Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or “All that you did and did not want to know about Nox inhibitory peptides”

Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms.     

Towards specific NADPH oxidase inhibition by small synthetic peptides

Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase is essential for host defenses against pathogens. ROS are very reactive with biological molecules such as lipids, proteins and DNA, potentially resulting in cell dysfunction and tissue insult. Excessive NADPH oxidase activation and ROS overproduction are believed to participate in disorders such as joint, lung, vascular and intestinal inflammation. NADPH oxidase is a complex enzyme composed of six proteins: gp91phox (renamed NOX2), p22phox, p47phox, p67phox, p40phox and Rac1/2. Inhibitors of this enzyme could be beneficial, by limiting ROS production and inappropriate inflammation. A few small non-peptide inhibitors of NADPH oxidase are currently used to inhibit ROS production, but they lack specificity as they inhibit NADPH oxidase homologues or other unrelated enzymes. Peptide inhibitors that target a specific sequence of NADPH oxidase components could be more specific than small molecules. Here we review peptide-based inhibitors, with particular focus on a molecule derived from gp91phox/NOX2 and p47phox, and discuss their possible use as specific phagocyte NADPH oxidase inhibitors.     

NADPH oxidases as therapeutic targets in ischemic stroke

Reactive oxygen species (ROS) act physiologically as signaling molecules. In pathological conditions, such as ischemic stroke, ROS are released in excessive amounts and upon reperfusion exceed the body's antioxidant detoxifying capacity. This process leads to brain tissue damage during reoxygenation. Consequently, antioxidant strategies have long been suggested as a therapy for experimental stroke, but clinical trials have not yet been able to promote the translation of this concept into patient treatment regimens. As an evolution of this concept, recent studies have targeted the sources of ROS generation-rather than ROS themselves. In this context, NADPH oxidases have been identified as important generators of ROS in the cerebral vasculature under both physiological conditions in general and during ischemia/reoxygenation in particular. Inhibition of NADPH oxidases or genetic deletion of certain NADPH oxidase isoforms has been found to considerably reduce ischemic injury in experimental stroke. This review focuses on recent advances in the understanding of NADPH oxidase-mediated tissue injury in the cerebral vasculature, particularly at the level of the blood-brain barrier, and highlights promising inhibitory strategies that target the NADPH oxidases.     

Targeting NADPH oxidases for the treatment of cancer and inflammation

NADPH oxidases are a family of oxidases that utilize molecular oxygen to generate hydrogen peroxide and superoxide, thus indicating physiological functions of these highly reactive and short-lived species. The regulation of these NADPH oxidases (nox) enzymes is complex, with many members of this family exhibiting complexity in terms of subunit composition, cellular location, and tissue-specific expression. While the complexity of the nox family (Nox1-5, Duox1, 2) is daunting, the complexity also allows for targeting of NADPH oxidases in disease states. In this review, we discuss which inflammatory and malignant disorders can be targeted by nox inhibitors, as well as clinical experience in the use of such inhibitors.     

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.     

Targeting NOX enzymes in pulmonary fibrosis

Oxidative stress has been associated with a number of human fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Oxidative stress is most often defined as an imbalance between the generation of reactive oxygen species (ROS) in excess of the capacity of cells/tissues to detoxify or scavenge them. Additionally, the regulated production of ROS participates in cellular signaling. Therapeutic strategies to treat IPF have, thus far, focused on augmenting anti-oxidant capacity. Recent studies have demonstrated a critical role for ROS-generating enzymatic systems, specifically, NADPH oxidase (NOX) family oxidoreductases in fibrotic processes. In this review, we examine the evidence for NOX isoforms in the generation and perpetuation of fibrosis, and the potential to target this gene family for the treatment of IPF and related fibrotic disorders.     

Regulation and termination of NADPH oxidase activity

NADPH oxidase of phagocytes plays a crucial role in host defense by producing reactive oxygen species (ROS) that are intended to kill invading microbes. Many other cells produce ROS for signaling purposes. The respiratory burst oxidase in human neutrophils is the main but not exclusive subject of this review, because it is archetypical and has been studied most extensively. The activity of this enzyme must be controlled in phagocytes to prevent collateral damage, and in non-phagocytic cells to perform its signaling role. With many stimuli, NADPH oxidase activity is transient. Various forms of evidence indicate that sustained NADPH oxidase activity requires continuous renewal of the enzyme complex, without which rapid deactivation occurs. This review considers mechanisms that have been proposed to terminate the phagocyte respiratory burst. Changes in the phosphorylation state of p47(phox) and in the species of nucleotide bound to Rac seem to be the dominant factors in deactivation.     

NOX enzymes: potential target for the treatment of acute lung injury

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), is characterized by acute inflammation, disruption of the alveolar-capillary barrier, and in the organizing stage by alveolar pneumocytes hyperplasia and extensive lung fibrosis. The cellular and molecular mechanisms leading to the development of ALI/ARDS are not completely understood, but there is evidence that reactive oxygen species (ROS) generated by inflammatory cells as well as epithelial and endothelial cells are responsible for inflammatory response, lung damage, and abnormal repair. Among all ROS-producing enzymes, the members of NADPH oxidases (NOXs), which are widely expressed in different lung cell types, have been shown to participate in cellular processes involved in the maintenance of lung integrity. It is not surprising that change in NOXs' expression and function is involved in the development of ALI/ARDS. In this context, the use of NOX inhibitors could be a possible therapeutic perspective in the management of this syndrome. In this article, we summarize the current knowledge concerning some cellular aspects of NOXs localization and function in the lungs, consider their contribution in the development of ALI/ARDS and discuss the place of NOX inhibitors as potential therapeutical target.     

Organogenesis from stem cells <Emphasis Type="Italic">in planta</Emphasis>: multiple feedback loops integrating molecular and mechanical signals

In multicellular organisms, the coordination of cell behaviors largely relies on biochemical and biophysical signals. Understanding how such signals control development is often challenging, because their distribution relies on the activity of individual cells and, in a feedback loop, on tissue behavior and geometry. This review focuses on one of the best-studied structures in biology, the shoot apical meristem (SAM). This tissue is responsible for the production of all the aerial parts of a plant. In the SAM, a population of stem cells continuously produces new cells that are incorporated in lateral organs, such as leaves, branches, and flowers. Organogenesis from stem cells involves a tight regulation of cell identity and patterning as well as large-scale morphogenetic events. The gene regulatory network controlling these processes is highly coordinated in space by various signals, such as plant hormones, peptides, intracellular mobile factors, and mechanical stresses. Many crosstalks and feedback loops interconnecting these pathways have emerged in the past 10 years. The plant hormone auxin and mechanical forces have received more attention recently and their role is more particularly detailed here. An integrated view of these signaling networks is also presented in order to help understanding how robust shape and patterning can emerge from these networks.     

Targeting NOX enzymes in the central nervous system: therapeutic opportunities

Among the pathogenic mechanisms underlying central nervous system (CNS) diseases, oxidative stress is almost invariably described. For this reason, numerous attempts have been made to decrease reactive oxygen species (ROS) with the administration of antioxidants as potential therapies for CNS disorders. However, such treatments have always failed in clinical trials. Targeting specific sources of reactive oxygen species in the CNS (e.g. NOX enzymes) represents an alternative promising option. Indeed, NOX enzymes are major generators of ROS, which regulate progression of CNS disorders as diverse as amyotrophic lateral sclerosis, schizophrenia, Alzheimer disease, Parkinson disease, and stroke. On the other hand, in autoimmune demyelinating diseases, ROS generated by NOX enzymes are protective, presumably by dampening the specific immune response. In this review, we discuss the possibility of developing therapeutics targeting NADPH oxidase (NOX) enzymes for the treatment of different CNS pathologies. Specific compounds able to modulate the activation of NOX enzymes, and the consequent production of ROS, could fill the need for disease-modifying drugs for many incurable CNS pathologies.     

Thyroid hormone actions in liver cancer

The thyroid hormone 3,3′,5-triiodo-l-thyronine (T₃) mediates several physiological processes, including embryonic development, cellular differentiation, metabolism, and the regulation of cell proliferation. Thyroid hormone receptors (TRs) generally act as heterodimers with the retinoid X receptor (RXR) to regulate target genes. In addition to their developmental and metabolic functions, TRs have been shown to play a tumor suppressor role, suggesting that their aberrant expression can lead to tumor transformation. Conversely, recent reports have shown an association between overexpression of wild-type TRs and tumor metastasis. Signaling crosstalk between T₃/TR and other pathways or specific TR coregulators appear to affect tumor development. Since TR actions are complex as well as cell context-, tissue- and time-specific, aberrant expression of the various TR isoforms has different effects during diverse tumorigenesis. Therefore, elucidation of the T₃/TR signaling mechanisms in cancers should facilitate the identification of novel therapeutic targets. This review provides a summary of recent studies focusing on the role of TRs in hepatocellular carcinomas (HCCs).     

From pluripotency to forebrain patterning: an in vitro journey astride embryonic stem cells

Embryonic stem cells (ESCs) have been used extensively as in vitro models of neural development and disease, with special efforts towards their conversion into forebrain progenitors and neurons. The forebrain is the most complex brain region, giving rise to several fundamental structures, such as the cerebral cortex, the hypothalamus, and the retina. Due to the multiplicity of signaling pathways playing different roles at distinct times of embryonic development, the specification and patterning of forebrain has been difficult to study in vivo. Research performed on ESCs in vitro has provided a large body of evidence to complement work in model organisms, but these studies have often been focused more on cell type production than on cell fate regulation. In this review, we systematically reassess the current literature in the field of forebrain development in mouse and human ESCs with a focus on the molecular mechanisms of early cell fate decisions, taking into consideration the specific culture conditions, exogenous and endogenous molecular cues as described in the original studies. The resulting model of early forebrain induction and patterning provides a useful framework for further studies aimed at reconstructing forebrain development in vitro for basic research or therapy.     

Epigenetic regulation of skin: focus on the Polycomb complex

Chromatin regulators have recently emerged as key players in the control of tissue development and tumorigenesis. One specific chromatin regulator, the Polycomb complex, has been shown to regulate the identity of embryonic stem cells, but its role in controlling fates of multipotent progenitors in developing tissues is still largely unknown. Recent findings have revealed that this complex plays a critical role in control of skin stem cell renewal and differentiation. Moreover, the expression of Polycomb complex components is often aberrant in skin diseases, including skin cancers. This review will detail recent findings on Polycomb control of skin and highlight critical unknown questions.     

Nitric oxide synthases: targets for therapeutic strategies in neurological diseases

Glutamate excitotoxicity, oxidative stress, and mitochondrial dysfunctions are common features leading to neuronal death in cerebral ischemia, traumatic brain injury, Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Nitric oxide (NO) alone or in cooperation with superoxide anion and peroxynitrite is emerging as a predominant effector of neurodegeneration The use of NO synthase (NOS) inhibitors and mutant mice lacking each NOS isoform have provided evidence for the injurious effects of NO derived from neuronal or inducible isoforms. New neuroprotective strategies have been proposed with selective NOS inhibitors for the neuronal (ARL17477) or the inducible (1400 W) isoforms or with compounds combining in one molecule selective nNOS inhibition and antioxidant properties (BN 80933), in experimental ischemia-induced acute neuronal damage. The efficacy of these new strategies is well established in acute neuronal injury but remains to be determined in more chronic neurological diseases.