Targeting oxidative stress in disease: promise and limitations of antioxidant therapy

Abstract

Oxidative stress is a component of many diseases, including atherosclerosis, chronic obstructive pulmonary disease, Alzheimer disease and cancer. Although numerous small molecules evaluated as antioxidants have exhibited therapeutic potential in preclinical studies, clinical trial results have been disappointing. A greater understanding of the mechanisms through which antioxidants act and where and when they are effective may provide a rational approach that leads to greater pharmacological success. Here, we review the relationships between oxidative stress, redox signalling and disease, the mechanisms through which oxidative stress can contribute to pathology, how antioxidant defences work, what limits their effectiveness and how antioxidant defences can be increased through physiological signalling, dietary components and potential pharmaceutical intervention.

Fig. 1. Reactive species in the extracellular space and defences by SOD or catalase mimics and NOX inhibitors.

Plasma membrane NADPH oxidase (NOX) production of superoxide (O₂^•−) outside cells may be prevented by NOX inhibitors. Dismutation of O₂^•− to hydrogen peroxide (H₂O₂) is accelerated by superoxide dismutase (SOD) mimics, preventing the formation of peroxynitrite (ONOO⁻), which spares nitric oxide (•NO). Reduction of H₂O₂ is accelerated by catalase mimics, preventing the formation of hypohalous acids (HOX) by myeloperoxidase (MPO) and hydroxyl radical (•OH) production via the Fenton reaction. Most SOD mimics appear to have catalase activity. Although NOX4, which is primarily in intracellular organelle membranes, has also been found in the plasma membrane, this has only been reported for one cell type and so its extracellular location remains debatable (indicated by the question mark). NOS, nitric oxide synthase.

Fig. 2. Glutathione metabolism and strategies to increase glutathione.

Glutathione (GSH) is synthesized through reactions catalysed by glutamate–cysteine ligase (GCL) and GSH synthetase (GS), with GCL as the rate-limiting enzyme and cysteine as the rate-limiting substrate. Both reduced GSH and glutathione disulfide (GSSG) are exported from cells through multidrug resistance protein (MRP), and extracellular GSH is sequentially metabolized by membrane-bound γ-glutamyl transpeptidase (GGT) into cysteinylglycine and γ-glutamyl products, and dipeptidase hydrolyses cysteinylglycine to cysteine and glycine. The amino acids are transported back into cells and participate in GSH synthesis. N-acetylcysteine (NAC) is deacetylated by esterase action into cysteine, while GSH esters (GSH-E) are directly converted by esterase into GSH. γ-Glutamylcysteine (γ-glu-cys) can bypass GCL, the rate-limiting step for GSH synthesis. Electrophiles cause the activation of NRF2, which regulates the transcription of the two subunits of GCL, and also GS. Some transporters have been identified: ASC, sodium-dependent alanine-serine-cysteine transporter; Xc⁻, system cystine/glutamate antiporter. Question marks denote the unidentified transporters/channels for GSH-E, γ-glu-cys and NAC.

Fig. 3. NRF2 signalling pathway and antioxidant therapeutic approaches.

(1) Transcription factor NRF2 is constantly synthesized in cells but its transport to the nucleus remains low under basal conditions. This is due to its degradation through association with Kelch-like ECH-associated protein 1 (KEAP1), which facilitates its degradation by the 26S proteasome. Boosting NRF2 synthesis represents a therapeutic antioxidant approach. (2) Upon exposure to electrophiles, KEAP1 is alkylated and loses its ability to cause degradation of NRF2. Using non-toxic electrophiles to alkylate KEAP1 represents another major therapeutic approach. For KEAP1, SH is the thiol form and SX denotes the adduct formed with the electrophile (X). (3) In a parallel pathway glycogen synthase kinase 3β (GSK3β) phosphorylates NRF2, which with β-transducin repeat-containing protein (βTrCP) is degraded by the proteasome, a process that is inhibited by oxidative inactivation of GSK3β. The interaction of NRF2 and βTrCP is disrupted owing to oxidant-mediated inhibition of GSK3β and the phosphorylation of NRF2 at the Deh6 domain. Inhibiting GSK3β is another potential therapeutic approach to modulate NRF2 signalling. (4) Oxidation-induced KEAP1 degradation also occurs through p62-mediated sequestration of KEAP1 and autophagy, a process initiated by phosphorylation of p62 via TANK-binding kinase 1 (TBK1) and mechanistic target of rapamycin complex 1 (mTORC1). p62 therefore provides another potential therapeutic target. Newly synthesized NRF2 that escapes degradation is translocated into the nucleus where it binds to EpRE sequences in the promoters of antioxidant genes and increases their expression. NRF2 activity is also positively regulated through NRF2 phosphorylation by protein kinase C (PKC) and its interaction with other proteins such as p21 (ref.) and BRCA1 (ref.). (5) In the nucleus, BACH1 negatively regulates NRF2 activity by competing to form heterodimers with small Maf (sMaf) or Jun proteins and binding to the electrophile response element (EpRE)^–. Thus, compounds that inhibit BACH1 offer an alternative therapeutic approach for increasing expression of some NRF2-regulated genes. Other negative regulators of NRF2, which represent potential therapeutic targets include HRD1, CRIF1, progerin and microRNA for NRF2 (ref.).