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Despite decades of promising preclinical validation and clinical translation, ischemic stroke still remains as one of the leading causes of death and disability worldwide. Within its complex pathophysiological signatures, thrombosis and inflammation, that is, thromboinflammation, are highly interconnected processes leading to cerebral vessel occlusion, inflammatory responses, and severe neuronal damage following the ischemic event. Hence, we here review the most recent updates on thromboinflammatory-dependent mediators relevant after stroke focusing on recent discoveries on platelet modulation, a potential regulation of the innate and adaptive immune system in thromboinflammation, utterly providing a thorough up-to-date overview of all therapeutic approaches currently undergoing clinical trial.

Nitric oxide (NO) is a widely distributed gasotransmitter binding to the heme of soluble guanylate cyclase (sGC) to stimulate the formation of the second messenger cyclic GMP. In disease, two pathomechanisms can occur, scavenging of NO by reactive oxygen species so that sGC is insufficiently stimulated, and oxidative damage of sGC resulting in heme loss and a NO-insensitive apo-form of sGC. To address this therapeutically, two pharmacological principles have entered the clinic or clinical development. sGC stimulators (sGCs) allosterically sensitize sGC for lower NO levels to reach physiological cGMP levels again; sGC activators (sGCa) bind to the empty heme pocket of apo-sGC to recover cGMP formation. Here we show in vitro and in vivo that this dichotomic pharmacology of sGCs/sGC and sGCa/apo-sGC is wrong. Instead apo-sGC is both activated by sGCa and allosterically modulated by sGC stimulators. The sGCs binding site in sGC and apo-sGC, however, is redox sensitive explaining why it has been previously missed in apo-sGC preparations generated for example by using heme oxidants such as 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Moreover, sGCs synergised with sGCa on apo-sGC in a supra-additive manner similar to the synergy between NO and sGCs on sGC. Our findings provide functional evidence including ischemic stroke, where apo-sGC rather than sGC is detectable, for identical modulation of sGC and apo-sGC by sGCs. This necessitates a revision of the dichotomic sGC/apo-sGC pharmacology and expands the therapeutic potential of sGCs and sGCa both individually and in combination.

Noncommunicable diseases (NCDs) have become globally abundant, yet the therapeutics we use for them are imprecise. In parallel, identifying new treatments has become more costly than ever due to the ever-aggravating efficacy crisis drug discovery faces. What unites these failures is our ontological classification of diseases, primarily based on descriptive terms. To achieve precision diagnosis and precision therapy in clinical practice, NCDs need to be redefined and subdivided based on their causal molecular mechanisms. However, the inconsistency and incompatibility of the current disease classification systems hinder data integration and analysis towards the characterization of such mechanisms. Here, we explain flaws in the current disease definitions and the dispersion among existing ontologies with the aim of establishing a mechanism-based classification of diseases hence, precision medicine.

Nitric oxide (NO) is a widely distributed gasotransmitter binding to the heme of soluble guanylate cyclase (sGC) to stimulate the formation of the second messenger cyclic GMP. In disease, two pathomechanisms can occur, scavenging of NO by reactive oxygen species so that sGC is insufficiently stimulated, and oxidative damage of sGC resulting in heme loss and a NO-insensitive apo-form of sGC. To address this therapeutically, two pharmacological principles have entered the clinic or clinical development. sGC stimulators (sGCs) allosterically sensitize sGC for lower NO levels to reach physiological cGMP levels again; sGC activators (sGCa) bind to the empty heme pocket of apo-sGC to recover cGMP formation. Here we show in vitro and in vivo that this dichotomic pharmacology of sGCs/sGC and sGCa/apo-sGC is wrong. Instead apo-sGC is both activated by sGCa and allosterically modulated by sGC stimulators. The sGCs binding site in sGC and apo-sGC, however, is redox sensitive explaining why it has been previously missed in apo-sGC preparations generated for example by using heme oxidants such as 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Moreover, sGCs synergised with sGCa on apo-sGC in a supra-additive manner similar to the synergy between NO and sGCs on sGC. Our findings provide functional evidence including ischemic stroke, where apo-sGC rather than sGC is detectable, for identical modulation of sGC and apo-sGC by sGCs. This necessitates a revision of the dichotomic sGC/apo-sGC pharmacology and expands the therapeutic potential of sGCs and sGCa both individually and in combination.

Rhian M. Touyz, Associate Editor, Reactive oxygen species (ROS) have been correlated with almost every human disease. Yet clinical exploitation of these hypotheses by pharmacological modulation of ROS has been scarce to nonexistent. Are ROS, thus, irrelevant for disease? No. One key misconception in the ROS field has been its consideration as a rather detrimental metabolic by-product of cell metabolism, and thus, any approach eliminating ROS to a certain tolerable level would be beneficial. We now know, instead, that ROS at every concentration, low or high, can serve many essential signaling and metabolic functions. This likely explains why systemic, nonspecific antioxidants have failed in the clinic, often with neutral and sometimes even detrimental outcomes. Recently, drug development has focused, instead, on identifying and selectively modulating ROS enzymatic sources that in a given constellation cause disease while leaving ROS physiologic signaling and metabolic functions intact. As sources, the family of NADPH oxidases stands out as the only enzyme family solely dedicated to ROS formation. Selectively targeting disease-relevant ROS-related proteins is already quite advanced, as evidenced by several phase II/III clinical trials and the first drugs having passed registration. The ROS field is expanding by including target enzymes and maturing to resemble more and more modern, big data–enhanced drug discovery and development, including network pharmacology. By defining a disease based on a distinct mechanism, in this case ROS dysregulation, and not by a symptom or phenotype anymore, ROS pharmacology is leaping forward from a clinical underperformer to a proof of concept within the new era of mechanism-based precision medicine., This study was supported by the European Research Council Advanced Investigator Grant/RadMed [Grant 294683], Proof-of-Concept Grant/SAVEBRAIN [Grant 737586], and H2020 Project REPO-TRIAL [Grant 777111] (to H.H.H.W.S.) and short-term scientific missions by the COST Actions EU-ROS and Open-MultiMed, and Kootstra Talented Fellowship (Maastricht University) (to A.I.C.).

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 mu M); NOX2, by VAS2870 (0.7 mu M); NOX4, by M13 (0.01 mu M) and NOX5, by ML090 (0.01 mu M). 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.

BACKGROUND Post-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit. METHODS WT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. RESULTS We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration. CONCLUSIONS These findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.

For complex diseases, most drugs are highly ineffective, and the success rate of drug discovery is in constant decline. While low quality, reproducibility issues, and translational irrelevance of most basic and preclinical research have contributed to this, the current organ-centricity of medicine and the ‘one disease–one target–one drug’ dogma obstruct innovation in the most profound manner. Systems and network medicine and their therapeutic arm, network pharmacology, revolutionize how we define, diagnose, treat, and, ideally, cure diseases. Descriptive disease phenotypes are replaced by endotypes defined by causal, multitarget signaling modules that also explain respective comorbidities. Precise and effective therapeutic intervention is achieved by synergistic multicompound network pharmacology and drug repurposing, obviating the need for drug discovery and speeding up clinical translation.

Background and purpose Ischaemic stroke is a leading cause of death, disability, and a high unmet medical need. Post-reperfusion inflammation and an up-regulation of toll-like receptor 4 (TLR4), an upstream sensor of innate immunity, are associated with poor outcome in stroke patients. Here, we identified the therapeutic effect of targeting the LPS/TLR4 signal transduction pathway. Experimental approach We tested the effect of the TLR4 inhibitor, eritoran (E5564) in different in vitro ischaemia-related models: human organotypic cortex culture, rat organotypic hippocampal cultures, and primary mixed glia cultures. We explored the therapeutic window of E5564 in the transient middle cerebral artery occlusion model of cerebral ischaemia in mice. Key results In vivo, administration of E5564 1 and 4 hr post-ischaemia reduced the expression of different pro-inflammatory chemokines and cytokines, infarct volume, blood-brain barrier breakdown, and improved neuromotor function, an important clinically relevant outcome. In the human organotypic cortex culture, E5564 reduced the activation of microglia and ROS production evoked by LPS. Conclusion and implications TLR4 signalling has a causal role in the inflammation associated with a poor post-stroke outcome. Importantly, its inhibition by eritoran (E5564) provides neuroprotection both in vitro and in vivo, including in human tissue, suggesting a promising new therapeutic approach for ischaemic stroke.

Background and purposePost-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit.MethodsWT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining.ResultsWe identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration.ConclusionsThese findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.

Approximately 44 million people worldwide live with Alzheimer’s disease (AD) or a related form of dementia. Aggregates of the microtubule-associated protein tau are a common marker of these neurodegenerative diseases collectively termed as tauopathies. However, all therapeutic attempts based on tau have failed, suggesting that tau may only indicate a higher-level causal mechanism. For example, increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation. Here we show that type 4 NADPH oxidase (NOX), the most abundant isoform of the only dedicated reactive oxygen producing enzyme family, is upregulated in dementia and AD patients and in a humanized mouse model of tauopathy. Both global knockout and neuronal knockdown of theNox4gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP). Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevented cognitive decline, suggesting a direct and causal role for neuronal NOX4. Thus, NOX4 is a previously unrecognized causal, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.Graphical abstract

Palladium-based electrocatalysts are widely used in alkaline direct alcohol fuel cells. The synthesis and characterization of carbon-supported bimetallic nanoparticles (NP) of AuPd and AgPd is described using pecan nutshell extract (Carya illinoinensis) which serves as both, reducing and the stabilizing agent. This environmentally friendly route generates bimetallic NP for a wide range of applications, including electrocatalysis; since particularly AuPd NP proved to be a potentially suitable electrode material for alkaline direct methanol fuel cells. The electrocatalytic activity of these nanomaterials was comparable to commercially available Pd/C 1% in the electro-oxidation of methanol in alkaline media.

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., 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.

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calciumactivated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenationinduced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.

Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monotherapy for the management of depression. A recent study by our group demonstrated that agmatine induces Nrf2 and protects against corticosterone effects in a hippocampal neuronal cell line. The present study is an extension of this previous study by assessing the antidepressant-like effect of agmatine in an animal model of depression induced by corticosterone in mice. Swiss mice were treated simultaneously with agmatine or imipramine at a dose of 0.1 mg/kg/day (p.o.) and corticosterone for 21 days and the daily administrations of experimental drugs were given immediately prior to corticosterone (20 mg/kg/day, p.o.) administrations. Wild-type C57BL/6 mice (Nrf2 (+/+)) and Nrf2 KO (Nrf2 (−/−)) were treated during 21 days with agmatine (0.1 mg/kg/day, p.o.) or vehicle. Twenty-four hours after the last treatments, the behavioral tests and biochemical assays were performed. Agmatine treatment for 21 days was able to abolish the corticosterone-induced depressive-like behavior and the alterations in the immunocontent of mature BDNF and synaptotagmin I, and in the serotonin and glutamate levels. Agmatine also abolished the corticosterone-induced changes in the morphology of astrocytes and microglia in CA1 region of hippocampus. In addition, agmatine treatment in control mice increased noradrenaline, serotonin, and dopamine levels, CREB phosphorylation, mature BDNF and synaptotagmin I immunocontents, and reduced pro-BDNF immunocontent in the hippocampus. Agmatine’s ability to produce an antidepressant-like effect was abolished in Nrf2 (−/−) mice. The present results reinforce the participation of Nrf2 in the antidepressant-like effect produced by agmatine and expand literature data concerning its mechanisms of action., This work was supported by CNPq, CAPES/PDSE, CAPES/PROCAD, Núcleo de Excelência em Neurociências Aplicadas de Santa Catarina (NENASC) Project/ PRONEX Program CNPq/FAPESC (Brazil) to ALSR. The 8th Convocatoria de proyectos de Cooperación Interuniversitaria UAM-Santander con America Latina, Spanish Ministry of Economy and Competence Ref. SAF2012-32223 to MGL and ALSR. This work was also supported by FIS CA12/00122 to ARN and FPU12/02220 to AW.

Unphysiological reactive oxygen species (ROS) formation is considered an important pathomechanism for several disease phenotypes with high unmet medical need. Therapeutically, antioxidants have failed multiple times. Instead, focusing on only disease-relevant, enzymatic sources of ROS appears to be a more promising and highly validated approach. Here the family of five NADPH oxidases (NOX) stands out as drug targets. Validation has been restricted, however, mainly to genetically modified rodents and is lacking in other species including human. It is thus unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five of the most advanced NOX inhibitors that indeed isoform selectivity can be achieved. 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). Conditions need to be carefully controlled though as previously unrecognized non-specific antioxidant and assay artefacts may limit the interpretation of data and this included, surprisingly, one of the most advanced NOX inhibitors, GKT136901. As proof-of-principle that now also pharmacological and non-rodent target validation of different NOX isoforms is possible, we used a human blood-brain barrier model and NOX inhibitor panel at IC50 concentrations. The protective efficacy pattern of this panel confirmed the predominant role of NOX4 in stroke from previous genetic models. Our findings strongly encourage further lead optimization efforts for isoform-selective NOX inhibitors and clinical development and provide an experimental alternative when genetic validation of a NOX isoform is not an option. Graphical abstract

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic options. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain-barrier. Its mechanism, however, is unknown. Here we identify type 5 NADPH oxidase (NOX5), a calcium-activated, reactive oxygen species (ROS)-forming enzyme as missing link. Using a humanised knock-in mouse model and in vitro in organotypic cultures, we find re-oxygenation or calcium overload to increase brain ROS levels in a NOX5-dependent manner. In vivo, post-ischemic ROS formation, infarct volume and functional outcomes were worsened in NOX5 knock-in mice. Of clinical and therapeutic relevance, in a human blood-barrier model pharmacological NOX inhibition also prevented acute re-oxygenation induced leakage. Our data therefore identify NOX5 as sufficient to induce acute post-reperfusion calcium-dependent blood-brain-barrier breakdown. We suggest urgent clinical validation by conducting protective post-stroke re-canalisation in the presence of a NOX inhibitor.

Protein tyrosine nitration is a hallmark of oxidative stress related disease states, commonly detected as anti-nitrotyrosine immunoreactivity. The precise reactive oxygen sources, mechanisms of nitration as well as the modified target proteins and functional consequences, however, remain often unclear. Here we explore protein tyrosine nitration under basal conditions and find surprisingly physiologically nitrated proteins. Upon purifying a prominent physiologically nitrotyrosine immunopositive in hearts from mouse, rat and pig, we identify it as lactate dehydrogenase (LDH). Mechanistically, LDH’s degree of basal nitration depended on two canonical sources, NO synthase (NOS) and myeloperoxidase (MPO), respectively. When validating the nitrated amino acid by MALDI-TOF mass spectrometry, we, surprisingly, located LDH nitration not to a tyrosine but the C-terminal tryptophan, Trp324. Molecular dynamics simulations suggested that Trp324 nitration restricts the interaction of the active site loop with the C-terminal α-helix essential for activity. This prediction was confirmed by enzyme kinetics revealing an apparent lower Vmax of nitrated LDH, although yet unidentified concurrent oxidative modifications may contribute. Protein nitration is, thus, not a by definition disease marker but reflects also physiological signaling by eNOS/NO, MPO/nitrite and possibly other pathways. The commonly used assay of anti-nitrotyrosine immunoreactivity is apparently cross-reactive to nitrotryptophan requiring a reevaluation of the protein nitration literature. In the case of LDH, nitration of Trp324 is aggravated under cardiac metabolic stress conditions and functionally limits maximal enzyme activity. Trp324-nitrated LDH may serve both as a previously not recognized disease biomarker and possibly mechanistic lead to understand the metabolic changes under these conditions.

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease–disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy., Systems based pharmacology: drug repurposing by diseasome Systems medicine utilizes common genetic origins and co-morbidities to uncover mechanistic links between diseases, which are summarized in the diseasome. Shared pathomechanisms may also allow for drug repurposing within these disease clusters. Here, Schmidt and co-workers show indeed that, based on this principle, a cardio-pulmonary drug can be surprisingly repurposed for a previously not recognised application as a direct neuroprotectant. They find that the cyclic GMP forming soluble guanylate cyclase becomes dysfunctional upon stroke but regains catalytic activity in the presence of specific activator compounds. This new mechanism-based therapy should be urgently validated clinically as a possible first-in-class treatment in stroke.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

Therapies that modulate cyclic guanosine-30-50-monophosphate (cGMP) have emerged as one of the most successful areas in recent drug discovery and clinical pharmacology. Historically, their focus has been on cardiovascular disease phenotypes; however, cGMP's relevance is likely to go beyond this rather limited organ-based set of indications. Moreover, the multitude of targets and their apparent interchangeability is a proof-of-concept of network pharmacology.

Significance Why the brain is uniquely sensitive to hypoxia and which cells are involved is incompletely understood. Here we identify that, upon ischemic stroke, in endothelial cells and neurons the reactive oxygen-forming NADPH oxidase 4 (NOX4) causes breakdown of the BBB and neuronal cell death. This mechanism is unique to the brain and not found in other forms of ischemia in the body. Genetic deletion of either cell type (endothelial or neuronal) or pharmacological inhibition of NOX4 leads to a significant reduction of infarct volume and direct neuroprotection. This mechanism explains the unique vulnerability of the hypoxic brain compared with other organs and provides a clear rationale for first-in-class neuroprotective therapies in stroke., Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood–brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

Promising results have been reported in preclinical stroke target validation for pharmacological principles that disrupt the N-methyl-D-aspartate receptor–post-synaptic density protein-95–neuronal nitric oxide synthase complex. However, post-synaptic density protein-95 is also coupled to potentially neuroprotective mechanisms. As post-synaptic density protein-95 inhibitors may interfere with potentially neuroprotective mechanisms and sufficient validation has often been an issue in translating basic stroke research, we wanted to close that gap by comparing post-synaptic density protein-95 inhibitors with NOS1−/− mice and a NOS inhibitor. We confirm the deleterious role of NOS1 in stroke both in vivo and in vitro, but find three pharmacological post-synaptic density protein-95 inhibitors to be therapeutically ineffective.

Analysis of neurotransmitters and their metabolites is useful for the diagnosis of central nervous system diseases. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation was developed to monitor levels of adrenaline (AD), noradrenaline (NA), glutamic acid (Glu), gamma-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in rat brain tissue. Isoprenaline was used as an internal standard (IS). Neurotransmitters and metabolites were eluted with a reverse phase column under gradient conditions through a mobile phase consisting of 0.2% formic acid water solution/acetonitrile. The compounds were detected and quantified by LC-MS/MS with positive or negative electrospray ionization, which operates in multiple-reaction monitoring mode. The method was linear or polynomial (R-2 > 0.99) for AD, NA, Glu, GABA, DA, 5-HT, 5-HIAA, and MHPG in the range of 0.25-200, 0.5-200, 250-20,000, 250-20,000, 0.25-200, 10-3000, 1-50, and 1-50 ng/mL, respectively. The validation assays for accuracy and precision, matrix effect, extraction recovery, stability and carry-over of the samples for neurotransmitters and metabolites were consistent with the requirements of regulatory agencies. The method enables rapid quantification of neurotransmitters and their metabolites and has been applied in the nuclear factor (erythroid 2-derived)-like 2 (Nrf2) knockout mouse model of depression.

The data presented in this article supports the rat brain sample pre- parathion procedure previous to its injection into the liquid chroma- tography–tandem mass spectrometry(LC–MS/MS) system to monitor levels of adrenaline, noradrenaline, glutamicacid, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine, 5-hydroxyindoleaceticacid, and 3-methoxy-4-hydroxyphenylglycol.In addition,we describe the method validation assays (such as calibration curve, lower limit of quantification, precision and accuracy intra-and inter-day,selectivity, extraction recovery and matrix effect, stability,and carry-over effect) according to the United States Food and Drug Administration and European Medicine Agency to measure in one step different neuro- transmitters and their metabolites.The data supplied in this articleis related to the research study entitled: “Simultaneous determination of 8 neurotransmitters and their metabolite levels in rat brainusing liquid chromatography in tandem with mass spectrometry: application to the murine Nrf2 model of depression” (Wojniczetal.2016), The authors wish to thank to Instituto-Fundación Teófilo Hernando for itscontinued financial support. This dataset was also supported by “FIS No.CA12/00122” to ARN ,Minstry of Education, Culture and Sport “FPU12/02220” to AW and the Ministry of Economy and Competitiveness “SAF2012-32223” to MGL

This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License.-- et al., [Significance]: Oxidative stress is suggested to be a disease mechanism common to a wide range of disorders affecting human health. However, so far, the pharmacotherapeutic exploitation of this, for example, based on chemical scavenging of pro-oxidant molecules, has been unsuccessful. [Recent Advances]: An alternative emerging approach is to target the enzymatic sources of disease-relevant oxidative stress. Several such enzymes and isoforms have been identified and linked to different pathologies. For some targets, the respective pharmacology is quite advanced, that is, up to late-stage clinical development or even on the market; for others, drugs are already in clinical use, although not for indications based on oxidative stress, and repurposing seems to be a viable option. [Critical Issues]: For all other targets, reliable preclinical validation and drug ability are key factors for any translation into the clinic. In this study, specific pharmacological agents with optimal pharmacokinetic profiles are still lacking. Moreover, these enzymes also serve largely unknown physiological functions and their inhibition may lead to unwanted side effects. [Future Directions]: The current promising data based on new targets, drugs, and drug repurposing are mainly a result of academic efforts. With the availability of optimized compounds and coordinated efforts from academia and industry scientists, unambiguous validation and translation into proof-of-principle studies seem achievable in the very near future, possibly leading towards a new era of redox medicine., Several authors of this review were supported by the European Cooperation in Science and Technology (COST Action BM1203/EU-ROS). H.H.H.W.S. is the recipient of an ERC Advanced Grant and Marie-Curie IRG and co-leads a EUROSTARS program. R.S. is supported by a Senior Principal Research Fellowship from the National Health and Medical Research Council of Australia N.K. is supported by an EFSD/Sanofi Award.

SIGNIFICANCE\ud \ud Enhanced levels of reactive oxygen species (ROS) have been associated with different disease states. Most attempts to validate and exploit these associations by chronic antioxidant therapies have provided disappointing results. Hence, the clinical relevance of ROS is still largely unclear.\ud \ud RECENT ADVANCES\ud \ud We are now beginning to understand the reasons for these failures, which reside in the many important physiological roles of ROS in cell signaling. To exploit ROS therapeutically, it would be essential to define and treat the disease-relevant ROS at the right moment and leave physiological ROS formation intact. This breakthrough seems now within reach.\ud \ud CRITICAL ISSUES\ud \ud Rather than antioxidants, a new generation of protein targets for classical pharmacological agents includes ROS-forming or toxifying enzymes or proteins that are oxidatively damaged and can be functionally repaired.\ud \ud FUTURE DIRECTIONS\ud \ud Linking these target proteins in future to specific disease states and providing in each case proof of principle will be essential for translating the oxidative stress concept into the clinic. Antioxid. Redox Signal. 23, 1171-1185.

Based on non-hypothesis-driven approaches genetic evidence suggests that diseases are interrelated differently to our current organ-based ontology. In fact, common effector mechanisms, when affected or triggered seem to produce pathophenotypes in diverse organs or co-morbidities. This will lead eventually to a revised disease nomenclature and opens up entirely new approaches for diagnosis and treatment. In this context, we noted that a common cardiovascular target, the cGMP-forming Fe(II) haem protein, soluble guanylate cyclase (sGC), appears to be situated in a common mechanism network that is prominently relevant to stroke. Ischemic stroke is the second leading cause of death worldwide and the leading cause of disability. Despite this high medical need only a single drug is available but due to its limited time window and risk of bleeding 85% of all patients are excluded from treatment. As a possible add-on, vasoactive drugs however typically dilate normal blood vessels and cause a paradoxical “steal phenomenon” by both shunting blood to healthy vascular beds and a systemic blood pressure drop. Upon middle cerebral artery occlusion sGC protein and nitric oxide-stimulated activity in the ischemic hemisphere were dramatically down-regulated leading to a high proportion of oxidized and/or haem-free apo-sGC activity. Pharmacological targeting of apo-sGC in vitro under oxygen and glucose deprivation conveyed strong neuroprotection via ERK/CREB signalling. In vivo, post-stroke apo-sGC activation by two distinct members of this compound class augmented cerebral blood-flow whilst leaving systemic blood pressure unaffected, reduced infarct size and increased survival. Different apo-sGC activators are in advanced stages of clinical development for different cardiovascular indications. Systems biology and network medicine and our preliminary target validation suggest that they should be urgently tested for repurposing as first-in-class, mechanism-based neuroprotective drugs in stroke.

Ischemic stroke is the second leading cause of death worldwide and the leading cause of disability. Despite this high medical need only a single drug is available but due to its limited time window and risk of bleeding 85% of all patients are excluded from treatment. Here we address three different new targets in stroke highly related to oxidative stress. NADPH oxidase is one of the most important sources of reactive oxygen species, which has been recently considered as a promising target in ischemic stroke. Neuronal nitric oxide synthase (NOS1) has been also suggested as a possible target so that its partial inhibition can lead to neuroprotective effects. Similarly, NOX possibly acts by scavenging or toxifying NO to nitrating species and oxidising the NO receptor, soluble guanylate cyclase (sGC). This then diminishes physiological NO-cGMP signalling, a process that could be reversed in mice by so-called sGC activator compounds. Pharmacological targeting of apo-sGC in vitro under oxygen and glucose deprivation conveyed strong neuroprotection via PKG/ERK/CREB signalling pathway. In vivo, post-stroke apo-sGC activation by two distinct members of this compound class augmented cerebral blood-flow whilst leaving systemic blood pressure unaffected, reduced infarct size and increased survival. Thus, both inhibiting NOX/NOS-dependent oxidative stress and augmenting cGMP signalling are neuroprotective in stroke. Current and future experiments are aimed at validating these finding in a second rodent species for further preclinical and clinical development as first-in-class neuroprotective drugs.