Abstract
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.”
Footnotes
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Send reprint requests to: Dr. Donald J. Wolff, Department of Pharmacology, UMDNJ, Robert Wood Johnson Medical School, Piscataway, NJ 08854.
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↵1 This work was supported by National Institutes of Health Grant HL54768.
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↵2 Compounds studied are named as derivatives of either aminoguanidine or S-methylisothiourea, both of which are established inhibitors of NOS. Consequently, naming does not necessarily follow standard conventions. For example, compound 3 could be more properly referred to as 1-amino-2-methylguanidine. Compound 9 could be described as 1-amino-2,3-dimethylpsuedourea or N-amino-N′,S-dimethylisothiourea but is more simply described as 3,4-dimethylisothiosemicarbazide.
- Abbreviations:
- AMITU
- 1-amino-S-methylisothiourea
- BH4
- (6R)-5,6,7,8-tetrahydro-l-biopterin
- CaM
- calmodulin
- DTT
- dithiothreitol
- NOS
- nitric oxide synthase
- eNOS
- endothelial nitric oxide synthase
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- iNOS
- inducible nitric oxide synthase
- LPS
- lipopolysaccharide
- NO
- nitric oxide
- nNOS
- neuronal nitric oxide synthase
- SEITU
- S-ethylisothiourea
- SIPITU
- S-isopropylisothiourea
- SMITU
- S-methylisothiourea
- Received March 28, 1997.
- Accepted June 3, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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