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.

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide.

The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H(4)Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H(4)Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H(4)Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydrooxazolo[1, 2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H(4)Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H(4)Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H(4)Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor N(omega)-nitro-L-arginine. Importantly, H(4)Bip was found to react chemically with superoxide (O(2)(-.)) and enzyme-bound H(4)Bip was consumed under O(2)(-.)-generating conditions in the absence of substrate. These results suggest that H(4)Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O(2)(-.).

Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.

Endothelial nitric-oxide synthase (NOS-III) is defined as being strictly dependent on Ca(2+)/calmodulin (CaM) for activity, although NO release from endothelial cells has been reported to also occur at intracellular free Ca(2+) levels that are substimulatory for the purified enzyme. We demonstrate here that NOS-III, but neither NOS-I nor -II, is rapidly and strongly activated and phosphorylated on both Ser and Thr in the presence of cGMP-dependent protein kinase II (cGK II) and the catalytic subunit of cAMP-dependent protein kinase (cAK) in vitro. Phosphopeptide analysis by mass spectrometry identified Ser(1177), as well as Ser(633) which is situated in a recently defined CaM autoinhibitory domain within the flavin-binding region of human NOS-III. Phosphoamino acid analysis identified a putative phosphorylation site at Thr(495) in the CaM-binding domain. Importantly, both cAK and cGK phosphorylation of NOS-III in vitro caused a highly reproducible partial (10-20%) NOS-III activation which was independent of Ca(2+)/CaM, and as much as a 4-fold increase in V(max) in the presence of Ca(2+)/CaM. cAK stimulation in intact endothelial cells also increased both Ca(2+/)CaM-independent and -dependent activation of NOS-III. These data collectively provide new evidence for cAK and cGK stimulation of both Ca(2+)/CaM-independent and -dependent NOS-III activity, and suggest possible cross-talk between the NO and prostaglandin I(2) pathways and a positive feedback mechanism for NO/cGMP signaling.

Inhibition of neuronal nitric oxide synthase by 4-amino pteridine derivatives: structure-activity relationship of antagonists of (6R)-5,6,7,8-tetrahydrobiopterin cofactor.

The family of nitric oxide synthases (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), an important cellular messenger molecule which has been implicated in the pathophysiology of septic shock and inflammatory and neurodegenerative disease states. NOS can be maximally activated by the ubiquitous cofactor, (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip), and antagonists of H(4)Bip may be of therapeutic importance to inhibit pathologically high NO formation. The 4-amino substituted analogue of H(4)Bip was reported to be a potent NOS inhibitor. Therefore, we developed a series of novel 4-amino pteridine derivatives, anti-pterins, to pharmacologically target the neuronal isoform of nitric oxide synthase (NOS-I). To functionally characterize the pterin/anti-pterin interaction and establish a structure-activity relationship (SAR), we systematically altered the substituents in the 2-, 4-, 5-, 6-, and 7-position of the pteridine nucleus. Varying the substitution pattern in the 2-, 5-, and 7-position resulted in no significant inhibitory effect on enzyme activity. In contrast, bulky substituents in the 6-position, such as phenyl, markedly increased the inhibitory potency of the reduced 4-amino-5,6,7,8-tetrahydropteridines, possibly as a consequence of hydrophobic interactions within NOS-I. However, this was not the case for the aromatic 4-amino pteridines. Interestingly, chemical modification of the 4-amino substituent by dialkyl/diaralkylation together with 6-arylation of the aromatic 2,4-diamino pteridine resulted in potent and efficacious inhibitors of NOS-I, suggesting possible hydrophilic and hydrophobic interactions within NOS-I. This SAR agrees with (a) the recently published crystal structure of the oxygenase domain of the inducible NOS isoform (NOS-II) and (b) the comparative molecular field analysis of selected NOS-I inhibitors, which resulted in a 3D-QSAR model of the pterin binding site interactions. Further optimization should be possible when the full length structure of NOS-I becomes available.

Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase.

The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity. We examined the effects of H(4)Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H(4)Bip in a competitive manner. Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min). NOS-associated H(4)Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control). A similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.

Autoinhibition of neuronal nitric oxide synthase: distinct effects of reactive nitrogen and oxygen species on enzyme activity.

Nitric oxide (NO) synthases (NOSs), which catalyse the oxidation of L-arginine to L-citrulline and an oxide of nitrogen, possibly NO or nitroxyl (NO-), are subject to autoinhibition by a mechanism that has yet to be fully elucidated. In the present study we investigated the actions of NO and other NOS-derived products as possible autoregulators of enzyme activity. With the use of purified NOS-I, L-arginine turnover was found to operate initially at Vmax (0-15 min, phase I) although, despite the presence of excess substrate and cofactors, prolonged catalysis (15-90 min, phase II) was associated with a rapid decline in L-arginine turnover. Taken together, these observations suggested that one or more NOS products inactivate NOS. Indeed, exogenously applied reactive nitrogen oxide species (RNSs) decreased Vmax during phase I, although with different potencies (NO->NO> ONOO-) and efficacies (NO>NO-=ONOO-). The NO scavengers oxyhaemoglobin (HbO2; 100 microM) and 1H-imidazol-1 - yloxy - 2 - (4-carboxyphenyl) - 4,5 - dihydro - 4,4,5,5 - tetramethyl - 3 -oxide (CPTIO; 10 microM) and the ONOO- scavenger GSH (7 mM) had no effect on NOS activity during phase I, except for an endogenous autoinhibitory influence of NO and ONOO-. However, superoxide dismutase (SOD; 300 units/ml), which is thought either to increase the half-life of NO or to convert NO- to NO, lowered Vmax in an NO-dependent manner because this effect was selectively antagonized by HbO2 (100 microM). This latter observation demonstrated the requirement of SOD to reveal endogenous NO-mediated autoinhibition. Importantly, during phase II of catalysis, NOS became uncoupled and began to form H2O2 because catalase, which metabolizes H2O2, increased enzyme activity. Consistent with this, exogenous H2O2 also inhibited NOS activity during phase I. Thus during catalysis NOS is subject to complex autoinhibition by both enzyme-derived RNS and H2O2, differentially affecting enzyme activity.

Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase.

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7, 8-tetrahydro-L-biopterin (H4Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H4Bip, has been attributed to enzyme-associated H4Bip. To elucidate further H4Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H4Bip and inhibited H4Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of Vmax) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H4Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H4Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H4Bip redox cycling in NOS. Rather, H4Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.