Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase

Abstract

The binding of tetrahydropteridines with 6-di- and trihydroxypropyl side chains to recombinant rat neuronal nitric oxide (NO) synthase (EC 1.14.13.39) was determined by competition with 6R-[3´-³H]-5,6,7,8-tetrahydro-L-erythro-biopterin (6R-[3´-³H]H₄biopterin). Although all but one of the derivatives exhibited only poor affinities (K_i 50 µM), the 4-amino analogue of 6R-H₄biopterin was a potent antagonist of 6R-H₄biopterin binding (K_i 13.2 nM). The 4-amino analogue of 6R-H₄ biopterin inhibited NO synthase stimulation by the natural cofactor 6R-H₄biopterin with an IC₅₀ of 1 µM without affecting the basal activity observed in the absence of added 6R-H₄biopterin. Because the 4-amino analogue of 6R-H₄biopterin also inhibited dihydropteridine reductase (EC 1.6.99.7; IC₅₀ 20 µM), our results support the hypothesis that redox cycling of H₄biopterin might be required for the NO synthase reaction.