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Review
. 2015 Aug 10;23(5):406-27.
doi: 10.1089/ars.2013.5814. Epub 2014 Feb 26.

Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

Sebastian Altenhöfer  1 , Kim A Radermacher  1 , Pamela W M Kleikers  1 , Kirstin Wingler  1 , Harald H H W Schmidt  1
Affiliations
Review

Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

Sebastian Altenhöfer et al. Antioxid Redox Signal. .

Abstract

Significance: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress.

Recent advances: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms.

Critical issues: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease.

Future directions: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition.

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Figures

<b>FIG. 1.</b>
FIG. 1.
The NADPH oxidase enzyme family. All NOX isoforms (yellow) are membrane proteins that are localized in the PM or cellular compartments' membranes (gray). Stabilizing or maturation factors of NOX are presented in brown, activating binding partners are in green, complex organizing binding partners are in blue, and destabilizing binding partners are in red. In addition to the PM localization, NOX1 was also found in caveolae (71). NOX2 is heavily expressed in the plasma membrane of phagocytic vesicles. NOX4 was found in several sub-cellular compartments' membranes, for example, mitochondria (1, 18, 91). A soluble NOX4 splice variant, NOX4D (14, 62, 130), lacking five out of six transmembrane domains, was suggested in the nucleus and nucleolus (4). Although localization of NOX4 (135) and NOX5 (9, 170) in the ER was suggested, a physiological localization or associated function was not shown until now. Activation of NOX1–3 depends on the formation of hetero oligomeric complexes. NOX1 is activated by the binding of its organizer NOXO1, which along with the small GTPase, Rac, enables the binding of the NOXA1 to fully activate the complex. NOX2 is activated in a similar manner by its organizer proteins, p47phox and Rac, that enable binding of the activator protein, p67phox. The activation of NOX2 can be further enhanced by the binding of p40phox to the complex. Although NOX3 requires NOXO1 for activation (87), its requirement of activator proteins is still under debate but likely. NOXA1 seems to be capable of activating NOX3, but its role still needs to be confirmed in vivo (31, 32, 121, 171, 172). NOX4 is the only NOX isoform that seems to be constitutively active in the absence of any cytosolic binding factor. However, its activity can be enhanced by binding proteins such as protein Poldip2 (102) and activated TLR4 (14, 130, 168). TLR-4 also seems to bind to the NOX4D splice variant (14). Recently, an analogue of NOXO1, the Tks4/5, and PDI have been found to bind and activate NOX1 and NOX4 (43, 57). Hsp90 was shown to enhance the activities of NOX1, NOX2, and NOX5; while Hsp70 binds to NOX2 and NOX5, leading to degradation of the protein by ubiquitination (28, 29). However, the roles of the latter two binding proteins need further confirmation. NOX5, NOX6/DUOX1, and NOX7/DUOX2 are mainly activated by calcium via their calcium binding sites. The calcium sensitivity of NOX5 can be enhanced by calmodulin (170) and Hsp90 (28). Up to date, no calcium sensitizing or other binding partners of DUOX1/NOX6 or DUOX2/NOX7 have been identified. DUOX, dual oxidase; ER, endoplasmic reticulum; Hsp, heat shock protein; NADPH, nicotinamide adenine dinucleotide phosphate; NOX, catalytic subunit of NADPH oxidases; NOXA1, NOX activator-1; NOXO1, NOX organizer-1; PDI, protein disulfide isomerase; PM, plasma membrane; Poldip2, polymerase (DNA-directed) delta-interacting protein 2; TKS4/5, tyrosine kinase substrate with 4/5 SH3 domains; TLR4, toll-like receptor-4. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 2.</b>
FIG. 2.
Mechanisms of NOX inhibition. The scheme shows the general structure of NAPDH oxidase complexes with the catalytic subunits, NOX1-7, in the plasma membrane, the membrane-bound binding partner, p22phox, one or two organizer binding proteins (Org I and Org II), the small GTPase, Rac, and one activator binding protein (Act). The cytosolic NOX C-termini have an FAD and an NAPDH binding domain. DUOX1/NOX6 and DUOX2/NOX7 have an additional transmembrane domain and an extracellular N-terminus. Suggested, but not completely validated or unspecific inhibitors of NAPDH oxidase activity are shown in red font, recommended inhibitors in white font on a red background, and partly recommended inhibitors in red font on a pale red background. Inhibitors are recommended if they are specific for NADPH oxidases and show efficacy in cell-free, cellular, and in vivo conditions. Placement of the inhibitors indicates their likely point of interaction, arrows indicate off-target effects, and arrows with question marks indicate insufficient characterization regarding off-target effects. The NOX2ds-tat peptide (145) and AEBSF (42) prevent complex assembly of the respective NOX isoform with its organizer subunit, in case of NOX2ds-tat, NOX2, and p47phox. AEBSF also inhibits serine proteases. Celastrol (76), ebselen (164), and apocynin (123, 166) inhibit the binding of the organizer proteins NOXO1 and p47phox to p22phox. Ebselen and apocynin are known ROS scavengers. The latter also inhibits rho kinases. Celastrol further inhibits topoisomerase II and the proteasome. NOXA1ds inhibits binding of the respective NOX activator, NOXA1 to NOX1. VAS2870 is very likely an assembly inhibitor with a yet unknown target domain (3). It was shown to alkylate cysteine residues in the RyR1 receptor (167). DPI is a flavoprotein inhibitor. Since ACD 084 inhibited ROS from the NOX4 dehydrogenase domain (89), it may act either on the FAD or NAPDH binding site or as a direct antioxidant. The Shionogi compounds are not NOX inhibitors but prevent the assembly of NADPH oxidase complexes indirectly by the inhibition of protein kinase C (55). Imipramin is a cation and can, therefore, not cross cell membranes. It most likely exerts its NOX inhibition extracellularly. S17834 and plumbagin are polyphenols and most likely scavenge ROS directly. The main target of S17834, however, seems to be AMPK. No mechanisms of action are published for the GKT compounds (GKT136901 and GKT137831) and ML171. GKT136901 scavenges peroxynitrite, and ML171 was reported to inhibit serotonin and adrenergic receptors with very low affinity and potency. AEBSF, 4-(2-aminoethyl)- benzenesulphonyl fluoride; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; DPI, diphenylene iodonium; FAD, flavin adenine dinucleotide; NOX2ds, NOX2 docking sequence; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 3.</b>
FIG. 3.
NOX inhibitor structures with scaffold similarities. Some of the novel NOX inhibitors share structural similarities. All inhibitors are planar molecules with heterocyclic indene cores (shown in red) with slightly different sets of nitrogen hetereo atoms. GKT136901 and GKT137831 are based on a pyrazolo pyridine scaffold; the Shionogi compound has a pyrazolo pyrimidine core; VAS2870 and VAS3947 consist of a triazolopyrimidine; while Ebselen and Fulvene-5 share an indoline-like core. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 4.</b>
FIG. 4.
NOX inhibitor structures with different scaffolds. ML171 is a planar phenothiazine; Celastrol is a triterpene isolated from the plant Tripterygium wilfordii Hook F; Grindelic acid is a diterpenoid isolated from the plant Grindelia integrifolia; and ACD 084 has a diarylheptanoid structure.
<b>FIG. 5.</b>
FIG. 5.
Quinoid structures. The suggested NOX inhibitors S17834 and the phenantridinone group inhibitors are polyphenols with quinoid structures similar to the antioxidant plumbagin.
<b>FIG. 6.</b>
FIG. 6.
Triphenylmethane derivatives. Gentian violet and Imipramin blue are planar cations with dimetylaniline side chains.
<b>FIG. 7.</b>
FIG. 7.
NOX enzymes as validated therapeutic targets. The validation status of NOX1–7 is presented based on partial validation of the mentioned disease model in knockout animals or by using NOX inhibitors (dashed lines), or—for full validation—based on both knockout animals and NOX inhibition or knowledge on mutations leading to human disease (full lines). Knockout mice and inhibition studies strongly suggested NOX1 (green lines) as a therapeutic target in diabetic atherosclerosis (64), ischemic retinopathy (179), and—in interaction with NOX4—liver fibrosis (5, 79, 129). A role in melanoma progression and tumor angiogenesis (54) needs to be confirmed in NOX1 knockout mice, while a role of NOX1 in heart I/R injury (21) needs to be confirmed by pharmacologic inhibition of NOX1. NOX2 (red lines) is suggested to be involved in almost every animal disease model, especially involving inflammatory components. The involvement in CGD is based on human disease and, therefore, validated. A likely role of NOX2 in atherosclerosis, endothelial dysfunction, and restenosis after arterial injury is based on both NOX2 knockout animals (30, 81) and studies with the NOX2ds-tat peptide (46, 74, 177, 194). NOX2 should, therefore, be further considered a target for atherosclerosis and restenosis after arterial injury. A minor role for NOX2 was found in I/R injury of several organs, including the brain [reviewed in ref. (140)], lung (178), and heart (51, 101), but this is currently insufficiently validated. The role of NOX2 in liver fibrosis (129) and allergic asthma (6) is awaiting confirmation with specific NOX2 inhibitors. NOX3's (brown line) physiological role in the inner ear can be considered fully validated in the mouse model, as NOX3 specific inhibition prevented cisplatin-induced hearing loss. NOX4 (blue lines) is a valid therapeutic target for stroke (88), diabetic nephropathy (78), liver fibrosis (5, 79), and osteoporosis (60), at least in mice. The validation of NOX4's role in lung fibrosis and heart failure needs to be confirmed using specific NOX inhibitors and knockout animals in a relevant model. For NOX5, a role in spermatozoa motility was suggested based on the inhibition with GKT136901 (118). DUOX1/NOX6 knockout animals do not show an obvious phenotype, but roles in the bladder (45) or in the lung host defence system were suggested (173). Dysfunctional DUOX2/NOX7 (purple line) due to bi-allelic mutations in humans leads to severe hypothyroidism. CGD, chronic granulomatous disease; I/R, ischemia-reperfusion. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
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