Characterisation of Nox4 Inhibitors from Edible Plants

Abstract

Introduction !ROS induce damage to biological systems and were shown to contribute to ageing in cells. Aberrant ROS levels are linked to a variety of cancers [1] and many other diseases, e.g., vascular disease [2][3][4][5][6]. Whereas ROS are obligatory byproducts of mitochondrial oxidative metabolism, Nox2, the founding member of the Nox family [7,8], was found responsible for the oxidative burst of phagocytes, where ROS are deliberately produced by Nox2 to enforce pathogen elimination [7,8]. In humans, the Nox family consists of seven enzymes, i.e., five NADPH oxidases (Nox1 through Nox5; for review, see [7,9]) and two Nox homologues (Duox1 and Duox2) [10], along with several subunits and regulatory proteins. All Nox proteins are transmembrane proteins with six (Nox1-Nox5) or seven (Duox1,2) transmembrane domains. In each case, four conserved histidines Abstract ! NADPH oxidases transport electrons from cytosolic NADPH through biological membranes to generate reactive oxygen species. NADPH oxidase 4, broadly expressed in humans, is an interesting pharmacological target, since its activity is deregulated in several diseases, including pulmonary fibrosis, diabetic nephropathy, and cardiac hypertrophy. Whereas several candidate NADPH oxidase 4 inhibitors were recently described, most of these compounds are either unspecific or toxic. Here we set out to identify new NADPH oxidase 4 inhibitors from edible plants, in an attempt to decrease the number of hits with toxic side effects. We screened a compound library prepared from edible plants for new bioactives with the ability to inhibit the activity of NADPH oxidase 4. Using both cell-based and cell-free assays, we identified several compounds with significant inhibitory activity towards NADPH oxidase 4. For selected compounds, the activity profile towards NADPH oxidase 2 and NADPH oxidase 5 was established, and controls were carried out to exclude general reactive oxygen species scavengers. A number of promising NADPH oxidase 4 inhibitors from edible plants was identified and characterised. Several new chemical entities are disclosed which act as NADPH oxidase 4 inhibitors, and the efficacies of our best hits, in particular several diarylheptanoids and lignans, are comparable to the best available pharmacological NADPH oxidase 4 inhibitors. These findings will provide valuable tools to study mechanisms of NADPH oxidase inhibition. Abbreviations

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

“…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).…”

“…While this might enable in vivo proof-of-concept studies for the respective indications, the unclear specificity and NOX isoform selectivity does not enable linking the effects of these compounds to NOX enzymes. (89). They identified diarylheptanoid structures with four-substituted phenolic structures as the main structural feature for NOX inhibition.…”

NADPH oxidases: key modulators in aging and age-related cardiovascular diseases?

“…Therefore, the development of selective and isoform-specific Nox inhibitors that preferably target only pathological Nox signalling has become a foremost objective for both academia and pharmaceutical companies. A number of different small-molecule inhibitors have become available that could become useful in their own right or by way of their derivatives [253–263]. Nox1/4 inhibitor GKT 136901 was reported to inhibit NADPH oxidase-dependent aortic ROS formation, attenuate CD44- and HA-dependent inflammatory cell recruitment and aortic lesion area in atherosclerotic lesions of ApoE −/− mice [264].…”

Nox Inhibitors & Therapies: Rational Design of Peptidic and Small Molecule Inhibitors

“…That being said, fulvenes should be classified as putative inhibitors until which time a mechanism of action is identified and their possible role as scavengers can be ruled out. Finally, the recently-identified diarylheptanoid group of compounds obtained from edible plants yielded a compound named ACD084, which inhibited Nox4 with an IC 50 of 3 µM without inhibition of either Nox2 or Nox5 [153]. Moreover, while other diaryl-heptanoids inhibited Nox4-derived ROS, they also were also capable of inhibiting Nox2.…”

“…However, limited scavenging analysis was performed on the identified compounds and the authors reportedly did not investigate the effects on Nox1-derived superoxide. While mechanistic information is lacking for the mode on Nox4 inhibition by ACD084, it is predicted to interfere with nucleotide binding given its ability to inhibit ROS production from purified Nox4-dehydrogenase domain samples [153]. It remains to be determined whether this could also be true for the other Nox isoforms.…”