Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Inhibition: An

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Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) inhibition, An Emerging Strategy in Cancer Therapy Zhengyu Jiang, Mengchen Lu, and Qi-Dong You J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01121 • Publication Date (Web): 16 Nov 2018 Downloaded from http://pubs.acs.org on November 17, 2018

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Journal of Medicinal Chemistry

Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) inhibition, An Emerging Strategy in Cancer Therapy

Zheng-Yu Jiang*,a,b‡, Meng-Chen Lu a,b,‡, Qi-Dong You*,a,b aState

Key Laboratory of Natural Medicines and Jiang Su Key Laboratory of Drug Design

and Optimization, China Pharmaceutical University, Nanjing 210009, China bDepartment

of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,

Nanjing 210009, China

ABSTRACT Nrf2 is a pleiotropic transcription factor, especially for its complex and dual effects in cancer. With the continuous growing research of the new regulatory modes and new functions of Nrf2 tumor promoting effects of Nrf2 in malignant transformed tumors has become increasingly clear. Accumulating evidence has established that Nrf2 contributes to the whole process of pathogenesis, progression, metastasis and prognosis of cancer, and Nrf2 could be a promising target in cancer therapy. However, the development of Nrf2 inhibitor is still limited. In this perspective, we will briefly describe the biological function and modulating network of Nrf2, stress its oncogenic role and point out possible ways to inhibit Nrf2, as well as summarize the reported Nrf2 inhibitors.

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1. Introduction Nuclear factor erythroid 2-related factor 2 (Nrf2) encoded by the NFE2L2 gene is a basic region-leucine zipper (bZIP) transcription factor, which forms heterodimers with small musculoaponeurotic fibrosarcoma (sMAF) protein.1 The heterodimer can induce target genes transcription by recognizing and binding to the antioxidant response element (ARE) that locates in their regulatory regions. Traditionally, Nrf2-regulated genes are mainly related with the oxidative stress regulation, including the glutathione (GSH)-based and thioredoxin (TXN)based antioxidant system, phase 1, 2 and 3 drug-metabolizing systems. By inducing the transcription of these genes, Nrf2 can activate the cellular defense system, protecting cells from exogenous and endogenous insults such as xenobiotics and reactive oxygen species (ROS). Thereby, Nrf2 is regarded as an important cytoprotective transcription factor that affects the fate of the cell.2 More recently, it has become apparent that Nrf2 is more than a cell protector. Nrf2 can regulate a much wider region of genes, especially the genes that control cellular metabolism.3 By regulating the expression of key metabolic enzymes, Nrf2 profoundly modulates the metabolism of lipids, carbohydrates, nucleotides and amino acids. Moreover, Nrf2 also controls several proteasome subunits and autophagy genes, which further enhances the great significance of Nrf2 in metabolic modulating network. Generally, Nrf2 enhances the expression of anabolic pathway genes, which is beneficial for the proliferation of cancer cells. These new findings further stressed the oncogenic role of Nrf2. Nrf2 not only enhances the survival of cancer cells by elevating the antioxidative and drug metabolizing capacity of the cell,4 but also participates in metabolic network topology changes to prefer the aerobic


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glycolysis.5 Not surprisingly, over activation of Nrf2 by diverse ways is being identified in more and more different types of cancer, including p62 accumulation induced Nrf2 activation in HCC, the mutation and hypermethylation of KEAP1 induced Nrf2 overactivation in nonsmall cell lung cancer (NSCLC) and promoter DNA demethylation of NFE2L2 induced Nrf2 elevation in CRC.6 Thus, developing Nrf2 inhibitors would be a potential way to combat cancer. However, discovery of drug-like inhibitors targeting a transcription factor is quite challenging, and the complicated modulating network of Nrf2 makes it more difficult. To date, the development of Nrf2 inhibitors is still in the early stage. In this perspective, we will briefly describe the biological function and modulating network of Nrf2, stress the oncogenic role of Nrf2, point out possible ways to inhibit Nrf2 and summarize the reported Nrf2 inhibitors. We hope this perspective could be beneficial for the further research of Nrf2 inhibitors.

2. Biological function of Nrf2 Our body is surrounded by constant oxidative and electrophilic insults from both extrinsic and intrinsic sources. To prevent threats, the human body has been equipped with a defense system through engagement of fine-tuned stress response pathways.7 Under stress conditions, the defense system can sense the abnormalities and activate the transcription of cytoprotective enzyme genes to antagonize harmful insults. Among these stress response pathways, Nrf2, which belongs to the cap-n-collar (CNC) subfamily of bZIP transcription factors, regulates the cellular responses against stress conditions.8 The Nrf2-sMAF heterodimer can activate the



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transcription of various cytoprotective enzyme genes by recognizing the cis-regulatory element sequence ARE (5’-GTGACnnnGC-3’) that locates in the upstream of these genes. Nrf2 simultaneously controls two most important antioxidant systems, the GSH-dependent and TXN-dependent antioxidant systems.9 In the GSH-based system, Nrf2 regulates GSH biosynthesis, usage and recycling enzymes. Nrf2 controls the biosynthesis of GSH by controlling the transcription of genes encoding glutamate-cysteine ligase catalytic (GCLC) and glutamate-cysteine ligase modifier (GCLM) subunits. GCLC and GCLM can form a heterodimer, catalyzing the rate-limiting step in GSH.9,

10

Nrf2 also regulates the GSH-

consuming process by regulating the expression of glutathione peroxidase (GPx) 2 and 4, which reduce the hydroperoxides by utilizing GSH as the reductant. GSR, encoding the key enzyme glutathione reductase (GSR) that is responsible for the regeneration of GSH from GSSG, the oxidized form of GSH, is also a target gene of Nrf2.

Figure 1. The biosynthesis and regeneration of glutathione. Nrf2 regulates glutathione biosynthesis, usage and recycling enzymes (labeled by red color).


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TXNs are ubiquitous antioxidant proteins which can promote the reduction of other proteins through cysteine thiol-disulfide exchange. The key components, TXN1, thioredoxin reductase 1 (TXNRD1), and sulfiredoxin 1 (SRXN1) are all regulated by Nrf2.11-14 Besides these antioxidant proteins and enzymes, Nrf2-ARE system also adjusts the transcription of various drug-metabolism enzymes, including Phase I, Phase II and Phase III drug-metabolizing enzymes. These drug-metabolizing enzymes catalyze a plethora of heterogeneous reactions, including reduction by Aldo-keto reductases (AKRs) and NAD(P)H: quinone oxidoreductase 1 (NQO1); conjugation by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs); oxidation by Cytochrome P450 proteins (CYPs), aldehyde dehydrogenases (ALDHs) and alcohol dehydrogenases (ADHs) and nucleophilic trapping reactions by Glutathione Stransferases (GSTs), nearly covering all kinds of drug metabolizing process.15,

16

Drug

transporters, also known as Phase III metabolism enzymes, which can transport drugs, toxins and conjugated metabolites across the cell membrane, are also affected by Nrf2. To date, multiple components in ATP-binding cassette family have been proven to be target genes of Nrf2.17, 18 Besides these antioxidant proteins and enzymes, Nrf2 regulates the regeneration process of NADPH,18 which is the most important reducing equivalents and serves as a cofactor in multiple antioxidant and metabolism pathways. Notably, almost all the NADPH-generating enzymes, including glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (PGD), malic enzyme 1 (ME1) and isocitrate dehydrogenase 1 (IDH1), are regulated by Nrf2 (Figure 2).16 G6PD and PGD are also the key enzymes in the pentose phosphate pathway (PPP), an aerobic glycolysis pathway generating NADPH and pentoses (5-



carbon sugars) as well as ribose 5-phosphate. Moreover, Nrf2 also regulates some other enzymes in PPP, including transketolase (Tkt) and transaldolase 1 (Taldo1).19 It indicates the pivotal role of Nrf2 in enhancing PPP. Nrf2 also activates genes involved in the de novo nucleotide synthesis, including methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and phosphoribosyl pyrophosphate amidotransferase (PPAT), promoting the production of purine nucleotides from glucose.19 Besides these genes involved in glucose metabolism, Nrf2 regulates distinct kinds of lipid metabolism related genes, positively regulating fatty acid βoxidation related genes, while restricting those involved in fatty acid synthesis and desaturation to spare NADPH (Figure 2).20, 21 In addition to these energy substances, Nrf2 also takes part in the heme and iron metabolism. HMOX1, encoding heme oxygenase 1 (HO-1) which is an essential enzyme in heme catabolism, cleaving heme to form biliverdin, carbon monoxide and ferrous iron, is an eminent Nrf2 target gene.22 HO-1 is an antioxidant related enzyme, which has known anti-inflammatory and cytoprotective functions against oxidative stresses.23, 24


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Figure 2. Nrf2 regulates NADPH and anabolic metabolism. Nrf2 reprograms the metabolic network to support cellular proliferation. Nrf2 can active the key enzymes (labeled by red color) in the pentose phosphate pathway (G6PD, PGD, TALDO1, TKT), nucleotide biosynthesis pathway (MTHFD2, PPAT), serine/glycine biosynthesis (PHGDH, PSAT1, SHMT), NADPH producing steps in TCA cycle (ME1, IDH1) and TCA cycle replenishing (GLS, PC). Nrf2 also represses fatty acid synthesis and desaturation (ACL, ACC1, FASN, SCD1) to spare NADPH (labeled by blue color). Nrf2 can activate the transcription of genes for some transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ)18, aryl hydrocarbon receptor (Ahr)25, retinoid X receptor α (RXRα)13 and Nrf2 itself26. Besides transcription factors, some co-factors



involved in transcription, including MafG protein and CCAAT/enhancer-binding protein (C/EBP)11, are also regulated by Nrf2. These findings indicate the complex cross-talk of Nrf2ARE signaling with these pathways. Recent studies have revealed that Nrf2 can act as a transcription repressor. Nrf2 can directly oppose transcriptional upregulation of pro-inflammatory cytokine genes, including IL1B(encoding Interleukin-1) and IL6 (encoding Interleukin-6).27 Nrf2 binds to ARE within regulatory domains of these proinflammatory cytokine genes and inhibits recruitment of RNA polymerase II to these loci, resulting in the rapid and direct suppression of pro-inflammatory cytokine genes.28

3. Regulation of Nrf2 Nrf2 is a modular protein composed of seven domains, Nrf2-ECH homology (Neh) domains 1-7, each with a distinct function (as shown in Figure 3). The Neh1 domain in Nrf2 is a typical CNC-bZIP region, which mediates heterodimerization with other bZIP proteins that is indispensable for transcription activation of target genes. The Neh2 domain in N-terminal mediates the interaction with Kelch-like ECH associated protein 1 (Keap1)29, the dominant regulator of Nrf2 ubiquitination. The C-terminal Neh3 domain is related with transactivation of target genes and interacts with the chromodomain helicase DNA-binding protein 6.30 Neh4 and Neh5 interact with transcription coactivator CREB binding protein to modulate the rate of gene transcription. Neh6 domain is involved in β-transducin repeat-containing protein (βTrCP)-dependent repression of Nrf2.31,

32

The Neh7 domain takes part in the transcription

activation modulation by interacting with the DNA-binding domain of RXRα.


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Figure 3. Domain structure of human Nrf2.

While there are multiple pathways to modulate Nrf2 activity, Nrf2 ubiquitination is the most dominant way to depress Nrf2 activity in normal conditions. The protein level of Nrf2 in cells is tightly regulated by cellular ubiquitin-proteasome system (UPS). To date, three E3 ligases, namely Keap1- Cullins3 (Cul3)- RING-box protein 1 (RBX1), β-TrCP-S-phase kinaseassociated protein 1 (SKP1)-Cul1-RBX1 and HMG-coA reductase degradation 1 homolog (Hrd1), have been identified to control the ubiquitination of Nrf2. Keap1-Cul3-RBX1 is the predominant regulator of Nrf2 ubiquitination. In this E3 ligase complex, Keap1 acts as an adaptor protein to recognize Nrf2 by a unique protein-protein interaction (PPI) mode. Currently, the detailed regulatory model of Keap1-Nrf2 has been intensively elucidated.33, 34In

general, Neh2 domain has two binding motifs for Keap1 with distinct binding strength, the

ETGE motif with high-affinity and the DLG motif with low-affinity.35 The Keap1-Nrf2 interaction follows a stoichiometric coefficient of 2:1, namely Keap1 dimer binds to one Nrf2 protein.36 Keap1-mediated Nrf2 ubiquitination occurs only when the two motifs in Nrf2



simultaneously bind to Keap1 dimer to form a stable complex, referring as the two site binding model37. Disrupting the formation of the complex inhibits the ubiquitination of Nrf2, resulting in the accumulation of Nrf2 protein and activation of Nrf2-ARE pathway. Under basal conditions, the newly synthesized Nrf2 protein can be ubiquitinated by the active Keap1-Cul3RBX1 E3 ligase, and then the ubiquitinated Nrf2 disassociates from the complex, resulting in the regeneration of the active E3 ligase. The recycling of E3 ligase can repress the consistently expressed Nrf2 protein at a low level. Under stress conditions, Keap1 sensors the microenvironment by the reactive cysteine residues, and ROS and electrophiles can covalently modify the cysteines in Keap138, impairing the E3 ligase activity. The Keap1 protein is locked by Nrf2 and the regeneration of Keap1 is inhibited, thus the newly produced Nrf2 protein can avoid the Keap1-dependent depression and translocate to nucleus to activate gene transcription.39, 40

Figure 4. Keap1-mediated regulation of Nrf2 under normal and stressed conditions.


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Another E3 ligase adapter β-TrCP, compromising β-TrCP-SKP1-Cul1-RBX1 E3 ligase, also mediates the ubiquitination of Nrf2.41 The two conserved peptide motifs DSGIS and DSAPGS in Nrf2 Neh6 domain mediates the interaction with β-TrCP.42 Unlike the recognition model of Nrf2-Keap1, the Nrf2-β-TrCP interaction is regulated by a phosphorylation-dependent way. The DSGIS motif can be phosphorylated by GSK-3, and this phosphorylation modification significantly enhances the ability of β-TrCP to suppress Nrf2.43, 44 More recently, Hrd1 has been identified as another E3 ligase responsible for suppressing Nrf2 activity. Hrd1 can interact with the Neh4 and Neh5 domains of Nrf2 and mediate the degradation of Nrf2 under ER stress.45 Besides the E3 ligase mediated protein degradation, Nrf2 activity can be regulated by a variety of mechanisms, which have been intensively reviewed elsewhere.8, 46-48

Figure 5. Regulation of Nrf2 ubiquitination by β-TrCP and Hrd1. GSK-3 mediated Nrf2 phosphorylation enhances the Nrf2-β-TrCP interaction, facilitating Nrf2 ubiquitination by βTrCP-SKP1-Cul1-RBX1 E3 ligase and degradation by proteasome. Increased endoplasmic reticulum (ER) stress activates Hrd1, resulting in the ubiquitination-dependent Nrf2 degradation.



4. Dual roles of Nrf2 in cancer The dual role of Nrf2 in cancer has been revealed for a long time;49, 50 activation of Nrf2 prevents the transformation of a normal cell to a cancerous one or may benefit the survival and proliferation of already transformed cancer cells. Nrf2 was firstly identified as a tumor suppressor. A lot of earlier studies showed that enhancing Nrf2 activity can inhibit carcinogenesis and genetic deletion of Nrf2 elevate susceptibility to cancer development by multiple carcinogen.51 Nrf2 prevents carcinogenesis by accelerating metabolism of chemical carcinogens, relieving oxidative injuries or quenching ROS and reactive nitrogen species (RNS) via the upregulation of its downstream targets.52 A lot of Nrf2 activators have been reported to prevent carcinogenesis induced by the chemical carcinogens and radiation (ionizing, ultraviolet) in Nrf2-/- mice.2, 53 As Nrf2 is a cellular protector that can enhance the survival of cells, it is logical to argue that Nrf2 activation could benefit cancer cells as well. Nrf2 significantly suppresses the oxidative stress that is important for the cytotoxic effects of chemotherapy.54, 55 Activation of Nrf2 can enhance drug detoxification through redox and conjugating mechanisms. The elevation of ATP-binding cassette family such as the multidrug resistance (MDR) system, can result in the lower concentration of drug level, which further explains the Nrf2 activation induced drug resistance. Thus, the activity of Nrf2 could be protective for cancer cells, especially for fully malignant cells. However, it cannot fully explain the aggressive proliferation effects of Nrf2 in cancer. Further intensive biological studies revealed that Nrf2 can nourish tumor cells through more complex and all-around mechanisms.56


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Among them, metabolic reprogramming effects of Nrf2 show promising characters in targeting cancer metabolism. The Warburg effect, a phenomenon that dividing cancer cells uptake more glucose and predominantly metabolize it by way of aerobic glycolysis, is a key feature for cancer cells. This metabolic iteration provides more anabolic precursors and reducing equivalents which are indispensable for rapid growth and proliferation of cancer cells. Although this metabolic character of cancer cells has been found for ages, little progress has been made in discovering therapeutic agents. Recently, transcription factor Nrf2 has been emerging as a key regulator of metabolic reprogramming in cancer cells. Overactivation of Nrf2 induces the metabolic reprogramming to anabolic pathways, guaranteeing the substance supply for cell proliferation. By controlling the expression of G6PD, PGD, TKT and TALDO1, Nrf2 redirects glucose to PPP, producing NADPH as reducing equivalents and ribose 5phosphate (R5P) as the key substrate for the nucleotide synthesis.19 Inactivation of Keap1 and PTEN in the mouse lung promotes adenocarcinoma formation and the tumorigenesis is associated with reprogramming of PPP.57 Moreover, as mentioned above, Nrf2 also enhances NADPH production by multiple steps. IDH1 and ME1, the other two enzymes involved in NADPH production, are also regulated by Nrf2. Besides PPP pathway, Nrf2 can directly promote the de novo nucleotide synthesis by regulating the transcription of MTHFD2 and PPAT.19, 58 Glutamine metabolism, an important source of both nitrogen and carbon in cancer metabolism59, 60, is also regulated by Nrf2 in several ways. Nrf2 can promote transport of glutamine by enhancing the expression of glutamine transporter, SLC1A5.61 Nrf2 regulates the expression of glutaminase, which converts glutamine to glutamate in the mitochondria.



Glutamate can be further metabolized by glutamate dehydrogenase or aminotransferases to α‐ketoglutarate, replenishing the TCA cycle.12 By cooperating with ATF4, Nrf2 also promotes the serine/glycine biosynthesis by enhancing the transcription of key genes in this process, including PHGDH encoding phosphoglycerate dehydrogenase, PSAT1 encoding phosphoserine aminotransferase and SHMT2 encoding mitochondrial serine hydroxymethyltransferase (Figure 3).62 Enhanced serine/glycine biosynthesis provides substances for glutathione synthesis and produces one-carbon unit for nucleotide production, thus promoting tumorigenesis. By regulating ATF4, Nrf2 also contributes to the addiction to the asparagine in NSCLC.63 Besides the amino acid metabolizing, Nrf2 regulates the translation process of mRNA. In some pancreatic cancer cells , Nrf2 deficiency resulted in defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, leading to impaired cap-dependent and cap-independent mRNA translation.64 These metabolic alternations not only directly nourish the proliferation of cancer cells but also create an environment favorable for survival of cancer cells. Besides the effect of tumor promotion, overexpression of Nrf2 may also lead to tumor initiation. Available lines of evidence suggested that extent of Nrf2 activation influences susceptibility to disease. Chemoprevention by Nrf2 activation could be context dependent and the U-shaped Nrf2-dose response curve for electrophilic and redox balance may best describe these influences. The physiological homeostatic range of optimal Nrf2 activation is between the biologically effective dose which minimally activates the Nrf2 pathway and a maximaltolerated dose which not only activates the Nrf2 pathway but also disturbs the homeostatic


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balance and may have deleterious consequences as well.65 The Phase I enzymes AKRs are among the most highly induced mRNA/proteins by Nrf2 activation.66 These enzymes can be cytoprotective by detoxifying reactive aldehydes and ketones. However, excess levels of electrophilic or redox-active agents, especially the lung carcinogens, can make AKR aggravate the oxidative stress. Based on context, AKR enzymes induced by Nrf2 activation can not only lead to chemotherapy resistance, but also produce active lung carcinogens such as polycyclic aromatic hydrocarbons.67 Another Nrf2-regulated metabolic enzyme, NQO1, has been proven to execute the activation of nitroarenes. In the context of air pollutants, nitroarenes can exert the genotoxicity through Nrf2-induced elevation of NQO1, which represents the “dark-side” of Nrf2 activation.68 Collectively, these results indicated that both too much and too little Nrf2 activity can cause adverse effects and disturb the homeostatic balance.

Figure 6. Roles of Nrf2 in blocking (yellow dotted lines) or promoting (blue dotted lines) the emergence of the hallmarks of cancer.



Overactivation of Nrf2 has been found in different cancers types. The gain-of-function mutations of NFE2L2 and loss-of-function mutations of KEAP1 have been intensively investigated in lung cancer. The first identified loss-of-function mutation of KEAP1 was found in two human lung adenocarcinoma cell lines, and the mutated Keap1 showed impaired affinity to Nrf2.69 Singh et. al reported a systematic analysis of the KEAP1 genomic locus in 12 cell lines and 54 NSCLC samples.70 Their sequencing results showed somatic mutations in KEAP1 in 6 cell lines and 10 tumor samples at a frequency of 50% and 19%. Moreover, their study showed that biallelic inactivation of KEAP1 induced by loss of heterozygosity at 19p13.2 in lung cancer is a more common event with allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. The analysis of different cohorts of patients gave similar results that loss-of-function mutations of KEAP1 activate Nrf2 and benefits the growth of lung cancer .71, 72

Besides the mutations of KEAP1, study from Rui Wang et. al first demonstrated that KEAP1

gene silencing may also be obtained by aberrant hypermethylation in the KEAP1 promoter. Decreased mRNA levels of Keap1 were found in 3 lung cancer cell lines and 5 cancer tissues compared to normal bronchial epithelial cells. Treatment with the DNA methyltransferase inhibitor 5-Aza-dC restored the expression of Keap1 in lung cancer cell lines, suggesting that promoter methylation may contribute to the Keap1 downregulation. Furthermore, they also demonstrated that the P1 region was highly methylated in lung cancer, and found that three specific CpG sites in P1 might be occupied by proteins to regulate Keap1 expression.73 Muscarella LA et. al carried out a comprehensive genetic and epigenetic analysis of KEAP1 in 47 NSCLC tissues and normal specimens, and promoter methylation of KEAP1 was observed in 22/47 NSCLC patients.74 Another study from Naoyuki Hanada et. al showed that aberrant


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methylation of the KEAP1 promoter was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical human colorectal cancer (CRC) specimens. It indicated that CRC tissue may exhibit increased expression of nuclear Nrf2 and ARE gene through increased KEAP1 promoter methylation. 75 These studies also found that low KEAP1 expression tends to be related with a worse prognosis and lower survival rates. In addition to NSCLC, loss-offunction mutations in the KEAP1 gene has been found in approximately 20% of KRAS-mutant lung adenocarcinoma (LUAD) tumors.76,

77

Somatic mutations also occur in the NEF2L2

coding region, especially in the Neh2 domain, and Nrf2-mutant cells constitutively express high levels of cytoprotective enzymes and drug efflux pumps, which are not affected by Keap1mediated regulation.78 Noteworthily, these mutations of Nrf2 and Keap1 have been found not only in the Keap1-Nrf2 interaction region but also in the whole sequence, indicating the complex modulating mechanism.70,

79

Somatic mutations in Nrf2 has been found through

whole-genome sequencing of hepatocellular carcinoma (HCC) patients. The enhanced Nrf2 activity is attributable to alterations in the ability to bind to its endogenous inhibitor Keap1.80 Notably, the mechanism studies by different groups showed that Nrf2 mutation and overactivation occurs at the early stage of rat hepatocarcinogenesis.81, 82 Particularly, papillary thyroid carcinoma sustains a remarkably high frequency (80.6%) of disruption to genes encoding the Nrf2 inhibitory complex components.83 Constitutive Nrf2 activation can cooperate with other oncogenic pathways to promote the carcinogenesis. Under the sustained activation of phosphoinositide 3-kinase (PI3K)-Akt pathway, Nrf2 promotes glutamine and glucose into anabolic metabolic pathways. The active PI3K-Akt pathway enhances the nuclear availability of Nrf2, leading to the Nrf2-mediated



promotion of metabolic activities that facilitate cell proliferation in addition to enhanced cytoprotection.19 Co-mutation of KRAS and KEAP1/NFE2L2 is an independent prognostic factor, predicting shorter survival, duration of response to initial platinum based chemotherapy and survival from start of immune therapy.84 More recently, a comprehensive analysis of oncogenic driver genes and mutations in >9,000 tumors across 33 cancer types further confirmed the cancer driver function of Nrf2 activation and identified more mutation sites in both NFE2L2 and KEAP1 genes.79 Enhancing Nrf2 activity also acts as an alternative way to promote proliferation of cancer cell, which affects the efficiency of kinase targeted therapy to lung cancer. Recent study showed that loss of Keap1 altered cell metabolism to support cell proliferation without mitogen-activated protein kinase (MAPK) signaling, and the aberration of the Keap1-Nrf2 system can facilitate cell survival to antagonize the agents targeting receptor tyrosine kinase (RTK)/Ras/MAPK pathway.85, 86 Besides genetic mutation or epigenetic silence of Nrf2 and Keap1, tumor cells utilize some alternative ways to enhance Nrf2 activity. Many oncogenic proteins, including K-RasG12D, BRafV619E and MycERT2, can increase the Nrf2 transcription to activate Nrf2-regulated cellular antioxidant and detoxification system, thereby downregulating the level of ROS and providing a more reduced microenvironment.87 These studies together showed that Nrf2 not only protects cancer cells from various forms of stress and maintain the cellular redox homoeostasis by regulating genes encoding antioxidants, detoxification enzymes, drug transporters, reductases and other defense proteins, but also takes


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part in the whole process of pathogenesis, progression, metastasis and prognosis of cancer. Thus, safe, specific and potent Nrf2 inhibitors could be a potential option for cancer therapy.

5. Possible ways to inhibit Nrf2 activity The E3 ligase mediated Nrf2 suppression by UPS provides convenient ways to develop small molecule Nrf2 activators. There are multiple ways to inhibit E3 ligase activities. Great achievements have been made to develop therapeutic agents to inhibit Keap1 activity by either covalently targeting Keap1 cysteines88 or competitively disrupting Keap1-Nrf2 PPI89. But for Nrf2 inhibition, especially for inhibiting constitutive activation of Nrf2 in tumor cells, it is the high-hanging fruit. Generally, downregulating protein levels of Nrf2 and/or repressing Nrf2 transcription activation activity is a possible way to inhibit Nrf2. Downregulating protein levels of Nrf2 can be achieved by inhibiting the transcription of NFE2L2 gene, decreasing the translation of Nrf2 mRNA or enhancing the degradation of Nrf2 protein. Nrf2 mRNA is expressed broadly and independently of inducers,15 and it is difficult to hinder the production of Nrf2 mRNA. Small interfering RNA targeting Nrf2 mRNA is a commonly used way to knockdown Nrf2 in cellular assays, and some miRNAs, including miR144, miR-28 and miR-93, have been known to downregulate Nrf2.90 However, the deficiency of drug-like properties of nucleic acid agents limit the further development. Enhancing Nrf2 ubiquitination is another way to inhibit Nrf2. Normally, Keap1-dependent ubiquitination and degradation of Nrf2 is the main way to restrict Nrf2 activity. Tumor cells utilize distinct mechanisms to deprive the function of Keap1. p62, an autophagy-adaptor protein, can activate Nrf2 at a Keap1-dependent manner. p62 contains a STGE motif that can



be recognized by Keap1,91 and the serine residue in this motif can be phosphorylated to further enhance the binding strength. The phosphorylated p62 has been reported to compete with Nrf2 for Keap1 binding.92 Moreover, the p62-Keap1 interaction can sequester Keap1 into the autophagosome, directly downregulating protein levels of Keap1.93 p62 accumulation induced Nrf2 activation has been found in HCC, which is important for the cancer initiation.94, 95 In such cases, inhibiting Nrf2 activity can be achieved by selectively disrupting the Keap1-p62 inhibition, and some pioneering work has been reported.96 Further work is stilled needed to ensure the compounds selectively impairing Keap1-p62 interaction. Recently, inhibitor of apoptosis stimulating protein of p53 (iASPP) has also been found to active Nrf2 system by competing Nrf2 with Keap1 binding, promoting the survival of cancer cells.97, 98 However, the major way to enhance Nrf2 activity is the genetic mutation. As mentioned above, a lot of mutation sites in both KEAP1 and NEF2L2 that impair the Keap1-dependent depression of Nrf2 have been identified. For these tumors, promoting the β-TrCP-dependent ubiquitination of Nrf2 is a possible way. Like many other substrates of β-TrCP, Nrf2 is recognized by β-TrCP in a phosphorylation-dependent manner, and GSK-3 has been validated as the key kinase to phosphorylate the β-TrCP-binding motif in Nrf2. Recent studies showed that the GSK-3 inhibitors can activate Nrf2 in different cell lines,99, 100 further stressing the importance of GSK-3 kinase activity in β-TrCP-dependent Nrf2 repression. Unfortunately, there is no reported direct activators of GSK-3 and discovery of a direct kinase activator is also challenging. The activity of GSK-3α and GSK-3β are negatively regulated by phosphorylation of their Ser21 and Ser9 that are executed by protein kinase B (PKB)/Akt, and using PKB/Akt inhibitor MK-2206 or upstream kinase PI3K inhibitor LY294002 to hinder the phosphorylation


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of the DSGIS motif can decrease protein levels of Nrf2.42 However, the complex role of upstream kinases of GSK-3, such as PI3K-PKB/Akt, p38MAPK and PKC,31 limits the further development of these kinase inhibitors as drug-like Nrf2 inhibitors. Recently, the rapid development of small-molecule-induced protein degradation, represented by proteolysistargeting chimaera (PROTAC) technology, may benefit the study of potent small-molecule transcription factor inhibitors.101 PROTAC is a bi-functional molecule, whereby one end binds to the target protein and the other hijacks E3 ligase to enable the ubiquitination-dependent degradation of the target protein. Currently, the lack of small-molecule Nrf2 ligands limits the discovery of Nrf2-targeting PROTACs. Besides the genetic mutation, the epigenetic modification of the NFE2L2 gene is also an effective approach to Nrf2 inhibition. Study from Ziming Li et. al showed that the histone acetyltransferase hMOF can bind to Nrf2 and acetylates Nrf2, maintaining the nucleus retention of Nrf2 and mediating its transcription, subsequently regulating lung cancer growth and drug resistance.102 Their detailed mechanism study showed that enhancer of zeste homolog 2 (EZH2), the H3K27 trimethyltransferase, can bind to NFE2L2 promoter and mediate the trimethylation of H3K27, which repressed Nrf2 transcription. In lung cancer tissues, loss of EZH2 function results in the reduction of H3K27me3 at the NFE2L2 promoter, resulting in the increase of Nrf2 transcription.103 Xiaoqian Zhao et. al explored the mechanisms underlying drug resistance in CRC cells to 5-Fluorouracil (5-FU), which is a widely used anticancer drug. Their study showed that Nrf2 protein levels, nuclear translocation as well as promoter binding were remarkably increased in fluorouracil-resistant CRC cells compared with normal CRC cells. Further methylation analysis indicated that fluorouracilinduced ROS production contributed to the upregulation of DNA demethylases, which



catalyzed the promoter DNA demethylation of NFE2L2, leading to elevated mRNA levels of Nrf2 in drug-resistant CRC cells.104 Taken together, these results suggested that epigenetic alteration in NFE2L2 could provide a promising way to combat anticancer drug resistance. Inhibiting Nrf2 transcription activity is an alternative way. There are some key points for the transcription inducing activity of Nrf2, including translocating to nucleus, forming heterodimer with sMAF protein and Nrf2-sMAF heterodimer binding to ARE. The nuclear localization of Nrf2 is also regulated by GSK-3 mediated phosphorylation. Nrf2 can be excluded from the nucleus by GSK-3β-mediated phosphorylation.105 Disrupting the PPI of Nrf2-sMAF is a reasonable way to inhibit Nrf2 activity. The formation of Nrf2-sMAF protein can activate the transcription of a number of cytoprotective genes, providing protection against cytotoxic anticancer drugs. Thereby, developing the Nrf2-sMAF PPI inhibitors is a promising strategy to discover novel Nrf2 inhibitors. However, the unknown of detailed binding information restricks the discovery of potential inhibitors. Some other PPIs in the nucleus also modulate Nrf2 transcription activity.106 RXRα can interact with the Neh7 domain of Nrf2 and formation of the complex significantly suppresses the Nrf2-ARE pathway by impairing the binding of Nrf2 to ARE107, diminishing the cytoprotective effects by Nrf2 and sensitizing cells to the cytotoxic therapeutics, and RXRα-specific ligands also show similar inhibitory effects.108, 109


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Figure 7. Possible ways and validated targets for inhibiting Nrf2 activity.

Compared to apparent ways to activate Nrf2, the way to selectively and effectively inhibit Nrf2 activity is still obscure. With the in-depth study of Nrf2 regulating network, many nodes can be targeted to inhibit Nrf2 activity, which provide many possibilities for developing Nrf2 inhibitors.

6. Discovery of Nrf2 inhibitors With more and more amount of attention to the oncogenic functions of Nrf2 activation, increasing efforts are being made to discover Nrf2 inhibitors. To date, a battery of molecules with distinct chemotypes and binding targets have been reported to show Nrf2 inhibitory



effects.110 Although most of them are reported with various biological activities, these agents provide useful information for the development of drug-like Nrf2 inhibitors. Natural products with Nrf2 inhibition activity Brusatol Donna D. Zhang’s group firstly identified brusatol as a potent inhibitor of Nrf2 pathway,111 Brusatol can sensitize a vast range of cancer cells and A549 xenografts to several different chemotherapeutic drugs. However, the further studies elucidated that the action mode of brusatol is not through directly inhibiting Nrf2 pathway, and brusatol can decrease the majority of detected proteins rapidly and potently, including Nrf2, in the cellular proteome analysis of KEAP1 mutant NSCLC cell line A549.112 The inhibition of both cap-dependent and capindependent protein translation mechanism of brusatol may affect many short-lived proteins, including Nrf2.113 This non-selective mechanism of action limits the further optimization of brusatol. Glucocorticoids Glucocorticoids, represented by dexamethasone, have been reported to repress Nrf2 activity at a glucocorticoid receptor (GCR)-dependent manner for long time.114 Unlike many other agents showing Nrf2 inhibition activity at micromolar range concentration, some glucocorticoids exhibit dramatic Nrf2 inhibition activity at nanomole concentration.115 E-J Choi et al. comprehensively evaluated the Nrf2 inhibition activity of various glucocorticoids and found that the Nrf2 inhibition activity of these compounds correlates with the GCR activation activity, further stressing the GCR-involved mechanism in Nrf2 inhibition effects of glucocorticoids. Their detailed mechanism study showed that clobetasol propionate prevents


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the elevation of Nrf2 in nuclear and promotes the GSK3- and β-TrCP-dependent ubiquitination and degradation of Nrf2 in a glucocorticoid receptor-dependent manner.116 Flavonoids Some natural flavonoids have been reported with Nrf2 inhibition activity, and interestingly, many of them show distinct effects on Nrf2 activity in different studies. Luteolin has been reported to down-regulate both the mRNA and the protein levels of Nrf2 by accelerating Nrf2 mRNA turnover in A549 cells.117 Another study also reported that luteolin can enhance the anti-cancer effects of cisplatin via inhibiting Nrf2 signaling on A549 cells-based xenograft model in Nrf2+/+ mice but not in Nrf2-/- mice.118 On the contrary, luteolin can enhance Nrf2ARE antioxidant defense system to ameliorate acute mercuric chloride exposure induced hepatotoxicity119 and confer a neuroprotective effect in traumatic brain injury models by activating the Nrf2-ARE pathway120. Although context-dependent dual effects of luteolin on Nrf2 were reported, it seems likely luteolin can inhibit Nrf2 in cancer cells, especially in those with constitutive Nrf2 activation while active Nrf2-mediated antioxidant system in the nontransformed cells. Similar results were also observed in the study of wogonin, which can sensitize HepG2 cells121 and MCF-7/DOX cells122 to chemotherapy drugs through inhibiting Nrf2-ARE pathway while inhibit inflammation-associated colorectal carcinogenesis partly by activating Nrf2 pathway.123 Ochratoxin A Ochratoxin A (OTA), a fungal species derived mycotoxin, also shows Nrf2 inhibition effects. Nrf2 inhibition activity of OTA is identified from its carcinogenicity mechanism study. OTA can reduce the Nrf2-regulated gene expression in kidney by a microarray study, and the



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reduction of Nrf2-regulated gene caused by OTA results in oxidative DNA damages both in vitro and in kidney.124 The kidney-specific Nrf2 inhibition of OTA takes part in the OTA nephrotoxicity and carcinogenicity. OTA can decrease the GST and SOD activity and enhance the ROS level,125 and Nrf2 inhibition activity of OTA may be mediated by impaired Nrf2 nuclear translocation and/or decreased Nrf2 mRNA.126, 127 Nevertheless, chronic exposure to OTA may enhance the somatic mutations in Nrf2 or Keap1 by acting as a selective pressure, leading to constitutive Nrf2 activation.128 The complex and unclear effects of OTA, together with its high toxicity limit the further development of OTA as a Nrf2 inhibitor. Camptothecin Some natural products-derived cytotoxic chemotherapy drugs also show Nrf2 inhibition activity. For example, camptothecin significantly suppresses the expression of Nrf2 and the Nrf2 transcriptional activity in various types of cancer cells including A549, HepG2 and SMMC-7721.

Consequently,

camptothecin

can

sensitize

these

cancer

cells

to

chemotherapeutic drugs in vitro and in xenograft models.129 All-trans retinoic acid All-trans retinoic acid (ATRA) also known as vitamine A acid or retinoic acid, and some other retinoic acid receptor alpha (RARα) agonists are a kind of Nrf2 inhibitors with clear mechanism. ATRA does not affect the protein level and the nuclear accumulation of Nrf2 but reduces the Nrf2-ARE binding as a result of forming a complex with RARα.130 RARα can bind to the Neh7 domain of Nrf2 and the formation of Nrf2-RARα complex significantly inhibits the binding of Nrf2 to ARE sequence.107 Bexarotene, an RXRα specific ligand, can also inhibit Nrf2 transcriptional activity at a RXRα-dependent manner.131 The Nrf2 inhibition of ATRA


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contributes to the efficacy of combined arsenic trioxide-ATRA treatment in the acute promyelocytic leukemia therapy.132

Figure 8. Representative natural products with Nrf2 inhibition activity. Ultra-small molecule drugs suppress Nrf2 activity Some well-known ultra-small molecule (MW lower than 200 g/mol) drugs have been reported to inhibit Nrf2-ARE pathway. Jingbo Pi’s group first reported that isoniazid, an antitubercular drug, can suppress Nrf2-ARE activity in diverse mouse and human cells. The inhibition of isoniazid on Nrf2-ARE pathway interrupts with the transcriptional network of adipogenesis, resulting in impaired adipogenic differentiation.133 Further study reveals that isoniazid can increase cytosolic protein level of Nrf2 while decrease nuclear protein level of



Nrf2 in Hep3B cells and this Nrf2 inhibitory mechanism may be associated with the extracellular signal-regulated kinase1 (ERK1) phosphorylation.134 Some isoniazid analogs, including isonicotinic acid amides and ethionamide, also show Nrf2 inhibition activity, and ethionamide can sensitize acute monocytic leukemia THP-1 cells to As2O3-induced cytotoxicity at a Nrf2-dependent manner.135 To date, the detailed Nrf2-ARE inhibitory mechanism of these antitubercular small-molecules is still obscure. These compounds, sharing a quite similar chemical structure, don’t affect both the mRNA and the protein levels of Nrf2, which needs more studies to clarify this unique mechanism. Metformin, the first-line medication for type 2 diabetes treatment, has also been reported to suppress Nrf2-ARE system. Minh Truong Do et al. found that metformin can reduce the mRNA and the protein levels of Nrf2 in HepG2 cells and the downregulation of Nrf2 is mediated by a Keap1-independent and AMPK-independent but Raf-ERK signaling attenuation involved mechanism.136 Their further study showed that miR-34a induced by metformin is important to suppress the Nrf2 pathway, which can sensitize wild-type p53 cancer cells to oxidative stress and apoptosis.137 Noteworthily, some studies also reported the Nrf2 activation effects of metformin, indicating the conditional effects of metformin on Nrf2 activity.138 In addition to these approved drugs, some other bioactive ultra-small molecules also show Nrf2-ARE inhibition activity. Trigonelline, a zwitterion formed by the methylation of the nitrogen atom of niacin (vitamin B3), inhibits Nrf2 activity by reducing nuclear accumulation of Nrf2. Prominently, the Nrf2-dependent expression of proteasomal genes can be inhibited by trigonelline, resulting in the reduction of proteasomal activity.139 These Nrf2 inhibitionmediated effects of trigonelline can sensitize tumor cells to chemotherapy drugs both in vitro


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and in vivo. All these ultra-small molecules have multiple targets and complex biological effects, and the high concentration of these compounds is required for the effective Nrf2 inhibition in vitro, which limit the further development of targeting Nrf2 inhibitors.

Figure 9. Ultra-small molecule with Nrf2 inhibition activity.

High throughput screening (HTS) to identify Nrf2 activators To date, there is no good protein target suitable for rational design of small molecule Nrf2 inhibitors. The transcription activity of Nrf2 makes it convenient for constructing a cellular phenotype-based screening assay to identify novel Nrf2 inhibitors. Considering that the expression of oncogene C-Myc can increase Nrf2 expression, cMyc was overexpressed in 3T3 mouse embryonic fibroblasts harboring a stably integrated ARE which drives the expression of firefly luciferase. The obtained cell-line (MYC-3T3-ARE-LUC cells) is suitable for HTS. A library containing about thirty thousand diverse heterocyclic compounds together with known biologically active small molecules was screened by this cell line for Nrf2-ARE inhibition activity. Among them, twenty-seven active hits containing three novel chemical scaffolds were discovered. Further optimization of the thienopyrimidine scaffold compound CBR-031-1 gave a preferable analog, AEM1, which shows an EC50 of ∼650 nM in inhibiting the ARE-LUC



signal. AEM1 can decrease the expression of Nrf2 target genes, sensitize A549 cells to different chemotherapeutic agents and inhibit the growth of A549 cells both in vitro and in vivo.140 This compound doesn’t affect the protein level of Nrf2 and doesn’t affect any known Nrf2-phosphorylation process.

Figure 10. Discovery of AEM1. In another study, ARE-driven luciferase reporter genes were stably transferred to HeLa cells, and this cell-line was used to identify a series of pyrazolyl hydroxamic acids with Nrf2-ARE inhibition activity. The representative compound 4f141 can downregulate the protein level of Nrf2 and increase the apoptosis of acute myeloid leukemia cells. The apoptosis promoting effects of 4f has been proven to be associated with Nrf2 downregulation and may be at least partly mediated by the reduction of both Bcl-2 expression and Bcl-2/Bax ratio. The mechanism of action of this series of compounds remains unknown.


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Figure 11. Discovery of 4f with pyrazolyl hydroxamic acid scaffold.

ML385, which was discovered by Shyam Biswal and co-workers, is the first reported Nrf2 inhibitor with direct Nrf2 binding mechanism.142 It was identified by an A549 cell based AREluciferase assay from the Molecular Libraries Small Molecule Repository Library. ML385 can concentration-dependently inhibit the binding of Nrf2-MafG complex to DNA, and the biotin derivate of ML385 (AB-ML385) shows Nrf2 binding activity in the cellular pull-down experiment. Moreover, the Nrf2 without the Neh1 domain (ΔNeh1) does not interact with ABML385. Collectively, the authors proposed that ML385 can inhibit the Nrf2-DNA interaction by directly binding to Nrf2 Neh1 domain. ML385 selectively inhibits the growth of NSCLC cells with Keap1 mutations and enhances the cyto-toxic effects of chemotherapy drugs both in vitro and in vivo. The further binding mode study of this Nrf2 inhibitor may promote the further optimization of ML385.



Figure 12. Discovery of ML385 with Nrf2 binding mechanism. Very recently, James H. Matthews et al. screened the Sigma LOPAC and Spectrum Collection libraries using the MDA-MB-231 cell-line expressing luciferase under the control of the ARE-containing NQO1 promoter, and they discovered three classes of compounds: cardiac glycosides, Stat3 inhibitors, and actin disrupting agents as Nrf2 inhibitors.143 In this study, the authors also screened a library of siRNAs targeting the possible druggable genomes to identify novel targets for Nrf2 inhibition, and found that the transcription factors TWIST1 and ELF4, the TAK1 kinase regulator TAB1, the protein kinase NEK8 and the dual specific phosphatase DUSP4 may be putative Nrf2 targets,143 which provided new insights into the discovery of Nrf2 inhibitors.

Figure 13. Discovery of emetine, quabain and stattic. Future Directions and Conclusions The dual function of Nrf2 in cancer has been proposed and investigated for a long time. With the continuous growing research of new regulatory modes and functions of Nrf2, the dual effects of Nrf2 in cancer has become increasingly clear. Although whether Nrf2 is an oncogene is still uncertain, the tumor promoting effects of Nrf2 has been intensively validated in different


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kinds of tumors. Nrf2 contributes to tumor cells in a lot of hallmarks of cancer, suggesting that inhibiting Nrf2 could be a good therapeutic approach. Compared to the rapid progress in the study of oncogenic effects of Nrf2, the development of Nrf2 inhibitors for cancer therapy is still limited. Most of currently available Nrf2 inhibitors lack detailed molecular mechanisms of action and few of them show direct and selective mechanism of action. Considering the inactivation of Keap1-dependent Nrf2 repression system is frequent in cancer, the panorama of Keap1-independent Nrf2 regulating network should be explored to identify druggable targets for Nrf2 inhibition. Moreover, inhibition of Keap1phosphorylated p62 PPI is also a promising way to restrict Nrf2 activity. However, the Nrf2 inhibition activity of Keap1-phosphorylated p62 PPI inhibitors has not yet been systematically evaluated, and the structure-activity relationship of the compounds for inhibition activity and selectivity of Keap1-p62 interaction merits further investigation. In addition, directly inhibiting Nrf2 transcription activity by targeting the Nrf2-involved PPIs in nucleus, especially disrupting Nrf2-sMaf PPI, is a promising strategy. However, the interface of Nrf2-sMaf PPI has not been clearly elucidated, limiting the discovery of targeting inhibitors. Further structural biology study of Nrf2-sMaf PPI will facilitate the development of modulating agents. We believe that with the efforts of many medicinal chemists, the specific and potent drug-like Nrf2 inhibitors will be identified in the next few years.

AUTHOR INFORMATION Corresponding Author * Prof. Zheng-Yu Jiang E-mail address: [email protected]



* Prof. Qi-Dong You E-mail address: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. ACKNOWLEDGMENT This study was supported by Projects 81602948, 81773581, 81773639 and 81803363 of the National Natural Science Foundation of China; the Natural Science Foundation of Jiangsu Province of China (No. BK20160746); China Postdoctoral Science Foundation-funded Project (1600010006 and 2018T110576); 2015ZX09101032 and 2017ZX09302003 of the National Major Science and Technology Project of China (Innovation and Development of New Drugs); the Priority Academic Program Development of Jiangsu Higher Education Institutions; Program for Outstanding Scientific and Technological Innovation Team of Jiangsu Higher Education (YY20180315004), 111 Project (no. B16046) and “Double First-Class” University project (CPU2018GY02). ABBREVIATIONS Nrf2, nuclear factor erythroid 2-related factor 2; bZIP, basic region-leucine zipper; sMAF, small musculoaponeurotic fibrosarcoma; ARE, antioxidant response element; ROS, reactive oxygen species; GSH, glutathione; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; GPx, glutathione peroxidase; GSR, glutathione reductase; TXN, thioredoxin; TXNRD, thioredoxin reductase; SRXN, sulfiredoxin; CYP,


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Cytochrome P450 protein; ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; NQO1, NAD(P)H:quinone oxidoreductase 1; AKR, Aldo-keto reductase; UGT, UDPglucuronosyltransferase; SULT, sulfotransferase; GST, Glutathione S-transferase; G6PD, glucose-6-phosphate dehydrogenase; PGD, 6-phosphogluconate dehydrogenase; IDH1, isocitrate dehydrogenase 1; ME1, malic enzyme 1; PPP, pentose phosphate pathway; Tkt, transketolase; Taldo1, transaldolase1; PPAT, phosphoribosyl pyrophosphate amidotransferase; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; HO-1, heme oxygenase 1; Ahr, aryl hydrocarbon receptor; PPARγ, peroxisome proliferator-activated receptor γ; RXRα, retinoid X receptor α; C/EBP, CCAAT/enhancer-binding protein; Neh, Nrf2-ECH homology; Keap1, Kelch-like ECH associated protein 1; β-TrCP, β-transducin repeat-containing protein; UPS, ubiquitin-proteasome system; Cul, Cullins; RBX1, RING-box protein 1; SKP1, S-phase kinase-associated protein 1; Hrd1, HMG-coA reductase degradation 1 homolog; PPI, proteinprotein interaction; RNS, reactive nitrogen species; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; PI3K, phosphoinositide 3-kinase; MAPK, mitogenactivated protein kinase; RTK, receptor tyrosine kinase; ATRA, All-trans retinoic acid; EZH2, enhancer of zeste homolog 2. BIOGRAPHY Zhengyu Jiang received his Bachelor’s degree from China Pharmaceutical University, where he also obtained his Ph.D. in Medicinal Chemistry under the supervision of Professor Qidong You. Currently, he is an Associate Professor of Medicinal Chemistry in the Department of Pharmacy, China Pharmaceutical University. His main research topics focus on the following:



(i)discovery and development of protein-protein interaction inhibitors, (ii) chemical biology study of Keap1-Nrf2 related system. Mengchen Lu earned her Ph.D. in medicinal chemistry from China Pharmaceutical University under the supervision of Professor Qidong You. She is currently a member of the Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University. Her research interests lie in the discovery and chemical biology study of the Keap1-Nrf2 protein–protein interactions inhibitors. Qidong You obtained his Bachelor’s degree from the Nanjing College of Pharmacy (the former of China Pharmaceutical university), China in 1982 and his Ph.D. in Medicinal Chemistry from Shanghai Institute of Pharmaceutical Industry in 1989. He did his research in medicinal chemistry as a visiting-scholar at Department of Pharmaceutical Sciences, Strathclyde University, Glasgow, UK between 1994 and 1995. Currently, he is a Professor of Medicinal Chemistry in the Department of Medicinal Chemistry, China Pharmaceutical University and the director of Jiang Su Key Laboratory of Drug Design and Optimization. His research interests mainly focus on small-molecule inhibitors of protein–protein interactions, anti-tumor natural products and targeted anticancer drugs.

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Figure 1. The biosynthesis and regeneration of glutathione. Nrf2 regulates glutathione biosynthesis, usage and recycling enzymes (labeled by red color). 177x65mm (300 x 300 DPI)



Figure 2. Nrf2 regulates NADPH and anabolic metabolism. Nrf2 reprograms the metabolic network to support cellular proliferation. Nrf2 can active the key enzymes (labeled by red color) in the pentose phosphate pathway (G6PD, PGD, TALDO1, TKT), nucleotide biosynthesis pathway (MTHFD2, PPAT), serine/glycine biosynthesis (PHGDH, PSAT1, SHMT), NADPH producing steps in TCA cycle (ME1, IDH1) and TCA cycle replenishing (GLS, PC). Nrf2 also represses fatty acid synthesis and desaturation (ACL, ACC1, FASN, SCD1) to spare NADPH (labeled by blue color). 177x135mm (300 x 300 DPI)


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Figure 3. Domain structure of human Nrf2. 177x68mm (300 x 300 DPI)



Figure 4. Keap1-mediated regulation of Nrf2 under normal and stressed conditions. 177x125mm (300 x 300 DPI)


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Figure 5. Regulation of Nrf2 ubiquitination by β-TrCP and Hrd1. GSK-3 mediated Nrf2 phosphorylation enhances the Nrf2-β-TrCP interaction, facilitating Nrf2 ubiquitination by β-TrCP-SKP1-Cul1-RBX1 E3 ligase and degradation by proteasome. Increased endoplasmic reticulum (ER) stress activates Hrd1, resulting in the ubiquitination-dependent Nrf2 degradation. 177x78mm (300 x 300 DPI)



Figure 6. Roles of Nrf2 in blocking (yellow dotted lines) or promoting (blue dotted lines) the emergence of the hallmarks of cancer. 177x128mm (200 x 200 DPI)


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Figure 7. Possible ways and validated targets for inhibiting Nrf2 activity. 177x126mm (300 x 300 DPI)



Figure 8. Representative natural products with Nrf2 inhibition activity. 177x154mm (300 x 300 DPI)


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Figure 9. Ultra-small molecule with Nrf2 inhibition activity. 83x41mm (300 x 300 DPI)



Figure 10. Discovery of AEM1. 83x57mm (300 x 300 DPI)


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Figure 11. Discovery of 4f with pyrazolyl hydroxamic acid scaffold. 83x46mm (300 x 300 DPI)



Figure 12. Discovery of ML385 with Nrf2 binding mechanism. 83x105mm (300 x 300 DPI)


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Figure 13. Discovery of emetine, quabain and stattic. 177x80mm (300 x 300 DPI)



TOC 112x55mm (300 x 300 DPI)


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Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Inhibition: An

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