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运用系统医学方法阐明转录因子NRF2作为慢性疾病的治疗靶标

发表于:2019-05-14   作者:Gina Manda   来源:PHARMACOLOGICAL REVIEWS   点击量:

Antonio Cuadrado et al., PHARMACOLOGICAL REVIEWS, 2018

  

 
缩写:
AD: 阿尔茨海默病;  ALS: 肌萎缩侧索硬化症;  AMPK,AMP活化蛋白激酶;  ARE: 抗氧化反应元件;  β-TrCP: 含有E3泛素蛋白连接酶的β-转导蛋白重复序列;  bZip: 碱性区域 - 亮氨酸拉链;  CDDO: 2-氰基-3,12-二氧代-油酸-1,9(11)- 二烯-28-酸;  CDDO-Me: CDDO-甲酯;  COPD:慢性阻塞性肺病;  CUL3,Cullin 3;  DC:树突状细胞;  DMF: 富马酸二甲酯;  EAE:实验性自身免疫性脑脊髓炎;  ECH:具有Cap'n'collar同源性的红系细胞衍生蛋白;  GCLC:g-谷氨酰半胱氨酸连接酶催化亚基;  GCLM:g-谷氨酰半胱氨酸连接酶调节剂亚基;  GI:胃肠道;  GSH:谷胱甘肽;  GSK-3:糖原合成酶激酶3;  HFD,高脂饮食;  HNSCC:头颈部鳞状细胞癌;  HO-1,血红素加氧酶-1;  IBD:炎症性肠病;  IκB:κ-B抑制剂;  IKK:IκB激酶;  IL:白细胞介素;  KEAP1:kelch样ECH相关蛋白1;  LDL:低密度脂蛋白;  LPS:脂多糖;  MAF:肌肉腱膜纤维肉瘤蛋白;  MMF:富马酸单甲酯;  MS:多发性硬化;  NASH:非酒精性脂肪性肝炎;  NF-κB:活化B细胞核因子κ-轻链增强子的p65亚基;  NQO1:NADPH:醌氧化还原酶;  NRF2:核因子(红细胞衍生的2)样2;  PD:帕金森病;  PI3K:磷脂酰肌醇3-激酶;  PPI:蛋白质-蛋白质相互作用;  PTEN:磷酸酶和张力蛋白同系物;  RA: 类风湿性关节炎;  RBX1:RING-box蛋白1;  RNS:活性氮物种; ROS:活性氧;  SFN:萝卜硫素; SLE:系统性红斑狼疮;  SNP:单核苷酸多态性;  SQSTM1:sequestosome 1;  STAT:信号转导和转录激活因子;  T2DM:2型糖尿病;  TGF:转化生长因子;  Th,T辅助子;  TNF:肿瘤坏死因子;  Treg:T调控。

 
 

运用系统医学方法阐明转录因子NRF2作为慢性疾病的治疗靶标


Antonio Cuadrado et al., PHARMACOLOGICAL REVIEWS, 2018

摘要

系统医学是基于疾病机理,而非基于具体症状或器官的疾病研究方法,并以非假设驱动的方式识别治疗靶标。本论文中,我们将系统医学方法应用于转录因子核因子( NRF2)的研究,通过交叉验证其在蛋白质-蛋白质相互作用网络(NRF2相互作用组)中的作用,该相互作用网络在功能上与低水平应激、慢性炎症、代谢改变和活性氧形成有关的细胞保护相关联。这些分子谱的多尺度网络分析表明,NRF2表达和活性的改变是疾病子网路中的常见机制(NRF2病变)。该相互作用网络关联了明显的异质性表型,如自身免疫、呼吸、消化、心血管、代谢和神经退行性疾病,以及癌症。重要的是,系统医学方法在计算机上匹配并证实了在临床开发的不同阶段在体内验证的NRF2调节药物的若干应用。从药理学上看,这些药物的特征多样,有亲电的富马酸二甲酯、合成的三萜类化合物如巴多索酮甲基和萝卜硫素、蛋白-蛋白或DNA -蛋白相互作用抑制剂,甚至有注册的药物如二甲双胍和他汀类药物,它们可以激活NRF2并且可能被重新用于指示 NRF2疾病表型群。因此,NRF2代表了经典和系统医学方法充分接受的第一类目标之一,这些方法通过关注一组似乎具有机械联系的疾病表型来促进药物开发和药物新用。 因此,由此产生的NRF2药物组可能会迅速为这类慢性疾病提供一些令人惊讶的临床选择。 

Ⅰ. 引言

在上个世纪,寿命几乎翻了一番,特定老龄化疾病现在变得普遍。然而,大多数疾病的病理机制很难理解,治疗方法也是通过纠正症状或危险因素来治疗。此外,与迄今为止只考虑一种疾病、一种药物的线性方法相反,慢性病显示出高度的关联性,需要更精确的、以机制为基础的疾病定义,而不是目前以器官和症状为基础的疾病定义。在人类基因组测序和分子网络的发展之后,一种新的疾病概念由此产生,疾病的诊断不仅是通过临床症状,而且主要是通过潜在的分子特征识别(Goh et al., 2007)。不同的病理表型具有共同的分子机制,这一事实也为总结为“多种疾病,一种药物”和药物新用的治疗新概念提供了理论基础。网络医学,即将网络概念应用于疾病和药物之间的动态联系分析,为开发这种新方法提供了新机会。老年人慢性病很可能的特征是衰老过程中体内平衡的丧失或环境因素的影响,所有这些都会通过病理性形成活性氧(ROS),慢性炎症和代谢失调导致低水平应激。基于网络医学方法,我们将在本文中提供大量证据,表明核因子(erythroid-derived 2)–like 2 (NRF2)作为多种细胞保护反应的主调控因子和特定疾病集群中的一个关键分子节点,为药物开发和药物新用提供了一种新的策略。

Ⅱ. 从NRF2相互作用组到NRF2疾病网络

A. NRF2作为一个细胞稳态的主要调节器

NRF2是一种碱性区域亮氨酸拉链(bZip)转录因子(图1),在细胞核内与小肌筋膜纤维瘤蛋白(MAF) K、G、F形成异二聚体。异二聚体识别一个称为抗氧化应答元件(ARE)的增强子序列,其存在于超过250个基因(ARE基因)的调节区域中(Ma,2013;Hayes and Dinkova-Kostova,2014)。这些基因编码参与I期、II期和III期生物转化反应和抗氧化机制的合作酶网络,产生NADPH、谷胱甘肽(GSH)和硫氧还蛋白反应;脂质和铁的分解代谢;以及与其他转录因子的相互作用等(Hayes and Dinkova-Kostova, 2014)。最近,NRF2也被发现可调控多个蛋白酶体亚基和自噬基因的表达,为其调控蛋白酶体平衡提供了额外的研究兴趣(Pajares et al., 2015, 2016, 2017;de la Vega等,2016)。

 
 
图1. NRF2作为细胞保护应答的主要调节因子。 (A)NRF2通过其bZip结构域与MAF家族成员异二聚体化。异二聚体与称为ARE的增强子序列结合,所述增强子序列存在于超过250个基因(ARE基因)的调节区中。 (B)如所示,这些基因参与氧化还原代谢,炎症和蛋白质稳态平衡的控制。 NFE2L2中易感性SNPs的存在,脑部尸检中其靶基因水平的升高以及临床前研究的阳性数据表明,NRF2激活可能抵消蛋白质稳态,氧化还原和炎症控制的不平衡。 AC1: ATP柠檬酸裂解酶; ACC1: 乙酰辅酶A羧化酶1; CALCOCO2: 钙结合和卷曲螺旋结构域2; cGS: c-谷氨酸半胱氨酸合成酶; FAS: 脂肪酸合成酶; G6PDH: 葡萄糖-6-磷酸脱氢酶; Gpx: 谷胱甘肽过氧化物酶; Gpx8: 谷胱甘肽过氧化物酶8; GR: 谷胱甘肽还原酶; HMOX1: 血红素加氧酶-1; IDH1: 异柠檬酸脱氢酶1; ME: 苹果酸酶; MTHFD2: 亚甲基四氢叶酸脱氢酶2; PGD: 磷酸葡萄糖酸脱氢酶; PPAT: 磷酸核糖焦磷酸酰胺转移酶; PSMB7: 蛋白酶体亚基b-7型; SCD1: 硬脂酰辅酶A去饱和酶; TrxR: 硫氧还蛋白还原酶; ULK1: unc-51像自噬激活激酶1。
 
NRF2在临床上的重要意义在于它可能具有药理学靶向性,对患者有益。调节NRF2转录活性的主要机制是通过E3连接酶适配器KEAP1(具有Cap'n'collar同源性(ECH)的Kelch样红系细胞衍生蛋白 - 相关蛋白1)来控制蛋白的稳定(图2)。KEAP1是同型二聚体蛋白,其将NRF2与Cullin 3和RING-box蛋白1(CUL3 / RBX1)形成的E3连接酶复合物连接。在稳态条件下,KEAP1同二聚体的N-末端结构域在两个氨基酸序列上与一个分子的NRF2结合,具有低(天冬氨酸,亮氨酸和甘氨酸; DLG)和高(谷氨酸,苏氨酸,甘氨酸和谷氨酸; ETGE)亲和力,因此,通过CUL3 / RBX1(Tong等人,2007)将NRF2呈现为泛素化,并随后通过蛋白酶体降解。KEAP1是一种氧化还原和亲电子传感器,在关键半胱氨酸修饰后失去其抑制NRF2的能力(图2;生物标志物作为NRF2特征并参与监测目标)。调节NRF2稳定性的另一种机制是由糖原合成酶激酶3(GSK-3)介导的磷酸化(图2)。这种激酶磷酸化NRF2(天冬氨酸,丝氨酸,甘氨酸,异亮氨酸,丝氨酸; DSGIS)的结构域,因此为含有E3泛素蛋白连接酶(b-TrCP)的E3连接酶接头b-转导蛋白重复序列产生识别基序,该基序将NRF2呈递给 CUL1 / RBX1复合物,导致NRF2泛素依赖性蛋白酶体降解的替代途径。因此,KEAP1和GSK-3 / b-TrCP分别在氧化还原稳态和细胞信号传导的情况下严格控制NRF2蛋白水平(Cuadrado,2015)。已经报道了NRF2在蛋白质,mRNA或基因水平上调节的其他机制(Hayes and Dinkova-Kostova,2014),但至少这两种机制适合于药理学调节。
 
 
 
图2. KEAP1和β-TrCP对NRF2稳定性的调节及其药理学靶向。(A)根据双重调节模型(Rada等,2011),NRF2的两个结构域,称为Neh2和Neh6,参与NRF2降解分别响应氧化还原和亲电子变化(KEAP1)和信号激酶(β-TrCP)。Neh2结构域结合E3连接酶适配子KEAP1,其呈递NRF2用于泛素化至CUL3 / RBX1复合物。 Neh6结构域需要GSK-3的先前磷酸化以结合E3连接酶适配子β-TrCP并随后通过CUL1/ RBX1复合物进行泛素化(详见文本)。(B)NRF2和KEAP1之间结合的细节以及当前针对这种相互作用的策略。KEAP1同型二聚体在Neh2结构域的两个基序处结合NRF2:低亲和力(29-DLG-31)和高亲和力(79-ETGE-82)结合位点。目前破坏这种相互作用的策略包括:改变半胱氨酸C151,273和288的巯基的亲电试剂; 改变NRF2与KEAP1的DC结构域的对接的PPI抑制剂。(C)NRF2和β-TrCP的假设结合,并提出了针对这种相互作用的策略。当它被GSK-3(Rada等,2011,2012)磷酸化时,β-TrCP同型二聚体在磷酸基序334-DSGIS-338处与Nhe6结构域结合,当与磷酸基序373-DSAPGS-378结合时独立于GSK-3(Chowdhry等,2013)。在该图中,我们假设,与KEAP1类似,一个β-TrCP同二聚体在两个磷酸基序上与一个NRF2分子相互作用,但仍缺乏实验证据。破坏这种相互作用的两种可能策略包括使用GSK-3抑制剂和PPI抑制剂。

B. NRF2及其调控途径在人类相互作用组和疾病组的定位

编码NRF2的基因,称为NFE2L2,具有高度多态性,诱变频率为每72 bp 1次。关于该主题的优秀综述在2015年报告了多达18个单核苷酸多态性(SNPs),其中大多数在5’调控区域和内含子1中(Cho等人,2015)。这些SNPs中的一些可能构成与慢性疾病发作或进展风险相关的功能性单倍型。功能性单倍型的变异可能对表现出特定疾病的临床症状的个体的比例具有微妙的影响,但是它们可能在群体水平上具有深远的影响,并且可能定义在精准医疗中靶向该基因的具体策略。
网络医学的最新进展提供了定量工具来表征基因与其相互作用(相互作用组)之间的相互作用如何与病理学相关(Barabasi等,2011; Vidal等,2011; Guney等,2016),分子网络失调在各种疾病中如何普遍(Menche等,2015),以及疾病如何在特定组织中表现(Kitsak等,2016)。为了从系统医学的角度理解NRF2在病理学中的相关性,首先我们已经生成了人类相互作用组图。我们整合并策划了与NRF2调节途径相关的蛋白质之间的物理相互作用的信息(Hayes and Dinkova-Kostova,2014; Cuadrado,2015)。交互数据来自最近发表的人类相互作用组,其汇编了几种蛋白质 - 蛋白质相互作用(PPI)资源的数据(Turei等,2013; Menche等,2015)。然而,目前可用于NRF2相互作用组开发的信息受限于以下事实:因为NRF2是非常短的半衰期蛋白质,所以可能未检测到一些有意义的相互作用。然而,在相互作用组中发现了一些与NRF2物理性相互作用的众所周知的蛋白质,包括KEAP1,b-TrCP和MAF。另一组NRF2相互作用蛋白对应于具有调节基因表达功能的核蛋白。这些包括与bZip转录因子,核受体或共激活因子相关的蛋白质,或涉及组蛋白乙酰化的蛋白质。因此,NRF2相互作用组证明除了与该转录因子直接相关的基因调控之外的其他基因调控机制。NRF2在几个残基处被磷酸化,因此预期它与几种激酶相互作用,所述激酶包括GSK-3和几种蛋白激酶C同种型。这些激酶是一些膜受体和衔接子/支架蛋白的下游。除了这种物理相互作用,我们已经确定了NRF2邻域中富集的几种生物功能,包括代谢过程,如戊糖,四吡咯,血红素,葡萄糖6-磷酸,半胱氨酸,GSH,甘油醛-3-磷酸和NADPH的生物合成。大多数这些调节蛋白不直接相互作用,而是通过作为介质的蛋白质连接(图3;特定相互作用分子的更详细描述可在这个网址找到http://sbi.imim.es/data/nrf2/)。
 
 
图3. 在人类相互作用组中定位NRF2调节途径。NRF2通过协调各种蛋白质的活性在病理性ROS形成以及炎症和代谢反应中起关键作用。这些相互作用构成分子相互作用网络,即NRF2相互作用组。参与NRF2调节途径的蛋白质之间的已知物理相互作用(即调节蛋白:棕色圆圈)和它们连接的蛋白质(即,介体蛋白质:绿色圆圈)用灰色连接显示。涉及多于一种介体蛋白连接到NRF2的调节蛋白用蓝色连接显示。调节蛋白质来自文献,它们的相互作用来自各种资源,包括IntAct,MINT,BioGRID,HPRD,KEGG和PhosphoSite。可在线获取人类相互作用组中与NRF2相互作用的所有蛋白质列表,网址为http://sbi.imim.es/data/nrf2/。
 
已经报道了几种疾病中NRF2相互作用组的扰动。我们基于DisGeNET (Pinero et al., 2017)和GeneCards (Stelzer et al., 2016)数据库,结合部分动物模型研究的知识,整理了37种nrf2相关疾病的列表,绘制了疾病组图。我们还使用DisGeNET,OMIM和GWAS数据库检索了这些病症的疾病 - 基因关联(Menche等,2015)(表1)。NRF2与每种NRF2相关疾病表型的已知疾病基因的基于相互作用组的接近度(Guney和Oliva,2014)显示在图4中。与随机选择的蛋白质相比,NRF2与消化系统和癌症(如前列腺,肝脏和肺肿瘤)的已知疾病基因显着接近,突出了NRF2在这些病理中的关键功能作用。此外,发现NRF2与代谢和心血管疾病相关的各种蛋白质均有近端表达,例如糖尿病,高血糖症,局部缺血,大脑中动脉梗塞和动脉粥样硬化。NRF2的蛋白质相互作用还将其与呼吸系统疾病相关的基因(如哮喘,肺纤维化和肺气肿)以及神经退行性疾病(如阿尔茨海默病(AD),帕金森病(PD)和肌萎缩侧索硬化症(ALS))联系起来。

表1.有NRF2相关证据的疾病群集

从DisGeNet数据库中选择具有与NRF2遗传相关的证据的疾病表型。DisGeNet整合来自各种资源的疾病基因关联信息,如UniProt,ClinVar,GWAS目录和比较毒物基因组学数据库,并根据资源数量和支持这些关联的出版物评估疾病基因关联。该清单的制定基于以下标准:1)仅选择在Pubmed中具有多于一个引用的病理表型; 2)可靠性评分设定为0.001的阈值; 3)具有非常相似的名称或重叠术语的疾病条目被简化为单个条目。



病理表型 可靠性分数 病理表型 可靠性分数
糖尿病肾病 0.2016 糖尿病性心肌病 0.0803
肝硬化 0.2005 大脑中动脉梗塞 0.0800
非酒精性脂肪性肝炎 0.2005 乳腺肿瘤 0.0087
急性肾损伤 0.2000 白癜风 0.0076
肺纤维化 0.2000 动脉粥样硬化 0.0067
非小细胞肺癌 0.1252 哮喘 0.0043
鳞状细胞癌 0.1243 白血病 0.0038
肝肿瘤 0.1238 结肠肿瘤 0.0038
高血糖 0.1208 胃肠疾病 0.0029
药物性肝损伤 0.1200 帕金森综合症 0.0026
前列腺肿瘤 0.1200 系统性红斑狼疮性肾炎 0.0026
慢性阻塞性肺疾病 0.0899 胶质瘤 0.0024
结直肠肿瘤 0.0847 肌萎缩侧索硬化症 0.0022
阿尔茨海默病 0.0837 缺血 0.0016
2型糖尿病 0.0814 肺气肿 0.0013
慢性肾病 0.0808 胰腺肿瘤 0.0013
糖尿病性视网膜病变 0.0805 血管疾病 0.0013
亨廷顿氏病 0.0805 脓血症 0.0013
 
 
 
 
图4. 从人类相互作用组的角度看NRF2的系统医学观点。疾病是由扰乱蛋白质及其相互作用的突变引发的,影响NRF2相互作用组中的某个邻域。基于相互作用组的接近度(proximity)测量基因与那些疾病邻域的距离。 条形图显示NRF2基因(NFE2L2)与参与NRF2相关病理表型的已知疾病基因的接近程度。这些条形图强调了NRF2在消化系统疾病和癌症中的作用。对于给定的疾病,接近度首先计算从NRF2到最接近的已知疾病基因的距离,然后将该距离与使用相互作用组中随机选择的蛋白质之间的平均距离估计的随机期望进行比较。基于相互作用组的接近度报告的z分数对应于NRF2与疾病基因之间观察到的距离的显着性。负值表示观察到的距离低于偶然预期的距离。根据z得分,条纹以不同的橙色调着色:分别为近端(深橙色),近端(橙色),非近端(浅橙色)。已知的疾病基因来自DisGeNet,OMIM和GWAS目录。
 
因此,基于相互作用组的接近度提供了关于NRF2如何与各种病理状况相关联的观点。之前已将疾病之间的关系概括为一种网络,成为疾病组,其基于遗传(Goh等人,2007)和临床(Hidalgo等人,2009; Zhou等人,2014)的共同性来连接它们。在图5中,我们基于共享基因,症状相似性和合并症定义了疾病网络,即NRF2疾病组。NRF2似乎连接基本上由炎症过程控制的疾病,例如急性肾损伤,肝硬化和动脉粥样硬化。此外,AD,PD,亨廷顿舞蹈病和ALS等神经退行性疾病构成一个集群,与最近的研究一致,也暗示NRF2在神经炎症过程中的作用(Rojo等,2010; Lastres-Becker等,2012,2014; Jung等,2017; Wang等,2017b)。通过激酶信号转导级联对NRF2的调控(Jung et al., 2017)可以解释密切相关的癌症簇,特别是支持NRF2参与结肠和乳腺肿瘤病理性ROS形成(Gonzalez-Donquiles et al., 2017;Lu et al., 2017)。
 
 
 
 
图5.  NRF2疾病组的当前状态。疾病之间的关系表示为一个网络,其中病理表型由共同的遗传和临床描述符连接。图中,节点(红色六角形)代表疾病,边缘根据共同的基因、共同的症状和共病(分别为灰色、橙色和蓝色线条)表示疾病之间的相似性。与疾病相关的基因和症状被用来识别具有显著遗传和症状重叠的疾病对,这些重叠是使用Jaccard指数计算出来的。在显着的疾病 - 疾病关系中(P <0.05,通过基于观察到的基因或症状重叠的Fisher精确检验评估),仅显示具有升高的重叠和共病的链接以消除潜在的虚假连接。因此,图中包含了至少10%的疾病相关基因和超过一半的相关症状。共病信息是从医疗保险索赔中提取的,代表人群中容易同时发生的疾病对(相对风险>2)。

Ⅲ. NRF2在人类疾病状态中的靶标验证

A. NRF2在炎症消退中的关键作用

持续性炎症是NRF2疾病组中发现的所有病理表型的标志。这很可能是因为炎症与活性氧(ROS)的局部和全身病理形成增加有关。事实上,ROS和活性氮物种(RNS)刺激和加重炎症反应,这些反应与转录因子NF-κB的激活机制相关的激活机制相关(NF-κB:激活的B细胞的核因子κ-轻链增强子的p65亚基)(Wenzel等,2017)。非常简化的,在静息免疫细胞中,NF-κB通过与核κ-B抑制因子(IκBα)的相互作用保留在细胞溶质中。源自微生物的病原体相关分子模式分子以及响应于组织损伤而释放的损伤相关分子模式分子刺激免疫细胞表达的同源受体,其导致IκB激酶(IKK)β的激活。该激酶使IκBα磷酸化,使其靶向降解并允许核转位和NF-κB活化(Napetschnig和Wu,2013)。这些事件通过IκBα的几种调节模式进行氧化还原控制(Bowie和O'Neill,2000; Morgan和Liu,2011; Siomek,2012),但最近描述的一种模式涉及KEAP1对IKKβ稳定性的调节。就像NRF2一样,IKKβ具有ETGE基序,使其能够与KEAP结合,进行泛素化和蛋白酶体降解。因此,在基础氧化还原条件下,活性KEAP1靶向IKKβ进行降解,然后IκBα抑制NF-κB。相反,在存在ROS的情况下,KEAP1被抑制并且IKKβ稳定,磷酸化IκBα并导致其降解并因此导致NF-κB的上调(Lee等人,2009)。

由于NRF2是氧化还原稳态的主要调节因子,因此它对NF-κB活性进行间接控制。脂多糖(LPS)同时激活快速、促炎性的NF-κB反应和缓慢的NRF2反应。当NRF2最大活性时,NF-κB响应随后被抑制(Cuadrado等,2014)。例如,Ras相关的C3肉毒杆菌毒素底物1(Rho家族的一个小G蛋白)激活NF-kB途径,并且NRF2过表达被阻断,而NRF2敲低增强NF-κB依赖性转录(Cuadrado等,2014)。一致地,在用LPS或肿瘤坏死因子(TNF)-α攻击的NRF2缺陷型(Nrf2-/-)小鼠中,IKK的活性恶化并导致IκB的磷酸化和降解增加(Thimmulappa等,2006a)。

NRF2还诱导抗炎表型,其调节CD8+T细胞(Sha等人,2015)以及巨噬细胞和小胶质细胞的功能(Rojo等人,2010,2014a; Brune等人,2013)。这是因为NRF2通过调节胱氨酸/谷氨酸转运蛋白和GSH合成酶ʏ-谷氨酰半胱氨酸连接酶调节器和催化亚基[ʏ-谷氨酰半胱氨酸连接酶调节剂亚基(GCLM)和ʏ-谷氨酰半胱氨酸连接酶催化亚基(GCLC)]来增加巨噬细胞中的半胱氨酸和GSH水平。相反,GSH耗竭使巨噬细胞对LPS激活NRF2敏感(Diotallevi等,2017)。所有这些研究都指出NRF2是一种抗炎因子,对控制炎症反应的强度和持续时间至关重要(图6)。
 
 
 
图6. 通过NRF2直接和间接调节炎症。直接作用机制包括抗炎基因的转录诱导以及促炎基因的转录抑制。 在第二种情况下,引号表示在此功能中需要进一步的工作来识别NRF2的bZip伙伴(如果有的话)。抵抗炎症的间接机制涉及ROS / RNS调节和抑制免疫细胞的迁移/浸润。总体而言,这些途径导致抗炎反应,有助于正确解决炎症。NFE2L2中多态性的存在与转录活性降低,患者中靶基因水平的改变以及来自临床前研究的有希望的数据相关,支持NRF2在炎症消退中的相关作用。
 
通过前馈和反馈机制产生NRF2和NF-κB串扰(图7)。在转录水平,由于NFE2L2基因的启动子区域中存在几个功能性结合位点,NF-κB激活NRF2表达,从而诱导负反馈环(Rushworth等,2012)。此外,NF-κB和NRF2转录因子都需要共激活因子CBP / p300,它是组蛋白乙酰转移酶,乙酰化并增加DNA结合能力。因此,NF-κB过表达阻碍了NRF2的CBP / p300的可用性,因此降低了其转录能力,而NF-kB敲低显示出相反的效果(Liu等人,2008)。另外,NF-κB可以促进组蛋白脱乙酰酶-3与MAF蛋白的相互作用,因此阻止它们与NRF2的二聚化(Liu等人,2008)。NF-κB将KEAP1结合并转移至细胞核,从而有利于NRF2在该细胞区室中的泛素化和降解(Yu等,2011)。 E3连接酶适配子β-TrCP标记IκBα(Winston等人,1999)和NRF2(Rada等人,2011,2012; Cuadrado,2015)用于蛋白酶体降解,因此它可以导致增加的NF-kB活性。
 
 


 
图7. NF-κB和NRF2之间的串扰发生在不同的水平。(A)已在NFE2L2的启动子区域中鉴定出响应元件。(B)NRF2和NF-κB转录因子都竞争结合转录共激活因子CREB结合蛋白(CBP / p300)。(C)NF-κB活化激酶IKKb含有ETGE基序,其允许KEAP1结合和随后的泛素蛋白-蛋白酶体降解。(D)据报道NF-κB结合并将KEAP1转移至细胞核,从而促进NRF2降解。(E)炎症过程中产生的ROS激活NF-κB和NRF2; 最后,NRF2减弱ROS并因此减弱NF-κB活性。(F)不同的促炎信号激活Rho GTP酶RAC1,导致NF-κB和NRF2活化。 然后NRF2抑制RAC1介导的NF-kB活化。
NRF2的抗炎活性被认为仅依赖于氧化还原代谢的调节或与NF-κB的串扰。然而,NRF2还可以在暴露于LPS后直接阻断巨噬细胞中促炎基因白细胞介素(IL)-6和IL-1β的转录(Kobayashi等,2016)。LPS暴露或NRF2的药理学活化导致其与这些促炎基因的近端启动子结合并阻断RNA pol II的募集。该机制似乎与NRF2与其成熟的ARE增强子的结合无关。在其他研究中,NRF2可以直接调节其他几种巨噬细胞特异性基因的表达,例如具有胶原结构的巨噬细胞受体,细菌吞噬作用所需的受体,或CD36,氧化低密度脂蛋白的清道夫受体(Harvey等,2011;Ishii和Mann,2014)。类似地,编码促炎细胞因子IL-17D的基因含有AREs,并且该NRF2-T辅助因子(Th)17轴似乎赋予针对肿瘤发生和病毒感染的保护作用(Saddawi-Konefka等,2016)。
慢性炎症过程涉及白细胞粘附到血管内皮并渗入受损组织。两种过程似乎都被NRF2与其编码血红素加氧酶-1(HO-1)的至少一种靶基因HMOX1一起调节。NRF2 / HO-1轴通过调节几种细胞粘附分子如血管细胞粘附分子1的表达来抑制炎性细胞与内皮的粘附(Banning和Brigelius-Flohe,2005; Wenzel等,2015)。另外,NRF2 / HO-1抑制巨噬细胞中的金属蛋白酶-9,这是组织内免疫细胞迁移所必需的(Bourdonnay等,2009)。
许多临床前研究报道,天然化合物(Satoh等,2013)或通过破坏其负调节因子KEAP1激活NRF2导致髓样白细胞(Kong等,2011)和巨噬细胞(Lin等, 2008)的强效抗炎作用。 在观察性研究中,NFE2L2中的多态性与转录活性降低相关,与炎症性肠病(Arisawa等,2008b)和慢性胃炎(Arisawa等,2007)的风险增加相关。NRF2的免疫调节作用的一个例子是在中枢神经系统。受损神经元释放出趋化因子,一种特异性激活小胶质细胞中磷脂酰肌醇3激酶/ AKT(PI3K / AKT)途径的趋化因子,导致GSK-3β的抑制和NRF2的上调(LastresBecker等,2014)。在这项研究中,来自AD和进行性核上性麻痹患者的尸检显示出趋化因子水平的补偿性增加以及上调的NRF2蛋白,表明该途径有助于限制病变大脑中的炎症反应。

B. NRF2在自身免疫性疾病中的作用

在NRF2疾病群的外围,我们发现了几种自身免疫疾病表型,如白癜风,哮喘,多发性硬化症(MS)和系统性红斑狼疮(SLE)。实际上,在实验性自身免疫性脑脊髓炎(EAE)和类风湿性关节炎(RA)的动物模型中的大量工作,以及MS和牛皮癣中的临床证据进一步指出NRF2在自身免疫疾病中的作用。氧化组织损伤和细胞凋亡可以增加自身抗原的产生,导致T细胞的活化和B细胞产生自身抗体,例如,如对3-硝基酪氨酸阳性蛋白所观察到的那样(Thomson等,2012)。此外,II相解毒酶的丢失,其中许多由NRF2转录调节,导致活性中间体的产生增加,这有助于形成半抗原或受损的大分子,有时变得具有免疫原性,从而增加引发自身免疫反应的自身抗原库。因为NRF2调节的酶在许多化学物质的解毒中起关键作用,因此可以认为NRF2可能是一种保护机制,可以抵抗环境对自身免疫发病机制的作用(Ma, 2013)。NRF2介导的自身免疫调节的潜在机制还涉及抑制促炎性Th1和Th17应答以及免疫抑制性T调节(Treg)和Th2应激的激活。还有越来越多的证据表明NRF2可以控制参与抗原呈递和适应性免疫应答调节的树突细胞(DC)和巨噬细胞的分化和功能。事实上,NRF2缺陷表明,通过增加共刺激分子的表达并因此增加抗原特异性T细胞反应性来改变DCs的功能和表型(Al-Huseini等,2013)。
MS是一种慢性炎性疾病,其特征在于自身反应性免疫细胞浸润到中枢神经系统中。NRF2的缺失加剧了EAE的发展,EAE是MS的小鼠模型(Johnson等人,2010)。与NRF2缺乏相关的部分影响可能与HO-1水平降低有关。因此,具有骨髓特异性HO-1缺陷的小鼠表现出较高的病变发生率,伴随着抗原呈递细胞的活化和炎性Th17和髓鞘特异性T细胞的浸润(Tzima等人,2009)。敲除KEAP1(Kobayashi等,2016)或用各种激活NRF2的小分子治疗(Buendia等,2016)抑制了疾病的发展和严重程度。NRF2在活跃的MS病变中强烈上调,并且NRF2应答基因的表达主要在初始髓鞘破坏的区域中被发现(Licht-Mayer等,2015)。在MS脑中,NRF2及其靶标NADPH:醌氧化还原酶(NQO1)和HO-1主要在浸润性巨噬细胞中表达,在较小程度上在星形胶质细胞中表达,最有可能作为对病理性ROS形成的代偿性反应。相反,在少突胶质细胞中缺乏NRF2和抗氧化基因表达,这可能是它们在MS中的损伤和损失的基础(van Horssen等,2010)。由于免疫和氧化还原稳态的改变,MS患者外周血单核细胞中HO-1表达降低,并且在疾病恶化期间下调(Fagone等,2013)。值得注意的是,干扰素-β治疗患者的基因表达谱分析鉴定出NRF2是长期抗氧化反应和神经元保存的潜在介质(Croze等,2013)。
SLE由高氧化环境、失调的细胞死亡和去除死细胞的缺陷而突出,这导致细胞坏死作为自身抗原的来源。NRF2缺陷的雌性小鼠随年龄增长而发展为类似于SLE的多器官自身免疫性疾病,其特征为DNA氧化增加,脂质过氧化,脾细胞凋亡,抗双链DNA抗体和史密斯抗原的存在,以及伴随重要的组织损伤(血管炎,肾小球肾炎,肝炎和心肌炎)(Li等,2004)。只有雌性小鼠出现SLE进展的事实表明,雌性特异性因子可能有助于打破对自身抗原的免疫耐受(Li等,2004)。NRF2缺乏还导致CD4 + T细胞的增殖反应增强、CD4 + / CD8 +比率改变、以及SLE中促炎性Th17的促进(Ma等人,2006; Zhao等人,2016)。事实上,NRF2耗竭与狼疮性肾炎发展过程中的Th17分化和功能有关,这似乎是通过调节细胞因子信号传导抑制因子3 /磷酸化信号转导和转录激活因子(STAT)3途径和IL-1b信号来调节的(Zhao 等,2016)。此外,Nrf2-/ -小鼠的唾液腺显示出强烈的淋巴细胞浸润,使人想起Sjögren综合征,这通常与SLE有关(Ma等,2006)。SLE患者表现出氧化性DNA损伤修复机制的改变(Evans等,2000),高血清氧化蛋白水平,载脂蛋白C3(Morgan等,2007),氧化磷脂和抗氧化修饰脂蛋白的自身抗体(Frostegard等,2005)。NRF2多态性尚未与SLE易感性相关,尽管SNP rs35652124与墨西哥女性儿童期发病肾炎风险增加有关(Cordova等,2010)。
类风湿关节炎(RA)是一种全身炎症性疾病,具有复杂但仍难以捉摸的自身免疫特性,在炎症关节中,中性粒细胞、巨噬细胞和淋巴细胞被积极募集和激活。这导致促炎介质如ROS / RNS,类二十烷酸,细胞因子(IL-17,TNF-α,干扰素-ʏ,IL-6和IL-1β)和分解代谢酶的分泌增加,这些促炎介质引发滑膜成纤维细胞的过度增殖, 关节肿胀,软骨和骨的逐渐破坏(Roberts等,2015)。NRF2基因的缺失增加了实验性RA模型中关节改变的易感性。例如,在表达T细胞受体KRN和主要组织相容性复合体II类分子A(g7) (K/BN关节炎模型)的小鼠中,以及在抗体诱导的关节炎中,NRF2缺乏加速了发病率并加重了疾病进程( Maicas等,2011; Wu等,2016b)。NRF2缺乏显着上调炎症细胞的迁移、环加氧酶-2和诱导型一氧化氮合酶的表达、ROS和RNS的产生以及促炎细胞因子和趋化因子的释放。此外,NRF2可能是关节炎中骨代谢的保护因子(Maicas等,2011),NRF2 / HO-1活化在RA动物模型和人滑膜成纤维细胞中发挥抗炎和抗氧化作用(Wu等,2016b)。有趣的是,抗风湿金(I)化合物通过激活NRF2和上调HO-1和GCLC来刺激抗氧化反应,证明其对RA的临床疗效(Kobayashi 等, 2016)。此外,NRF2 / HO-1激活介导了滑膜细胞凋亡的诱导和西洛他唑对促炎细胞因子产生的抑制(Park等,2010)以及H2S和相关化合物的抗炎作用,它们能通过对KEAP1的半胱氨酸残基进行硫化物修饰(Wu等,2016b)。其他诱导NRF2和HO-1信号传导的药物,如瑞巴派特,可以转导人和小鼠CD4+ T细胞向免疫抑制性Treg表型的分化,并通过特异性抑制STAT3来抑制TCD4+细胞向炎性Th17细胞的分化(Moon等,2014)。发炎的滑膜内过量的ROS产生似乎有助于RA的发病机制,因为患者的ROS形成,脂质过氧化,蛋白质氧化,DNA损伤和抗氧化防御机制活性降低显着增加,所有这些都促成了组织损伤和疾病进展(Datta等,2014)。响应于病理性ROS的形成,NRF2途径在RA患者的滑膜细胞和抗体诱导的关节炎小鼠的关节中被激活,但是该反应显然不足以抵消疾病进展(Wu等,2016b)。
白癜风是一种皮肤炎症性疾病,其特征在于表皮中ROS的积累,其参与黑素细胞的死亡。这些分子修饰DNA和黑素体蛋白,形成自身抗原并激活针对黑素细胞的自身免疫应答(Xie等,2016)。 遗传学研究发现NRF2启动子SNPs与白癜风易感性有关,如-650位点的SNP (Guan等, 2008),而rs35652124的C等位基因在汉族人群中被证明具有保护作用(Song等, 2016)。NRF2及其下游解毒靶基因NQO1,GCLC和GCLM在白癜风患者的表皮中上调,表明这种防御机制的激活不足(Natarajan等,2010)。

C. NRF2在慢性呼吸系统疾病中的作用

NRF2在呼吸系统疾病中的相关性在2010年得到了回顾(Cho和Kleeberger,2010),在这项工作中,我们将仅重点介绍最相关的发现(图8)。香烟烟雾是慢性阻塞性肺病(COPD)的主要危险因素。COPD患者具有功能失调的肺泡巨噬细胞,其导致不受控制的ROS产生,促炎介质,吞噬作用缺陷和一系列参与组织损伤的金属蛋白酶。慢性阻塞性肺病(COPD)患者的肺泡巨噬细胞功能失调,导致ROS生成失控、促炎介质、有缺陷的吞噬作用以及一系列参与组织损伤的金属蛋白酶。事实上,肺气肿肺组织显示实质中肺泡巨噬细胞密度与肺破坏严重程度之间存在直接关系(Finkelstein等,1995)。肺泡巨噬细胞的吞噬活性受损是引起COPD急性加重的细菌和病毒复发的主要原因,并且是发病率和死亡率的主要来源。Nrf2-/- 小鼠对香烟烟雾诱发的肺气肿表现出增强的易感性(Rangasamy等,2004)。重要的是,用异硫氰酸酯萝卜硫素(SFN)激活NRF2可恢复对细菌的识别和吞噬作用,增强肺泡巨噬细胞对肺细菌的清除,并减少野生型小鼠的炎症,但对于暴露于接触香烟烟雾的Nrf2-/-小鼠不会发生(Harvey等,2011)。在人类中,与吸烟和非吸烟的非肺气肿患者相比,吸烟相关性肺气肿患者肺泡巨噬细胞中NRF2的转录特征降低(Goven等, 2008)。NRF2表达降低与脂质过氧化产物4-羟基壬烯醛巨噬细胞表达增加有关。在NFE2L2启动子中,由三个SNPs和一个三联体重复构成的功能性单倍型,产生低至中等NRF2表达的多态性(Yamamoto等,2004),与发生COPD的风险增加有关(Hua等,2010)。低表达单倍型是发展呼吸衰竭的重要预测因子。因此,SNP rs6721961的-617A等位基因发生急性肺损伤的风险显着较高(Marzec等,2007)。
病理性ROS形成可能在慢性肺纤维化的发病机制中起作用。早期研究表明,博来霉素诱导的肺纤维化在Nrf2-/- 中比在野生型小鼠中更严重(Cho等,2004)。事实上,野生型小鼠通过上调NRF2诱导抗氧化和抗炎反应,而这在Nrf2-/-小鼠中无法实现。后来证实,患有特发性肺纤维化或慢性结节病/过敏性肺炎的患者表现出NRF2的表达增加,并且支气管肺泡灌洗液中的低摩尔.wt.抗氧化剂水平增加,如尿酸,抗坏血酸,视黄醇和α-生育酚 ,表明对ROS挑战的适应性反应不成功(Markart等,2009)。机制地,NRF2缺乏增加肌成纤维细胞分化,而用SFN药理诱导NRF2导致肌成纤维细胞数量减少和转化生长因子-β(TGF-β)的促纤维化作用减弱(Artaud-Macari等人,2013)。
 
  
 
图8. NRF2在慢性病的常见机制和病理表型中的作用。该图提供了从图5的NRF2疾病组中提取的一些实例。这些疾病的常见病理机制包括异常高的ROS水平和参与组织损伤的低度慢性炎症。NRF2通过调节许多细胞保护基因的表达,提供针对这些和其他组织特异性改变的细胞保护特征。

D. 消化系统中的NRF2

NRF2疾病组中消化系统病理表型的显着地位突出了NRF2的转录特征作为一种对异生素引发的慢性氧化损伤和炎症应激的有效适应机制的相关性。在胃肠道(GI)中,长期接触异生素可引发肠腔微生物群与免疫系统之间功能失调的相互作用(Aviello和Knaus,2017)。这可导致胃肠道的慢性疾病,如包括克罗恩病和溃疡性结肠炎的炎性肠病(IBD)表型,其中存在激活保护性NRF2应答的证据。例如,在来自IBD患者的结肠组织中,结肠上皮细胞通过与蛋白酶体蛋白表达增加相关的NRF2依赖性适应对炎症信号起反应(Kruse等,2016)。来自IBD患者的单核细胞衍生的巨噬细胞证明了特异性NRF2依赖性基因表达谱,其响应于LPS而加重,进一步表明这是一种减轻炎症反应的尝试(Baillie等, 2017)。在遗传水平上,NFE2L2基因的特定基因型(-686-684)与日本队列研究中的溃疡性结肠炎的发展相关,尤其是在女性中(Arisawa等,2008a)。事实上,GI中的病理过程高度依赖于宿主的遗传背景,与肠腔微生物群和免疫系统之间的功能失调相互作用有关(Aviello和Knaus,2017)。
GI的微生物群的共生作用之一是释放适量的ROS,其引起由NRF2介导的上皮细胞和浸润的免疫细胞中的细胞保护反应(Jones等,2015)。此外,在真核生物中受NRF2转录控制的细胞保护分子也可以由共生细菌产生。例如,微生物群中的HO-1同源物可能对胃肠道内稳态有很大的贡献,这可以用于治疗一氧化碳在肠道的局部递送(Onyiah等, 2014)。
NRF2在维持胃肠道稳态方面的作用已被证实,使NRF2这一转录因子成为IBD中有希望的治疗靶点。因此,几种化合物和膳食补充剂可能表现出有益效果,如褪黑激素,3-(3-吡啶基亚甲基)-2-吲哚满酮,丁酸盐,干酪乳杆菌,左旋肉碱,4-乙烯基-2,6-二甲氧基苯酚(卡诺尔), 乳枸杞(脱脂牛奶中枸杞的配方产品)等(Orena等,2015)。因此,明确NRF2在消化道慢性和急性疾病中的参与情况,对于更好地指导NRF2通路的调节治疗方法至关重要。
肝脏也是抵抗食物异生素的第一道防线。因此,NRF2疾病组强调该转录因子在与肝损伤相关的病理表型中的相关性也就不足为奇了。早期使用Nrf2-/-小鼠模型证明其对对乙酰氨基酚诱导的肝细胞损伤、苯并[a]芘诱导的肿瘤形成以及Fas-和TNF-α介导的肝细胞凋亡具有保护作用(Aleksunes和Manautou,2007)。Nrf2-/-小鼠对化学毒性的较高敏感性与解毒酶的基础表达和诱导表达降低相关。在人类中,导致NRF2表达降低的三种NRF2启动子SNPs的功能性单倍型与胃粘膜炎症的发展显著相关,无论是独立地还是通过与幽门螺杆菌感染相互作用(Arisawa等,2007)。对原发性胆汁性胆管炎患者NRF2转录特征的分析表明,这些患者表现出NRF2表达降低以及HO-1和GCLC蛋白水平低,这些损伤在肝硬化患者中更为严重(Wasik等,2017)。
病理性ROS形成是非酒精性脂肪性肝炎(NASH)患者肝细胞损伤和疾病进展的关键机制(图8)。该疾病分两个阶段发展,一个是肝细胞中脂肪酸的逐渐积累,另一个是肝损伤和炎性病理性ROS的形成(Wang等,2018)。因此,喂食高脂肪饮食(HFD)的小鼠出现了单纯的脂肪变性,其特征是肝脏脂肪沉积增加而没有炎症或纤维化,但Nrf2-/-小鼠出现加剧的肝脏脂肪变性和大量炎症,与NASH一致(Reccia等,2017)。然而有趣的是,肝细胞特异性KEAP1的缺失在减少肝脏脂肪变性的同时,并没有改变NASH发展过程中的炎症反应,提示其具有代偿机制(Ramadori等, 2016)。至少在NASH的大鼠模型中,饮食NRF2激活剂减弱了肝纤维化的进展(Shimozono等,2013)。在NASH患者的肝脏活组织检查中病理性ROS形成的标志物增加,并且NRF2特征增加,表明其试图减少氧化剂和炎症负荷(Takahashi等人,2014)。

E. 心血管系统中的NRF2

NRF2疾病群指出心血管系统对细胞氧化还原平衡的变化以及众所周知的动脉粥样硬化、高血压和糖尿病等共病的发展具有高度敏感性(Griendling and FitzGerald, 2003a,b;Harrison等,2003;Jay等,2006)(图8)。NRF2在预防这些病理表型方面的作用已经在NRF2 -/-小鼠中得到证实,NRF2 -/-小鼠在慢性耐力运动中表现出心脏结构受损(更多的重塑事件)和功能受损(较少缩短分数)(Shanmugam等, 2017a)。它们也更容易在心肌梗塞后发生心力衰竭(Strom和Chen,2017)。相反,NRF2的组成型活化产生还原状态,其特征书是心脏GSH /谷胱甘肽二硫化物比率增加和ROS形成和丙二醛水平降低(Shanmugam等,2017b)。在人类中,Tako-Tsubo心肌病的微阵列分析表明,在这种收缩功能障碍的急性期,病理性ROS水平增加,NRF2代偿性上调(Nef等,2008)。最近,血液透析患者的全身性炎症和病理性ROS形成与NRF2的下调相关(Pedruzzi等,2015),并且两种启动子多态性(rs35652124和rs6721961)与这些患者的死亡风险增加相关(Shimoyama等,2014)。
NRF2在内皮稳态中最相关的靶点之一是HO-1,其通常与铁蛋白的上调相平行,因此降低游离铁水平,并阻止芬顿型反应。由HO-1和胆绿素还原酶的联合活性产生的胆红素是清除ROS / RNS的最强大的内源性抗氧化剂之一(Jansen等,2010),并且在体外预防脂质过氧化方面非常有效( Stocker等,1987)。Hmox1-/-小鼠显示出慢性缺氧引起的肺动脉高压升高(Christou等,2000),药理学HO-1诱导可改善糖尿病并发症(Kruger等,2006),以及硝酸甘油诱导的血管功能障碍( 硝酸盐耐受性)(Wenzel等,2007)。最近,Hmox1-/-小鼠在血管紧张素II响应中显示NADPH氧化酶-2上调、血管病理ROS形成、炎症标志物、内皮功能障碍和血压升高(Wenzel et al., 2015)。事实上,高血清胆红素水平与冠状动脉疾病的发病率呈负相关(Hopkins等,1996)。胆红素阻止血管NADPH氧化酶的活化(Kwak等,1991),其参与心血管疾病的发展(Griendling和FitzGerald,2003a,b; Harrison等,2003; Jay等,2006)。外周动脉疾病是动脉粥样硬化的常见表现,患有外周动脉疾病的患者HO-1水平降低(Signorelli等, 2016)。

F. 代谢性疾病中的NRF2

2型糖尿病(T2DM)是最常见的慢性代谢疾病之一,并且在NRF2疾病组中尤为突出。胰岛素敏感组织以及胰腺中的病理性ROS形成已在T2DM患者中发现,导致胰腺β细胞分泌胰岛素和外周组织胰岛素作用严重受损(Uruno等,2015)(图 8)。同样,由于蛋白质的非酶促糖化,病理性ROS水平也有助于糖尿病并发症的发病机理。这已在糖尿病肾病中得到证实,其中肾小球表现出病理性ROS水平和NRF2的代偿性升高(Jiang等人,2010)。自2007年以来,一些研究利用动物模型和细胞系研究了NRF2在T2DM及其并发症中的作用。体外对人体细胞的研究表明,NRF2的激活是在急性高糖环境下实现的,而较长时间的培育时间或振荡的葡萄糖浓度均不能激活NRF2 (Ungvari等, 2011;Liu等, 2014)。因此,这些研究指出NRF2活化依赖于葡萄糖浓度和动力学。相反,NRF2在前驱糖尿病和糖尿病患者的外周血单核细胞中下调,表明NRF2可能是重要的治疗靶点(Jimenez-Osorio等,2014)。
NRF2缺乏对高血糖的影响首先在Nrf2-/-小鼠中显示,其中氧化和亚硝化改变增强并导致早期肾损伤(Yoh等,2008)。 在随后的研究中,链脲佐菌素诱导的糖尿病Nrf2-/-小鼠表现出加剧的肾小球损伤,同时ROS产生增加和促纤维化标志物TGF-β和纤连蛋白表达的增加(Jiang等人,2010)。在这个糖尿病模型中,NRF2对血视网膜屏障功能障碍和糖尿病视网膜病变的进展具有保护作用(Xu等, 2014)。同样,HFD诱导的血管ROS水平的增加在Nrf2-/-小鼠中显着加剧,并伴有严重的内皮功能障碍,表现为乙酰胆碱诱导的主动脉松弛减弱和细胞间粘附分子-1和TNF-α表达增加( Ungvari等,2011)。
NRF2在组织特异性胰岛素抵抗中起复杂作用。因此,与野生型小鼠相比,HFD喂养的Nrf2-/-小鼠由于肝脏和骨骼肌中胰岛素信号的增强而表现出更好的胰岛素敏感性,但相反,这些小鼠由于与病理ROS形成相关的肝脂毒性过高而产生严重的NASH (Meakin等, 2014)。因此,该研究将肝胰岛素抵抗与NASH的发展分离。根据这些数据,随后的研究表明,由于CYP2A5酶的表达减弱,HFD喂养的Nrf22-/-小鼠的肝脏由于GSH的显着消耗而表现出更高的病理性ROS形成(Cui等人,2013)。NRF2在肝细胞中的敲低增强了棕榈酸诱导的细胞凋亡,棕榈酸是一种在胰岛素抵抗性肥胖患者中高度升高的脂肪酸。这种效应与病理性ROS的产生增加相关,再次强化了NRF2在NASH进展中的关键作用(Pilar Valdecantos等,2015)。
为了进一步研究NRF2在代谢综合征中的作用,我们在瘦素缺乏(ob/ob)小鼠模型中去除NRF2,该模型具有极好的正能量平衡(Xue等, 2013)。有趣的是,全身ob / ob / Nrf2-/-小鼠或脂肪细胞特异性ob/ob/ Nrf2-/-小鼠显示白色脂肪量减少,显示NRF2是脂肪生成的关键参与者。这些小鼠具有更严重的代谢综合征,其特征在于高脂血症,加重的胰岛素抵抗和高血糖,表明代谢综合征与病理性ROS形成之间的机制联系。
另一组研究评估了持续诱导NRF2在葡萄糖代谢中的作用。NRF2基因诱导利用Keap1的一个亚型等位基因(Keap1flox/- 突变体)降低肥胖糖尿病db/db小鼠的血糖,是通过抑制肝细胞中cAMP-CREB信号传导以及其他糖异生基因(例如过氧化物酶体增殖激活受体辅激活因子-1α)来抑制肝葡萄糖6磷酸酶(Uruno等,2013)。此外,Keap11敲低小鼠中NRF2活性的增强增加了肝脏中AMP活化蛋白激酶(AMPK)的磷酸化,以及骨骼肌中的胰岛素信号传导,导致葡萄糖耐量的显着改善(Xu等,2013)。由于NRF2在T2DM背景下的多效活性,所有这些和其他研究的结果证明需要设计多种遗传和药理学策略来阐明参与全身葡萄糖稳态控制的组织中的全部NRF2功能。
除了年龄,体重和血糖等糖尿病因素外,与NRF2相关的遗传因素在人类中研究较少。在中国人群中,SNP rs6721961与病理性ROS形成和新诊断的T2DM风险有关,并且还可能导致胰岛素分泌能力受损和胰岛素抵抗增加(Wang等,2015)。在墨西哥混血男性中,相同的SNP与糖尿病相关(Jimenez-Osorio等,2017)。在一项汉族志愿者参与的病例对照研究中,发现T2DM患者无论有无并发症,包括周围神经病变、肾病、视网膜病变、足溃疡和微血管病变,NFE2L2基因4个SNPs的基因型和等位频率存在显著差异(Xu等, 2016b)。

G. 神经退行性疾病中的NRF2

NRF2疾病组提供NRF2参与几种神经退行性疾病的证据,包括AD和PD,其代表老年人最普遍的认知和运动障碍。在神经退行性疾病中,低级病理性ROS形成与蛋白质稳态之间的联系尤为重要,因为大多数这些病理表型的特征在于特定蛋白质的异常聚集(图8)。在细胞和动物模型中提供了指向蛋白病变中病理ROS形成以及NRF2作为蛋白酶体和自噬调节因子的证据(Pajares等, 2017)。最初,据报道自噬货物蛋白质sequestosome 1(SQSTM1)与NRF2竞争结合KEAP1。最初,有报道称自噬货物蛋白sequestosome 1 (SQSTM1)与NRF2竞争与KEAP1的结合。SQSTM1将KEAP1置于自噬体降解途径,因此上调NRF2(Komatsu等,2010)。SQSTM1将KEAP1置于自噬体降解途径,因此上调NRF2(Komatsu等,2010)。最近,发现NRF2调节参与自噬起始、货物识别、延伸和自溶酶体清除的自噬基因的表达(Pajares等,2016)。在本研究中,Nrf2-/-小鼠中,转基因过表达人突变淀粉样前体蛋白和tau蛋白导致淀粉样病变和tau蛋白病加重。NRF2缺乏症和神经退行性变之间的联系被越来越多的动物模型证据所支持(Johnson and Johnson, 2015)。一般的观点是受损的神经元试图激活NRF2依赖性转录,可能是为了增加它们自身的存活率。此外,星形胶质细胞中NRF2的上调参与代谢补偿,包括增加GSH的供应以增强其增殖能力(Bolanos,2016),而小胶质细胞中的NRF2上调使该免疫细胞恢复到静止状态(Rojo等,2014a)。
Ramsey等人(2007)证明了NRF2在PD患者的多巴胺能神经元中的核定位。其他研究发现淀粉样蛋白前体蛋白和tau损伤神经元表达NRF2及其靶SQSTM1水平升高,可能是通过自噬清除这些毒性蛋白的补偿机制(Lastres-Becker等,2014; Pajares等,2016)。与这些结果一致,AD和PD脑中HO-1、NQO1、GCLM和SQSTM1水平升高(van Muiswinkel等, 2004;Cuadrado等,2009;Schipper等,2009;LastresBecker等,2016)。此外,与NRF2表达相关的细胞保护蛋白,如NQO1和SQSTM1,在路易体中被部分隔离,表明PD患者中NRF2特征的神经保护能力受损(Lastres-Becker等,2016)。这种差异的一种可能解释是NRF2及其靶基因水平可能在衰老和疾病进展过程中发生变化。
NFE2L2的一些SNP单倍型与ALS,AD或PD的风险降低或延迟发作相关。在关于先前与高基因表达相关的三种功能性启动子SNPs的两项研究中分析了ALS的发病。有趣的是,这种单倍型与ALS发病延迟4年有关(Bergstrom等,2014),但另一项研究未发现明确的相关性(LoGerfo等,2014)。关于AD,一个单倍型等位基因与AD的2年早期发病相关,表明NFE2L2基因的变体可能影响AD进展(von Otter等,2010b)。已经更详细地分析了NFE2L2与PD的遗传关联。NFE2L2启动子中的三个SNPs(rs6721961,rs6706649和rs35652124)在病例对照研究中被证明为保护性单倍型(von Otter等,2010a)。这种单倍型在瑞典队列中延迟了疾病的发作,甚至降低了波兰队列中PD的风险。这些结果得到了四项新的独立的欧洲病例对照研究的支持(von Otter等,2014),但未在台湾人群中复制(Chen等,2013),这表明种族和环境因素存在差异。另一种方法是将来自嗅黏膜的PD细胞暴露于烟提物或杀虫剂中,以评估基因-环境的相互作用,并且鉴定了几种影响对这些毒素的易感性的SNPs(Todorovic等,2015)。总而言之,NRF2的轻微激活(例如对于NFE2L2基因的某些功能性单倍型所发现的)应该足以触发大脑中的保护机制。

IV. 癌症中Keap1的悖论

NRF2在肿瘤发生和进一步肿瘤进展中的作用似乎存在明显的二分法。一方面,通过激活生物转化反应,NRF2可以防止化学诱导的癌症发生。临床前研究表明NRF2在大鼠体内经药理激活后,对黄曲霉毒素B(1)诱导的肝癌具有完全的保护作用(Johnson等, 2014)。相反,NRF2引发的保护性反应在确定的癌症中提供了生长优势,这将是本节的重点。
不断增加的ROS水平可以通过改变基因组稳定性来维持肿瘤发生,同时激活特定的氧化还原信号转导和促进肿瘤细胞存活和增殖的炎症过程(Reuter等,2010)。因此,NRF2的上调代表癌细胞适应的机制,以耐受推动肿瘤进展的高ROS水平(Schumacker,2006)以及维持导致肿瘤复发和远处转移形成的癌症干细胞(Ryoo等,2016)。例如,来自人类结肠直肠肿瘤的癌症干细胞中的NRF2特征指出了由高水平的GCLC,谷胱甘肽过氧化物酶和硫氧还蛋白还原酶-1介导的保护机制,这是这些细胞抵抗应激物和化学治疗剂的能力的基础(Emmink等,2013)。从这个角度来看,NRF2在癌细胞中表现得像一个致癌基因,通过诱导ARE介导的细胞保护反应的慢性激活,可以帮助其适应其氧化环境(Panieri和Santoro,2016)。已经报道了NRF2的恶性激活的几种机制,包括体细胞突变,表观遗传学和致癌信号传导改变。
据报道,沿着NFE2L2的编码序列在癌症中接近600个体细胞突变(Gao等人,2017)。在图9中,我们显示了来自10,000名癌症患者的数据集的结果(Zehir等人,2017)。在大多数情况下,这些突变改变了NRF2的DLG和ETGE基序与KEAP1的相互作用,从而诱导NRF2在几种实体瘤(包括食道癌,皮肤癌,肺癌和喉癌)中的过度活化(Kim等,2010b; Taguchi和 Yamamoto,2017)。例如,在晚期食管鳞状细胞癌中,NRF2功能获得性突变与肿瘤复发和预后不良相关,其原因包括增殖增加、独立依附生存以及对化疗和放疗的抵抗(Shibata 等, 2011)。KEAP1基因中的功能丧失突变在一些实体肿瘤例如肺癌中也是常见的(Singh等人,2006)。基于这一通路调控β-TrCP/NRF2的有力证据,奇怪的是在NRF2与β-TrCP的交界面未发现干扰体突变。这一事实表明,由于未知的原因,这些突变是不可生存的,或者β-TrCP逃逸导致NRF2水平的升高不足以驱动致癌性。
尽管如此,体细胞突变仅在一小部分癌症患者中引起长期/慢性NRF2激活。在基因表达水平上,有趣的是位于人NRF2基因的ARE增强子的SNP rs6721961(-617C>A)的等位基因消除了NRF2的自身诱导,这与这些癌症患者的显着存活相关(Okano等,2013)。已经在肺肿瘤中描述了由于KEAP1的三个CpG位点的启动子高甲基化引起的表观遗传变化,导致随后的NRF2活化,其可以通过5-氮杂-2’-脱氧胞苷处理逆转NRF2的活化(Wang等,2008)。最近已经回顾了miRNAs在NRF2水平的转录后调节中的作用(Kurinna和Werner,2015)。简而言之,miR200a靶向人乳腺癌细胞中的KEAP1 mRNA,导致其降解并随后激活NRF2(Eades等,2011)。反过来,miR28促进NRF2 mRNA的降解(Yang等人,2011)。
致癌基因或突变的肿瘤抑制因子可以增强癌症中NRF2的活化。KRAS,BRAF或c-MYC的内源性致癌等位基因上调NRF2,可能是通过致癌基因介导的ROS和随后的KEAP1慢性失活(DeNicola等,2011)。肿瘤抑制因子p53的突变形式,通过增强营养摄取和构建块的合成来维持癌细胞的生长,也可以通过与结合NRF2启动子的Sp1转录因子的串扰来上调NRF2(Tung等,2015)。
各种蛋白激酶对NRF2(磷酸-NRF2)的磷酸化是107种肝细胞癌中潜在的激活机制。增加的磷酸化NRF2水平与表现出这种独特表型的患者的KEAP1表达降低和5年总体存活率差有关(Chen等,2016)。此外,磷酸酶和张力蛋白同源物(PTEN)肿瘤抑制因子的突变维持过度活跃的和致癌的磷脂酰肌醇-3-激酶(PI3K)-AKT信号传导,并由于为NRF2蛋白酶体降解的PTEN / GSK-3 / β-TrCP途径的下调而导致NRF2活性增加。(Rada等,2011,2012; Cuadrado,2015)。针对PTEN / GSK-3 / β-TrCP途径的治疗干预措施应考虑到GSK-3既可作为肿瘤抑制因子又可作为肿瘤启动子,并且还与癌症干细胞的产生有关(McCubrey等,2014)。
一些应激诱导的蛋白质与KEAP1相互作用,因此在癌细胞中,这些蛋白与KEAP1结合的NRF2竞争。因此,NRF2逃脱了KEAP1介导的降解。NRF2与KEAP1结合的竞争对手之一是自噬衔接蛋白SQSTM1的磷酸化形式,该蛋白发生在癌细胞用于维持自身生长的选择性自噬过程中(Shimizu等, 2016)。还证明了促进癌症干细胞中细胞周期停滞的细胞周期蛋白依赖性激酶抑制剂p21通过其KRR基序与NRF2中的DLG和ETGE基序的相互作用抑制NRF2与KEAP1的结合(Chen等人,2009)。最近,显示具有ETGE基序的二肽基肽酶3可以与NRF2竞争结合KEAP1(Hast等人,2013)。可能由氧化还原状态的长期改变诱导的二肽基肽酶3的过表达与ARE基因的表达增加和预后不良相关,特别是在雌激素受体阳性乳腺癌中(Lu等人,2017)。
NRF2诱导导致癌症进展的代谢变化。例如,一项多平台非靶向代谢组学研究确定了乳腺肿瘤样本中代谢物变化的模式(Tang等,2014)。他们发现GSH和3-(4-羟基苯基)乳酸通过与NRF2的相互作用与BRCA1参与氧化还原稳态呈正相关。代谢组学研究还表明,NRF2可以增加癌细胞中的有氧糖酵解,以支持其高能量需求。这通过NRF2介导的锰-超氧化物歧化酶表达的诱导发生,导致线粒体过氧化氢的产生升高和AMPK的活化。该过程由caveolin-1调节,其直接结合NRF2和KEAP1,并阻碍NRF2活化并因此阻碍糖酵解转变。这显然解释了为什么通常更具侵略性的糖酵解肿瘤具有caveolin-1低/ Mn超氧化物歧化酶高的表型(Hart等,2016)。NRF2还可以驱动葡萄糖和谷氨酰胺向肿瘤细胞增殖所需的合成代谢途径(Mitsuishi等,2012)。在PI3K-AKT信号通路活跃和KEAP1活性缺失的情况下,NRF2被证明可以诱导癌症中葡萄糖代谢从糖酵解向合成代谢途径(嘌呤合成)的转移(Mitsuishi等, 2012;Xu等, 2016a)。以上这一点被强调通过NRF2介导的参与磷酸戊糖途径的基因转录和NADPH(葡萄糖6磷酸脱氢酶;磷酸葡萄糖酸脱氢酶,苹果酸酶1,异柠檬酸脱氢酶1,转酮醇酶和转醛酶)的产生以及涉及嘌呤核苷酸合成(磷酸核糖焦磷酸酰胺转移酶,亚甲基四氢叶酸脱氢酶2)的相关基因。
NRF2的激活赋予癌细胞生长优势的事实可能会争辩说,在这项工作中提到的慢性疾病NRF2的药理学活化可能意味着患癌症的高风险。然而,必须考虑到NRF2的致癌活性需要其基因或KEAP1发生突变,从而导致NRF2信号的持续高水平诱导。但在药理学治疗中并非如此,在药理学治疗中,调节药物剂量和NRF2活性是可能的。此外,经验证据表明,参与NRF2激活剂临床试验的受试者没有显示出更高的癌症风险。自从2013年获得监管机构批准以来,已经服用NRF2活化剂富马酸二甲酯数年的MS患者就是最好的例证。相反,在癌症患者中使用NRF2抑制剂可能导致NRF2疾病组中描述的病理表型的表现。当NRF2抑制剂到达临床时,这种可能性需要进一步研究。

V. NRF2药物组

  本节试图开发一种NRF2药物组,该药物组可用于未来的临床指导,以NRF2疾病组的病理表型为中心靶向NRF2进行治疗。如图2B中所述,通过靶向KEAP1来正在寻求NRF2的药理学活化以增加其稳定性。这些策略基于发现改变KEAP1结构的亲电化合物或阻止NRF2与KEAP1对接的小分子。虽然尚未经验证实,但GSK-3抑制剂应阻止b-TrCP识别NRF2,并且可发现一些化合物以防止NRF2与b-TrCP的结合。用靶向bZip二聚化结构域的化合物分析NRF2的抑制作用,以防止形成活性NRF2 / MAF异二聚体。通过与其他转录因子比较,可能发现阻碍NRF2 / MAF异二聚体与ARE结合的小分子(图9C)。本节总结了新药物发现和重新调整药物用途的转化观点中最重要的发现。

 
 
图9. 在MSK IMPACT临床测序组(MSKCC)研究(Zehir等,2017)的肿瘤中发现的体细胞突变和抑制NRF2的药理学策略。(A)具有NRF2突变的肿瘤的百分比。 (B)沿NRF2多肽的突变分布。(C)NRM2 / MAFF异二聚体与ARE元件之间相互作用的PyMOL表示。 蓝色:NRF2; 粉红色:MAFF。 红色箭头表示可能通过小分子抑制NRF2的机制,这些小分子可以靶向NRF2和MAF蛋白之间相互作用的bZip结构域(PPI抑制剂)或NRF2-MAF异二聚体与ARE相互作用的界面[DNA-蛋白质相互作用(DPI)]

A. 亲电NRF2诱导剂

大多数已知的生理学或药理学NRF2诱导剂是亲电分子,其通过氧化或烷基化共价修饰富含硫醇的KEAP1蛋白中存在的半胱氨酸残基(Hur和Gray,2011; Satoh等,2013)。KEAP1是最适合作为亲电子或氧化还原传感器的蛋白质之一,因为它在人体中含有27个半胱氨酸残基,并起到亲电子陷阱的作用。KEAP1的半胱氨酸C151,C273和C288似乎是最容易发生亲电子反应的(图2B),尽管存在一些特异性(Yamamoto等,2008; Saito等,2015)。亲电子加合物以两种不同的方式抑制KEAP1。 一种是诱导KEAP1的构象变化,这将导致其对NRF2的结合能力丧失。另一个是阻断KEAP1和CUL3 / RBX1之间的相互作用,导致KEAP1与NRF2的隔离以及新合成的NRF2的进一步稳定化(Rachakonda等,2008; Baird和Dinkova-Kostova,2013; Cleasby等, 2014; Saito等,2015)。
NRF2调制剂的至少30项近期专利在世界国际产权组织中被收录。这些专利正在保护查尔酮衍生物,新型酰胺三萜衍生物,氘取代的富马酸衍生物,3-烷基氨基-1H-吲哚丙烯酸酯衍生物,甲醇酰胺,含有活化乙烯基的苄基衍生物,穿心莲内酯或[S]+阿扑吗啡,以及倍半萜内酯衍生物(Sun等,2017)。尽管从临床前概念验证的角度来看,大多数这些化合物在某种程度上被证明是有用的,但它们的临床价值迄今通常非常有限。其中只有少数药物进入了临床试验,而食品和药物管理局(Food and Drug Administration)或欧洲药品管理局(European Medicines Agency)等监管机构批准的药物更少。我们在这项研究中讨论了转化管道中最发达的NRF2激活剂。
富马酸酯是KEAP1改性剂的最突出的例子,并且富马酸二甲酯(DMF)是迄今为止唯一的食品和药物管理局和欧洲药品管理局批准的药物,其注册为NRF2活化剂。单酯形式的DMF,富马酸单甲酯(MMF)被描述为其活性代谢物。DMF和MMF是迈克尔受体,其直接与KEAP1中存在的半胱氨酸残基反应(Lin等,2011)。
从NRF2的功能尚不清楚开始,DMF和其他富马酸酯已经用于治疗牛皮癣超过50年。该化合物在欧洲以商品名Fumaderm获得许可。临床试验显示,在DMF治疗12-16周后,银屑病面积和严重程度指数降低至50%-80%(Altmeyer等,1994; Mrowietz等,1998)。最近,在III期试验(BRIDGE)中,DMF已证明其在治疗患有中度至重度慢性斑块状银屑病的成人中的功效(Mrowietz等,2017)。富马酸盐在银屑病病变缓解中的作用机制包括外周T细胞数量的减少以及从Th1向Th2免疫应答的转变(Ghoreschi等,2011; Tahvili等,2015)。在另一种自身免疫性疾病SLE中,富马酸酯已被用作治疗严重、广泛和难治性皮肤表现的全身联合疗法(Saracino和Orteu,2017)。
2013年,DMF以商品名Tecfidera被批准用于治疗MS(Xu等,2015)。在MS患者中使用DMF是通过在EAE的MS小鼠模型中获得的阳性结果推动的。对疾病进程和组织学的显着治疗效果与脊髓中巨噬细胞介导的炎症显着减少有关。血液中的多重细胞因子分析证明DMF处理的动物中抗炎细胞因子IL-10的增加(Schilling等人,2006)。此外,DMF还改善了野生型中髓鞘,轴突和神经元的保存,但在Nrf2-/-小鼠中没有(Ellrichmann等,2011)。在人类中,DMF显示MS病变和年复发率显着降低(Schimrigk等,2006)。两项III期临床试验DEFINE和CONFIRM证实了这些结果(Fox等,2012; Gold等,2012)。因此,DMF目前被用作复发缓解型MS的第一线治疗,其不能通过传统疗法治疗。新的DMF配方正在测试和申请专利,以提高药物的生物利用度和功效(Sun等,2017)。例如,MMF已被用于开发第二代NRF2诱导物作为前药(Zeidan等,2014)。铅化合物ALKS-8700,一种MMF的2-(2,5-二氧代-1-吡咯烷基)乙酯衍生物,在体内迅速转化为MMF,因此提高了其生物利用度并减少了胃肠道副作用。ALKS-8700目前正在进行III期临床试验(EVOLVE MS)。
DMF和MMF调节免疫应答。例如,它们通过减少炎性细胞因子的释放并因此减少DCs处理抗原的能力来抑制DCs的成熟。此外,DMF和MMF激活天然杀伤细胞以裂解DCs并增强DCs和T细胞的凋亡(Ghoreschi等,2011; Al-Jaderi和Maghazachi,2015)。因此,DMF和MMF阻碍T细胞介导的自身反应。一些研究表明,DMF还通过触发GSH耗竭诱导II型DCs,这导致HO-1活性增强和STAT1磷酸化抑制。这些经典的II型DCs抑制Th1和Th17介导的反应,支持Th2的反应。此外,通过DCs增加IL-10的产生有利于CD4+T细胞向抑制性Treg表型的分化(Ockenfels等,1998; Ghoreschi等,2011)。DMF还抑制NF-κB的核转位(Peng等,2012),从而在小胶质细胞和星形胶质细胞(Brennan等,2017)以及外周血单核细胞(Eminel等,2017)中产生炎症介质,如TNF-α,IL-1β,IL-6,趋化因子,粘附分子和一氧化氮。此外,DMF发挥抗血管生成作用,其依赖于内皮细胞中血管内皮生长因子受体-2表达的下调(Meissner等,2011)。最近的研究结果表明,DMF减少了CD4+,CD8+,Th1和Th17细胞的数量,而CD4+/CD8+比例和Th2亚群在这些患者的血液中增加。有趣的是,DMF/MMF对T细胞活化的抑制作用主要局限于记忆T细胞(Wu等, 2017)。DMF或MMF的这些免疫调节活性对于保护少突胶质细胞免受ROS诱导的细胞毒性具有重要意义(Scannevin等,2012)。
其他机制可以解释NF-κB的抑制,而与NRF2活化无关。因此,DMF可能与几种调节NF-kB信号传导的蛋白质中的半胱氨酸残基相互作用(Blewett等,2016)。此外,DMF可以抑制泛素偶联酶,从而防止NF-κB的IkB抑制因子在IL-1β或Toll样受体激动剂的作用下降解(McGuire等, 2016)。此外,DMF直接结合蛋白激酶C-θ中的特定半胱氨酸残基,这是参与T细胞受体信号传导的关键激酶(Blewett等,2016)。此外,MMF和DMF激活羟基羧酸受体-2,导致NF-κB的抑制和促炎细胞因子和粘附分子的下调(Chen等,2014; Gillard等,2015)并导致中性粒细胞浸润减少 (Chen等,2014)。尽管这些NRF2非依赖性作用与EAE的急性炎症期相关,但DMF在慢性自身免疫性脱髓鞘中的神经保护功效取决于NRF2活化(Linker等,2011)。在Nrf2-/-和野生型小鼠中DMF治疗的临床益处与炎性Th1和Th17细胞的减少以及抗炎M2单核细胞的诱导相关。同时,在野生型中观察到CD80和CD86共刺激分子的表达降低,但在Nrf2-/-小鼠中未观察到,表明至少这些作用是NRF2依赖性的(Schulze-Topphoff等,2016)。
DMF用于治疗自身免疫疾病的成功表明,具有由慢性,低级炎症和病理性ROS形成强调的共同病理机制的其他疾病可能受益于该药物的重新定位。在亨廷顿舞蹈病的小鼠模型中,DMF治疗保留了存活率、肌肉功能和体重,这与完整神经元数量的增加有关(Ellrichmann等,2011)。此外,在最近的PD临床前研究中,使用该疾病的α-突触核蛋白病模型,由于自噬诱导受损,DMF在野生型中具有神经保护作用,但在Nrf2-/-小鼠中则不具有神经保护作用(Lastres-Becker等,2016)。
DMF被证明可预防糖尿病小鼠的内皮功能障碍和心血管病理性ROS形成和炎症(Sharma等,2017),并且在注射链脲佐菌素后载脂蛋白E缺陷小鼠中动脉粥样硬化、肾功能不全和其他糖尿病并发症减少(Tan等,2014)。此外,一些研究表明,DMF可能通过抑制NF-κB途径发挥抗肿瘤活性,因此在治疗侵袭性癌症中增加了治疗价值(Kastrati等,2016)。DMF是网络药理学方法中老药新用概念的相关实例。
合成三萜类化合物是2-氰基-3,12-二氧代 - 油酸-1,9(11) - 二烯-28-酸盐(CDDO; 巴多索隆,RTA401)的衍生物,其类似于天然产物齐墩果酸。它们通过其α-β不饱和支架表现出迈克尔受体活性,并且代表了最有效的NRF2诱导剂(Sun等,2017)。它们与KEAP1的C151相互作用并阻碍其与CUL3的相互作用,从而导致NRF2激活(Cleasby等,2014)。原理证明研究强烈支持合成的三萜类化合物用于退行性疾病,并且正在成为Reata / Abbott作为炎症抗氧化剂调节剂的深入研究的焦点。例如,CDDO-咪唑(CDDO-Im RTA403)在野生型而非Nrf2-/-小鼠的腹膜中性粒细胞中诱导各种抗氧化基因(Hmox1,Gclc,Gclm和Nqo1)的表达,并减弱LPS诱导的ROS产生和产生和促炎细胞因子的产生,从而降低死亡率(Thimmulappa等,2006b)。CDDO-乙基酰胺(RTA405)和CDDO-CDDO-三氟乙基酰胺(RTA 404)在毒素诱导的PD模型(1-甲基-4-苯基-1,2,3,6-四氢吡啶)中测量的所有终点均具有显着作用 (Kaidery等,2013)。在MS的EAE模型中,CDDOCDDO-三氟乙基酰胺抑制炎症、病理性ROS形成和髓鞘变性(Pareek等,2011)。
CDDO-甲酯(CDDO-Me,RTA 402)是在用于治疗糖尿病肾病的临床试验中达到的第一个CDDO(Pergola等,2011)。虽然第二阶段的结果非常令人鼓舞,但由于心血管安全问题(Zhang,2013),CDDO-Me后来在第三阶段(BEACON试验)被撤回,这与NRF2无关,但最有可能是脱靶改变内皮素信号传导(de Zeeuw等,2013; Chin等,2014)。目前,CDDO-Me正在进行临床研究,作为Alport综合征和肺动脉高压的潜在治疗方法(表2)。为了改善其安全性,进一步的研究导致了CDDO-二氟丙酰胺(RTA-408, omaveloxone)的开发,该药物目前正在进行二期试验,用于治疗弗里德雷希的共济失调、眼部炎症和眼部手术后疼痛。
奥替普拉是一种有机硫化合物,用作抗血吸虫病药物,目前正在进行III期试验,用于治疗非酒精性脂肪性肝炎。用于治疗亨廷顿氏病的高级临床试验正在开发中,米诺环素是一种抗生素,由于NRF2活化而具有神经保护作用(Kuang等,2009)。用于治疗急性肾病的I期临床研究中的另一种NRF2诱导剂是CXA-10,一种通过激活NRF2具有抗炎特性的硝基脂肪酸(Batthyany和Lopez,2015)。近年来,许多具有相同作用机制的NRF2诱导剂已被描述(Buendia等, 2015a,b, 2016),其中一些还处于临床前研究阶段,如化合物VEDA-1209,它是一种查尔酮衍生物,具有良好的抗炎作用,可用于溃疡性结肠炎的治疗。
SFN是由有机硫化合物萝卜硫苷的酶促裂解产生的异硫氰酸酯,其存在于西兰花、卷心菜和其他十字花科植物的芽苗中。催化反应由植物中发现的黑芥子酶和胃肠道的微生物群驱动(Kensler等,2013)。催化反应由植物中发现的黑芥子酶和胃肠道的微生物群驱动(Kensler等,2013)。最近,SFN已经通过化学合成获得(Kim等,2015)。通过向患有T2DM的患者施用含有SFN的花椰菜芽粉,实现了SFN向临床的转化(Bahadoran等,2012)。西兰花粉降低血浆丙二醛和氧化低密度脂蛋白(LDL),提高总抗氧化能力。心血管危险因素如血清甘油三酯,氧化LDL/LDL比率和血浆致动脉粥样硬化指数(甘油三酯对数/高密度脂蛋白比率)也降低。此外,促炎标志物如C-反应蛋白和IL-6减少。在最近的一项研究中,SFN作为浓缩西兰花芽提取物使用,通过NRF2的核转位抑制肝细胞葡萄糖的生成,并降低参与糖异生的关键酶的表达。此外,SFN降低了患有T2DM的肥胖患者的空腹血糖和糖化血红蛋白(Axelsson等,2017)。SFN诱导的NRF2活化通过降低ROS负荷和抑制NF-κB和TGF-β1信号传导途径来保护肾细胞免于狼疮性肾炎(Jiang等,2014a)。
关于神经退行性疾病,已经表明SFN穿过血脑屏障并提供足够的脑生物利用度来激活NRF2特征并减少LPS诱发的神经炎症,这反映在促炎性标志物(诱导型一氧化氮合酶、IL-6、TNF-α)的减少中和海马中的小神经胶质细胞增生(Innamorato等,2008)。SFN还保护多巴胺能神经元免受帕金森病毒1-甲基-4-苯基-1,2,3,6-四氢吡啶的影响,并减弱星形胶质细胞增生和小神经胶质细胞增生(Jazwa等,2011)。与这些发现一致,SFN降低了磷酸化tau水平并增加了Beclin-1和LC3-II,表明NRF2活化可能通过大脑中的自噬促进这种毒性蛋白的降解(Jo等,2014)。SFN处理的脊髓损伤大鼠炎症因子水平明显降低,挫伤体积减小,协调性改善(Wang等, 2012)。该药还通过保留血脑屏障和减少病理性ROS形成和炎症细胞数量来改善EAE(Li等,2013)。SFN迄今已用于至少32项针对慢性疾病的临床研究,如癌症、哮喘、慢性肾病、T2DM、囊性纤维化、自闭症和精神分裂症(Duran等,2016; Houghton等,2016)(表2)。
 
表2. 选择的NRF2诱导剂作为KEAP1的亲电子修饰剂
该引用对应于ClinicalTrials.gov中的代码。
 
 
 
 
 
 

 
总之,这些观察结果为其他SFN衍生化合物的开发铺平了道路,这些化合物表现出更好的药代动力学特征。SFN是一种在亲水性介质中稳定性较差的油性物质。其物理化学特征促使Evgen Pharma(Wilmslow,Cheshire,England)开发出一种环糊精复合物制剂Sulforadex,该制剂正在进行用于治疗蛛网膜下腔出血的II期临床试验。SFN还与褪黑激素杂交以产生ITH12674,该化合物被设计为具有用于治疗脑缺血的双重药物-前药作用机制(Egea等人,2015)。

姜黄素是姜黄中发现的主要类姜黄素,已被用于治疗肥胖症、代谢综合征和前驱糖尿病。采用气相色谱-电子冲击质谱法对姜黄素对大鼠肝脏的影响进行了非靶向代谢组学研究。间断摄入姜黄素可上调NRF2,并在抗肝损伤方面发挥抗氧化和抗炎作用(Qiu等,2016)。口服姜黄素可有效降低血清甘油三酯、IL-1β、IL-4和血管内皮生长因子,以及增加血液中脂联素水平。在T2DM患者中,姜黄素降低空腹血糖,糖化血红蛋白,血清游离脂肪酸,甘油三酯和尿酸的水平,并增加脂蛋白脂肪酶的水平(Na等,2013; Chuengsamarn等,2014)。

白藜芦醇是一种保护植物免受真菌感染的多酚,存在于葡萄皮、红葡萄酒、浆果和许多其他植物中。白藜芦醇通过下调KEAP1表达和激活蛋白去乙酰化酶sirtuin-1来激活NRF2信号通路,从而发挥抗氧化作用(Ungvari等, 2010)。在健康受试者中,白藜芦醇的饮食施用防止了血浆中胆固醇、内毒素、促氧化剂和炎性标记物(p47phox,KEAP1,IL-1β和TNF-α)的升高。这些事件与NRF2活性的升高相关,NRF2活性升高是由NRF2靶标NQO1和谷胱甘肽S-转移酶的表达增强所确定的(Ghanim等,2011)。在T2DM患者中,治疗4周后胰岛素敏感性得到改善,这通过AKT增强胰岛素信号传导,病理性ROS形成减少和糖化血红蛋白水平降低来确定(Brasnyo等,2011; Bhatt等,2012)。总的来说,白藜芦醇在动物模型和患者中可预防高血压、高胆固醇血症、动脉粥样硬化、缺血性心脏病、糖尿病和代谢综合征等主要心血管、炎症、氧化和代谢并发症(Xia等, 2017)。

经常被忽视的问题是亲电KEAP1抑制剂缺乏选择性。亲电试剂与细胞中不同的亲核试剂发生反应,从而表现出非靶向和非预期的副作用。例如,CDDO-Im可与500多种不同的目标相互作用(Yore等,2011)。通常,几种蛋白磷酸酶在其催化中心含有对氧化还原敏感的半胱氨酸,并且一些KEAP1抑制剂可以修饰和灭活这些磷酸酶,因此扰乱信号传导网络。这些磷酸酶之一是PTEN(Lee等,2002; Kitagishi和Matsuda,2013; Han等,2015)。PTEN的催化C124残基可以通过与强亲电试剂例如CDDO-Im(Pitha-Rowe等,2009)和叔丁基氢醌(Rojo等,2014b)形成加合物来修饰。然后,PI3K/AKT通路的激活增加涉及GSK-3的抑制和NRF2随后的稳定(图2C) (Rada等, 2011, 2012)。此外,KEAP1与其他同样含有高亲和力结合基序ETGE的蛋白相互作用(Hast等, 2013),如Bcl-2和IKKβ (Kim等, 2010a;Cazanave等,2014)。因此,从KEAP1缺陷细胞获得的一些结果可能不一定与NRF2活化有关。

B. 用于NRF2激活的蛋白质-蛋白质相互作用抑制剂

为了克服选择性的缺陷,出现了一类阻止NRF2与KEAP1对接的新型NRF2诱导剂(Richardson等,2015)。通过先前阐明KEAP1(Padmanabhan等人,2006)的X射线晶体结构与含有NRF2的高亲和力结合ETGE基序的肽结合,已经实现了PPI抑制剂的使用(Lo等,2006)。KEAP1包含六叶β螺旋桨,具有特定的疏水和亲水残基,参与采用β-发夹结构的EGGE基序的对接。对接主要受KEAP1的几个精氨酸与ETGE基序中的两个谷氨酸之间的静电相互作用的支持(Lo等人,2006; Padmanabhan等人,2006)。NRF2的低亲和力DLG基序与KEAP1的对接也被表征(Tong等, 2007)。基于这些相互作用,拟肽化合物是PPI抑制剂的第一个例子,其对亲电试剂的选择性显着提高(Hu等,2013; Marcotte等,2013; Winkel等,2015)。这些抑制剂在细胞中表现出较弱的活性,现在已经报道了一种新的基于循环肽的激发策略。这些肽之一显示出对KEAP1的高结合亲和力和NRF2的激活,并在小鼠巨噬细胞中引发抗炎作用(Lu等,2018)。

最近对KEAP1/NRF2相互作用的新多肽和小分子抑制剂的发现进行了综述(Abed等, 2015;Jiang等, 2016)。简而言之,最初使用表面等离振子共振和荧光偏振测定评估一系列截短的NRF2肽作为PPI的直接抑制剂(Hu等,2013)。抑制能力最小的肽序列为LDE-ETGE-FL的9-mer序列(Chen等, 2011;Inoyama等,2012)。与此同时,Wells和合作者(Hancock等,2013)使用噬菌体展示文库结合高通量荧光偏振测定法搜索新推定的肽配体。他们发现,与单独的天然肽相比,基于NRF2和SQSTM1的ETGE基序的杂合肽对KEAP1具有更高的结合活性。为了促进细胞摄取,设计一种肽,其中ETGE基序与HIV-Tat蛋白的细胞转导结构域和钙蛋白酶的切割序列(DEETGE-Cal-Tat)融合。该肽在脑缺血的小鼠模型中显示出神经保护和认知保护作用(Tu等,2015)。

已经描述了五个PPI抑制剂家族:四氢异喹啉(Jnoff等,2014; Richardson等,2015),硫嘧啶(Marcotte等,2013),萘(Jiang等,2014b),咔唑酮(Ranjan等,2014)和尿素衍生物(Sato等,2013)。表3汇总了最近针对这些小分子的专利。 尽管这些化合物非常有前途,但仍需要证明它们对KEAP1/NRF2相互作用具有选择性,因为KEAP1也至少靶向Bcl2和IKK(Kim等,2010a; Hast等,2013; Cazanave等,2014)。
从可用文库中索引的大量化合物中,化合物LH601,苯磺酰基嘧啶酮2,N-苯基-苯磺酰胺和一系列1,4-二苯基-1,2,3-三唑可能是KEAP1抑制PPI非常适合的候选者(Hu等,2013; Jnoff等,2014; Bertrand等,2015; Wen等,2015; Nasiri等,2016)。这些研究详细描述了与KEAP1的原子相互作用、亲和力和结合的热力学参数。这些化合物的治疗功效将在未来的工作中进行分析,其中应该解决安全性,效力和血脑屏障渗透性。
 
表3. 选择的NRF2诱导剂充当NRF2-KEAP1蛋白质 - 蛋白质相互作用抑制剂

 
 
 

C. 用于NRF2激活的Keap1以外的药物靶

蛋白激酶GSK-3磷酸化NRF2的DSGIS序列中的两个丝氨酸残基以产生一个磷酸化依赖性降解基序或磷酸化二核(图2)。该磷酸二核被E3连接酶适配子β-TrCP识别,导致NRF2的泛素依赖性蛋白酶体降解。因此,GSK-3抑制剂应通过阻止这种磷酸二核的产生来阻止NRF2降解。GSK-3是AD和其他病理表型中的重要激酶。它使细胞骨架蛋白tau磷酸化,促进神经原纤维缠结的形成,神经原纤维缠结是病理性细胞内聚集体,其干扰轴突运输并导致神经元死亡(Silva等,2014)。因此,推测GSK-3抑制可能具有防止神经原纤维缠结形成和NRF2降解的双重益处。不幸的是,大多数用于开发GSK-3抑制剂的管道由于无效而已经停止,尽管在大多数情况下没有很好的证据表明靶标调节(Palomo和Martinez,2017)。

从概念上讲,β-TrCP-磷酸化NRF2相互作用的抑制剂也应该导致NRF2活化,因为它们应该破坏NRF2降解的这一分支(图2)。β-TrCP的β-螺旋和含有NRF2磷酸二核的肽之间的分子相互作用已经通过NMR解决(Rada等,2012)。正如KEAP1/EGTE所发生的那样,最相关的氨基酸似乎是β-TrCP的几个精氨酸残基,其与DpSGIpS基序的两个磷酸丝氨酸相互作用。然而,能够抑制β-TrCP-磷酸化NRF2相互作用的小分子的发现仍在进行中。
已经开发了其他策略来抑制NRF2抑制因子BACH1,这是一种bZip蛋白,可与MAF蛋白形成异二聚体并阻断ARE基因的表达。已经在体外描述了HPP-4382化合物对BACH1的有效抑制(Attucks等,2014),但是,在完整的临床试验之前,必须在体内证明HPP-4382的安全性和功效特征。考虑到其他途径也可能影响NRF2的活性,我们有理由推测,组合方法将是激活该转录因子的最佳途径。

D. NRF2抑制剂

NRF2在构成性和高度过表达时具有与其致癌活性相关的“阴暗面”。因此,已提出NRF2抑制作为使癌细胞对化学治疗药物或放射疗法敏感的机制(Milkovic等,2017)。可以设想两种策略用小分子抑制NRF2:破坏NRF2和MAFs之间的bZip相互作用的PPI抑制剂,以及阻断NRF2-MAF与ARE结合的DNA-蛋白质相互作用抑制剂(图9C)。这两种策略都受到了阻碍,因为这类药物需要克服蛋白质-蛋白质之间以及蛋白质-DNA界面(在较小程度上)之间的大量自由能。 然而,已发现其他bZip转录因子如STAT3-STAT3,MYC-MAX和JUNFOS(Yap等,2011),并且正在描述NRF2-MAF的新的小分子。例如,malabaricone-A是一种促氧化合物,通过靶向NRF2克服了白血病抗性(Manna等,2015)。抗坏血酸(维生素C),一种著名的ROS清除剂,被发现通过降低NRF2/ARE复合物水平,降低GCLC基因的表达,降低GSH水平,来增强伊马替尼耐药癌细胞的敏感性(Tarumoto等,2004)。全反式维甲酸是NRF2抑制剂的另一个实例,其在体外和体内显着降低有效的亲电NRF2诱导物对NRF2的活化。它激活视黄酸受体α,其与NRF2形成复合物,因此阻碍转录因子与ARE基因的结合(Wang等,2007)。

天然产物如鸦胆子苦醇(Ren等,2011; Olayanju等,2015),赭曲霉毒素A(Tarumoto等,2004; Limonciel和Jennings,2014)和葫芦巴碱(Arlt等,2013)也有被发现抑制NRF2。然而,他们的作用机制还没有完全明白。事实上,与现有化合物相关的一个重要问题是它们可能具有的深远的脱靶效应。例如,最近发现鸦胆子苦醇对蛋白质合成具有普遍和非特异性抑制作用,导致NRF2水平下降,而且许多其他快速转换蛋白下降,因此最近不鼓励使用鸦胆子苦醇(Harder et al。,2017))。类似地,兽医实践中使用的抗原虫剂卤虫酮通过抑制NRF2积累来增强癌细胞的化学敏感性,但这种效应似乎是间接的,它通过抑制核糖基转移RNA的合成,这是NRF2以及许多其他含脯氨酸的蛋白质的核糖体转化所强烈需要的(Tsuchida等,2017)。

最近报道了一种识别选择性NRF2抑制剂的新方法,即使用小分子抑制剂的高通量定量筛选(Singh等, 2016)。作者确定了一种名为ML385的一流化合物,它最有可能阻止NRF2与其他bZip共激活因子的结合。该化合物阻断NRF2转录活性并使KEAP1缺陷细胞对卡铂和其他化学治疗药物敏感。需要进一步的研究来确认ML385是否对NRF2具有选择性,或者它是否也抑制其他bZip转录因子。

鉴于NRF2在多种肿瘤病理表型中具有良好的全身效应,用小分子抑制剂特异性靶向NRF2似乎提供了一种良好的临床途径。然而,有必要确定用NRF2抑制剂治疗癌症是否会增加NRF2疾病组中的其他病理表型的风险。


E. 药物新用代替新药物的发现与开发

如前所述,许多化合物正在开发中,为与NRF2疾病组相关的病理表型提供益处。另一种方法是给已经在临床中用于某种病理机制的药物一个新的用途,用于治疗与NRF2有关的其他病理机制。本节根据一些常用药物在NRF2调控中的作用,为其重新定位提供依据。

二甲双胍是T2DM的一线单药治疗。根据图6,它为与葡萄糖代谢相关的病理表型的NRF2亚群提供治疗益处。事实上,SFN可降低肝脏葡萄糖的产生,并改善T2DM患者的血糖控制(Axelsson等,2017)。有趣的是,一些证据表明,二甲双胍可能有效预防NRF2疾病组中的其他非糖尿病病理表型,包括心血管疾病(Nesti和Natali,2017),呼吸系统疾病(Sato等,2016),消化系统(Bauer和Duca,2016),神经退行性疾病(Markowicz-Piasecka等,2017),自身免疫(Schuiveling等,2017)和肿瘤(Heckman-Stoddard等,2017)疾病。二甲双胍的作用机制尚不完全清楚,但它涉及抑制线粒体复合物I,从而增加AMP/ATP比值(El-Mir等,2000; Owen等,2000)并导致能量传感器AMPK的激活(Hardie,2004; Rena等,2017)。重要的是,AMPK激活NRF2(Wang等,2017a; Zhao等,2017),并且该轴的药理学靶向可减轻中风后的炎症(Wang等,2017c)或内毒素暴露(Ci等,2017; Lv等,2017)。事实上,二甲双胍以AMPK依赖的方式激活NRF2,从而抑制临床前啮齿动物短暂性全脑缺血模型的炎症反应(Ashabi等, 2015;Kaisar等,2017)。葡萄糖代谢和炎症可能不是二甲双胍/NRF2作用的唯一病理机制。实际上,已经描述了氧化还原的其他有益效果(Kocer等,2014; Kelleni等,2015)和蛋白质稳态(Tsai等,2017)。

他汀类药物可预防和减少心血管病变表型。除了降脂作用外,他汀类药物似乎可以预防与NRF2网络相关的病理机制,如炎症(Pantan等,2016; Wu等,2016a; Hwang等,2017)和病理性ROS形成( Abdanipour等,2014)。它们是3-羟基-3-甲基-戊二酰-CoA还原酶的竞争性抑制剂,其催化胆固醇合成中的限速反应。其他多效性影响包括转录因子Krüppel样因子2的上调,其在肝硬化进展期间早期诱导,并减轻肝血管功能障碍的发展(Marrone等,2015)。最近的证据表明,至少一些他汀类药物会激活NRF2。在分离的肝细胞中进行的一项研究中,高浓度的辛伐他汀激活了NRF2,可能作为一种防御机制(Cho等,2013)。用洛伐他汀预处理神经干细胞激活NRF2途径并引发针对过氧化氢诱导的细胞死亡的保护(Abdanipour等,2014)。在肝硬化中,辛伐他汀激活由Krüppel样因子2和NRF2形成的轴,以减少星状细胞的氧化负荷和炎症反应,改善肝纤维化,内皮功能障碍和门静脉高压。辛伐他汀激活NRF2的机制尚不完全清楚,但似乎涉及NRF2相互作用组中发现的元素,如丝裂原活化蛋白激酶、PI3K/AKT途径(Jang等,2016)和GSK-3(Lin等,2016)。

图4的NRF2相互作用组可以推断出药物新用的其他病例,特别是信号激酶。如图2C所示,GSK-3磷酸化NRF2的Neh6结构域,导致β-TrCP的识别和进一步泛素依赖性蛋白酶体降解。GSK-3在没有刺激的情况下是活跃的,而在激活AKT和其他激酶的信号级联导致GSK-3在其N端伪底物域磷酸化时是不活跃的。因此,已知靶向信号激酶的药物可用于上调(GSK-3抑制剂)或下调(PI3K/AKT抑制剂)NRF2特征。

GSK-3参与NRF2疾病组中发现的至少一些病理表型,例如糖尿病和神经退行性疾病(Beurel等,2015; Maqbool和Hoda,2017)。从天然和合成来源发现了广泛的GSK-3抑制剂(Khan等,2017),但将GSK-3抑制剂重新利用来增加NRF2活性的最佳证据可能源于临床使用锂作为情绪稳定剂(Chiu等,2013)。虽然此时在NRF2疾病组中未发现双相情感障碍和抑郁,但很明显它们表现出神经炎症和退行性病变表型,至少在小鼠模型中暗示NRF2失调(Martin-de-Saavedra等,2013; Freitas等,2016; Yao等,2016)。

NRF2相互作用组也为阻断信号激酶从而激活GSK-3的癌症药物抑制NRF2提供了一个理由。例如,表皮生长因子受体抑制剂厄洛替尼导致NRF2抑制,参与非小细胞肺癌中的肿瘤细胞感觉(Xiaobo等,2016)。用于治疗肝细胞癌的激酶级联抑制剂索拉非尼也导致NRF2及其下游靶标金属硫蛋白-1(Houessinon等,2016)和亚甲基四氢叶酸脱氢酶1的抑制(Lee等人,2017)。

最后,在两项相关研究中,迄今为止一直在寻找可能影响NRF2调节的再利用药物。使用基于荧光相关光谱的筛选系统,1633种药物中的两种显着增加HepG2细胞中的NRF2蛋白水平:叶绿酸和bonaphton(Yoshizaki等,2017)。在另一项研究中,分析了连接图数据库,其包含用1309种试剂处理的人细胞系的基因表达谱(Lamb等,2006; Iorio等,2010)(Zhang等,2017),通过激活NRF2以寻找有潜力的氧化还原调节剂(Xiong等,2014)。该研究发现阿司咪唑是一种有效的抗组胺药,用于过敏性疾病,是一种新型的NRF2激活剂。
 

VI. 生物标志物作为NRF2特征参与监测目标

由于ROS的半衰期短,在毫秒或纳秒的范围内,对患者或群体研究中的氧化还原状态的评估受到阻碍(Ghezzi等,2017b)。因此,病理性ROS形成的生物标志物基于测量ROS留下的痕迹,ROS通常是细胞分子的末端氧化产物,其中许多是非特异性的(Frijhoff等,2015)。相反,NRF2的激活和其靶基因的后续表达是对生物体对病理性ROS形成的总暴露的间接但可靠的估计。由于NRF2激活是对环境应激源的一种成熟的细胞反应,所以它被认为是暴露于异生素的生物标志物。在肺部,通过对多个转录研究的数据挖掘,并结合独创性通路分析,报道了NRF2信号在健康吸烟者中上调,因此表明NRF2调控的抗氧化基因在预防烟草烟雾的毒性作用中发挥核心作用(Comandini等, 2010)。类似地,NRF2调控的一种酶NQO1在过量服用对乙酰氨基酚患者的肝组织中含量是正常水平的15倍(Aleksunes等, 2006)。疾病与营养之间的关联通常基于不可靠的自我报告(Archer等,2015)。通过激活NRF2来测量对营养素响应的生物标志物,据称具有有益效果,这可以提供一种可靠的方法来验证营养研究。但是,这种可能性仍未得到探索。
与NRF2转录相关的变化可用作监测药物功效的生物标志物,所述药物旨在通过黄嘌呤氧化酶和NADPH氧化酶抑制剂减少病理性ROS形成。同样,可以通过定义全局蛋白和基因表达谱来检测和监测环境化学品的暴露情况(Ghezzi等,2017a)。这种方法类似于第一阶段药物代谢酶的使用,其中细胞色素P450是各种异种生物通过Ah受体诱导的,可以作为海洋污染的指标(Cajaraville等, 2000)。12周内每日口服富马酸酯与银屑病患者皮肤中NRF2靶基因表达增加有关(Onderdijk等,2014)。同样,在接受每日剂量CDDO-Me治疗3周的癌症患者外周血单核细胞中,NQO1的mRNA水平增加了5倍(Hong等, 2012)。

使用NRF2的转录特征作为生物标记需要很好地了解ARE基因活化所涉及的机制,因为大多数NRF2靶标受另外的转录因子调节。因此,分析几种ARE基因的表达很重要。例如,一项使用NRF2作为肺鳞癌治疗反应预测因子的研究提出使用28个基因来定义NRF2激活谱(Cescon等, 2015)。
 

VII. 结论

系统医学和网络药理学共同强调了NRF2在慢性疾病中起基础性作用的一组病理表型。这些疾病具有共同的机制,包括氧化,炎症和代谢改变。 本研究中提出的NRF2相互作用组,NRF2疾病组和NRF2药物组仍处于早期发展阶段,但它们代表了将NRF2构建为一种常见的治疗和系统医学方法的首次尝试。 即将完善的现有数据库和即将公布的临床结果数据将进一步提高这一新方法的准确性,这一新方法是以药理学和机制为基础的药物新用。本文为药物发现激活或抑制NRF2的综合策略提供了路线图,并强调需要为新药的开发或针对NRF2作为慢性病常见成分的药物的重新利用的转化而努力。
 

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