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.
Reversed-phase high-performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors.
MACCALLINI, Cristina;DI MATTEO, MAURO;AMMAZZALORSO, Alessandra;D'ANGELO, ALESSANDRA;DE FILIPPIS, Barbara;DI SILVESTRE, SARA;FANTACUZZI, MARIALUIGIA;GIAMPIETRO, Letizia;PANDOLFI, Assunta;AMOROSO, Rosa
2014-01-01
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.