Structural basis for pterin antagonism in nitric-oxide synthase. Development of novel 4-oxo-pteridine antagonists of (6R)-5,6,7,8-tetrahydrobiopterin

• Published in: The Journal of biological chemistry Vol. 276; no. 52; p. 49133
• Main Authors: Kotsonis, P, Fröhlich, L G, Raman, C S, Li, H, Berg, M, Gerwig, R, Groehn, V, Kang, Y, Al-Masoudi, N, Taghavi-Moghadam, S, Mohr, D, Münch, U, Schnabel, J, Martásek, P, Masters, B S, Strobel, H, Poulos, T, Matter, H, Pfleiderer, W, Schmidt, H H
• Format: Journal Article
• Language: English
• Published: United States 28-12-2001
• Subjects: Animals; Antioxidants - chemistry; Antioxidants - metabolism; Binding Sites; Biopterins - analogs & derivatives; Biopterins - chemistry; Biopterins - metabolism; Cerebellum - enzymology; Humans; Isoenzymes - chemistry; Isoenzymes - metabolism; Models, Molecular; Molecular Structure; Nitric Oxide Synthase - antagonists & inhibitors; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase - metabolism; Protein Binding; Protein Structure, Tertiary; Structure-Activity Relationship; Swine
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M011469200

Pathological nitric oxide (NO) generation in sepsis, inflammation, and stroke may be therapeutically controlled by inhibiting NO synthases (NOS). Here we targeted the (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)Bip)-binding site of NOS, which, upon cofactor binding, maximally increases enzyme activity and NO production from substrate l-arginine. The first generation of H(4)Bip-based NOS inhibitors employed a 4-amino pharmacophore of H(4)Bip analogous to antifolates such as methotrexate. We developed a novel series of 4-oxo-pteridine derivatives that were screened for inhibition against neuronal NOS (NOS-I) and a structure-activity relationship was determined. To understand the structural basis for pterin antagonism, selected derivatives were docked into the NOS pterin binding cavity. Using a reduced 4-oxo-pteridine scaffold, derivatives with certain modifications such as electron-rich aromatic phenyl or benzoyl groups at the 5- and 6-positions, were discovered to markedly inhibit NOS-I, possibly due to hydrophobic and electrostatic interactions with Phe(462) and Ser(104), respectively, within the pterin binding pocket. One of the most effective 4-oxo compounds and, for comparisons an active 4-amino derivative, were then co-crystallized with the endothelial NOS (NOS-III) oxygenase domain and this structure solved to confirm the hypothetical binding modes. Collectively, these findings suggest (i) that, unlike the antifolate principle, the 4-amino substituent is not essential for developing pterin-based NOS inhibitors and (ii), provide a steric and electrostatic basis for their rational design.