Identification and molecular mechanisms of novel antioxidant peptides from fermented broad bean paste: A combined in silico and in vitro study.

This study aimed to investigate the antioxidative and cytoprotective activity of antioxidant peptides from fermented broad bean paste (FBBP) and explore their potential molecular mechanisms using a combined in silico and in vitro approach. Seven novel antioxidant peptides (VSRRFIYYL, SPAIPLP, PVPPPGG, KKDGYWWAKFK, LAWY, LGFMQF, and LPGCP) identified by integrated approaches of peptidomics and in silico bioinformatic analysis were synthesized, exhibiting strong antioxidant potential against in vitro radicals. Molecular docking results suggested that these peptides could form stable hydrogen bonds and solvent-accessible surface with key amino acid residues of Keap1, thus potentially regulating the Keap1-Nrf2 pathway by occupying the Nrf2-binding site on Keap1. Additionally, they exhibited strong cellular antioxidant activity and could protect HepG2 cells from AAPH-induced oxidative injury by reducing reactive oxygen species and MDA accumulation. This study firstly unraveled the molecular mechanisms of antioxidant peptides from FBBP, and provided a new theoretical basis for the high-value utilization of FBBP.

Inhibiting the Keap1/Nrf2 Protein-Protein Interaction with Protein-Like Polymers.

Successful and selective inhibition of the cytosolic protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein 1 (Keap1) can enhance the antioxidant response, with the potential for a therapeutic effect in a range of settings including in neurodegenerative disease (ND). Small molecule inhibitors have been developed, yet many have off-target effects, or are otherwise limited by poor cellular permeability. Peptide-based strategies have also been attempted to enhance specificity, yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, these barriers are overcome utilizing a polymer-based proteomimetics. The protein-like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1-binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell-penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2-inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as NDs.

Computational Prediction of Cyclic Peptide Structural Ensembles and Application to the Design of Keap1 Binders.

The Nrf2 transcription factor is a master regulator of the cellular response to oxidative stress, and Keap1 is its primary negative regulator. Activating Nrf2 by inhibiting the Nrf2-Keap1 protein-protein interaction has shown promise for treating cancer and inflammatory diseases. A loop derived from Nrf2 has been shown to inhibit Keap1 selectively, especially when cyclized, but there are no reliable design methods for predicting an optimal macrocyclization strategy. In this work, we employed all-atom, explicit-solvent molecular dynamics simulations with enhanced sampling methods to predict the relative degree of preorganization for a series of peptides cyclized with a set of bis-thioether "staples". We then correlated these predictions to experimentally measured binding affinities for Keap1 and crystal structures of the cyclic peptides bound to Keap1. This work showcases a computational method for designing cyclic peptides by simulating and comparing their entire solution-phase ensembles, providing key insights into designing cyclic peptides as selective inhibitors of protein-protein interactions.

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

Computational Screening for the Anticancer Potential of Seed-Derived Antioxidant Peptides: A Cheminformatic Approach.

Some seed-derived antioxidant peptides are known to regulate cellular modulators of ROS production, including those proposed to be promising targets of anticancer therapy. Nevertheless, research in this direction is relatively slow owing to the inevitable time-consuming nature of wet-lab experimentations. To help expedite such explorations, we performed structure-based virtual screening on seed-derived antioxidant peptides in the literature for anticancer potential. The ability of the peptides to interact with myeloperoxidase, xanthine oxidase, Keap1, and p47phox was examined. We generated a virtual library of 677 peptides based on a database and literature search. Screening for anticancer potential, non-toxicity, non-allergenicity, non-hemolyticity narrowed down the collection to five candidates. Molecular docking found LYSPH as the most promising in targeting myeloperoxidase, xanthine oxidase, and Keap1, whereas PSYLNTPLL was the best candidate to bind stably to key residues in p47phox. Stability of the four peptide-target complexes was supported by molecular dynamics simulation. LYSPH and PSYLNTPLL were predicted to have cell- and blood-brain barrier penetrating potential, although intolerant to gastrointestinal digestion. Computational alanine scanning found tyrosine residues in both peptides as crucial to stable binding to the targets. Overall, LYSPH and PSYLNTPLL are two potential anticancer peptides that deserve deeper exploration in future.

EndoBind detects endogenous protein-protein interactions in real time.

We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.

New insights into the mechanism of Keap1-Nrf2 interaction based on cancer-associated mutations.

AIMS: Keap1-Nrf2 signaling pathway is one of the most important antioxidant signaling pathways, and its abnormal activation is related to cancer metastasis and drug resistance. Many studies have shown Keap1 and Nrf2 mutations are closely associated with cancer occurrence. However, few studies focus on Keap1-Nrf2 binding characteristics of cancer-associated mutations. The study investigated the molecular mechanism between Keap1/Nrf2 mutations and cancer. MAIN METHODS: We have determined the crystal structure of the Keap1-Kelch domain with Nrf2 25-mer peptide. What's more, we clarified the molecular effects of Nrf2Thr80 and Nrf2Pro85 on the binding of Keap1 by the method isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF) and electrophoretic mobility shift assay (EMSA). Especially, we confirmed the effect of Thr80 and Pro85 mutations on Keap1/Nrf2 signaling pathway in HEK293T cells by RT-PCR and western blot (WB). Finally, we verified the effect of six cancer-related high-frequency somatic mutations Keap1G364C, Keap1D422N, Keap1R470C, Keap1G480W, Keap1E493Q and Keap1R601L on binding with Nrf2 through ITC experiments. KEY FINDINGS: Nrf2Thr80 and Nrf2Pro85 play a vital role in the Keap1-Nrf2 interaction. Mutant or modification at position Thr80 will disrupt the interaction. Especially, Nrf2Thr80 and Nrf2Pro85 mutations activate the expression of cytoprotective genes in HEK293T cells. As for Keap1, except G364C, the binding affinity of other cancer-related mutants to Nrf2 hardly changed, which means that Keap1 mutants can activate Nrf2 without disrupting the binding to Nrf2. SIGNIFICANCE: The study provides new insight into Keap1/Nrf2 signaling pathway and cancer.

Recapitulating the Binding Affinity of Nrf2 for KEAP1 in a Cyclic Heptapeptide, Guided by NMR, X-ray Crystallography, and Machine Learning.

Macrocycles, including macrocyclic peptides, have shown promise for targeting challenging protein-protein interactions (PPIs). One PPI of high interest is between Kelch-like ECH-Associated Protein-1 (KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided by X-ray crystallography, NMR, modeling, and machine learning, we show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be recapitulated in a cyclic 7-mer peptide, c[(D)-ß-homoAla-DPETGE]. This compound was identified from the Nrf2-derived linear peptide GDEETGE (KD = 4.3 µM) solely by optimizing the conformation of the cyclic compound, without changing any KEAP1 interacting residue. X-ray crystal structures were determined for each linear and cyclic peptide variant bound to KEAP1. Despite large variations in affinity, no obvious differences in the conformation of the peptide binding residues or in the interactions they made with KEAP1 were observed. However, analysis of the X-ray structures by machine learning showed that locations of strain in the bound ligand could be identified through patterns of subangstrom distortions from the geometry observed for unstrained linear peptides. We show that optimizing the cyclic peptide affinity was driven partly through conformational preorganization associated with a proline substitution at position 78 and with the geometry of the noninteracting residue Asp77 and partly by decreasing strain in the ETGE motif itself. This approach may have utility in dissecting the trade-off between conformational preorganization and strain in other ligand-receptor systems. We also identify a pair of conserved hydrophobic residues flanking the core DxETGE motif which play a conformational role in facilitating the high-affinity binding of Nrf2 to KEAP1.

Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury.

Background and Purpose: Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from Ilex pubescens, has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA. Methods: The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA. Results: PBA protected cardiomyocytes against OGD/R in vitro and LAD-induced MI/RI in vivo. PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1. Conclusions: Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.

H2S probe CPC inhibits autophagy and promotes apoptosis by inhibiting glutathionylation of Keap1 at Cys434.

H2S is actual an endogenous signaling gas molecule and involved in a range of cell physiological processes. However, the mechanism of endogenous H2S regulating autophagy and apoptosis has not been thoroughly investigated. Here, we try to address this issue by using a H2S probe, (E)-2-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)-piperazin-1-yl)-styryl)-1, 3, 3-trimethyl-3H-indol-1-ium iodide (CPC), which could react with endogenous H2S. Herein, we reported that CPC inhibited autophagy and decreased the expression and activity of NF-E2-related factor 2 (Nrf2), then induced cell apoptosis. CPC inhibited autophagy and promoted apoptosis by inhibiting Nrf2 activation, which was H2S dependent. Furthermore, we found that CPC inhibited Nrf2 nucleus translocation by inhibiting glutathionylation of Kelch-like ECH-associated protein 1 (Keap1) at the Cys434 residue. CPC also inhibited various cancer cell growth, but had no effect on normal cell growth in vitro, and inhibited A549 cancer growth, but did not affect normal angiogenesis in vivo. Therefore, we not only found a new inhibitor of autophagy and Nrf2, but also suggested a novel mechanism that endogenous H2S could regulate autophagy, apoptosis and Nrf2 activity through regulating glutathionylation of Keap1 at the Cys434 residue.

Structural insights into the multiple binding modes of Dimethyl Fumarate (DMF) and its analogs to the Kelch domain of Keap1.

The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription function has been implicated in the protection of neurodegenerative diseases. The cytoplasmic protein, Kelch-like ECH-associated protein 1 (Keap1), negatively regulates Nrf2. The Keap1-Nrf2 pathway is a potential therapeutic target for tackling free-radical damage. Dimethyl fumarate (DMF) is currently an approved drug for the treatment of relapsing multiple sclerosis. Recent studies showed that DMF modifies the reactive cysteines in the BTB domain of Keap1 and thus activates Nrf2 transcription function. Intriguingly, our crystal structure studies revealed that DMF also binds to the ß-propeller domain (Keap1-DC) of Keap1. The crystal structure of the complex, refined to 1.54 Å resolution, revealed unexpected features: DMF binds (a) to the Nrf2-binding site (bottom region of Keap1-DC, site 1) with moderate interaction, and (b) to the top region of Keap1-DC, near to the blade II (site 2). The specificity of the binding 'site 2' was found to be unique to blade II of the ß-propeller domain. The newly identified 'site 2' region in Keap1-DC may have a different functional role to regulate Nrf2. Moreover, the crystal structures of Keap1-DC in complex with the DMF analogs, including monoethyl fumarate, fumarate, and itaconate, also exhibited similar binding modes with Keap1-DC. Binding studies confirmed that DMF binds, in a nanomolar range, to the Keap1-DC region as well as the BTB domain of Keap1. Furthermore, the competitive binding assay in the presence of the Nrf2 peptide affirmed the direct binding of DMF at the Nrf2-binding region of Keap1-DC. Overall, our studies suggest that the drug molecule, DMF, binds at multiple sites of Keap1 and thus potentially activates Nrf2 function through covalent as well as the noncovalent mode of action, to combat oxidative stress. DATABASE: Structural data are available in RCSB-protein data bank database(s) under the accession numbers 6LRZ, 7C60, and 7C5E.

The Nrf2 in the pathophysiology of the intestine: Molecular mechanisms and therapeutic implications for inflammatory bowel diseases.

Nrf2 (nuclear factor erythroid 2-related factor 2) is a stress-responsive transcription factor, associated with cellular homeostasis. Under normal conditions Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1) which facilitates its degradation. Meanwhile, oxidative or electrophilic stress trigger Keap1 dissociation from the Nrf2/Keap1 complex and Nrf2 translocation to the nucleus where it induces the expression of numerous anti-oxidative and anti-inflammatory genes. The Nrf2/Keap1 axis plays a crucial role in the development of gastrointestinal (GI) tract and the maintenance of its proper functionality. This axis also seems to be a promising candidate for prevention of inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), as well as their severe complications such as intestinal fibrosis and colorectal cancer. This review focuses on the role of Nrf2/Keap1 in 1) the development and proper functionality of GI tract, 2) the pathophysiology of GI diseases and their long-term complications, 3) the effectiveness of currently used drugs and non-conventional treatments which influence Nrf2/Keap1 and are potentially effective in IBD treatment, as well as 4) the effect of gut microbiota on Nrf2/Keap1 pathway in IBD.

An Overview of Nrf2 Signaling Pathway and Its Role in Inflammation.

Inflammation is a key driver in many pathological conditions such as allergy, cancer, Alzheimer's disease, and many others, and the current state of available drugs prompted researchers to explore new therapeutic targets. In this context, accumulating evidence indicates that the transcription factor Nrf2 plays a pivotal role controlling the expression of antioxidant genes that ultimately exert anti-inflammatory functions. Nrf2 and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH- associated protein 1 (Keap1), play a central role in the maintenance of intracellular redox homeostasis and regulation of inflammation. Interestingly, Nrf2 is proved to contribute to the regulation of the heme oxygenase-1 (HO-1) axis, which is a potent anti-inflammatory target. Recent studies showed a connection between the Nrf2/antioxidant response element (ARE) system and the expression of inflammatory mediators, NF-κB pathway and macrophage metabolism. This suggests a new strategy for designing chemical agents as modulators of Nrf2 dependent pathways to target the immune response. Therefore, the present review will examine the relationship between Nrf2 signaling and the inflammation as well as possible approaches for the therapeutic modulation of this pathway.

Discovery of polypodiside as a Keap1-dependent Nrf2 activator attenuating oxidative stress and accumulation of extracellular matrix in glomerular mesangial cells under high glucose.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes mellitus. High glucose has resulted in oxidative stress and following renal fibrosis as the crucial nodes of this disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating transcription of many antioxidant genes and suppressing synthesis of extracellular matrix. To discover Nrf2 activators targeting DN, we have evaluated polypodiside using cell-based assays. The results showed polypodiside inhibited the high glucose-induced self-limited proliferation of glomerular meangial cells. Activation of Nrf2 and enhanced transcription to antioxidant response elements were observed in the presence of polypodiside. Oxidative stress and accumulation of extracellular matrix induced by high glucose in glomerular meangial cells have been ameliorated by polypodiside. Further investigations revealed the effects of polypodiside on glomerular meangial cells were associated with activation of Nrf2. Co-immunoprecipitation of Nrf2 disclosed polypodiside disrupted the Kelch-like ECH-associated protein-1 (Keap1)-Nrf2 interaction. Molecular docking elucidated polypodiside could enter the Nrf2 binding cavity of Keap1 via interacting with the residues encompassing that cavity. These findings indicate polypodiside is a Keap1-dependent Nrf2 activator affording the catabatic effects against oxidative stress and accumulation of extracellular matrix in glomerular meangial cells under high glucose.

CDDO-imidazolide Targets Multiple Amino Acid Residues on the Nrf2 Adaptor, Keap1.

Synthetic triterpenoids including CDDO, its methyl ester (CDDO-Me, bardoxolone methyl), and its imidazolide (CDDO-Im) enhance Nrf2-mediated antioxidant and anti-inflammatory activity in many diseases by reacting with thiols on the adaptor protein, Keap1. Unlike monofunctional CDDO-Me, the bifunctional analog, CDDO-Im, has a second reactive site (imidazolide) and can covalently bind to amino acids other than cysteine on target proteins such as glutathione S-transferase pi (GSTP), serum albumin, or Keap1. Here we show for the first time that bifunctional CDDO-Im (in contrast to CDDO-Me), as low as 50 nM, can covalently transacylate arginine and serine residues in GSTP and cross-link them to adjacent cysteine residues. Moreover, we show that CDDO-Im binds covalently to Keap1 by forming permanent Michael adducts with eight different cysteines, and acyl adducts with lysine and several tyrosine residues. Modeling studies suggest that the Tyr 85 adduct stabilizes the Keap1-Cul3 complex, thereby enhancing the potency of CDDO-Im.

Discovery of 4-Anilinoquinolinylchalcone Derivatives as Potential NRF2 Activators.

Activation of nuclear factor erythroid-2-related factor 2 (NRF2) has been proven to be an effective means to prevent the development of cancer, and natural curcumin stands out as a potent NRF2 activator and cancer chemopreventive agent. In this study, we have synthesized a series of 4-anilinoquinolinylchalcone derivatives, and used a NRF2 promoter-driven firefly luciferase reporter stable cell line, the HaCaT/ARE cells, to screen a panel of these compounds. Among them, (E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one (13b) significantly increased NRF2 activity in the HaCaT cell with a half maximal effective concentration (EC50) value of 1.95 µM. Treatment of compound 13b upregulated HaCaT cell NRF2 expression at the protein level. Moreover, the mRNA level of NRF2 target genes, heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD) were significantly increased in HaCaT cells upon the compound 13b treatment. The molecular docking results exhibited that the small molecule 13b is well accommodated by the bound region of Kelch-like ECH-associated protein 1 (Keap1)-Kelch and NRF2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. Compound 13b has been identified as the lead compound for further structural optimization.

Recent progress in Keap1-Nrf2 protein-protein interaction inhibitors.

Therapeutic targeting the protein-protein interaction (PPI) of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its main regulator, Kelch-like ECH-Associating protein 1 (Keap1) has been emerged as a feasible way to combat oxidative stress related diseases, due to the key role of Nrf2 in oxidative stress regulation. In recent years, many efforts have been made to develop potent Keap1-Nrf2 inhibitors with new chemical structures. Various molecules with diverse chemical structures have been reported and some compounds exhibit high potency. This review summarizes peptide and small molecule Keap1-Nrf2 inhibitors reported recently. We also highlight the pharmacological effects and discuss the possible therapeutic application of Keap1-Nrf2 inhibitors.

KEAP1, a cysteine-based sensor and a drug target for the prevention and treatment of chronic disease.

Redox imbalance and persistent inflammation are the underlying causes of most chronic diseases. Mammalian cells have evolved elaborate mechanisms for restoring redox homeostasis and resolving acute inflammatory responses. One prominent mechanism is that of inducing the expression of antioxidant, anti-inflammatory and other cytoprotective proteins, while also suppressing the production of pro-inflammatory mediators, through the activation of transcription factor nuclear factor-erythroid 2 p45-related factor 2 (NRF2). At homeostatic conditions, NRF2 is a short-lived protein, which avidly binds to Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 functions as (i) a substrate adaptor for a Cullin 3 (CUL3)-based E3 ubiquitin ligase that targets NRF2 for ubiquitination and proteasomal degradation, and (ii) a cysteine-based sensor for a myriad of physiological and pharmacological NRF2 activators. Here, we review the intricate molecular mechanisms by which KEAP1 senses electrophiles and oxidants. Chemical modification of specific cysteine sensors of KEAP1 results in loss of NRF2-repressor function and alterations in the expression of NRF2-target genes that encode large networks of diverse proteins, which collectively restore redox balance and resolve inflammation, thus ensuring a comprehensive cytoprotection. We focus on the cyclic cyanoenones, the most potent NRF2 activators, some of which are currently in clinical trials for various pathologies characterized by redox imbalance and inflammation.

The role of natural products in revealing NRF2 function.

Covering: up to 2020The transcription factor NRF2 is one of the body's major defense mechanisms, driving transcription of >300 antioxidant response element (ARE)-regulated genes that are involved in many critical cellular processes including redox regulation, proteostasis, xenobiotic detoxification, and primary metabolism. The transcription factor NRF2 and natural products have an intimately entwined history, as the discovery of NRF2 and much of its rich biology were revealed using natural products both intentionally and unintentionally. In addition, in the last decade a more sinister aspect of NRF2 biology has been revealed. NRF2 is normally present at very low cellular levels and only activated when needed, however, it has been recently revealed that chronic, high levels of NRF2 can lead to diseases such as diabetes and cancer, and may play a role in other diseases. Again, this "dark side" of NRF2 was revealed and studied largely using a natural product, the quassinoid, brusatol. In the present review, we provide an overview of NRF2 structure and function to orient the general reader, we will discuss the history of NRF2 and NRF2-activating compounds and the biology these have revealed, and we will delve into the dark side of NRF2 and contemporary issues related to the dark side biology and the role of natural products in dissecting this biology.

4ß-Hydroxywithanolide E from Goldenberry (Whole Fruits of Physalis peruviana L.) as a Promising Agent against Chronic Obstructive Pulmonary Disease.

Environmental toxicant- and oxidant-induced [e.g., cigarette smoke (CS)] respiratory oxidative stress and inflammatory response play a vital role in the onset and progression of COPD. The nuclear factor erythroid 2-related factor 2 (Nrf2) represents an important mechanism for regulating intracellular oxidative stress and inflammatory response and is a promising target for developing agents against COPD. Herein, a bioactivity-guided purification of goldenberry (whole fruits of Physalis peruviana L.) led to the isolation of a novel and potent Nrf2 activator 4ß-hydroxywithanolide E (4ß-HWE). Our study indicated that (i) 4ß-HWE activated the Nrf2-mediated defensive response through interrupting Nrf2-Keap1 protein-protein interaction (PPI) via modification of Cys151 and Cys288 cysteine residues in Keap1 and accordingly suppressing the ubiquitination of Nrf2. (ii) 4ß-HWE enhanced intracellular antioxidant capacity and inhibited oxidative stress in normal human lung epithelial Beas-2B cells and wild-type AB zebrafish. (iii) 4ß-HWE blocked LPS-stimulated inflammatory response and inhibited LPS-stimulated NF-κB activation in RAW 264.7 murine macrophages. (iv) 4ß-HWE effectively suppressed oxidative stress and inflammatory response in a CS-induced mice model of pulmonary injury. Collectively, these results display the feasibility of using 4ß-HWE to prevent or alleviate the pathological progression of COPD and suggest that 4ß-HWE is a candidate or a leading molecule against COPD.