Blocked dihydropteridines as nitric oxide synthase activators

It has been shown that 6-acetyl-7,7-dimethyl-5,6,7,8-tetrahydropteridin-4(3H)-one can act as a competent cofactor for the production of nitric oxide by neuronal nitric oxide synthase (nNOS). More information was sought on the structural features that could contribute to strong binding within the enzyme whilst maintaining a fast electron transfer rate. This study was concerned with expansion at the C2-position of the pteridine scaffold. The evidence suggests that expansion at the C2-position had a deleterious effect with respect to Km and as a consequence electron transfer rate. Unexpectedly, several lines of evidence suggested that a methyl substituent on nitrogen at C2 reduced the electron density in the pyrimidine and dihydropterin rings

Tetrahydrobiopterin and electron transfer in NO synthase

Mammalian NO synthase requires the cofactor tetrahydrobiopterin (H4B) to act as an electron donor during the activation of molecular oxygen at the heme site. After donating an electron, the resultant H4B radical is then required to abstract an electron from the ferrous NO complex, which is generated at the end of the catalytic reaction, in order to facilitate NO release. We have recently explored the structural requirements of NO synthase for the H4B cofactor by studying a range of novel cofactor analogues with highly modified structures. Substituents on the C6 and C7 positions of H4B are tolerated well, with surprisingly bulky pterins being able to bind and drive NO synthesis. The modified pterins have a wide range of activities and binding constants, but the main function of the cofactors in activating molecular oxygen appears to be independent of C6 and C7 modification as shown by rapid reaction studies. We have also assessed the possibility of direct electron transfer across the dimer interface between H4B molecules in the two NO synthase subunits. The H4B cofactors are within the range for facile electron transfer and present a possible mechanism for NO synthase to escape from the unreactive ferrous-NO complex, which is known to originate from product inhibition

6-Acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin is an activator of nitric oxide synthases

6-Acetyl-7,7-dimethyl-7,8-dihydropterin 3 has been shown to be able to substitute for the natural cofactor of nitric oxide synthases, tetrahydrobiopterin 1, in cells and tissues that contain active nitric oxide synthases (NOSs). In both macrophages, which produce iNOS, and endothelial cells, which produce eNOS, in which tetrahydrobiopterin biosynthesis has been blocked by inhibition of GTP cyclohydrolase 1, dihydropterin 3 restored production of nitric oxide by these cells. In tissues, 3 caused relaxation in preconstricted rat aortic rings, again in which tetrahydrobiopterin biosynthesis had been inhibited, an effect that was blocked by the NOS inhibitor, l-NAME. However, dihydropterin 3 was not itself an active cofactor in purified NOS (nNOS) preparations free of tetrahydrobiopterin suggesting that intracellular reduction to 6-acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin 4 is required for activity. Compound 4 was prepared by reduction of the corresponding 7,8-dihydropterin with sodium cyanoborohydride and has been shown to be a competent cofactor for nitric oxide production by nNOS. Together, the results show that the 7,7-dimethyl-7,8-dihydropterin is a novel structural framework for effective tetrahydrobiopterin analogues