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.

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Antioxidant Response Activating nanoParticles (ARAPas) localize to atherosclerotic plaque and locally activate the Nrf2 pathway

10.1101/2021.09.16.460323 ◽

2021 ◽

Author(s):

Sophie Maiocchi ◽

Ana Cartaya ◽

Sydney Thai ◽

Adam Akerman ◽

Edward Bahnson

Keyword(s):

Atherosclerotic Plaque ◽

Targeted Delivery ◽

Polymeric Nanoparticles ◽

Effective Means ◽

Antioxidant Response ◽

Related Factor ◽

Drug Molecules ◽

Classically Activated Macrophages ◽

Nrf2 Activator

Atherosclerotic disease is the leading cause of death world-wide with few novel therapies available despite the ongoing health burden. Redox dysfunction is a well-established driver of atherosclerotic progression; however, the clinical translation of redox-based therapies is lacking. One of the challenges facing redox-based therapies is their targeted delivery to cellular domains of redox dysregulation. In the current study, we sought to develop Antioxidant Response Activating nanoParticles (ARAPas), encapsulating redox-based interventions, that exploit macrophage biology and the dysfunctional endothelium in order to selectively accumulate in atherosclerotic plaque. We employed flash nanoprecipitation (FNP) to synthesize bio-compatible polymeric nanoparticles encapsulating the hydrophobic Nrf2 activator drug, CDDO-Methyl (CDDOMe-ARAPas). Nuclear factor erythroid 2-related factor 2 (Nrf2)-activators are a promising class of redox-active drug molecules whereby activation of Nrf2 results in the expression of several antioxidant and cyto-protective enzymes that can be athero-protective. In this study, we characterize the physiochemical properties of CDDOMe-ARAPas as well as confirm their in vitro internalization by murine macrophages. Drug release of CDDOMe was determined by Nrf2-driven GFP fluorescence. Moreover, we show that these CDDOMe-ARAPas exert anti-inflammatory effects in classically activated macrophages. Finally, we show that CDDOMe-ARAPas selectively accumulate in atherosclerotic plaque of two widely-used murine models of atherosclerosis: ApoE−/− and LDLr−/− mice, and are capable of increasing gene expression of Nrf2-transcriptional targets in the atherosclerotic aortic arch. Future work will assess the therapeutic efficacy of intra-plaque Nrf2 activation with CDDOMe-ARAPas to inhibit atherosclerotic plaque progression. Overall, our present studies underline that targeting of atherosclerotic plaque is an effective means to enhance delivery of redox-based interventions.

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Regulation of heme oxygenase-1 expression by demethoxy curcuminoids through Nrf2 by a PI3-kinase/Akt-mediated pathway in mouse β-cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00111.2007 ◽

2007 ◽

Vol 293 (3) ◽

pp. E645-E655 ◽

Cited By ~ 75

Author(s):

Subbiah Pugazhenthi ◽

Leonid Akhov ◽

Gopalan Selvaraj ◽

Maorong Wang ◽

Jawed Alam

Keyword(s):

Signaling Pathways ◽

Heme Oxygenase ◽

Nuclear Translocation ◽

Regulatory Subunit ◽

Luciferase Reporter ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Mrna Levels ◽

Phase 2 ◽

Β Cells

Curcumin (diferuloylmethane), a component of turmeric, has been shown to have therapeutic properties. Induction of phase 2 detoxifying enzymes is a potential mechanism through which some of the actions of curcumin could proceed. Heme oxygenase-1 (HO-1), an antioxidant phase 2 enzyme, has been reported to have cytoprotective effects in pancreatic β-cells. Curcumin on further purification yields demethoxy curcumin (DMC) and bisdemethoxy curcumin (BDMC). The objective of the present study was to determine the mechanism by which these purified curcuminoids induce HO-1 in MIN6 cells, a mouse β-cell line. Demethoxy curcuminoids induced HO-1 promoter linked to the luciferase reporter gene more effectively than curcumin. The induction was dependent on the presence of antioxidant response element (ARE) sites containing enhancer regions (E1 and E2) in HO-1 promoter and nuclear translocation of nuclear factor-E2-related factor (Nrf2), the transcription factor that binds to ARE. Curcuminoids stimulated multiple signaling pathways that are known to induce HO-1. Inhibition of specific signaling pathways with pharmacological inhibitors and cotransfection experiments suggested the involvement of phosphotidylinositol 3-kinase and Akt. Real-time quantitative RT-PCR analysis showed significant elevation in the mRNA levels of HO-1 and two other phase 2 enzymes, the regulatory subunit of glutamyl cysteine ligase, which is needed for the synthesis of glutathione, and NAD(P)H:quinone oxidoreductase, which detoxifies quinones. DMC and BDMC induced the expression of HO-1 and translocated Nrf2 to nucleus in β-cells of mouse islets. Our observations suggest that demethoxy curcuminoids could be used to induce a cellular defense mechanism in β-cells under conditions of stress as seen in diabetes.

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Allyl Isothiocyanate Protects Acetaminophen-Induced Liver Injury via NRF2 Activation by Decreasing Spontaneous Degradation in Hepatocyte

Nutrients ◽

10.3390/nu12113585 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3585

Author(s):

Min Woo Kim ◽

Ju-Hee Kang ◽

Hyun Jin Jung ◽

Se Yong Park ◽

Thu Han Le Phan ◽

...

Keyword(s):

Target Genes ◽

Nuclear Translocation ◽

Biological Effects ◽

Allyl Isothiocyanate ◽

Heme Oxygenase 1 ◽

Toxic Metabolite ◽

Related Factor ◽

Dependent Manner ◽

Nrf2 Activation ◽

Nrf2 Target Gene

Acetaminophen (APAP) is one of the most frequently prescribed analgesic and anti-pyretic drugs. However, APAP-induced hepatotoxicity is a major cause of acute liver failure globally. While the therapeutic dose is safe, an overdose of APAP produces an excess of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI), subsequently resulting in hepatotoxicity. Allyl isothiocyanate (AITC), a bioactive molecule in cruciferous plants, is reported to exert various biological effects, including anti-inflammatory, anti-cancer, and anti-microbial effects. Notably, AITC is known for activating nuclear factor erythroid 2-related factor 2 (NRF2), but there is limited evidence supporting the beneficial effects on hepatocytes and liver, where AITC is mainly metabolized. We applied a mouse model in the current study to investigate whether AITC protects the liver against APAP-induced injury, wherein we observed the protective effects of AITC. Furthermore, NRF2 nuclear translocation and the increase of target genes by AITC treatment were confirmed by in vitro experiments. APAP-induced cell damage was attenuated by AITC via an NRF2-dependent manner, and rapid NRF2 activation by AITC was attributed to the elevation of NRF2 stability by decreasing its spontaneous degradation. Moreover, liver tissues from our mouse experiment revealed that AITC increases the expression of heme oxygenase-1 (HO-1), an NRF2 target gene, confirming the potential of AITC as a hepatoprotective agent that induces NRF2 activation. Taken together, our results indicate the potential of AITC as a natural-product-derived NRF2 activator targeting the liver.

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Astragaloside IV ameliorates preeclampsia-induced oxidative stress through the Nrf2/HO-1 pathway in a rat model

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00357.2020 ◽

2020 ◽

Vol 319 (5) ◽

pp. E904-E911 ◽

Cited By ~ 1

Author(s):

Shuangyan Yang ◽

Ruixue Zhang ◽

Baoheng Xing ◽

Ling Zhou ◽

Peipei Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Rat Model ◽

Target Genes ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Astragaloside Iv ◽

Pregnant Rats ◽

Beneficial Effects ◽

Antioxidative Effects ◽

Protein Serum

Preeclampsia (PE) can cause serious health problems for pregnant women and their infants. Astragaloside IV has been shown to exert cardioprotective, anti-inflammatory, and antioxidative effects on various disorders. We aimed to study the effects of Astragaloside IV on PE symptoms using an NG-nitro-l-arginine methyl ester (l-NAME)-induced rat model of PE. The pregnant rats’ physiological features, including blood pressure, urine protein, serum soluble fms-like tyrosine kinase- 1 ( sFlt - 1)/placental growth factor (PlGF) ratio, and weight of placenta, as well as the weight, length, and survival of pups, were documented. The expression levels of target genes were analyzed by Western blot and qRT-PCR assays. The levels of target secreted proteins were determined by ELISA. We demonstrated that the administration of Astragaloside IV might exert a multitude of beneficial effects on attenuated PE symptoms in a rat model of PE. We further revealed that the effects of Astragaloside IV on PE rats were achieved, at least partially, through elimination of oxidative stress and stimulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Our study indicated that Astragaloside IV may serve as a promising candidate for the development of new therapeutic methods for patients with PE.

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Inchinkoto, a herbal medicine, and its ingredients dually exert Mrp2/MRP2-mediated choleresis and Nrf2-mediated antioxidative action in rat livers

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00302.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. G1450-G1463 ◽

Cited By ~ 58

Author(s):

Kosuke Okada ◽

Junichi Shoda ◽

Masahito Kano ◽

Sachiko Suzuki ◽

Nobuhiro Ohtake ◽

...

Keyword(s):

Herbal Medicine ◽

Biliary Excretion ◽

Mrna Level ◽

Heme Oxygenase 1 ◽

Magic Bullet ◽

Related Factor ◽

Mrna Levels ◽

Chimeric Mice ◽

Rat Livers ◽

Antioxidative Action

Inchinkoto (ICKT), a herbal medicine, has been recognized in Japan and China as a “magic bullet” for jaundice. To explore potent therapeutic agents for cholestasis, the effects of ICKT or its ingredients on multidrug resistance-associated protein 2 (Mrp2/ MRP2)-mediated choleretic activity, as well as on antioxidative action, were investigated using rats and chimeric mice with livers that were almost completely repopulated with human hepatocytes. Biliary excretion of Mrp2 substrates and the protein mass, subcellular localization, and mRNA level of Mrp2 were assessed in rats after 1-wk oral administration of ICKT or genipin, a major ingredient of ICKT. Administration of ICKT or genipin to rats for 7 days increased bile flow and biliary excretion of bilirubin conjugates. Mrp2 protein and mRNA levels and Mrp2 membrane densities in the bile canaliculi and renal proximal tubules were significantly increased in ICKT- or genipin-treated rat livers and kidneys. ICKT and genipin, thereby, accelerated the disposal of intravenously infused bilirubin. The treatment also increased hepatic levels of heme oxygenase-1 and GSH by a nuclear factor-E2-related factor (Nrf2)-dependent mechanism. Similar effects of ICKT on MRP2 expression levels were observed in humanized livers of chimeric mice. In conclusion, these findings provide the rationale for therapeutic options of ICKT and its ingredients that should potentiate bilirubin disposal in vivo by enhancing Mrp2/MRP2-mediated secretory capacities in both livers and kidneys as well as Nrf2-mediated antioxidative actions in the treatment of cholestatic liver diseases associated with jaundice.

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The TBX1/miR-193a-3p/TGF-β2 Axis Mediates CHD by Promoting Ferroptosis

Oxidative Medicine and Cellular Longevity ◽

10.1155/2022/5130546 ◽

2022 ◽

Vol 2022 ◽

pp. 1-13

Author(s):

Li Zhong ◽

Huiqin Yang ◽

Binlu Zhu ◽

Xueqi Zhao ◽

Meijun Xie ◽

...

Keyword(s):

Protein Expression ◽

Western Blotting ◽

Cell Counting ◽

Luciferase Reporter ◽

Deletion Syndrome ◽

H9c2 Cell ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Messenger Ribonucleic Acid ◽

Nadph Oxidase 4

Congenital heart disease (CHD) is the most common noninfectious cause of death during the neonatal stage. T-box transcription factor 1 (TBX1) is the main genetic determinant of 22q11.2 deletion syndrome (22q11.2DS), which is a common cause of CHD. Moreover, ferroptosis is a newly discovered kind of programmed cell death. In this study, the interaction among TBX1, miR-193a-3p, and TGF-β2 was tested using quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and dual-luciferase reporter assays. TBX1 silencing was found to promote TGF-β2 messenger ribonucleic acid (mRNA) and protein expression by downregulating the miR-193a-3p levels in H9c2 cells. In addition, the TBX1/miR-193a-3p/TGF-β2 axis was found to promote ferroptosis based on assessments of lipid reactive oxygen species (ROS) levels, Fe2+ concentrations, mitochondrial ROS levels, and malondialdehyde (MDA) contents; Cell Counting Kit-8 (CCK-8) assays and transmission electron microscopy; and Western blotting analysis of glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), NADPH oxidase 4 (NOX4), and acyl-CoA synthase long-chain family member 4 (ACSL4) protein expression. The protein expression of NRF2, GPX4, HO-1, NOX4, and ACSL4 and the level of MDA in human CHD specimens were also detected. In addition, TBX1 and miR-193a-3p expression was significantly downregulated and TGF-β2 levels were high in human embryonic CHD tissues, as indicated by the H9c2 cell experiments. In summary, the TBX1/miR-193a-3p/TGF-β2 axis mediates CHD by inducing ferroptosis in cardiomyocytes. TGF-β2 may be a target gene for CHD diagnosis and treatment in children.

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BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress

Molecular and Cellular Biology ◽

10.1128/mcb.00700-06 ◽

2006 ◽

Vol 26 (21) ◽

pp. 7942-7952 ◽

Cited By ~ 124

Author(s):

Jianyong Zhang ◽

Tsutomu Ohta ◽

Atsushi Maruyama ◽

Tomonori Hosoya ◽

Keizo Nishikawa ◽

...

Keyword(s):

Oxidative Stress ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Target Genes ◽

Inducible Expression ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Gene Encoding ◽

Z Dna ◽

Responsive Element

ABSTRACT NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.

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Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex regulating the response to ROS homeostasis

Cell Death and Disease ◽

10.1038/cddis.2013.448 ◽

2013 ◽

Vol 4 (11) ◽

pp. e921-e921 ◽

Cited By ~ 34

Author(s):

S Tanigawa ◽

C H Lee ◽

C S Lin ◽

C C Ku ◽

H Hasegawa ◽

...

Keyword(s):

Oxidative Stress ◽

Transcriptional Activation ◽

Leucine Zipper ◽

Target Genes ◽

Critical Role ◽

Notch Receptor ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Mobility Shift ◽

Basic Leucine Zipper

Abstract Oxidative stress and reactive oxygen species (ROS) are associated with diseases such as cancer, cardiovascular complications, inflammation and neurodegeneration. Cellular defense systems must work constantly to control ROS levels and to prevent their accumulation. We report here that the Jun dimerization protein 2 (JDP2) has a critical role as a cofactor for transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and small Maf protein family K (MafK) in the regulation of the antioxidant-responsive element (ARE) and production of ROS. Chromatin immunoprecipitation–quantitative PCR (qPCR), electrophoresis mobility shift and ARE-driven reporter assays were carried out to examine the role of JDP2 in ROS production. JDP2 bound directly to the ARE core sequence, associated with Nrf2 and MafK (Nrf2–MafK) via basic leucine zipper domains, and increased DNA-binding activity of the Nrf2–MafK complex to the ARE and the transcription of ARE-dependent genes. In mouse embryonic fibroblasts from Jdp2-knockout (Jdp2 KO) mice, the coordinate transcriptional activation of several ARE-containing genes and the ability of Nrf2 to activate expression of target genes were impaired. Moreover, intracellular accumulation of ROS and increased thickness of the epidermis were detected in Jdp2 KO mice in response to oxidative stress-inducing reagents. These data suggest that JDP2 is required to protect against intracellular oxidation, ROS activation and DNA oxidation. qPCR demonstrated that several Nrf2 target genes such as heme oxygenase-1, glutamate–cysteine ligase catalytic and modifier subunits, the notch receptor ligand jagged 1 and NAD(P)H dehydrogenase quinone 1 are also dependent on JDP2 for full expression. Taken together, these results suggest that JDP2 is an integral component of the Nrf2–MafK complex and that it modulates antioxidant and detoxification programs by acting via the ARE.

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Nuclear factor erythroid 2-related factor 2 rescues the oxidative stress induced by di-N-butylphthalate in testicular Leydig cells

Human & Experimental Toxicology ◽

10.1177/0960327114530744 ◽

2014 ◽

Vol 34 (2) ◽

pp. 145-152 ◽

Cited By ~ 10

Author(s):

B Shen ◽

W Wang ◽

L Ding ◽

Y Sao ◽

Y Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Leydig Cells ◽

Target Genes ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Nuclear Factor ◽

Quinone Oxidoreductase ◽

Antioxidant Genes ◽

Protein Levels

Aim: This study aimed to determine whether nuclear factor erythroid 2-related factor 2 antagonized the oxidative stress induced by di- N-butylphthalate (DBP) in testicular Leydig cells. Methods: Mouse TM3 testicular Leydig cells were treated with Nrf2 knockdown (KD) or overexpression in the presence and absence of DBP. Oxidative profiles were examined. Nrf2 target antioxidant genes were studied, and the effects of Nrf2 inducer sulphoraphane (SFN) were tested. Results: DBP induced intracellular oxidative stress to a similar extent with Nrf2 KD. Expression and protein levels of Nrf2 were increased together with its target genes, namely heme oxygenase 1, nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 and peroxiredoxin 6, following DBP stimulation. Use of SFN not only restored the intracellular oxidative toxicity but also cell proliferation and testosterone secretion in response to DBP. Conclusion: Increased Nrf2 activity, for example, by SFN can effectively antagonize the oxidative stress in testicular Leydig cells caused by DBP.

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Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00402.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. H1133-H1140 ◽

Cited By ~ 106

Author(s):

Zoltan Ungvari ◽

Lora Bailey-Downs ◽

Tripti Gautam ◽

Rosario Jimenez ◽

Gyorgy Losonczy ◽

...

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

High Glucose ◽

Target Genes ◽

Cell Model ◽

Luciferase Reporter ◽

Standard Diet ◽

Related Factor ◽

Antioxidant Genes ◽

Gene Assay

Hyperglycemia in diabetes mellitus promotes oxidative stress in endothelial cells, which contributes to development of cardiovascular diseases. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a transcription factor activated by oxidative stress that regulates expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes. This study was designed to elucidate the homeostatic role of adaptive induction of Nrf2-driven free radical detoxification mechanisms in endothelial protection under diabetic conditions. Using a Nrf2/antioxidant response element (ARE)-driven luciferase reporter gene assay we found that in a cultured coronary arterial endothelial cell model hyperglycemia (10–30 mmol/l glucose) significantly increases transcriptional activity of Nrf2 and upregulates the expression of the Nrf2 target genes NQO1, GCLC, and HMOX1. These effects of high glucose were significantly attenuated by small interfering RNA (siRNA) downregulation of Nrf2 or overexpression of Keap-1, which inactivates Nrf2. High-glucose-induced upregulation of NQO1, GCLC, and HMOX1 was also prevented by pretreatment with polyethylene glycol (PEG)-catalase or N-acetylcysteine, whereas administration of H2O2 mimicked the effect of high glucose. To test the effects of metabolic stress in vivo, Nrf2+/+ and Nrf2−/− mice were fed a high-fat diet (HFD). HFD elicited significant increases in mRNA expression of Gclc and Hmox1 in aortas of Nrf2+/+ mice, but not Nrf2−/− mice, compared with respective standard diet-fed control mice. Additionally, HFD-induced increases in vascular ROS levels were significantly greater in Nrf2−/− than Nrf2+/+ mice. HFD-induced endothelial dysfunction was more severe in Nrf2−/− mice, as shown by the significantly diminished acetylcholine-induced relaxation of aorta of these animals compared with HFD-fed Nrf2+/+ mice. Our results suggest that adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions.

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