Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets

Abstract

NADPH oxidases are a family of enzymes that generate reactive oxygen species (ROS). The NOX1 (NADPH oxidase 1) and NOX2 oxidases are the major sources of ROS in the artery wall in conditions such as hypertension, hypercholesterolaemia, diabetes and ageing, and so they are important contributors to the oxidative stress, endothelial dysfunction and vascular inflammation that underlies arterial remodelling and atherogenesis. In this Review, we advance the concept that compared to the use of conventional antioxidants, inhibiting NOX1 and NOX2 oxidases is a superior approach for combating oxidative stress. We briefly describe some common and emerging putative NADPH oxidase inhibitors. In addition, we highlight the crucial role of the NADPH oxidase regulatory subunit, p47phox, in the activity of vascular NOX1 and NOX2 oxidases, and suggest how a better understanding of its specific molecular interactions may enable the development of novel isoform-selective drugs to prevent or treat cardiovascular diseases.

Figure 1. Subunit composition of the seven mammalian NADPH oxidase isoforms

The catalytic core subunits of the enzymes (NADPH oxidase 1 (NOX1)–NOX5, dual oxidase 1 (DUOX1) and DUOX2) are shown in green; NOX and DUOX maturation and stabilization partners (p22phox, DUOX activator 1 (DUOXA1) and DUOXA2) are shown in red; cytosolic organizers (p40phox, NOX organizer 1 (NOXO1) and p47phox) are shown in orange; cytosolic activators (p67phox and NOX activator 1 (NOXA1)) are shown in green; and small GTPases (RAC1 and RAC2) are shown in blue. Also shown (in pink) is polymerase δ-interacting protein 2 (POLDIP2), which is thought to regulate NOX4 activity and link production of reactive oxygen species by this isoform with cytoskeletal organization. EF hand motifs (yellow circles) are also shown, which bind to Ca²⁺ and thereby regulate the activity of NOX5, DUOX1 and DUOX2 oxidases. The figure also illustrates the putative additional amino-terminal transmembrane domain and extracellular peroxidase-like region (shown in purple) on DUOX1 and DUOX2. Although NOX3 oxidase activity is enhanced by the expression of organizer and activator proteins, the enzyme displays constitutive activity in their absence.

Figure 2. Cellular and subcellular expression of NADPH oxidase isoforms in the blood vessel wall

a | Schematic diagram showing cellular localization (endothelial cells, vascular smooth muscle cells, fibroblasts, macrophages and T cells) of NADPH oxidase isoforms (NOX1 oxidase, NOX2 oxidase, NOX4 oxidase and NOX5 oxidase) through a cross-section of an artery. b | Schematic diagram of a hypothetical cell in which all of the vascular NADPH oxidase isoforms (starting with NOX1 oxidase in the left hand column and finishing with NOX5 oxidase in the right hand column) are expressed in each of their possible subcellular locations. H₂O₂, hydrogen peroxide; NOXA1, NADPH oxidase activator 1; O₂^•−, superoxide; p22, p22phox; p40, p40phox; p47, p47phox; p67, p67phox; POLDIP2, polymerase δ-interacting protein 2.

Figure 3. Schematic diagram showing p47phox as the central organizer of the vascular NOX1 and NOX2 oxidases

a | This figure shows the conformation of p47phox in resting cells in which the protein forms a complex with other cytosolic regulatory subunits via an interaction between its carboxy-terminal proline-rich region (PRR) and an Src homology 3 (SH3) domain on one of two potential activator proteins, p67phox or NADPH oxidase activator 1 (NOXA1) subunit (site 1). In the resting state, p47phox adopts a closed conformation in which its tandem-repeat SH3 and phox homology (PX) domains are ensconced and unable to interact with the membrane components. This conformation is largely achieved via intramolecular interactions of both the polybasic autoinhibitory region (AIR) and the PX domain with the tandem-repeat SH3 domain (site 2). b | Phosphorylation of sulfhydryl groups (SH) on crucial cysteine residues within the AIR destabilizes these intramolecular interactions and causes p47phox to unfold. This exposes the tandem-repeat SH3 domain of p47phox, thereby allowing it to associate with a PRR on the amino terminus of membrane-bound p22phox (site 3), and the PX domain of p47phox, which interacts with membrane phospholipids including phosphatidylinositol-3,4-biphosphate (PtdIns(3,4)P₂) and phosphatidic acid (PA) (site 4). The association of the cytosolic complex with the membrane subunits is further stabilized by direct protein–protein interactions between p47phox and the NOX2 subunit (site 5). It is unclear whether p47phox undergoes similar protein–protein interactions with the NOX1 subunit. Also shown are likely target sites for conventional NADPH oxidase inhibitors. PKC, protein kinase C.

Figure 4. The p47phox tandem-repeat SH3 domain as a potential drug target

Nuclear magnetic resonance solution structure of the p22phox proline-rich region (PRR)-binding domain formed by the tandem repeat Src homology (SH3) domain of p47phox before (a) and after (b) molecular modelling to rotate a putative dynamic tryptophan residue (Trp193) within its core. Note that rotation of Trp193 results in two smaller pockets (red and orange) combining to form a single large pocket that is potentially druggable.