The NRF2 Pathway: Mechanisms, Targets, and Therapeutic Applications

The Keap1-Nrf2 signaling pathway represents one of the most critical cellular defense mechanisms against oxidative and electrophilic stresses in the human body. This pathway has garnered significant scientific attention due to its fundamental role in maintaining cellular homeostasis and its therapeutic potential across diverse pathological conditions. Before discussing the molecular intricacies of this pathway, it is important to understand that Nuclear factor erythroid 2-related factor 2 (Nrf2) functions as the master regulator of cytoprotective responses, orchestrating cellular adaptation to various stressors and playing a pivotal role in preventing numerous diseases associated with oxidative stress and inflammation.

Molecular Structure and Components of the NRF2 Pathway

Nrf2 is a basic leucine zipper (bZIP) transcription factor with a 66 kDa cap'n'collar (CNC) structure that is ubiquitously expressed across human tissues. Its expression is particularly pronounced in organs exposed to the external environment (gastrointestinal tract, respiratory system), metabolically active tissues (endocrine organs, heart, skeletal muscle, brain), and detoxification organs (liver and kidneys)3. The protein's functional versatility stems from its complex domain architecture, comprising seven Nrf2-erythroid cell-derived protein with CNC homology (Neh) domains, each serving distinct regulatory functions.

The Neh1 domain contains the CNC-bZIP region that enables Nrf2 to heterodimerize with small musculoaponeurotic fibrosarcoma (sMaf) proteins, essential for DNA binding3. The Neh2 domain is critical for interaction with Keap1 (Kelch-like ECH-associated protein 1), the primary negative regulator of Nrf2, and contains seven lysine residues that serve as degrons for Keap1-dependent ubiquitination3. The Neh3, Neh4, and Neh5 domains function as transactivation domains, facilitating transcriptional activity. Meanwhile, the Neh6 domain contains another degron involved in redox-insensitive degradation of Nrf2, while the Neh7 domain mediates repression of Nrf2 activity by retinoid X and retinoic acid receptors3.

Keap1, the cytosolic repressor of Nrf2, belongs to the BTB-kelch protein family and consists of three primary structural components: the BTB domain (amino acids 50-179), which facilitates homodimerization and Cullin 3 binding; the IVR (intervening linker region, amino acids 180-314), containing critical cysteine residues Cys273 and Cys288; and the DGR (double-glycine repeat)/Kelch domain (amino acids 327-611), which contains the Nrf2 binding domain3. These structural elements enable Keap1 to function as both a sensor of oxidative stress and a substrate adaptor for Nrf2 degradation.

Molecular Mechanisms of NRF2 Regulation

Under homeostatic conditions, the Keap1-Nrf2 system operates through an elegantly designed regulatory mechanism often referred to as the "Hinge-Latch model." In this model, the Keap1 homodimer binds to two motifs in the Neh2 domain of Nrf2: the high-affinity ETGE motif (functioning as the "Hinge") and the lower-affinity DLG motif (serving as the "Latch")3. This binding configuration positions Nrf2 optimally for ubiquitination by the Keap1-Cullin 3 E3 ligase complex, leading to rapid proteasomal degradation of Nrf2, which typically has a half-life of only 15-20 minutes under basal conditions8.

The mechanism shifts dramatically under conditions of oxidative or electrophilic stress. Keap1 functions as a redox sensor through its multiple reactive cysteine residues, particularly Cys151 in the BTB domain and Cys273 and Cys288 in the IVR domain3. When these cysteines undergo modification by oxidants or electrophiles, Keap1 undergoes conformational changes that disrupt its ability to target Nrf2 for ubiquitination. Consequently, newly synthesized Nrf2 evades degradation, accumulates within the cytosol, and subsequently translocates to the nucleus8. There, Nrf2 heterodimerizes with small Maf proteins and binds to antioxidant response elements (AREs), also called electrophile response elements, in the promoter regions of its target genes3.

It's important to note that Nrf2 activity is not solely regulated by the Keap1-dependent mechanism described above. Several Keap1-independent regulatory pathways have been identified, including phosphorylation by protein kinase C (PKC), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), and glycogen synthase kinase 3β (GSK-3β)3. Additionally, Nrf2 can undergo autoregulation, as its own promoter contains an ARE-like sequence, allowing for self-activation and amplification of its response3. MicroRNAs also participate in Nrf2 regulation by targeting either Nrf2 itself or Keap1, adding another layer of complexity to this pathway3.

Downstream Targets and Physiological Functions

Upon nuclear translocation and binding to AREs, the Nrf2-sMaf complex activates transcription of an extensive array of cytoprotective genes. These include antioxidant enzymes such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD), NAD(P)H:quinone oxidoreductase-1 (NQO1), and glutathione cysteine ligase (GCL), which collectively bolster cellular defenses against oxidative damage3. Beyond these canonical antioxidant targets, Nrf2 regulates genes involved in glutathione synthesis and regeneration, xenobiotic metabolism, NADPH regeneration, and various aspects of cellular metabolism.

The scope of Nrf2's influence extends well beyond simple antioxidant defense. It regulates glucose metabolism by modulating expression of glucose-metabolizing enzymes and influencing insulin sensitivity3. In lipid metabolism, Nrf2 affects fatty acid synthesis, oxidation, and transport3. Nrf2 also participates in iron metabolism, thereby influencing processes ranging from erythropoiesis to mitochondrial function317. Additionally, Nrf2 exerts potent anti-inflammatory effects by suppressing pro-inflammatory cytokine production and inhibiting NF-κB signaling, a major pathway driving inflammation3.

The interplay between Nrf2 and NF-κB is particularly noteworthy. While NF-κB can transcriptionally activate Nrf2 expression, the two pathways also engage in mutual inhibition. NF-κB and Nrf2 compete for binding to the co-activator CREB-binding protein (CBP)/p300, and NF-κB can promote recruitment of histone deacetylase 3 (HDAC3) to repress Nrf2-driven gene expression3. Conversely, Nrf2 inhibits NF-κB activity both indirectly by suppressing reactive oxygen species (ROS) and directly by binding to the p65 subunit of NF-κB3.

Role of NRF2 in Disease Pathogenesis and Progression

The Nrf2 pathway's involvement in disease pathology is multifaceted and sometimes paradoxical, functioning as a double-edged sword in certain contexts. In cancer, for instance, while Nrf2 activation generally protects normal cells from carcinogenic insults, constitutive activation of Nrf2 in established tumors can enhance cancer cell survival and contribute to chemoresistance1. Indeed, mutations in KEAP1 or NFE2L2 (the gene encoding Nrf2) that lead to constitutive Nrf2 activation have been identified in various human malignancies, including lung, liver, and thyroid cancers111.

In neurodegenerative and neurological disorders, Nrf2 typically plays a protective role. In neuropathic pain, Nrf2 activation reduces oxidative damage, inflammation, and mitochondrial impairment, presenting a promising therapeutic target2. In Friedreich's ataxia, a rare genetic neurodegenerative disorder, Nrf2 activation improves mitochondrial function and restores redox balance, which has led to the recent approval of the first Nrf2 activator (omaveloxolone) for this condition1013.

For liver diseases, the Keap1-Nrf2 system represents a critical defense mechanism against various hepatic insults. Nrf2 activation has been shown to attenuate drug-induced liver injury, viral hepatitis, alcoholic hepatitis, and non-alcoholic fatty liver disease (NAFLD)1419. In the context of NAFLD, adequate selenium supplementation has been demonstrated to improve hepatic injury and insulin resistance by promoting selenoprotein P1 synthesis, which regulates the Keap1-Nrf2 pathway to defend against hepatocyte oxidative stress19.

In inflammatory and autoimmune conditions, Nrf2 generally exerts protective effects. It modulates immune cell function and suppresses production of pro-inflammatory mediators such as cytokines, cyclooxygenases, and matrix metalloproteinases3. For instance, in traumatic brain injury, Nrf2 functions as a pyroptosis-related mediator, with serum Nrf2 levels negatively correlating with Glasgow Coma Scale scores in severe cases6.

Clinically Proven NRF2 Activators and Interventions

The therapeutic potential of Nrf2 activation has been validated through various clinical studies, with several Nrf2 activators progressing through the drug development pipeline. The most notable success is omaveloxolone (also known as RTA 408), which received FDA approval for treatment of Friedreich's ataxia in 2023. In a pivotal phase 2 clinical trial involving 103 patients with Friedreich's ataxia, omaveloxolone demonstrated significant efficacy in improving neurological function as measured by the modified Friedreich's Ataxia Rating Scale (mFARS)13. Patients receiving 150 mg daily of omaveloxolone showed a 2.40-point mean difference in mFARS scores compared to placebo after 48 weeks of treatment (p=0.014)13. Clinical experience suggests that omaveloxolone provides "a significant advance in the treatment of FRDA that is likely to be beneficial in a majority of the FRDA population" with "relatively modest" adverse effects10.

Bardoxolone methyl represents another promising Nrf2 activator with substantial clinical evidence. In a multinational phase III clinical trial enrolling 2,185 patients with type 2 diabetes mellitus and stage 4 chronic kidney disease, bardoxolone methyl significantly increased estimated glomerular filtration rate, indicating improved kidney function15. However, the study also documented increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase, which were maximal after 4 weeks of treatment and subsequently trended back toward baseline through week 4815. Importantly, these enzyme elevations were not associated with liver injury markers like increased bilirubin, and experimental evidence suggests they may result from Nrf2-mediated induction of aminotransferase expression rather than hepatotoxicity15.

Dimethyl fumarate (DMF, commercially known as Tecfidera) is an FDA-approved Nrf2 activator used primarily for multiple sclerosis treatment. Clinical studies indicate that DMF inhibits expression of pro-inflammatory cytokines like IL-6 and IL-1 in experimental models of multiple sclerosis and other autoimmune diseases3. In vitro research demonstrates that DMF inhibits microglial and astrocytic inflammation by suppressing synthesis of nitric oxide, IL-1β, TNF-α, and IL-63.

Experimental and Less Established NRF2-Targeting Approaches

Several compounds are in various stages of investigation for their Nrf2-activating properties, though they lack the robust clinical validation of the aforementioned drugs. Sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, has shown promise in preclinical studies and early clinical trials. However, a recent double-blind randomized controlled trial investigating stabilized synthetic sulforaphane (S-SFN, 300 mg once daily for 14 days) in patients hospitalized with COVID-19 found that while S-SFN treatment increased Nrf2 target TGFalpha significantly at day 15 compared to placebo (p=0.004), it did not improve clinical status at the primary endpoint4. This suggests that despite biochemical evidence of Nrf2 activation, the clinical benefit of sulforaphane may be context-dependent or insufficient in acute severe conditions like COVID-19.

Deferoxamine (DFO), an iron chelator traditionally used to treat iron overload, has been investigated for its potential to inhibit ferroptosis and activate the Nrf2 pathway. In a study of osteoarthritis, DFO was found to alleviate cartilage degradation by inhibiting chondrocyte ferroptosis and activating the Nrf2 pathway16. This suggests potential application in degenerative joint diseases, though clinical studies specifically targeting Nrf2 with DFO are still lacking.

IMR-261 (previously known as CXA-10) represents a novel oral Nrf2 activator that has advanced to Phase 2 clinical trials. Preclinical studies in mouse models of sickle cell disease and β-thalassemia demonstrated that IMR-261 increases fetal hemoglobin and total hemoglobin, decreases markers of hemolysis and adhesion, reduces vaso-occlusive crises, and improves ineffective erythropoiesis17. It has also shown promise in regulating iron homeostasis by activating Bmp6-mediated synthesis of hepcidin, the master regulator of iron metabolism17. While these results are encouraging, definitive clinical efficacy awaits completion of ongoing trials.

Selenium supplementation represents an indirect approach to enhancing Nrf2 activity. Studies indicate that selenium can promote selenoprotein P1 synthesis, which regulates the Keap1-Nrf2 pathway19. In a mouse model of NAFLD induced by a high-fat diet, adequate selenium supplementation significantly improved hepatic injury and insulin resistance, decreased fat accumulation, inhibited reactive oxygen species production, inhibited apoptosis, and restored mitochondrial number and membrane potential19. These effects were mediated through the Keap1-Nrf2 pathway, suggesting that nutritional approaches may complement pharmacological Nrf2 activation in certain conditions.

Conclusion: Current Status and Future Directions

The Nrf2 pathway stands as a central cellular defense mechanism with broad implications for health and disease. From a mechanistic perspective, this pathway operates through an intricate regulatory network centered on the Keap1-Nrf2 interaction, with multiple layers of control including redox sensing, protein-protein interactions, post-translational modifications, and transcriptional feedback. Its downstream effects extend beyond classical antioxidant functions to encompass metabolism, inflammation, and tissue homeostasis.

The therapeutic exploitation of Nrf2 has yielded several clinically validated interventions, most notably omaveloxolone for Friedreich's ataxia and bardoxolone methyl for chronic kidney disease. These successes demonstrate the viability of targeting Nrf2 for disease management. However, the pathway's context-dependent roles, particularly in cancer where it can promote both prevention and progression, highlight the need for careful consideration of when and how to modulate Nrf2 activity.

Future research directions should focus on developing more selective Nrf2 modulators that can target specific tissues or disease contexts while minimizing unwanted effects. Additionally, combination approaches that pair Nrf2 activators with other therapeutic agents may enhance efficacy while mitigating potential drawbacks. As our understanding of the complex interplay between Nrf2 and other signaling networks continues to evolve, so too will our ability to harness this pathway for improved health outcomes across a spectrum of conditions.