1400W IS A SLOW, TIGHT-BINDING, AND HIGHLY SELECTIVE INHIBITOR OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN-VITRO AND IN-VIVO

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitric-oxide synthase (iNOS), The slow o nset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s(-1) and a binding constant of 2.0 mu M. Inhib ition was dependent on the cofactor NADPH. L-Arginine was a competitiv e inhibitor of 1400W binding with a K-s value of 3.0 mu M Inhibited en zyme did not recover activity after 2 h. Thus, 1400W was either an irr eversible inhibitor or an extremely slowly reversible inhibitor of hum an iNOS with a K-d value less than or equal to 7 nM. In contrast, inhi bition of human neuronal NOS and endothelial NOS (eNOS) was relatively weaker, rapidly reversible, and competitive with L-arginine, with Ri values of 2 mu M and 50 mu M, respectively, Thus, 1400W was at least 5 000-fold selective for MOS versus eNOS. This selectivity was similar t o that observed in rat aortic rings, in which 1400W was greater than 1 000-fold more potent against rat iNOS than eNOS. Finally, 1400W was gr eater than 50-fold more potent against iNOS than eNOS in a rat model o f endotoxin-induced vascular injury. Thus, the potency and selectivity of 1400W inhibition of iNOS both in vitro and in vivo were far greate r than of any previously described iNOS inhibitor.