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The 1998 Nobel Prize in Medicine and Physiology for the discovery of nitric oxide, a nitrogen containing reactive oxygen species (also termed reactive nitrogen or reactive nitrogen/oxygen species) stirred great hopes. Clinical applications, however, have so far pertained exclusively to the downstream signaling of cGMP enhancing drugs such as phosphodiesterase inhibitors and soluble guanylate cyclase stimulators. All clinical attempts, so far, to inhibit NOS have failed even though preclinical models were strikingly positive and clinical biomarkers correlated perfectly. This rather casts doubt on our current way of target identification in drug discovery in general and our way of patient stratification based on correlating but not causal biomarkers or symptoms. The opposite, NO donors, nitrite and enhancing NO synthesis by eNOS/NOS3 recoupling in situations of NO deficiency, are rapidly declining in clinical relevance or hold promise but need yet to enter formal therapeutic guidelines, respectively. Nevertheless, NOS inhibition in situations of NO overproduction often jointly with enhanced superoxide (or hydrogen peroxide production) still holds promise, but most likely only in acute conditions such as neurotrauma (Stover et al., J Neurotrauma 31(19):1599-1606, 2014) and stroke (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019). Conversely, in chronic conditions, long-term inhibition of NOS might be too risky because of off-target effects on eNOS/NOS3 in particular for patients with cardiovascular risks or metabolic and renal diseases. Nitric oxide synthases (NOS) and their role in health (green) and disease (red). Only neuronal/type 1 NOS (NOS1) has a high degree of clinical validation and is in late stage development for traumatic brain injury, followed by a phase II safety/efficacy trial in ischemic stroke. The pathophysiology of NOS1 (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016) is likely to be related to parallel superoxide or hydrogen peroxide formation (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 114(46):12315-12320, 2017; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019) leading to peroxynitrite and protein nitration, etc. Endothelial/type 3 NOS (NOS3) is considered protective only and its inhibition should be avoided. The preclinical evidence for a role of high-output inducible/type 2 NOS (NOS2) isoform in sepsis, asthma, rheumatic arthritis, etc. was high, but all clinical development trials in these indications were neutral despite target engagement being validated. This casts doubt on the role of NOS2 in humans in health and disease (hence the neutral, black coloring).

Reactive oxygen species (ROS) are ubiquitous metabolic products and important cellular signaling molecules that contribute to several biological functions. Pathophysiology arises when ROS are generated either in excess or in cell types or subcellular locations that normally do not produce ROS or when non-physiological types of ROS (e.g., superoxide instead of hydrogen peroxide) are formed. In the latter scenario, antioxidants were considered as the apparent remedy but, clinically, have consistently failed and even sometimes induced harm. The obvious reason for that is the non-selective ROS scavenging effects of antioxidants which interfere with both qualities of ROS, physiological and pathological. Therefore, it is essential to overcome this "antidote or neutralizer" strategy. We here review the most promising alternative approach by identifying the disease-relevant enzymatic sources of ROS, target these selectively, but leave physiological ROS signaling through other sources intact. Among all ROS sources, NADPH oxidases (NOX1-5 and DUOX1-2) stand out as their sole function is to produce ROS, whereas most other enzymatic sources only produce ROS as a by-product or upon biochemical uncoupling or damage. This qualifies NOXs as the main potential drug-target candidates in diseases associated with dysfunction in ROS signaling. As a reflection of this, the development of several NOX inhibitors has taken place. Recently, the WHO approved a new stem, "naxib," which refers to NADPH oxidase inhibitors, and thereby recognized NOX inhibitors as a new therapeutic class. This has been announced while clinical trials with the first-in-class compound, setanaxib (initially known as GKT137831) had been initiated. We also review the differences between the seven NOX family members in terms of structure and function in health and disease and then focus on the most advanced NOX inhibitors with an exclusive focus on clinically relevant validations and applications. Therapeutically relevant NADPH oxidase isoforms type 1, 2, 4, and 5 (NOX1, NOX2, NOX4, NOX5). Of note, NOX5 is not present in mice and rats and thus pre-clinically less studied. NOX2, formerly termed gp91

Most diseases are defined by a symptom, not a mechanism. Consequently, therapies remain symptomatic. In reverse, many potential disease mechanisms remain in arbitrary search for clinical relevance. Reactive oxygen species (ROS) are such an example. It is an attractive hypothesis that dysregulation of ROS can become a disease trigger. Indeed, elevated ROS levels of various biomarkers have been correlated with almost every disease, yet after decades of research without any therapeutic application. We here present a first systematic, non-hypothesis-based approach to transform this field as a proof of concept for biomedical research in general. We selected as seed proteins 9 families with 42 members of clinically researched ROS-generating enzymes, ROS-metabolizing enzymes or ROS targets. Applying an unbiased network medicine approach, their first neighbours were connected, and, based on a stringent subnet participation degree (SPD) of 0.4, hub nodes excluded. This resulted in 12 distinct human interactome-based ROS signalling modules, while 8 proteins remaining unconnected. This ROSome is in sharp contrast to commonly used highly curated and integrated KEGG, HMDB or WikiPathways. These latter serve more as mind maps of possible ROS signalling events but may lack important interactions and often do not take different cellular and subcellular localization into account. Moreover, novel non-ROS-related proteins were part of these forming functional hybrids, such as the NOX5/sGC, NOX1,2/NOS2, NRF2/ENC-1 and MPO/SP-A modules. Thus, ROS sources are not interchangeable but associated with distinct disease processes or not at all. Module members represent leads for precision diagnostics to stratify patients with specific ROSopathies for precision intervention. The upper panel shows the classical approach to generate hypotheses for a role of ROS in a given disease by focusing on ROS levels and to some degree the ROS type or metabolite. Low levels are considered physiological; higher amounts are thought to cause a redox imbalance, oxidative stress and eventually disease. The source of ROS is less relevant; there is also ROS-induced ROS formation, i.e. by secondary sources (see upwards arrow). The non-hypothesis-based network medicine approach uses genetically or otherwise validated risk genes to construct disease-relevant signalling modules, which will contain also ROS targets. Not all ROS sources will be relevant for a given disease; some may not be disease relevant at all. The three examples show (from left to right) the disease-relevant appearance of an unphysiological ROS modifier/toxifier protein, ROS target or ROS source.

Several low molecular weight compounds are capable of activating soluble guanylyl cyclase. Recent evidence suggests that some of these are formed under physiological conditions: the nitric oxide radical, carbon monoxide and the hydroxyl radical. Thus, multiple signal transduction pathways appear to exist that form a family of guanylyl cyclase activating factors and thereby regulate the intracellular cyclic guanosine 3′, 5′-monophosphate level.

Cyclic guanosine 3', 5'-monophosphate (cGMP) plays an integral role in the control of vascular function. Generated from guanylate cyclases in response to the endogenous ligands, nitric oxide (NO) and natriuretic peptides (NPs), cGMP influences a number of vascular cell types and regulates vasomotor tone, endothelial permeability, cell growth and differentiation, as well as platelet and blood cell interactions. Reciprocal regulation of the NO-cGMP and NP-cGMP pathways is evident in the vasculature such that one cGMP generating system may compensate for the dysfunction of the other. Indeed, aberrant cGMP production and/or signalling accompanies many vascular disorders such as hypertension, atherosclerosis, coronary artery disease and diabetic complications. This chapter highlights the main vascular functions of cGMP, its role in disease and the resulting current and potential therapeutic applications. With respect to pulmonary hypertension, heart failure and erectile dysfunction, as well as cGMP signal transduction, the reader is specifically referred to other dedicated chapters.

We have used a polyclonal monospecific antibody to rat cerebellum nitric oxide synthase type I (NOS-I, 160 kD) in combination with reduced NADPH-diaphorase histochemical reaction (NADPH-d) to verify colocalization of both NOS protein and NOS enzymatic activity in the rat spinal cord autonomic system. Strong immunoreactivity (IR) of NOS-I was clearly detected in the four main thoracolumbar autonomic nuclei in spinal cord layers of Rexed's laminae VI, VII and X. In all labelled neurones, NOS-I-IR colocalized with NADPH-d activity, suggesting coexistence of brain-specific NOS-I-like protein and its associated enzyme activity. For these neurones the new term 'nitroxergic' (i.e. NO-generating) neurones appears to be justified. Spinal cord nitroxergic neurones are part of a NO-mediated signal transduction pathway for control of the sympathetic 'outflow' to various peripheral target organs.