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 onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 microM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 microM. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a Kd value </= 7 nM. In contrast, inhibition of human neuronal NOS and endothelial NOS (eNOS) was relatively weaker, rapidly reversible, and competitive with L-arginine, with Ki values of 2 microM and 50 microM, respectively. Thus, 1400W was at least 5000-fold selective for iNOS versus eNOS. This selectivity was similar to that observed in rat aortic rings, in which 1400W was greater than 1000-fold more potent against rat iNOS than eNOS. Finally, 1400W was greater than 50-fold more potent against iNOS than eNOS in a rat model of endotoxin-induced vascular injury. Thus, the potency and selectivity of 1400W inhibition of iNOS both in vitro and in vivo were far greater than of any previously described iNOS inhibitor.
Garvey
E P
EP
Institute of Biochemistry, Glaxo Wellcome, Research Triangle Park, North Carolina 27709, USA. Ed_Garvey@glaxo.com
Oplinger
J A
JA
Furfine
E S
ES
Kiff
R J
RJ
Laszlo
F
F
Whittle
B J
BJ
Knowles
R G
RG
D000578:Amidines / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001596:Benzylamines / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D001808:Blood Vessels / Q000187:drug effects / Q000473:pathology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats
D000578:Amidines; D001596:Benzylamines; D004791:Enzyme Inhibitors; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D019001:Nitric Oxide Synthase