Biochemistry of novel tetrahydrobiopterin derivatives and analogues in vitro and in vivo

Nitric oxide synthases are a class of enzymes catalyzing the conversion of L-arginine to citrulline and nitric oxide (NO). Nitric oxide thus formed is responsible for a variety of physiologically important reactions. These include regulation and effector functions of the host`s immune response to pathogens, neuronal signalling and regulation of the blood pressure via endothelium-derived vascular relaxation. For the role of NO in endothelium-derived vascular relaxation, the last year`s Nobel Price of Medicine has been awarded. The enzymatic reaction leading to the formation of NO from L-arginine has a complex mechanism and requires the participation of a surprisingly rich selection of cofactors. Our work focusses on one of these, tetrahydrobiopterin. In previous work we were able to show that intracellular concentrations of tetrahydrobiopterin modulate the amount of nitric oxide formed by intact cells. In one part of the project, we would like to characterize the mechanism of action of tetrahydrobiopterin in NO synthases using novel tetrahydrobiopterin derivatives. Comparison of the results obtained with effects of these derivatives on phenylalanine hydroxylase and dihydropteridine reductase should provide clues on the role of tetrahydrobiopterin in NO synthesis. Phenylalanine hydroxylase is another important tetrahydrobiopterin-dependent enzyme. Dihydropteridine reductase recycles the cofactor from the oxidised form produced by the phenylalanine hydroxylase reaction back to the active tetrahydro state. Apart from this investigation of the mechanism of the stimulation of NO synthases by tetrahydrobiopterin derivatives, the present project focusses on the action of our new tetrahydrobiopterin derivatives and analogues on the immune response. In pilot studies, we observed surprinsingly favourable effects of at least one of these novel compounds, the 4-amino analogue of tetrahydrobiopterin, on survival in septic shock and on suppression of allograft rejection. In the present project we would like to substantiate these results and unravel the mode of action of the novel tetrahydrobiopterin derivatives. As a major tool we would like to take advantage of the latest powerful techniques of molecular biology for the search of genes affected by the treatment with the compounds and for unravelling the exact target of their action. We expect from these studies an improved knowledge on the regulation of the immune response, in particular on the role of NO in the cytokine network. We feel that our work has the potential to contribute to the development of a new class of immunosuppressive drugs based on tetrahydrobiopterin derivatives and analogues.

Tetrahydrobiopterin is a compound with structural similarity to the vitamins folic acid and riboflavin. In contrast to these, tetrahydrobiopterin is synthesized by mammals. Tetrahydrobiopterin is required as cofactor for the metabolism of aromatic amino acids and for nitric oxide synthases. Nitric oxide synthases are a recently discovered family of proteins catalyzing the formation of nitric oxide from L-arginine. Nitric oxide regulates important physiological functions such as host defense to pathogens, blood pressure and neurotransmission. This project succeeded in getting new insights into the mechanism of stimulation of nitric oxide synthases by tetrahydrobiopterin by the use of novel tetrahydrobiopterin derivatives as unique tools. We could show that tetrahydrobiopterin stimulates nitric oxide synthases by a novel mechanism that involves the formation of a radical derived from tetrahydrobiopterin. This novel mechanism proved to be clearly different from the mechansim of stimulation of phenylalanine hydroxylase by tetrahydrobiopterin.We could also demonstrate that one of the derivatives used, the 4-amino analogue of tetrahydrobiopterin, is a compound with promising pharmacological action. This compound is able to counteract the deadly side effects of septic shock and is an immunosuppressant, which efficiently blocks the rejection of allografts.