TY - JOUR T1 - Inactivation of Nitric Oxide Synthase by Substituted Aminoguanidines and Aminoisothioureas JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 265 LP - 273 VL - 283 IS - 1 AU - Donald J. Wolff AU - Douglas S. Gauld AU - Matthew J. Neulander AU - Garry Southan Y1 - 1997/10/01 UR - http://jpet.aspetjournals.org/content/283/1/265.abstract N2 - 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, exhibitingvs. iNOS an apparent KI value of 120 μM as measured at 100 μM arginine and akinact max value of 0.48 min−1 and thus an apparent second-order rate constant for inactivation of 4.0 mM−1min−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 μM extracellular arginine with an apparent KI value of 55 μM and a kinact 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.” The American Society for Pharmacology and Experimental Therapeutics ER -