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. Significance Current one drug–one target–one disease approaches in drug discovery have become increasingly inefficient. Network pharmacology defines disease mechanisms as networks best targeted by multiple, synergistic drugs. Using the high unmet medical need indication stroke, we here develop an integrative in silico approach based on a primary target, NADPH oxidase type 4, to identify a mechanistically related cotarget, NO synthase, for network pharmacology. Indeed, we validate both in vivo and in vitro, including humans, that both NOX4 and NOS inhibition is highly synergistic, leading to a significant reduction of infarct volume, direct neuroprotection, and blood–brain-barrier stabilization. This systems medicine approach provides a ground plan to decrease current failure in the field by being implemented in other complex indications.

Dysfunctional reactive oxygen species (ROS) signaling is considered an important disease mechanism. Therapeutically, non-selective scavenging of ROS by antioxidants, however, has failed in multiple clinical trials to provide patient benefit. Instead, pharmacological modulation of disease-relevant, enzymatic sources of ROS appears to be an alternative, more promising and meanwhile successfully validated approach. With respect to targets, the family of NADPH oxidases (NOX) stands out as main and dedicated ROS sources. Validation of the different NOX isoforms has been mainly through genetically modified rodent models and is lagging behind in other species. It is unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five widely used NOX inhibitors that isoform selectivity can be achieved, although individual compound specificity is as yet insufficient. NOX1 was most potently (IC₅₀) targeted by ML171 (0.1 μM); NOX2, by VAS2870 (0.7 μM); NOX4, by M13 (0.01 μM) and NOX5, by ML090 (0.01 μM). In addition, some non-specific antioxidant and assay artefacts may limit the interpretation of data, which included, surprisingly, the clinically advanced NOX inhibitor, GKT136901. In a human ischemic blood-brain barrier hyperpermeability model where genetic target validation is not an option, we provide proof-of-principle that pharmacological target validation for different NOX isoforms is possible by applying an inhibitor panel at IC₅₀ concentrations. Moreover, our findings encourage further lead optimization and development efforts for isoform-selective NOX inhibitors in different indications.

Reactive oxygen species (ROS) have been mainly viewed as unwanted by-products of cellular metabolism, oxidative stress, a sign of a cellular redox imbalance, and potential disease mechanisms, such as in diabetes mellitus (DM). Antioxidant therapies, however, have failed to provide clinical benefit. This paradox can be explained by recent discoveries that ROS have mainly essential signaling and metabolic functions and evolutionally conserved physiological enzymatic sources. Disease can occur when ROS accumulate in nonphysiological concentrations, locations, or forms. By focusing on disease-relevant sources and targets of ROS, and leaving ROS physiology intact, precise therapeutic interventions are now possible and are entering clinical trials. Their outcomes are likely to profoundly change our concepts of ROS in DM and in medicine in general.

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 Nox5 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. Nox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.

Background: Most human disease definitions, except for rare and communicable diseases, are based on symptoms in specific organs, not on causal molecular mechanisms. This limits treatments to imprecise symptomatic approaches with high numbers needed to treat. Systems medicine, instead, has a holistic approach and defines diseases in an organ‐agnostic manner on the basis of associated risk genes, their encoded proteins, and protein–protein interactions. Dysregulation of such disease modules is best corrected by multitarget, synergistic network pharmacology. Here we test this principle in acute ischemic stroke, a highly unmet medical indication without any approved neuroprotective drug so far. Methods: We extend 3 validated risk genes, neuronal nitric oxide synthase ( NOS1 ), NADPH oxidase 5 ( NOX5 ), and soluble guanylate cyclase ( sGC ), to a single disease module. For preclinical validation, we used C57/Bl6 mice and humanized NOX5 –knock‐in mice because NOX5 is not present in the mouse genome despite playing a key role in early stroke. Because up to 70% of patients with stroke have diabetes or prediabetes as an aggravating comorbidity, we also induced diabetes in these mice to model the increased clinical risk for hemorrhagic transformation. Results: We found that a triple‐drug combination of a NOX inhibitor, a nitric oxide synthase inhibitor, and an sGC activator reduced infarct size and, in diabetic animals, also prevented hemorrhagic transformation. Reducing each individual compound dose to subthreshold levels still resulted in full protection when combined, typical for supra‐additive network pharmacology. To examine clinical safety, 3 drugs, either marketed for sGC or repurposed for nitric oxide synthase and NADPH oxidase, were administered to healthy volunteers in a phase I trial. Conclusions: Our data establish that a mechanism‐based network pharmacology approach is effective and clinically safe, warranting a currently ongoing first‐in‐class neuroprotective phase II interventional trial. Registration: URL: https://clinicaltrials.gov/study/NCT05762146?term=repo‐stroke&rank=1 ; Unique Identifier: NCT05762146.