Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas.

S-Ethylisothiourea was a potent competitive inhibitor of human nitric oxide synthase (NOS), with Ki values of 17, 36, and 29 nM for the inducible (i), endothelial (e), and neuronal (n) isozymes, respectively. Unlike some potent inhibitors of NOS, no time dependence was observed. S-Ethylisothiourea was not a detectable substrate for eNOS. S-Ethylisothiourea was also a potent inhibitor of mouse iNOS (Ki value of 5.2 nM), and its binding perturbed the spectrum of iNOS consistent with its altering the environment of the bound heme. The optimum binding of S-ethyl- and S-isopropylisothiourea relative to 70 other analogs suggested that these alkyl substitutions fit into a small hydrophobic pocket. Most isothioureas were 2-6-fold selective for the human iNOS (Ki for iNOS versus Ki for eNOS), with one being 19-fold selective. The cyclized mimics of S-ethylisothiourea, 2-NH2-thiazoline, and 2-NH2-thiazole, were also competitive inhibitors of human NOS. A third structural class of inhibitors, bisisothioureas, were, in general, the most selective in their inhibition of human iNOS. S,S'-(1,3-Phenylenebis(1,2-ethanediyl))bisisothiourea was 190-fold selective (Ki value of 0.047 microM against iNOS versus 9.0 microM against eNOS). These results demonstrate that potent and selective inhibition of human NOS isozymes is achievable.

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 (nNOS) 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-ethyl-L-thiocitrulline (Et-TC) inhibited the oxidation of L-arginine and the L-arginine-independent oxidation of NADPH by nNOS from human brain. Me-TC and Et-TC were slow, tight binding inhibitors of nNOS with second-order association rate constants (kon) 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 (koff) were 3 x 10(-4) s-1 and 0.7 x 10(-4) s-1. Thus, the Kd values calculated from koff/kon were 1.2 and 0.5 nM, respectively. L-Arginine was a competitive inhibitor of Me-TC and Et-TC binding with competition constant (Ks) values of 2.2 and 2.7 microM, respectively. The Km of nNOS for L-arginine was 1.6 microM. The active site concentration of nNOS was estimated by titration with Et-TC. Based on this active site concentration, a kcat of 0.4 s-1 for the oxidation of L-arginine, was calculated. Me-TC and Et-TC were less potent inhibitors of human iNOS (Ki values of 34 and 17 nM, respectively) and human eNOS (Ki values of 11 and 24 nM). Thus, Me-TC and Et-TC were 10- and 50-fold, respectively, more potent inhibitors of nNOS than eNOS. Furthermore, Me-TC was also 17-fold selective for rat nNOS in neuronal tissue compared to rat eNOS in vascular endothelium, suggesting that Me-TC may be selective for nNOS in vivo and therefore, may be therapeutically useful to treat neurodegenerative diseases.

Selective inhibition of constitutive nitric oxide synthase by L-NG-nitroarginine.

L-NG-Nitroarginine (NA) inhibited both the L-arginine oxidation and the L-arginine-independent NADPH oxidation reactions catalyzed by the calcium/calmodulin-dependent constitutive nitric oxide synthase (cNOS) from bovine brain. NA binding did not require calmodulin, calcium, or NADPH. The onset of inhibition was slow with a second-order association rate constant (k(on) of 4.4 x 10(4) M-1 s-1. The dissociation rate constant (k(off) was 6.5 x 10(-4) s-1. The Kd value (k(off)/k(on)) of bovine brain cNOS for NA was 15 nM. L-Arginine was a competitive inhibitor of NA binding with a Ks value of 0.8 microM. The Km for L-arginine in the cNOS reaction was 1.2 microM. The NA binding sites of cNOS were titrated with NA, which enabled a kcat of 0.7 s-1, for the oxidation of L-arginine, to be calculated. Finally, a brain cNOS-(3H)NA complex was isolated. In contrast to the potent and slow onset of NA inhibition of brain cNOS, NA inhibition of inducible mouse macrophage NOS (iNOS) was weaker (Ki = 4.4 microM) and rapidly reversible. Thus, NA was a 300-fold more potent inhibitor of bovine brain cNOS than mouse macrophage iNOS.