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Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein–protein interactions but also metabolite-dependent interactions. Based on this protein–metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4. Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood–brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein–metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.

According to the recent data, nitric oxide (NO) is a chemical messenger that mediates functions such as vasodilation and neurotransmission, as well as displaying antimicrobial and antitumoral activities. NO has been implicated in the neurotoxicity associated with stroke and neurodegenerative diseases; neural regulation of smooth muscle, including peristalsis; and penile erections. We searched for full-text English publications from the past 15 years in Pubmed and SNPedia databases using keywords and combined word searches (nitric oxide, single nucleotide variants, single nucleotide polymorphisms, genes). In addition, earlier publications of historical interest were included in the review. In our review, we have summarized information regarding all NOS1, NOS2, NOS3, and NOS1AP single nucleotide variants (SNVs) involved in the development of mental disorders and neurological diseases/conditions. The results of the studies we have discussed in this review are contradictory, which might be due to different designs of the studies, small sample sizes in some of them, and different social and geographical characteristics. However, the contribution of genetic and environmental factors has been understudied, which makes this issue increasingly important for researchers as the understanding of these mechanisms can support a search for new approaches to pathogenetic and disease-modifying treatment.

Background: Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Results: Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-10), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1β. The increase of the antiinflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-10, and IL-10 following CFA, overall corroborating the inhibitor data. Conclusion: These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

Objective Nitrinergic control is important in meal-induced satiety. The aim of this study was to assess functional polymorphisms in nitric oxide synthase (NOS) genes in the susceptibility to functional dyspepsia (FD). Methods Genomic DNA from 89 patients with FD and 180 healthy subjects matched for age and gender were typed for the gene of neuronal NOS ( nNOS , rs2682826), inducible NOS ( iNOS , rs2297518) and a variable number tandem repeat in intron 4 of endothelial NOS ( eNOS ). Patients ingested 500 mL of Ensure ® during a 20 min period and dyspeptic symptoms were scored. Results Genotype frequencies of eNOS and iNOS were not significantly different between FD patients and controls. The frequency of the T allele in nNOS was significantly higher in FD patients compared to the controls (49 vs. 16 %; odds ratio 5.01; 95 % confidence interval 2.83–9.01; p  < 0.05). Patients with the T allele in the nNOS polymorphism reported a higher satiation score than those with the CC genotype during the nutrition drink test (median 179 vs. 117; p  < 0.05). Conclusion The nNOS gene polymorphism is associated with susceptibility to FD and influences satiation in FD patients. Our data support the importance of NOS gene polymorphisms in the pathogenesis of FD.

Nitric oxide synthase (NOS) genes ( NOS1 , NOS2A , and NOS3 ) may create excess nitric oxide that contributes to neurodegeneration in Parkinson’s disease (PD). NOS genes might also interact with one another or with environmental factors in PD. Coding and tagging single nucleotide polymorphisms (SNPs) (27 NOS1 , 18 NOS2A , and five NOS3 SNPs) were genotyped in families with PD (1,065 cases and 1,180 relative and other controls) and were tested for allelic associations with PD using the association in the presence of linkage test and the pedigree disequilibrium test (PDT), allelic associations with age-at-onset (AAO) using the quantitative transmission disequilibrium test, and interactions using the multifactor dimensionality reduction—PDT. Gene–environment interactions involving cigarette smoking, caffeine, nonsteroidal anti-inflammatory drugs, and pesticides were examined using generalized estimating equations in participants with environmental data available. Significant associations with PD were detected for the NOS1 SNPs rs3782218, rs11068447, rs7295972, rs2293052, rs12829185, rs1047735, rs3741475, and rs2682826 (range of p  = 0.00083–0.046) and the NOS2A SNPs rs2072324, rs944725, rs12944039, rs2248814, rs2297516, rs1060826, and rs2255929 (range of p  = 0.0000040–0.047) in earlier-onset families with sporadic PD, and some SNPs were also associated with earlier AAO. There was no compelling statistical evidence for gene–gene interactions. However, of the significantly associated SNPs, interactions were found between pesticides and the NOS1 SNPs rs12829185, rs1047735, and rs2682826 (range of p  = 0.012–0.034) and between smoking and the NOS2A SNPs rs2248814 ( p  = 0.021) and rs1060826 ( p  = 0.013). These data implicate NOS1 and NOS2A as genetic risk factors for PD and demonstrate that their interactions with established environmental factors may modulate the environmental effects.

Patients with tension-type headache (TTH) have an increased risk of developing arterial hypertension (AH), while hypertensive subjects do seem to have an increased risk of TTH. We searched for full-text English publications in databases using keywords and combined word searches over the past 15 years. In addition, earlier publications of historical interest were included in the review. In our review, we summed up the single nucleotide variants (SNVs) of Nitric Oxide Synthases (NOSs) genes involved in the development of essential AH and TTH. The results of studies we discussed in this review are contradictory. This might be due to different designs of the studies, small sample sizes in some of them, as well as different social and geographical characteristics. However, the contribution of genetic and environmental factors remains understudied. This makes the issue interesting for researchers, as understanding these mechanisms can contribute to a search for new approaches to pathogenetic and disease-modifying treatment of the AH and TTH phenotype. New drugs against AH and TTH can be based on inhibition of nitric oxide (NO) production, blockade of steps in the NO-cGMP pathway, or NO scavenging. Indeed, selective neuronal NOS (n-NOS) and inducible NOS (i-NOS) inhibitors are already in early clinical development.

Aim: To investigate the effect of icariin on erectile function and the expression of nitric oxide synthase (NOS) isoforms in castrated rats. Methods: Thirty‐two adult male Wistar rats were randomly divided into one sham‐operated group (A) and three castrated groups (B, C and D). One week after surgery, rats were treated with normal saline (groups A and B) or oral icariin (1 mg/[kg·day] for group C and 5 mg/[kg·day] for group D) for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure (ICP) during electrostimulation of the cavernosal nerve. The serum testosterone (ST) levels, the percent of smooth muscle (PSM) in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and phosphodiesterase V (PDE5) in corpus cavernosum (CC) were also evaluated. Results: ICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A (P < 0.01). However, ICP, PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B (P < 0.05). Changes in ST and the expression of eNOS and PDE5 were not significant (P > 0.05) in groups C and D compared with those in group B. Conclusion: Oral treatment with icariin (> 98.6 % purity) for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.

Hypertension is the most important cause of death and disability in the elderly. In 9 out of 10 cases, the molecular cause, however, is unknown. One mechanistic hypothesis involves impaired endothelium-dependent vasodilation through reactive oxygen species (ROS) formation. Indeed, ROS forming NADPH oxidase ( Nox ) genes associate with hypertension, yet target validation has been negative. We re-investigate this association by molecular network analysis and identify NOX5, not present in rodents, as a sole neighbor to human vasodilatory endothelial nitric oxide (NO) signaling. In hypertensive patients, endothelial microparticles indeed contained higher levels of NOX5—but not NOX1, NOX2, or NOX4—with a bimodal distribution correlating with disease severity. Mechanistically, mice expressing human N ox5 in endothelial cells developed—upon aging—severe systolic hypertension and impaired endothelium-dependent vasodilation due to uncoupled NO synthase (NOS). We conclude that NOX5-induced uncoupling of endothelial NOS is a causal mechanism and theragnostic target of an age-related hypertension endotype. N ox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.

Background Due to the pleiotropic effects of nitric oxide (NO) within the lungs, it is likely that NO is a significant factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to test for association between single nucleotide polymorphisms (SNPs) in three NO synthase (NOS) genes and lung function, as well as to examine gene expression and protein levels in relation to the genetic variation. Methods One SNP in each NOS gene (neuronal NOS ( NOS1 ), inducible NOS ( NOS2 ), and endothelial NOS ( NOS3 )) was genotyped in the Lung Health Study (LHS) and correlated with lung function. One SNP (rs1800779) was also analyzed for association with COPD and lung function in four COPD case–control populations. Lung tissue expression of NOS3 mRNA and protein was tested in individuals of known genotype for rs1800779. Immunohistochemistry of lung tissue was used to localize NOS3 expression. Results For the NOS3 rs1800779 SNP, the baseline forced expiratory volume in one second in the LHS was significantly higher in the combined AG + GG genotypic groups compared with the AA genotypic group. Gene expression and protein levels in lung tissue were significantly lower in subjects with the AG + GG genotypes than in AA subjects. NOS3 protein was expressed in the airway epithelium and subjects with the AA genotype demonstrated higher NOS3 expression compared with AG and GG individuals. However, we were not able to replicate the associations with COPD or lung function in the other COPD study groups. Conclusions Variants in the NOS genes were not associated with lung function or COPD status. However, the G allele of rs1800779 resulted in a decrease of NOS3 gene expression and protein levels and this has implications for the numerous disease states that have been associated with this polymorphism.

ABSTRACT Human seminal vesicles present basal release of epithelium‐derived 6‐nitrodopamine (6‐ND) and this catecholamine potentiates noradrenaline‐induced contractions. Since nitric oxide synthase (NOS) activation is a determining factor involved in 6‐ND biosynthesis, this study aimed to investigate which NOS isoform is responsible for the 6‐ND release in mouse isolated seminal vesicles (MISV). Male control, eNOS−/−, NOS−/−, iNOS−/−, and e/n/iNOS−/− mice were used. 6‐ND release was evaluated by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). MISV contractility was assessed following electric‐field stimulation (EFS) or construction of concentration‐response curves to catecholamines. MISV from control, eNOS−/−, nNOS−/−, iNOS−/−, and e/n/iNOS−/− mice exhibited basal release of 6‐ND, but 6‐ND release from eNOS−/− and e/n/iNOS−/− was significantly reduced compared to control and nNOS−/− mice. Epithelium removal in MISV from control mice significantly reduced 6‐ND levels. EFS (2–32 Hz) induced frequency‐dependent MISV contractions in all groups, but the contractions from eNOS−/− and e/n/iNOS−/− mice were significantly decreased compared to control groups. In vitro l‐NAME treatment or epithelium removal significantly reduced EFS‐induced contractions. Pre‐incubation of MISV with 6‐ND (1–100 nM) significantly potentiated both EFS‐ and noradrenaline‐induced contractions, whereas pre‐incubation with noradrenaline, adrenaline, and dopamine did not affect the EFS‐induced responses. Immunohistochemistry and fluorescence in situ hybridization (FISH) assays revealed positivity for tyrosine hydroxylase and eNOS in the epithelium and endothelium of the seminal vesicles. In conclusion, MISV releases epithelium‐derived 6‐ND, and its biosynthesis involves eNOS activation. The finding that 6‐ND markedly amplified the noradrenaline‐induced contractions indicates that epithelium‐derived 6‐ND acts as a major modulator of MISV contractility.