thiouracil and sapropterin

6-n-Propyl-2-thiouracil (6-PTU), the antithyroid agent, produces a time-, concentration-, and turnover-dependent inactivation of the NO synthetic capability of the neuronal nitric oxide synthase isoform irreversible by either arginine or (6R)-5,6,7,8-tetrahydro-L-biopterin. By contrast 6-PTU produces an inhibition of the cytokine-inducible and endothelial nitric oxide synthases fully reversible by arginine. The inactivation of neuronal nitric oxide synthase by 6-PTU follows first order kinetics, and is inhibited competitively by both arginine and (6R)-5,6,7,8-tetrahydro-L-biopterin, but is not accompanied by either a loss of heme-CO binding, heme fluorescence, or disassembly of dimeric structure. 2-Thiouracil behaves qualitatively identically to 6-PTU. Turnover-dependent inactivation of neuronal nitric oxide synthase by [2-14C]-2-thiouracil is accompanied by incorporation of radioactivity into the polypeptide chain. Ca2+-dependent NO formation by GH3 pituitary cells is inhibited by 6-PTU in a manner enhanced by depletion of either extracellular arginine or intracellular (6R)-5,6,7,8-tetrahydro-L-biopterin. These observations establish that 6-PTU is an alternate substrate, mechanism-based inactivator of the neuronal nitric oxide synthase isoform with the ability to suppress cellular NO formation.

Topics: Animals; Antithyroid Agents; Arginine; Biopterins; Calcium; Calmodulin; Carbon Monoxide; Carbon Radioisotopes; Citrulline; Enzyme Activation; Enzyme Inhibitors; Heme; Kinetics; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Pituitary Neoplasms; Propylthiouracil; Rats; Thiouracil; Tumor Cells, Cultured

Two established antithyroid drugs, 6-propyl-2-thiouracil and 6-methyl-2-thiouracil, as well as S-methylthiouracil, are shown to be competitive inhibitors of nitric oxide synthase (NOS) (K(I) values ranging from 14 to 60 microM), with moderate selectivity for the neuronal isoform. Other thioureylene and thioamide-containing heterocyclic systems proved virtually ineffective as NOS inhibitors. Besides offering novel useful leads for inhibitor design as well as to probe the active site of neuronal NOS, the results of this study may have interesting implications in relation to the antithyroid activity of thiouracils and their possible neurological effects.

Topics: Animals; Antithyroid Agents; Binding Sites; Binding, Competitive; Biopterins; Cerebellum; Enzyme Inhibitors; Heme; In Vitro Techniques; Models, Biological; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Rats; Structure-Activity Relationship; Thiouracil

2-thiouracil (TU), an established antithyroid drug and melanoma-seeker, was found to selectively inhibit neuronal nitric oxide synthase (nNOS) in a competitive manner (K(i)=20 microM), being inactive on the other NOS isoforms. The drug apparently interfered with the substrate- and tetrahydrobiopterin (BH(4))-binding to the enzyme. It caused a 60% inhibition of H(2)O(2) production in the absence of L-arginine and BH(4), and antagonised BH(4)-induced dimerisation of nNOS, but did not affect cytochrome c reduction. These results open new perspectives in the understanding of the antithyroid action of TU and provide a new lead structure for the development of selective nNOS inhibitors to elucidate the interdependence of the substrate and pteridine sites and to modulate pathologically aberrant NO formation.

Topics: Animals; Antithyroid Agents; Arginine; Binding, Competitive; Biopterins; Cerebellum; Citrulline; Cytochrome c Group; Dimerization; Enzyme Activation; Enzyme Inhibitors; Hydrogen Peroxide; Kinetics; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Rats; Thiouracil