Structural basis for pterin antagonism in nitric-oxide synthase. Development of novel 4-oxo-pteridine antagonists of (6R)-5,6,7,8-tetrahydrobiopterin.
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.
Kotsonis
P
P
Department of Pharmacology and Toxicology, Julius-Maximilians University, Versbacher Strasse 9, Würzburg 97078, Germany. peter.kotsonis@pharma.novartis.com
Fröhlich
L G
LG
Raman
C S
CS
Li
H
H
Berg
M
M
Gerwig
R
R
Groehn
V
V
Kang
Y
Y
Al-Masoudi
N
N
Taghavi-Moghadam
S
S
Mohr
D
D
Münch
U
U
Schnabel
J
J
Martásek
P
P
Masters
B S
BS
Strobel
H
H
Poulos
T
T
Matter
H
H
Pfleiderer
W
W
Schmidt
H H
HH
D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D002531:Cerebellum / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013552:Swine
D000975:Antioxidants; D007527:Isoenzymes; D001708:Biopterins; D019001:Nitric Oxide Synthase; C003402:sapropterin