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Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of a cerebral palsy phenotype in an animal model.

ABSTRACT: Selective inhibition of the neuronal isoform of nitric oxide synthase NOS (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. However, given the high active site conservation among all three NOS isoforms, the design of selective inhibitors is an extremely challenging problem. Here we present the structural basis for why novel and potent nNOS inhibitors exhibit the highest level of selectivity over eNOS reported so far (approximately 3,800-fold). By using a combination of crystallography, computational methods, and site-directed mutagenesis, we found that inhibitor chirality and an unanticipated structural change of the target enzyme control both the orientation and selectivity of these novel nNOS inhibitors. A new hot spot generated as a result of enzyme elasticity provides important information for the future fragment-based design of selective NOS inhibitors.

SUBMITTER: Delker SL

PROVIDER: S-EPMC3202968 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of a cerebral palsy phenotype in an animal model.

Delker Silvia L SL Ji Haitao H Li Huiying H Jamal Joumana J Fang Jianguo J Xue Fengtian F Silverman Richard B RB Poulos Thomas L TL

Journal of the American Chemical Society 20100401 15

Selective inhibition of the neuronal isoform of nitric oxide synthase NOS (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. However, given the high active site conservation among all three NOS isoforms, the design of selective inhibitors is an extremely challenging problem. Here we present the structural basis for why novel and potent nNOS inhibitors exhibit the highest level of selectivity over eNOS reported so far (approxim ...[more]

PMID: 20337441

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Project description:Cerebral palsy and death are serious consequences of perinatal hypoxia-ischemia (HI). Important concepts can now be tested using an animal model of cerebral palsy. We have previously shown that reactive oxygen and nitrogen species are produced in antenatal HI. A novel class of neuronal nitric oxide synthase (nNOS) inhibitors have been designed, and they ameliorate postnatal motor deficits when administered prior to the hypoxic-ischemic insult. This study asks how the new class of inhibitors, using JI-8 (K(i) for nNOS: 0.014 ?M) as a representative, compare with the frequently used nNOS inhibitor 7-nitroindazole (7-NI; K(i): 0.09 ± 0.024 ?M). A theoretical dose equivalent to 75 K(i) of JI-8 or equimolar 7-NI was administered to pregnant rabbit dams 30 min prior to and immediately after 40 min of uterine ischemia at 22 days gestation (70% term). JI-8 treatment resulted in a significant decrease in NOS activity (39%) in fetal brain homogenates acutely after HI, without affecting maternal blood pressure and heart rate. JI-8 treatment resulted in 33 normal kits, 2 moderately and 13 severely affected kits and 5 stillbirths, compared with 8 normal, 3 moderately affected and 5 severely affected kits and 10 stillbirths in the 7-NI group. In terms of neurobehavioral outcome, 7-NI was not different from saline treatment, while JI-8 was superior to saline and 7-NI in its protective effect (p < 0.05). In the surviving kits, JI-8 significantly improved the locomotion score over both saline and 7-NI scores. JI-8 was also significantly superior to saline in preserving smell, muscle tone and righting reflex function, but 7-NI did not show significant improvement. Furthermore, a 100-fold increase in the dose (15.75 ?mol/kg) of 7-NI significantly decreased systolic blood pressure in the dam, while JI-8 did not. The new class of inhibitors such as JI-8 shows promise in the prevention of cerebral palsy and is superior to the previously more commonly used nNOS inhibitor.

Project description:Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.

Dataset Information

Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of a cerebral palsy phenotype in an animal model.

Publications

Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of a cerebral palsy phenotype in an animal model.

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