS inactivators. The five areas of proposed research are as follows: (1) Studies will be carried out on the mechanisms of inactivation of nitric oxide synthase by amidine and substituted arginine inactivators. (2) Structure-based de novo design of analogues of our most potent and selective new series of compounds obtained from computer modeling will be carried out and synthesized. (3) Selective nNOS inhibitors for human nNOS will be designed based on homology modeling of human nNOS into the crystal structure of rat nNOS and on site-directed mutagenesis of a key residue difference in rat versus human nNOS. The homology model will be supported by inhibition studies using human and lower animal nitric oxide synthases. Attempts at obtaining a crystal structure of human nitric oxide synthases are being made by a collaborator. (4) Novel subunit dimerization inhibitors of neuronal nitric oxide synthase will be designed and synthesized. (5) Various bioavailability studies will be carried out, including in vivo studies to determine if our selective inhibitors cross the blood-brain barrier; stability studies of the compounds with microsomes; Caco-2 cell permeability studies; substrate activity studies for the P-glycoprotein efflux transporter. Prodrug analogues will be synthesized to increase bioavailability.