INACTIVATION OF NITRIC-OXIDE SYNTHASE BY SUBSTITUTED AMINOGUANIDINES AND AMINOISOTHIOUREAS

A series of substituted aminoguanidines and amino-substituted isothiou reas have been examined as inhibitors of nitric oxide (NO) synthase (N OS) isoforms. Each of the agents produced a time-and concentration-dep endent inactivation of the NO-forming activity of the affinity-purifie d NOS isoforms. These inactivations required exposure of NOS to the dr ug under conditions that supported catalysis, consistent with the prop osal that they act as alternate substrate, mechanism-based inactivator s. Of the aminoguanidines examined, 2-ethylaminoguanidine was the most efficient inactivator, exhibiting vs. iNOS an apparent K-l value of 1 20 mu M as measured at 100 mu M arginine and a k(inact max) value of 0 .48 min(-1) and thus an apparent second-order rate constant for inacti vation of 4.0 mM(-1)min(-1). 2-Ethylaminoguanidine displayed a high is oform selectivity for the iNOS compared with the nNOS and eNOS isoform s. 2-Ethylaminoguanidine inactivated NO synthetic activity in cytokine -induced RAW 264.7 cells as measured at 100 mu M extracellular arginin e with an apparent K-l value of 55 mu M 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 cyc loheximide 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 c ontrast to S-isopropylisothiourea, which was found to be both cell pen etrant and reversible, 1-amino-S-methylisothiourea appeared cell imper meable and inhibited NOS enzyme ''irreversibly.''