Inactivation of nitric oxide synthase by substituted aminoguanidines and aminoisothioureas.
A series of substituted aminoguanidines and amino-substituted isothioureas have been examined as inhibitors of nitric oxide (NO) synthase (NOS) isoforms. Each of the agents produced a time- and concentration-dependent inactivation of the NO-forming activity of the affinity-purified NOS isoforms. These inactivations required exposure of NOS to the drug under conditions that supported catalysis, consistent with the proposal that they act as alternate substrate, mechanism-based inactivators. Of the aminoguanidines examined, 2-ethylaminoguanidine was the most efficient inactivator, exhibiting vs. iNOS an apparent KI value of 120 microM as measured at 100 microM arginine and a k(inact max) value of 0.48 min(-1) and thus an apparent second-order rate constant for inactivation of 4.0 mM(-1)min(-1). 2-Ethylaminoguanidine displayed a high isoform selectivity for the iNOS compared with the nNOS and eNOS isoforms. 2-Ethylaminoguanidine inactivated NO synthetic activity in cytokine-induced RAW 264.7 cells as measured at 100 microM extracellular arginine with an apparent KI value of 55 microM and a k(inact max) value of 0.09 min(-1). The inactivated RAW 264.7 cell NO synthetic capability was restored over a 3-hr period after drug removal to a value 60% of its pretreatment value. This recovery occurred despite the presence of cycloheximide sufficient to inhibit protein synthesis by >99%. 1-Amino-S-methylisothiourea by contrast with the aminoguanidines was identified as a mechanism-based inactivator selective for the nNOS isoform. In contrast to S-isopropylisothiourea, which was found to be both cell penetrant and reversible, 1-amino-S-methylisothiourea appeared cell impermeable and inhibited NOS enzyme "irreversibly."
Wolff
D J
DJ
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854, USA.
Gauld
D S
DS
Neulander
M J
MJ
Southan
G
G
D000818:Animals; D002417:Cattle; D016207:Cytokines / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D013890:Thiourea / Q000494:pharmacology*
D016207:Cytokines; D006146:Guanidines; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019255:NADPH Oxidases; D013890:Thiourea; C004479:pimagedine