Inhibition of human vascular NADPH oxidase by apocynin derived oligophenols

Abstract

Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC₅₀ = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47^phox and p22^phox. To that end, while apocynin was unable to block the interaction of his-tagged p47^phox with a surface immobilized biotinylated p22^phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC₅₀ = 1.6 μM). These results provide evidence that peroxidase-generated AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase.

Introduction

Recent years have seen substantial improvement in our understanding of the role of superoxide anion (${}^{}{\text{O}}_2^{-}$) in eliciting oxidative stress and vascular diseases.1, 2, 3, 4, 5 The production of ${}^{}{\text{O}}_2^{-}$ is catalyzed by a variety of enzymes, including xanthine oxidase, cytochromes P450, lipoxygenase, enzymes in the mitochondrial respiratory chain, and NADPH oxidases.⁶ The latter, in particular, have been identified as the major source of ${}^{}{\text{O}}_2^{-}$ in vascular endothelial cells (VECs). Excessive production of ${}^{}{\text{O}}_2^{-}$ in VECs leads to increased oxidative stress and endothelial dysfunction. This in turn can result in a diverse array of cardiovascular diseases, including atherosclerosis, hypertension, diabetes, heart failure, stroke, and restenosis.1, 7, 8, 9, 10, 11

The most well-studied NADPH oxidase in humans is found in neutrophils. In the resting state, the neutrophil enzyme consists of at least six partially dissociated components.¹² Tight regulation of NADPH oxidase activity is achieved by at least two mechanisms; the association of the cytosolic subunits and the modulation of reversible protein–protein and protein–membrane interactions.¹³ Despite near 100% homology with the neutrophil NADPH oxidase,14, 15 the exact assembly and activation of VEC NADPH oxidase is poorly understood.¹⁶ Nevertheless, current evidence suggests that phosphorylation of key serine residues in p47^phox facilitates interaction of a Src homology 3 (SH3) domain of this protein with a proline-rich region (PRR) of p22^phox, thereby forming the active membrane-associated NADPH oxidase complex17, 18 (Scheme 1).

The catalytic subunit of NADPH oxidase (Nox) has several isoforms and, at least three of them are expressed in VEC (Nox1, Nox2, and Nox4). Their precise activation mechanisms and cellular regulation remain unclear.19, 20 Nox1 appears to be of only minor importance in the generation of VEC reactive oxygen species (ROS). Nox4, however, is abundantly expressed in endothelial cells, more than Nox2.²¹ Nevertheless, Li et. al.²¹ showed that, despite low expression relative to Nox4, under starvation conditions Nox2 was upregulated ∼8-fold and, as a consequence of this nutrient deprivation-induced oxidative stress, the production of ${}^{}{\text{O}}_2^{-}$ increased ∼2.3-fold. Importantly, Nox2 requires assembly of p47^phox with other cytosolic subunits prior to translocation to the membrane to form the active NADPH oxidase complex.22, 23

Due to the key role VEC NADPH oxidase appears to play in vascular diseases, identification of selective inhibitors is of great interest. Along these lines, several inhibitors have been identified, including nitrovasodilators,²⁴ the flavonoid derivative 6,8-diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-methoxyphenyl)-1-H-benzo-[b]-pyran-4-one,²⁵ and peptides such as the antibiotic PR-39.²⁶ Interestingly, PRR regions are also known to bind polyphenols (such as flavonoids).²⁷ It is not surprising, therefore, that phenolics have been found to have NADPH oxidase inhibitory activity.

Apocynin (4′-hydroxy-3′-methoxyacetophenone)28, 29 is a particularly interesting phenol that has been used as inhibitor of NADPH oxidase. While apocynin itself was found to have low activity in vitro29, 30 metabolism in vivo converts the phenol into active metabolites that inhibit the enzyme.30, 31, 32, 33, 34 This may be due to peroxidase catalysis29, 30, 35, 36 leading to disruption of the p47^phox–p22^phox interaction, which is required for translocation of the cytosolic enzyme components to the membrane leading to activation of the enzyme complex. In the current work, we demonstrate that several oligomeric apocynin oxidation products generated by peroxidases are extremely potent inhibitors of VEC NADPH oxidase in vitro. Moreover, a strong correlation exists between the inhibition of VEC NADPH oxidase in endothelial cell-based assays and disruption of the interaction of EC p47^phox–p22^phox in cell-free assays. These results provide additional mechanistic insight into the nature and function of active metabolites of apocynin.