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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.

Aims To investigate the effect of 16 weeks of aerobic training performed at two different intensities on nitric oxide (tNO x ) availability and iNOS/nNOS expression, oxidative stress (OS) and inflammation in obese humans with or without type 2 diabetes mellitus (T2DM). Methods Twenty-five sedentary, obese (BMI > 30 kg/m 2 ) males (52.8 ± 7.2 years); 12 controls versus 13 T2DM were randomly allocated to four groups that exercised for 30 min, three times per week either at low (Fat-Max; 30–40 % V O 2max ) or moderate ( T vent ; 55–65 % V O 2max ) intensity. Before and after training, blood and muscle samples (v. lateralis) were collected. Results Baseline erythrocyte glutathione was lower (21.8 ± 2.8 vs. 32.7 ± 4.4 nmol/ml) and plasma protein oxidative damage and IL-6 were higher in T2DM (141.7 ± 52.1 vs. 75.5 ± 41.6 nmol/ml). Plasma catalase increased in T2DM after T vent training (from 0.98 ± 0.22 to 1.96 ± 0.3 nmol/min/ml). T2DM groups demonstrated evidence of oxidative damage in response to training (elevated protein carbonyls). Baseline serum tNO x were higher in controls than T2DM (18.68 ± 2.78 vs. 12.34 ± 3.56 μmol/l). Training at T vent increased muscle nNOS and tNO x in the control group only. Pre-training muscle nNOS was higher in controls than in T2DMs, while the opposite was found for iNOS. No differences were found after training for plasma inflammatory markers. Conclusion Exercise training did not change body composition or aerobic fitness, but improved OS markers, especially when performed at T vent . Non-diabetics responded to T vent training by increasing muscle nNOS expression and tNO x levels in skeletal muscle while these parameters did not change in T2DM, perhaps due to higher insulin resistance (unchanged after intervention).

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice—elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using N ω -nitro- l -arginine methyl ester ( l -NAME)—recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S -nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α–XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF. iNOS-driven dysregulation of the IRE1α–XBP1 pathway leads to cardiomyocyte dysfunction in mice and recapitulates the systemic and cardiovascular features of human heart failure with preserved ejection fraction.

NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms— inducible, endothelial, and neuronal NOS—that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.

Polymorphisms in Nos3 increase risk for glaucoma, the leading cause of irreversible blindness worldwide. A key modifiable risk factor for glaucoma is intraocular pressure (IOP), which is regulated by NO - a product of nitric oxide synthase 3 (encoded by Nos3) - in Schlemm's canal of the conventional outflow pathway. We studied the effects of a conditional, endothelial cell-specific postnatal deletion of Nos3 (Endo-SclCre-ERT;Nos3fl/fl) on tissues of the outflow pathway. We observed that Cre-ERT expression spontaneously and gradually increased with time in vascular endothelia including in Schlemm's canal, beginning at P10, with complete Nos3 deletion occurring around P90. Whereas outflow resistance was reduced in global Nos3-KO mice, outflow resistance and IOP in Endo-SclCre-ERT;Nos3fl/fl mice were normal. We observed - coincident with Nos3 deletion - recruitment of macrophages to and induction of both ELAM1 and NOS2 expression by endothelia in the distal portion of the outflow pathway, which increased vessel diameter. These adjustments reduced outflow resistance to maintain IOP in these Endo-SclCre-ERT;Nos3fl/fl mice. Selective inhibition of iNOS by 1400W resulted in narrowing of distal vessels and IOP elevation. Together, the results emphasize the pliability of the outflow system and the importance of NO signaling in IOP control, and imply an substantial role for macrophages in IOP homeostasis.

Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior. Evidence suggests that stress during development might lead to sexual dysfunction. Here, authors show that pubertal stress disrupted female sexual behavior by reducing activation of nitric oxide synthase-expressing neurons in response to male cues.

Nitric oxide (NO) is a ubiquitous gas with free radical groups that is soluble in water, and which is involved in numerous physiological functions including inflammatory and immune responses. However, the role of NO in tumor biology is controversial and misunderstood. NO has been shown to have both anti-cancer and carcinogenic effects, which are dependent on the time, location, and concentration of NO. This duality presents a double challenge to determine the net impact of NO on cancer and to define the therapeutic role of NO-centered anti-cancer strategies. Nevertheless, it is believed that a comprehensive and dynamic understanding of the cascade of molecular and cellular events underlying tumor biology that are affected by NO will allow researchers to exploit the potential anti-tumor properties of drugs that interfere with NO metabolism.

Gastric ulcers occur due to an imbalance between harmful and protective factors. This study aimed to examine the effect of L-arginine on ethanol-induced gastric ulcers in rats. In this study, 30 male Wistar rats were divided into five groups: a control group that received saline, group 1 which was treated with 70% ethanol to induce gastric ulcers, group 2 that received saline along with L-arginine at a dose of 500 mg/kg administered intraperitoneally, group 3 that was given ethanol along with L-arginine at the same dose but administered orally, and group 4 which received ethanol together with omeprazole at a dose of 10 mg/kg administered intraperitoneally. After the ethanol treatment induced ulceration, gastric lesions were evaluated, and assays were performed to measure antioxidant enzyme levels, malondialdehyde (MDA) levels, and the gene expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). Treatment with L-arginine or omeprazole reduced ethanol-induced gastric damage by lowering the ulcer index, increasing gastric pH, improving antioxidant status (decreased MDA, increased SOD and CAT), and modulating NO and iNOS levels. L-arginine showed protective effects comparable to those of omeprazole. This study shows that L-arginine can effectively reduce gastric mucosal injury caused by ethanol, achieving results comparable to those of omeprazole. The protective effects of L-arginine are likely due to its ability to enhance antioxidant defenses, regulate gastric acidity, and modulate nitric oxide pathways. These findings highlight the potential therapeutic role of L-arginine in managing gastric ulcers.

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

The nutraceutical COMP-4 –consisting of L-citrulline, ginger extract, and herbal components Paullinia cupana and muira puama–has been shown previously to stimulate the production of nitric oxide (NO) in a variety of tissue types. We hypothesized that COMP-4 may have a protective, stimulatory effect on the vascular endothelial cell. Human umbilical arterial endothelial cells were incubated for 24 hours with or without COMP-4 and, to replicate impairment of endothelial function, co-incubated with or without H 2 O 2 . NO intracellular content, nitrite formation and cGMP content in culture media, nitric oxide synthase (NOS) isoforms and mRNA content, pro-inflammatory cytokines, and PAI-1 expression and activity were measured. COMP-4 increased endothelial cell production of NO and cGMP and the expression of both endothelial NOS (eNOS) and inducible NOS (iNOS), in tandem with a reduction in cytokine expression and activity of PAI-1. Co-incubation of COMP-4 with H 2 O 2 reversed detrimental effects of H 2 O 2 on endothelial function, evidenced by improvement in NO availability and abrogation of the pro-inflammatory milieu. These results suggest that COMP-4 exerts a stimulatory effect on endothelial cell eNOS and iNOS to increase NO bioavailability, leading to a reduction in pro-inflammatory cytokines, particularly the prothrombotic PAI-1.