Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole.
7-Nitro-indazole (7-NI) has been described as novel nitric oxide synthase (NOS) inhibitor with in vivo selectivity for the neuronal isozyme [Moore et al. Br. J. Phaarmac. 110, 219-224 (1993)]. In the present study we have used purified porcine brain NOS to investigate the molecular mechanisms of enzyme inhibition by 7-NI. The drug was competitive with L-arginine, exhibited a kinetic KI of 2.8 microM, and additionally induced a slight reduction in Vmax. As a cytochrome P-450, NOS catalyzes a heme-mediated reduction of molecular oxygen, resulting in the formation of H2O2 in the absence of L-arginine. 7-NI turned out as a potent inhibitor of H2O2 formation (IC50 = 0.28 +/- 0.096 microM) but did not affect flavin-mediated electron transfer. Thus, 7-NI resembled imidazole, a known heme-site inhibitor of NOS. We found that imidazole was a purely competitive inhibitor of L-citrulline formation (KI = 263 microM) and blocked H2O2 formation at similar concentrations (IC50 = 280 +/- 38 microM). In accordance with their L-arginine-competitive effects in the citrulline assay, both drugs antagonized binding of radiolabeled NG-nitro-L-arginine (L-NNA), a high affinity probe for reversible labelling of the substrate site of NOS [Klatt et al., J. Biol. Chem. 269, 14781-14787 (1994)]. The calculated KI values for 7-NI and imidazole were 0.09 +/- 0.024 microM and 200 +/- 63 microM, respectively. Finally, binding of radiolabelled tetrahydrobiopterin, a NOS cofactor with unknown function, was also antagonized by 7-NI with a KI of 0.12 +/- 0.023 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Mayer
B
B
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
Klatt
P
P
Werner
E R
ER
Schmidt
K
K
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors*; D000700:Analgesics / Q000494:pharmacology*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D001708:Biopterins / Q000378:metabolism; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D002956:Citrulline / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007191:Indazoles / Q000494:pharmacology*; D007700:Kinetics; D009569:Nitric Oxide / Q000037:antagonists & inhibitors; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D011485:Protein Binding; D013552:Swine
D000700:Analgesics; D004791:Enzyme Inhibitors; D007191:Indazoles; D019335:Nitroarginine; D001708:Biopterins; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C080122:7-nitroindazole