Synthesis,molecular modeling and NAD(P)H:quinone oxidoreductase 1 inducer activity of novel 2-phenylquinazolin-4-amine derivatives

Reactive oxygen species (ROS) play an integral role in the pathogenesis of most diseases. This work presents the design and synthesis of novel 2-phenylquinazolin-4-amine derivatives (2–12) and evaluation of their NAD(P)H:quinone oxidoreductase 1 (NQO1) inducer activity in murine cells. Also, molecular docking of all the new compounds was performed to assess their ability to inhibit Keap1–Nrf2 protein–protein interaction through occupying the Keap1–Nrf2-binding domain which biologically leads to a consequent Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed that all compounds have the ability to interact with Keap1; however compound 7, the most active compound in this study, showed more interactions with key amino acids.     

Keap1 Cysteine 288 as a Potential Target for Diallyl Trisulfide-Induced Nrf2 Activation

Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.     

Identification of a novel small-molecule Keap1–Nrf2 PPI inhibitor with cytoprotective effects on LPS-induced cardiomyopathy

A new Keap1–Nrf2 protein–protein interaction (PPI) inhibitor ZJ01 was identified from our compound library by fluorescence polarization assay, surface plasmon resonance, molecular docking and molecular dynamics simulation. ZJ01 could in vitro trigger Nrf2 nuclear translocation, subsequently resulting in increased mRNA levels of Nrf2 target genes HO-1 and NQO1. Meanwhile, ZJ01 suppressed LPS-induced production of ROS and the mRNA levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in H9c2 cardiac cells. Moreover, in an in vivo mouse model of septic cardiomyopathy induced by intraperitoneal injection of lipopolysaccharide, ZJ01 demonstrated a cytoprotective effect, upregulated Nrf2 protein nuclear accumulation, and remarkably suppressed the abovementioned cytokine levels in cardiomyocytes. The results presented herein provided a novel chemotype for the development of direct Keap1–Nrf2 PPI inhibitors and suggested that compound ZJ01 is a promising drug lead for septic cardiomyopathy treatment.

ZJ01 was identified as a new Keap1–Nrf2 PPI inhibitor and drug lead for septic cardiomyopathy treatment by in vitro and in vivo experiments.     

miR-19通过Keap-Nrf2/HO-1信号通路对卵巢癌细胞增殖的影响

摘要 目的：研究卵巢癌组织和细胞中miR-19的表达,探讨其异常表达对卵巢癌细胞Kelch样环氧氯丙烷相关蛋白-1（Kelch-like epichlorohydrin-associated protein1,Keap1）--核因子E2相关因子2（nuclearfactor-E2-relatedfactor2,Nrf2） /血红素氧合酶-1（heme oxygenase1,HO-1）信号通路及卵巢癌细胞增殖的影响。方法：回顾性收集2019年1月至2020年12月于我院就诊的患者经病理切片诊断为卵巢癌上皮细胞的手术标本30例,卵巢良性肿瘤标本30例,正常卵巢组织标本30例。免疫组化检测不同标本中Keap1、Nrf2、HO-1的表达,检测卵巢组织及细胞中miR-19、Keap1、Nrf2、HO-1的mRNA表达水平,及卵巢癌细胞中Keap1、Nrf2、HO-1的蛋白表达水平。在OVCAR-3细胞中沉默miR-19后,Western Blot检测细胞内Keap1、Nrf2、HO-1蛋白表达水平,收集沉默miR-19,对照组,沉默Nrf2、对照组的OVCAR-3细胞,继续培养0 h、24 h、48 h后,检测细胞增殖和凋亡。结果：Keap1蛋白在卵巢癌组织中的阳性表达显著低于良性卵巢肿瘤组织及正常卵巢组织;Nrf2和HO-1蛋白在卵巢癌组织中的阳性表达显著低于良性卵巢肿瘤组织及正常卵巢组织（P＜0.05）;沉默miR-19抑制其表达后,细胞内Keap1 mRNA、蛋白表达水平明显升高,Nrf2、HO-1 mRNA表达水平无明显变化,蛋白表达水平明显降低（P＜0.05）;沉默miR-19 组、沉默Nrf2组与转染阴性对照组相比,增殖能力明显降低,凋亡能力明显升高（P＜0.05）。结论：卵巢癌细胞中,miR-19表达水平升高,可通过调控Keap1-Nrf2/HO-1信号通路影响卵巢癌细胞的增值、凋亡能力。     

NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes

The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway responds to oxidative stress via control of several antioxidant defense gene expressions. Recent efforts demonstrate that Nrf2 modulates development of adiposity and adipogenesis. One of the major Nrf2-regulated proteins, NAD(P)H:quinone oxidoreductase 1 (NQO1), is implicated in the development of adipose tissue and obesity. However, little is known about in situ disposition of Nrf2, Keap1, and NQO1 during adipogenesis in isolated adipocytes. Based on literature data, we hypothesized that adipocyte differentiation would increase expression of the Nrf2/Keap1 pathway and NQO1. Using murine 3T3-L1 preadipocytes, we mapped an increase in NQO1 protein at limited clonal expansion and postmitotic growth arrest (Days 1-3) stages and a decrease in terminally differentiated (Day 8) adipocytes that lasted for several days afterward. Conversely, NQO1, Nrf2, and Keap1 mRNA expressions were all increased in differentiated adipocytes (Days 11-14), indicating a discrepancy between steady-state mRNA levels and resulting protein. Treatment of differentiated 3T3-L1 adipocytes with glycogen synthase kinase-3β (GSK-3β) inhibitor, LiCl, led to 1.9-fold increase in NQO1 protein. Sulforaphane enhanced NQO1 protein (10.5-fold) and blunted triglyceride and FABP4 accumulation. The decrement in triglyceride content was partially reversed when NQO1 activity was pharmacologically inhibited. These data demonstrate a biphasic response of Nrf2 and NQO1 during adipocyte differentiation that is regulated by Keap1- and GSK-3β-dependent mechanisms, and that hypertrophy is negatively regulated by NQO1 activity.     

阿里红多糖通过激活Nrf2/ARE通路改善阿尔茨海默病大鼠海马及脑皮层的氧化应激损伤

探讨阿里红多糖(Fomes offficinalis Ames polysaccharides,FOPS)抗氧化应激的作用,并从Nrf2/ARE信号通路研究其作用机制.72只健康雄性SD大鼠称体质量并按随机原则分为空白组、模型组、盐酸多奈哌齐组(0.5 mg/kg)、阿里红多糖高、中、低剂量组(100、50、25 mg...