POTENT AND SELECTIVE-INHIBITION OF HUMAN NITRIC-OXIDE SYNTHASES - SELECTIVE-INHIBITION OF NEURONAL NITRIC-OXIDE SYNTHASE BY S-METHYL-L-THIOCITRULLINE AND S-ETHYL-L-THIOCITRULLINE

Potent and selective inhibition of neuronal nitric oxide synthase (nNO S) compared to endothelial NOS (eNOS) and inducible NOS (iNOS) may be useful to treat cerebral ischemia (stroke) and other neurodegenerative diseases. S-Methyl-L-thiocitrulline (Me-TC) and S-ethyll-thiocitrulli ne (Et-TC) inhibited the oxidation of L-arginine and the L-arginine-in dependent oxidation of NADPH by nNOS from human brain. Me-TC and Et-TC were slow, tight binding inhibitors of nNOS with second-order associa tion rate constants (k(on)) of 2.6 x 10(5) M(-1) s(-1) and 1.3 x 10(5) M(-1) s(-1), respectively. The respective dissociation rate constants (k(off)) were 3 x 10(-4) s(-1) and 0.7 x 10(-4) s(-1). Thus, the K-d values calculated from k(off)/k(on) were 1.2 and 0.5 nM, respectively. L-Arginine was a competitive inhibitor of Me-TC and Et-TC binding wit h competition constant (K-s) values of 2.2 and 2.7 mu M respectively. The K-m of nNOS for L-arginine was 1.6 mu M. The active site concentra tion of nNOS was estimated by titration with Et-TC. Based on this acti ve site concentration, a k(cat) of 0.4 s(-1) for the oxidation of L-ar ginine, was calculated. Me-TC and Et-TC were less potent inhibitors of human iNOS (K-i values of 34 and 17 nM, respectively) and human eNOS (K-i values of 11 and 24 nM). Thus, Me-TC and Et-TC were 10- and 50-fo ld, respectively, more potent inhibitors of nNOS than eNOS. Furthermor e, Me-TC was also 17-fold selective for rat nNOS in neuronal tissue co mpared to rat eNOS in vascular endothelium, suggesting that Me-TC may be selective for nNOS in vivo and therefore, may be therapeutically us eful to treat neurodegenerative diseases.