title: Reversed-phase high-performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors.
abstract: Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of L-[(3)H]-citrulline produced from L-[(3)H]-arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed-phase high-performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of L-citrulline with o-phthaldialdehyde/N-acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N-[3-(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well-established radiometric assay.
authors.affiliation: Dipartimento di Farmacia, Università degli Studi "G. d'Annunzio", Chieti, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Maccallini
authors.forename: Mauro
authors.initials: M
authors.lastname: Di Matteo
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ammazzalorso
authors.forename: Alessandra
authors.initials: A
authors.lastname: D'Angelo
authors.forename: Barbara
authors.initials: B
authors.lastname: De Filippis
authors.forename: Sara
authors.initials: S
authors.lastname: Di Silvestre
authors.forename: Marialuigia
authors.initials: M
authors.lastname: Fantacuzzi
authors.forename: Letizia
authors.initials: L
authors.lastname: Giampietro
authors.forename: Assunta
authors.initials: A
authors.lastname: Pandolfi
authors.forename: Rosa
authors.initials: R
authors.lastname: Amoroso
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D052247:Nitric Oxide Synthase Type II
keywords: Fluorescence detection; High-performance liquid chromatography; Inhibitors; Nitric oxide synthases; l-Citrulline