Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity.

Abstract

1. The induction of a calcium-independent isoform of nitric oxide (NO) synthase (iNOS) and a subsequent enhanced formation of NO has been implicated in the pathophysiology of a variety of diseases including inflammation and circulatory shock. Here we demonstrate that the S-substituted isothioureas, S-methylisothiourea (SMT), S-(2-aminoethyl)isothiourea (aminoethyl-TU), S-ethylisothiourea (ethyl-TU) and S-isopropylisothiourea (isopropyl-TU) potently inhibit iNOS activity in J774.2 macrophages activated with bacterial endotoxin with EC50 values 8-24 times lower than that of NG-methyl-L-arginine (MeArg) and 200-times lower than that of NG-nitro-L-arginine (L-NO2Arg). 2. The inhibition of iNOS activity by these S-substituted isothioureas is dose-dependently prevented by excess of L-arginine suggesting that these isothioureas are competitive inhibitors of iNOS at the L-arginine binding site. 3. Ethyl-TU and isopropyl-TU are 4-6 times more potent than MeArg in inhibiting the constitutive NOS activity in homogenates of bovine aortic endothelial cells (eNOS) and are more potent pressor agents than MeArg in the anaesthetized rat. SMT is equipotent with MeArg, whereas aminoethyl-TU is 6-times less potent in inhibiting eNOS activity in vitro. Both SMT and aminoethyl-TU, however, elicit only weak pressor responses (approximately 15 mmHg at 10 mg kg-1, i.v.) in vivo. 4. A comparison of the potencies of ethyl-, iso-propyl-, n-propyl-, t-butyl- and n-butyl-isothioureas on iNOS activity shows that the inhibitory activity of S-substituted isothioureas declines sharply if the side chain exceeds 2 carbon atoms in length. Similarly, substitution of the ethylene side chain of ethyl-TU also results in a diminished potency. Substitution of either one or both nitrogens of SMT with either amino or alkyl groups also substantially reduces its NOS inhibitory potency.5. In conclusion, isothioureas represent a new class of NOS inhibitors which includes the most potent inhibitors of iNOS activity reported to date. Some members of this class (ethyl-TU and isopropyl-TU)are potent inhibitors of eNOS and iNOS with little selectivity towards either isoform, while others (SMT and aminoethyl-TU) are relatively selective inhibitors of iNOS activity. These latter agents may become useful tools for studying the role of iNOS in various disease models and may be useful in the therapy of diseases that are associated with an enhanced formation of NO due to iNOS induction, such as inflammation, circulatory shock or cancer.