Trends in Pharmacological Sciences
ReviewToward clinical application of the Keap1–Nrf2 pathway
Section snippets
Inducible cellular defense system
Mammalian cells are frequently exposed to extrinsically and intrinsically generated toxic substances and to oxidative insults that disrupt their normal function by damaging nucleic acids, proteins, and membrane lipids. To overcome such insults, cells are equipped with elaborate defense systems. The basal level expression of detoxification enzymes and xenobiotic transporters appears to be sufficient to protect cells against low level chemical and/or oxidative stresses. The body is also equipped
Target genes of Nrf2
Nrf2 dimerizes with members of the small Maf family and binds to antioxidant or electrophile response elements (AREs/EpREs) located in the regulatory regions of cellular defense enzyme genes [1]. Several Nrf2 target genes have been identified, and the number has increased through the analysis of the gene expression profiles of Nrf2-deficient mice [2]. Recent technical advances have enabled us to analyze the genome-wide distribution of Nrf2 and have provided us with comprehensive unbiased
Keap1 negatively regulates Nrf2
Keap1 is an adaptor protein for a Cul3-based ubiquitin E3 ligase 6, 7. Under normal conditions, Keap1 binds to Nrf2 in the cytoplasm and promotes the ubiquitination of Nrf2. Subsequently, Nrf2 protein is degraded by the proteasome. In the presence of Nrf2-inducing chemicals, the ubiquitin E3 ligase activity of the Keap1–Cul3 complex declines, and Nrf2 is stabilized (Figure 1). The stabilized Nrf2 accumulates in the nucleus and activates its target genes.
The function of Keap1 as a negative
Keap1 as a sensor of chemical and oxidative stresses
Many Nrf2-activating chemicals [e.g., diethyl maleate (DEM), tert-butylhydroquinone (tBHQ), sulforaphane (SFN), and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2)] are electrophilic and are capable of reacting with nucleophilic thiols, including cysteine sulfhydryl groups [10]. Several independent research groups have employed mass spectrometry to identify the specific pattern of Keap1 cysteine modification generated by each Nrf2 inducer [11]. The modification of Keap1 at cysteine residues results
Molecular mechanisms of stress sensing by the Keap1–Nrf2 system
One of the most interesting mechanisms for the regulation of Nrf2 by Keap1 proposed to date is the ‘hinge and latch model’ [22] (Figure 1). This model is supported by single-particle electron microscopy data, which indicate that the overall structure of the Keap1 dimer resembles a cherry-bob with two globular units (the double glycine repeat and the C-terminal or DC domains) connected to a stem [the homodimerizing BTB (bric-a-brac, tramtrack, and broad complex) domains] [23]. The Keap1
Activation of Nrf2 as a potential therapeutic strategy
Targeted Nrf2 knockout (Nrf2−/−) mice exhibit markedly lower expression levels for cellular defense genes in various tissues [29]. Accordingly, Nrf2−/− mice are inherently more susceptible to drug-induced toxicity and oxidative stress-induced diseases, including acute lung injury, chronic obstructive pulmonary diseases, diabetic nephropathy, heart failure, and cancer [29]. In addition, the activation of Nrf2 by pharmacological (i.e., pretreatment with Nrf2 inducers) or genetic (i.e., the
Development of Nrf2 activators
Because many studies have demonstrated Nrf2 plays important roles in the protection against various diseases, including cancer, neurodegenerative disease, cardiovascular disease, acute lung injury, chronic obstructive pulmonary diseases, autoimmune disease, and inflammation [29], there is substantial interest in identifying and developing Nrf2 activators for therapeutic use. Some of the most promising Nrf2 inducers are a series of triterpenoids derived from oleanolic acid, which itself has
Search for novel Nrf2-activating drugs
The search for novel Nrf2 inducers will be accelerated by the development of screening systems that provide direct indications of Nrf2 activation or Keap1 modification. An example of such an assay is a reporter assay in which the reporter is fused to the N terminus of Nrf2 including Neh2 and Neh6 degron domains; this reporter is highly responsive to molecules that activate Nrf2 34, 35, 36, 37. In addition, in silico screening for chemicals capable of modifying Keap1, and thus likely to activate
Adverse effects due to the activation of Nrf2
Nrf2 activation may have a therapeutic potential. However, the lethality of Keap1 knockout mice indicates that constitutive activation of Nrf2 in the upper digestive tract tissues can result in serious adverse effects 8, 9. Multiple studies have demonstrated a link between oncogenesis and mutations in the Keap1–Nrf2 pathway that result in the constitutive activation of Nrf2 40, 41, 42. The resistance of cancer to chemotherapy has been attributed to an increase in Nrf2 signaling.
Role of Nrf2 in oncogenesis
Elevated NRF2 levels have been detected in various cancer tissues [41]. Somatic mutations in the KEAP1 gene, including missense mutations, insertions and deletions, have been identified in tumor tissues, most often in heterozygous form. Importantly, these mutations are associated with the stabilization of NRF2. As shown in Figure 3, a genetically engineered mouse model demonstrated that heterozygous, dominant-negative mutations in KEAP1 in cancer cells could lead to increases in NRF2 activity
Small-molecule inhibitors of Nrf2
In the context of the adverse roles that Nrf2 plays in diseases, the pharmacological inhibition of Nrf2 signaling could provide a promising strategy to improve the efficacy of treatment. Retinoic acid receptor α agonists have been shown to inhibit Nrf2 activity through their physical interaction with Nrf2, thus preventing Nrf2 from binding to the ARE and activating its target genes [58]. Similarly, brusatol, a component of the Brucea javanica shrub, was identified in a screen of natural
Unresolved questions and future studies
Recent studies have proven that Keap1 senses a wide range of Nrf2 activators 15, 16. However, the way in which Keap1 utilizes distinct sensing mechanisms through different cysteine residues is currently unknown. Further studies on the individual sensing mechanisms should provide the answer and give valuable insight to guide drug development.
With regard to the clinical application of Nrf2 activators, adverse effects still need to be rigorously explored. There is a growing concern that Nrf2
Concluding remarks
There have been significant advances in our understanding of the function and regulation of Nrf2. In particular, analyses of Nrf2-knockout mice [1] and the identification of Keap1 [6] have provided invaluable insights into the contribution of the Keap1–Nrf2 system to the stress response. This review has highlighted the potential therapeutic benefits of targeting Nrf2. Although a large body of evidence indicates that the activation of Nrf2 protects animals against a variety of diseases [29], the
Acknowledgments
This work was supported in part by Grants-in-Aids for Creative Scientific Research and Scientific Research from Japan Society for the Promotion of Science (JSPS), the Target Protein Program from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), the Tohoku University Global COE Program for Conquest of Signal Transduction Diseases with ‘Network Medicine’, CREST from Japan Science and Technology Agency (JST), and the NAITO foundation.
References (60)
An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements
Biochem. Biophys. Res. Commun.
(1997)Cysteine-based regulation of the CUL3 adaptor protein Keap1
Toxicol. Appl. Pharmacol.
(2010)Validation of the multiple sensor mechanism of the Keap1–Nrf2 system
Free Radic. Biol. Med.
(2012)The critical role of nitric oxide signaling, via protein S-guanylation and nitrated cyclic GMP, in the antioxidant adaptive response
J. Biol. Chem.
(2010)Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response
Mol. Cell
(2009)The Keap1–Nrf2 cell defense pathway – a promising therapeutic target?
Adv. Pharmacol.
(2012)Sickle hemoglobin confers tolerance to Plasmodium infection
Cell
(2011)Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators
Chem. Biol.
(2011)Discovery of inhibitors of microglial neurotoxicity acting through multiple mechanisms using a stem-cell-based phenotypic assay
Cell Stem Cell
(2012)Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling
Cancer Cell
(2011)