Minimal amidine structure for inhibition of nitric oxide biosynthesis

Pharmacological modulation of nitric oxide synthase activity has been achie ved using structural analogs of arginine. In the present studies, we demons trated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cu ltures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a con centration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expre ssing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidi ne had no effect on gamma -interferon-induced expression of nitric oxide sy nthase protein, its enzymatic activity was blocked. Kinetic analysis reveal ed that formamidine acts as a simple competitive inhibitor with respect to arginine (K-i formamidine similar to 800 muM). Using a polarographic micros ensor to measure real-time flux of nitric oxide release from RAW 264.7 macr ophages, formamidine was found to require 30-90 min to inhibit enzyme activ ity, suggesting that cellular uptake of the drug may limit its biological a ctivity. Our data indicate that formamidine is an effective inhibitor of ni tric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased pro duction of nitric oxide. (C) 2001 Elsevier Science Inc. All rights reserved .