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    運用系統醫學方法闡明轉錄因子NRF2作為慢性疾病的治療靶標

    發表于:2019-05-14   作者:admin   來源:本站   點擊量:4406

    Antonio Cuadrado et al., PHARMACOLOGICAL REVIEWS, 2018

      

     
    縮寫:
    AD: 阿爾茨海默病;  ALS: 肌萎縮側索硬化癥;  AMPK,AMP活化蛋白激酶;  ARE: 抗氧化反應元件;  β-TrCP: 含有E3泛素蛋白連接酶的β-轉導蛋白重復序列;  bZip: 堿性區域 - 亮氨酸拉鏈;  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:樹突狀細胞;  DMF: 富馬酸二甲酯;  EAE:實驗性自身免疫性腦脊髓炎;  ECH:具有Cap'n'collar同源性的紅系細胞衍生蛋白;  GCLC:g-谷氨酰半胱氨酸連接酶催化亞基;  GCLM:g-谷氨酰半胱氨酸連接酶調節劑亞基;  GI:胃腸道;  GSH:谷胱甘肽;  GSK-3:糖原合成酶激酶3;  HFD,高脂飲食;  HNSCC:頭頸部鱗狀細胞癌;  HO-1,血紅素加氧酶-1;  IBD:炎癥性腸病;  IκB:κ-B抑制劑;  IKK:IκB激酶;  IL:白細胞介素;  KEAP1:kelch樣ECH相關蛋白1;  LDL:低密度脂蛋白;  LPS:脂多糖;  MAF:肌肉腱膜纖維肉瘤蛋白;  MMF:富馬酸單甲酯;  MS:多發性硬化;  NASH:非酒精性脂肪性肝炎;  NF-κB:活化B細胞核因子κ-輕鏈增強子的p65亞基;  NQO1:NADPH:醌氧化還原酶;  NRF2:核因子(紅細胞衍生的2)樣2;  PD:帕金森病;  PI3K:磷脂酰肌醇3-激酶;  PPI:蛋白質-蛋白質相互作用;  PTEN:磷酸酶和張力蛋白同系物;  RA: 類風濕性關節炎;  RBX1:RING-box蛋白1;  RNS:活性氮物種; ROS:活性氧;  SFN:蘿卜硫素; SLE:系統性紅斑狼瘡;  SNP:單核苷酸多態性;  SQSTM1:sequestosome 1;  STAT:信號轉導和轉錄激活因子;  T2DM:2型糖尿病;  TGF:轉化生長因子;  Th,T輔助子;  TNF:腫瘤壞死因子;  Treg:T調控。

     
     

    運用系統醫學方法闡明轉錄因子NRF2作為慢性疾病的治療靶標


    Antonio Cuadrado et al., PHARMACOLOGICAL REVIEWS, 2018

    摘要

    系統醫學是基于疾病機理,而非基于具體癥狀或器官的疾病研究方法,并以非假設驅動的方式識別治療靶標。本論文中,我們將系統醫學方法應用于轉錄因子核因子( NRF2)的研究,通過交叉驗證其在蛋白質-蛋白質相互作用網絡(NRF2相互作用組)中的作用,該相互作用網絡在功能上與低水平應激、慢性炎癥、代謝改變和活性氧形成有關的細胞保護相關聯。這些分子譜的多尺度網絡分析表明,NRF2表達和活性的改變是疾病子網路中的常見機制(NRF2病變)。該相互作用網絡關聯了明顯的異質性表型,如自身免疫、呼吸、消化、心血管、代謝和神經退行性疾病,以及癌癥。重要的是,系統醫學方法在計算機上匹配并證實了在臨床開發的不同階段在體內驗證的NRF2調節藥物的若干應用。從藥理學上看,這些藥物的特征多樣,有親電的富馬酸二甲酯、合成的三萜類化合物如巴多索酮甲基和蘿卜硫素、蛋白-蛋白或DNA -蛋白相互作用抑制劑,甚至有注冊的藥物如二甲雙胍和他汀類藥物,它們可以激活NRF2并且可能被重新用于指示 NRF2疾病表型群。因此,NRF2代表了經典和系統醫學方法充分接受的第一類目標之一,這些方法通過關注一組似乎具有機械聯系的疾病表型來促進藥物開發和藥物新用。 因此,由此產生的NRF2藥物組可能會迅速為這類慢性疾病提供一些令人驚訝的臨床選擇。 

    Ⅰ. 引言

    在上個世紀,壽命幾乎翻了一番,特定老齡化疾病現在變得普遍。然而,大多數疾病的病理機制很難理解,治療方法也是通過糾正癥狀或危險因素來治療。此外,與迄今為止只考慮一種疾病、一種藥物的線性方法相反,慢性病顯示出高度的關聯性,需要更精確的、以機制為基礎的疾病定義,而不是目前以器官和癥狀為基礎的疾病定義。在人類基因組測序和分子網絡的發展之后,一種新的疾病概念由此產生,疾病的診斷不僅是通過臨床癥狀,而且主要是通過潛在的分子特征識別(Goh et al., 2007)。不同的病理表型具有共同的分子機制,這一事實也為總結為“多種疾病,一種藥物”和藥物新用的治療新概念提供了理論基礎。網絡醫學,即將網絡概念應用于疾病和藥物之間的動態聯系分析,為開發這種新方法提供了新機會。老年人慢性病很可能的特征是衰老過程中體內平衡的喪失或環境因素的影響,所有這些都會通過病理性形成活性氧(ROS),慢性炎癥和代謝失調導致低水平應激?;诰W絡醫學方法,我們將在本文中提供大量證據,表明核因子(erythroid-derived 2)–like 2 (NRF2)作為多種細胞保護反應的主調控因子和特定疾病集群中的一個關鍵分子節點,為藥物開發和藥物新用提供了一種新的策略。

    Ⅱ. 從NRF2相互作用組到NRF2疾病網絡

    A. NRF2作為一個細胞穩態的主要調節器

    NRF2是一種堿性區域亮氨酸拉鏈(bZip)轉錄因子(圖1),在細胞核內與小肌筋膜纖維瘤蛋白(MAF) K、G、F形成異二聚體。異二聚體識別一個稱為抗氧化應答元件(ARE)的增強子序列,其存在于超過250個基因(ARE基因)的調節區域中(Ma,2013;Hayes and Dinkova-Kostova,2014)。這些基因編碼參與I期、II期和III期生物轉化反應和抗氧化機制的合作酶網絡,產生NADPH、谷胱甘肽(GSH)和硫氧還蛋白反應;脂質和鐵的分解代謝;以及與其他轉錄因子的相互作用等(Hayes and Dinkova-Kostova, 2014)。最近,NRF2也被發現可調控多個蛋白酶體亞基和自噬基因的表達,為其調控蛋白酶體平衡提供了額外的研究興趣(Pajares et al., 2015, 2016, 2017;de la Vega等,2016)。

     
     
    圖1. NRF2作為細胞保護應答的主要調節因子。 (A)NRF2通過其bZip結構域與MAF家族成員異二聚體化。異二聚體與稱為ARE的增強子序列結合,所述增強子序列存在于超過250個基因(ARE基因)的調節區中。 (B)如所示,這些基因參與氧化還原代謝,炎癥和蛋白質穩態平衡的控制。 NFE2L2中易感性SNPs的存在,腦部尸檢中其靶基因水平的升高以及臨床前研究的陽性數據表明,NRF2激活可能抵消蛋白質穩態,氧化還原和炎癥控制的不平衡。 AC1: ATP檸檬酸裂解酶; ACC1: 乙酰輔酶A羧化酶1; CALCOCO2: 鈣結合和卷曲螺旋結構域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-磷酸脫氫酶; Gpx: 谷胱甘肽過氧化物酶; Gpx8: 谷胱甘肽過氧化物酶8; GR: 谷胱甘肽還原酶; HMOX1: 血紅素加氧酶-1; IDH1: 異檸檬酸脫氫酶1; ME: 蘋果酸酶; MTHFD2: 亞甲基四氫葉酸脫氫酶2; PGD: 磷酸葡萄糖酸脫氫酶; PPAT: 磷酸核糖焦磷酸酰胺轉移酶; PSMB7: 蛋白酶體亞基b-7型; SCD1: 硬脂酰輔酶A去飽和酶; TrxR: 硫氧還蛋白還原酶; ULK1: unc-51像自噬激活激酶1。
     
    NRF2在臨床上的重要意義在于它可能具有藥理學靶向性,對患者有益。調節NRF2轉錄活性的主要機制是通過E3連接酶適配器KEAP1(具有Cap'n'collar同源性(ECH)的Kelch樣紅系細胞衍生蛋白 - 相關蛋白1)來控制蛋白的穩定(圖2)。KEAP1是同型二聚體蛋白,其將NRF2與Cullin 3和RING-box蛋白1(CUL3 / RBX1)形成的E3連接酶復合物連接。在穩態條件下,KEAP1同二聚體的N-末端結構域在兩個氨基酸序列上與一個分子的NRF2結合,具有低(天冬氨酸,亮氨酸和甘氨酸; DLG)和高(谷氨酸,蘇氨酸,甘氨酸和谷氨酸; ETGE)親和力,因此,通過CUL3 / RBX1(Tong等人,2007)將NRF2呈現為泛素化,并隨后通過蛋白酶體降解。KEAP1是一種氧化還原和親電子傳感器,在關鍵半胱氨酸修飾后失去其抑制NRF2的能力(圖2;生物標志物作為NRF2特征并參與監測目標)。調節NRF2穩定性的另一種機制是由糖原合成酶激酶3(GSK-3)介導的磷酸化(圖2)。這種激酶磷酸化NRF2(天冬氨酸,絲氨酸,甘氨酸,異亮氨酸,絲氨酸; DSGIS)的結構域,因此為含有E3泛素蛋白連接酶(b-TrCP)的E3連接酶接頭b-轉導蛋白重復序列產生識別基序,該基序將NRF2呈遞給 CUL1 / RBX1復合物,導致NRF2泛素依賴性蛋白酶體降解的替代途徑。因此,KEAP1和GSK-3 / b-TrCP分別在氧化還原穩態和細胞信號傳導的情況下嚴格控制NRF2蛋白水平(Cuadrado,2015)。已經報道了NRF2在蛋白質,mRNA或基因水平上調節的其他機制(Hayes and Dinkova-Kostova,2014),但至少這兩種機制適合于藥理學調節。
     
     
     
    圖2. KEAP1和β-TrCP對NRF2穩定性的調節及其藥理學靶向。(A)根據雙重調節模型(Rada等,2011),NRF2的兩個結構域,稱為Neh2和Neh6,參與NRF2降解分別響應氧化還原和親電子變化(KEAP1)和信號激酶(β-TrCP)。Neh2結構域結合E3連接酶適配子KEAP1,其呈遞NRF2用于泛素化至CUL3 / RBX1復合物。 Neh6結構域需要GSK-3的先前磷酸化以結合E3連接酶適配子β-TrCP并隨后通過CUL1/ RBX1復合物進行泛素化(詳見文本)。(B)NRF2和KEAP1之間結合的細節以及當前針對這種相互作用的策略。KEAP1同型二聚體在Neh2結構域的兩個基序處結合NRF2:低親和力(29-DLG-31)和高親和力(79-ETGE-82)結合位點。目前破壞這種相互作用的策略包括:改變半胱氨酸C151,273和288的巰基的親電試劑; 改變NRF2與KEAP1的DC結構域的對接的PPI抑制劑。(C)NRF2和β-TrCP的假設結合,并提出了針對這種相互作用的策略。當它被GSK-3(Rada等,2011,2012)磷酸化時,β-TrCP同型二聚體在磷酸基序334-DSGIS-338處與Nhe6結構域結合,當與磷酸基序373-DSAPGS-378結合時獨立于GSK-3(Chowdhry等,2013)。在該圖中,我們假設,與KEAP1類似,一個β-TrCP同二聚體在兩個磷酸基序上與一個NRF2分子相互作用,但仍缺乏實驗證據。破壞這種相互作用的兩種可能策略包括使用GSK-3抑制劑和PPI抑制劑。

    B. NRF2及其調控途徑在人類相互作用組和疾病組的定位

    編碼NRF2的基因,稱為NFE2L2,具有高度多態性,誘變頻率為每72 bp 1次。關于該主題的優秀綜述在2015年報告了多達18個單核苷酸多態性(SNPs),其中大多數在5’調控區域和內含子1中(Cho等人,2015)。這些SNPs中的一些可能構成與慢性疾病發作或進展風險相關的功能性單倍型。功能性單倍型的變異可能對表現出特定疾病的臨床癥狀的個體的比例具有微妙的影響,但是它們可能在群體水平上具有深遠的影響,并且可能定義在精準醫療中靶向該基因的具體策略。
    網絡醫學的最新進展提供了定量工具來表征基因與其相互作用(相互作用組)之間的相互作用如何與病理學相關(Barabasi等,2011; Vidal等,2011; Guney等,2016),分子網絡失調在各種疾病中如何普遍(Menche等,2015),以及疾病如何在特定組織中表現(Kitsak等,2016)。為了從系統醫學的角度理解NRF2在病理學中的相關性,首先我們已經生成了人類相互作用組圖。我們整合并策劃了與NRF2調節途徑相關的蛋白質之間的物理相互作用的信息(Hayes and Dinkova-Kostova,2014; Cuadrado,2015)。交互數據來自最近發表的人類相互作用組,其匯編了幾種蛋白質 - 蛋白質相互作用(PPI)資源的數據(Turei等,2013; Menche等,2015)。然而,目前可用于NRF2相互作用組開發的信息受限于以下事實:因為NRF2是非常短的半衰期蛋白質,所以可能未檢測到一些有意義的相互作用。然而,在相互作用組中發現了一些與NRF2物理性相互作用的眾所周知的蛋白質,包括KEAP1,b-TrCP和MAF。另一組NRF2相互作用蛋白對應于具有調節基因表達功能的核蛋白。這些包括與bZip轉錄因子,核受體或共激活因子相關的蛋白質,或涉及組蛋白乙?;牡鞍踪|。因此,NRF2相互作用組證明除了與該轉錄因子直接相關的基因調控之外的其他基因調控機制。NRF2在幾個殘基處被磷酸化,因此預期它與幾種激酶相互作用,所述激酶包括GSK-3和幾種蛋白激酶C同種型。這些激酶是一些膜受體和銜接子/支架蛋白的下游。除了這種物理相互作用,我們已經確定了NRF2鄰域中富集的幾種生物功能,包括代謝過程,如戊糖,四吡咯,血紅素,葡萄糖6-磷酸,半胱氨酸,GSH,甘油醛-3-磷酸和NADPH的生物合成。大多數這些調節蛋白不直接相互作用,而是通過作為介質的蛋白質連接(圖3;特定相互作用分子的更詳細描述可在這個網址找到http://sbi.imim.es/data/nrf2/)。
     
     
    圖3. 在人類相互作用組中定位NRF2調節途徑。NRF2通過協調各種蛋白質的活性在病理性ROS形成以及炎癥和代謝反應中起關鍵作用。這些相互作用構成分子相互作用網絡,即NRF2相互作用組。參與NRF2調節途徑的蛋白質之間的已知物理相互作用(即調節蛋白:棕色圓圈)和它們連接的蛋白質(即,介體蛋白質:綠色圓圈)用灰色連接顯示。涉及多于一種介體蛋白連接到NRF2的調節蛋白用藍色連接顯示。調節蛋白質來自文獻,它們的相互作用來自各種資源,包括IntAct,MINT,BioGRID,HPRD,KEGG和PhosphoSite??稍诰€獲取人類相互作用組中與NRF2相互作用的所有蛋白質列表,網址為http://sbi.imim.es/data/nrf2/。
     
    已經報道了幾種疾病中NRF2相互作用組的擾動。我們基于DisGeNET (Pinero et al., 2017)和GeneCards (Stelzer et al., 2016)數據庫,結合部分動物模型研究的知識,整理了37種nrf2相關疾病的列表,繪制了疾病組圖。我們還使用DisGeNET,OMIM和GWAS數據庫檢索了這些病癥的疾病 - 基因關聯(Menche等,2015)(表1)。NRF2與每種NRF2相關疾病表型的已知疾病基因的基于相互作用組的接近度(Guney和Oliva,2014)顯示在圖4中。與隨機選擇的蛋白質相比,NRF2與消化系統和癌癥(如前列腺,肝臟和肺腫瘤)的已知疾病基因顯著接近,突出了NRF2在這些病理中的關鍵功能作用。此外,發現NRF2與代謝和心血管疾病相關的各種蛋白質均有近端表達,例如糖尿病,高血糖癥,局部缺血,大腦中動脈梗塞和動脈粥樣硬化。NRF2的蛋白質相互作用還將其與呼吸系統疾病相關的基因(如哮喘,肺纖維化和肺氣腫)以及神經退行性疾?。ㄈ绨柎暮D。ˋD),帕金森?。≒D)和肌萎縮側索硬化癥(ALS))聯系起來。

    表1.有NRF2相關證據的疾病群集

    從DisGeNet數據庫中選擇具有與NRF2遺傳相關的證據的疾病表型。DisGeNet整合來自各種資源的疾病基因關聯信息,如UniProt,ClinVar,GWAS目錄和比較毒物基因組學數據庫,并根據資源數量和支持這些關聯的出版物評估疾病基因關聯。該清單的制定基于以下標準:1)僅選擇在Pubmed中具有多于一個引用的病理表型; 2)可靠性評分設定為0.001的閾值; 3)具有非常相似的名稱或重疊術語的疾病條目被簡化為單個條目。



    病理表型 可靠性分數 病理表型 可靠性分數
    糖尿病腎病 0.2016 糖尿病性心肌病 0.0803
    肝硬化 0.2005 大腦中動脈梗塞 0.0800
    非酒精性脂肪性肝炎 0.2005 乳腺腫瘤 0.0087
    急性腎損傷 0.2000 白癜風 0.0076
    肺纖維化 0.2000 動脈粥樣硬化 0.0067
    非小細胞肺癌 0.1252 哮喘 0.0043
    鱗狀細胞癌 0.1243 白血病 0.0038
    肝腫瘤 0.1238 結腸腫瘤 0.0038
    高血糖 0.1208 胃腸疾病 0.0029
    藥物性肝損傷 0.1200 帕金森綜合癥 0.0026
    前列腺腫瘤 0.1200 系統性紅斑狼瘡性腎炎 0.0026
    慢性阻塞性肺疾病 0.0899 膠質瘤 0.0024
    結直腸腫瘤 0.0847 肌萎縮側索硬化癥 0.0022
    阿爾茨海默病 0.0837 缺血 0.0016
    2型糖尿病 0.0814 肺氣腫 0.0013
    慢性腎病 0.0808 胰腺腫瘤 0.0013
    糖尿病性視網膜病變 0.0805 血管疾病 0.0013
    亨廷頓氏病 0.0805 膿血癥 0.0013
     
     
     
     
    圖4. 從人類相互作用組的角度看NRF2的系統醫學觀點。疾病是由擾亂蛋白質及其相互作用的突變引發的,影響NRF2相互作用組中的某個鄰域?;谙嗷プ饔媒M的接近度(proximity)測量基因與那些疾病鄰域的距離。 條形圖顯示NRF2基因(NFE2L2)與參與NRF2相關病理表型的已知疾病基因的接近程度。這些條形圖強調了NRF2在消化系統疾病和癌癥中的作用。對于給定的疾病,接近度首先計算從NRF2到最接近的已知疾病基因的距離,然后將該距離與使用相互作用組中隨機選擇的蛋白質之間的平均距離估計的隨機期望進行比較?;谙嗷プ饔媒M的接近度報告的z分數對應于NRF2與疾病基因之間觀察到的距離的顯著性。負值表示觀察到的距離低于偶然預期的距離。根據z得分,條紋以不同的橙色調著色:分別為近端(深橙色),近端(橙色),非近端(淺橙色)。已知的疾病基因來自DisGeNet,OMIM和GWAS目錄。
     
    因此,基于相互作用組的接近度提供了關于NRF2如何與各種病理狀況相關聯的觀點。之前已將疾病之間的關系概括為一種網絡,成為疾病組,其基于遺傳(Goh等人,2007)和臨床(Hidalgo等人,2009; Zhou等人,2014)的共同性來連接它們。在圖5中,我們基于共享基因,癥狀相似性和合并癥定義了疾病網絡,即NRF2疾病組。NRF2似乎連接基本上由炎癥過程控制的疾病,例如急性腎損傷,肝硬化和動脈粥樣硬化。此外,AD,PD,亨廷頓舞蹈病和ALS等神經退行性疾病構成一個集群,與最近的研究一致,也暗示NRF2在神經炎癥過程中的作用(Rojo等,2010; Lastres-Becker等,2012,2014; Jung等,2017; Wang等,2017b)。通過激酶信號轉導級聯對NRF2的調控(Jung et al., 2017)可以解釋密切相關的癌癥簇,特別是支持NRF2參與結腸和乳腺腫瘤病理性ROS形成(Gonzalez-Donquiles et al., 2017;Lu et al., 2017)。
     
     
     
     
    圖5.  NRF2疾病組的當前狀態。疾病之間的關系表示為一個網絡,其中病理表型由共同的遺傳和臨床描述符連接。圖中,節點(紅色六角形)代表疾病,邊緣根據共同的基因、共同的癥狀和共病(分別為灰色、橙色和藍色線條)表示疾病之間的相似性。與疾病相關的基因和癥狀被用來識別具有顯著遺傳和癥狀重疊的疾病對,這些重疊是使用Jaccard指數計算出來的。在顯著的疾病 - 疾病關系中(P <0.05,通過基于觀察到的基因或癥狀重疊的Fisher精確檢驗評估),僅顯示具有升高的重疊和共病的鏈接以消除潛在的虛假連接。因此,圖中包含了至少10%的疾病相關基因和超過一半的相關癥狀。共病信息是從醫療保險索賠中提取的,代表人群中容易同時發生的疾病對(相對風險>2)。

    Ⅲ. NRF2在人類疾病狀態中的靶標驗證

    A. NRF2在炎癥消退中的關鍵作用

    持續性炎癥是NRF2疾病組中發現的所有病理表型的標志。這很可能是因為炎癥與活性氧(ROS)的局部和全身病理形成增加有關。事實上,ROS和活性氮物種(RNS)刺激和加重炎癥反應,這些反應與轉錄因子NF-κB的激活機制相關的激活機制相關(NF-κB:激活的B細胞的核因子κ-輕鏈增強子的p65亞基)(Wenzel等,2017)。非常簡化的,在靜息免疫細胞中,NF-κB通過與核κ-B抑制因子(IκBα)的相互作用保留在細胞溶質中。源自微生物的病原體相關分子模式分子以及響應于組織損傷而釋放的損傷相關分子模式分子刺激免疫細胞表達的同源受體,其導致IκB激酶(IKK)β的激活。該激酶使IκBα磷酸化,使其靶向降解并允許核轉位和NF-κB活化(Napetschnig和Wu,2013)。這些事件通過IκBα的幾種調節模式進行氧化還原控制(Bowie和O'Neill,2000; Morgan和Liu,2011; Siomek,2012),但最近描述的一種模式涉及KEAP1對IKKβ穩定性的調節。就像NRF2一樣,IKKβ具有ETGE基序,使其能夠與KEAP結合,進行泛素化和蛋白酶體降解。因此,在基礎氧化還原條件下,活性KEAP1靶向IKKβ進行降解,然后IκBα抑制NF-κB。相反,在存在ROS的情況下,KEAP1被抑制并且IKKβ穩定,磷酸化IκBα并導致其降解并因此導致NF-κB的上調(Lee等人,2009)。

    由于NRF2是氧化還原穩態的主要調節因子,因此它對NF-κB活性進行間接控制。脂多糖(LPS)同時激活快速、促炎性的NF-κB反應和緩慢的NRF2反應。當NRF2最大活性時,NF-κB響應隨后被抑制(Cuadrado等,2014)。例如,Ras相關的C3肉毒桿菌毒素底物1(Rho家族的一個小G蛋白)激活NF-kB途徑,并且NRF2過表達被阻斷,而NRF2敲低增強NF-κB依賴性轉錄(Cuadrado等,2014)。一致地,在用LPS或腫瘤壞死因子(TNF)-α攻擊的NRF2缺陷型(Nrf2-/-)小鼠中,IKK的活性惡化并導致IκB的磷酸化和降解增加(Thimmulappa等,2006a)。

    NRF2還誘導抗炎表型,其調節CD8+T細胞(Sha等人,2015)以及巨噬細胞和小膠質細胞的功能(Rojo等人,2010,2014a; Brune等人,2013)。這是因為NRF2通過調節胱氨酸/谷氨酸轉運蛋白和GSH合成酶?-谷氨酰半胱氨酸連接酶調節器和催化亞基[?-谷氨酰半胱氨酸連接酶調節劑亞基(GCLM)和?-谷氨酰半胱氨酸連接酶催化亞基(GCLC)]來增加巨噬細胞中的半胱氨酸和GSH水平。相反,GSH耗竭使巨噬細胞對LPS激活NRF2敏感(Diotallevi等,2017)。所有這些研究都指出NRF2是一種抗炎因子,對控制炎癥反應的強度和持續時間至關重要(圖6)。
     
     
     
    圖6. 通過NRF2直接和間接調節炎癥。直接作用機制包括抗炎基因的轉錄誘導以及促炎基因的轉錄抑制。 在第二種情況下,引號表示在此功能中需要進一步的工作來識別NRF2的bZip伙伴(如果有的話)。抵抗炎癥的間接機制涉及ROS / RNS調節和抑制免疫細胞的遷移/浸潤??傮w而言,這些途徑導致抗炎反應,有助于正確解決炎癥。NFE2L2中多態性的存在與轉錄活性降低,患者中靶基因水平的改變以及來自臨床前研究的有希望的數據相關,支持NRF2在炎癥消退中的相關作用。
     
    通過前饋和反饋機制產生NRF2和NF-κB串擾(圖7)。在轉錄水平,由于NFE2L2基因的啟動子區域中存在幾個功能性結合位點,NF-κB激活NRF2表達,從而誘導負反饋環(Rushworth等,2012)。此外,NF-κB和NRF2轉錄因子都需要共激活因子CBP / p300,它是組蛋白乙酰轉移酶,乙?;⒃黾覦NA結合能力。因此,NF-κB過表達阻礙了NRF2的CBP / p300的可用性,因此降低了其轉錄能力,而NF-kB敲低顯示出相反的效果(Liu等人,2008)。另外,NF-κB可以促進組蛋白脫乙酰酶-3與MAF蛋白的相互作用,因此阻止它們與NRF2的二聚化(Liu等人,2008)。NF-κB將KEAP1結合并轉移至細胞核,從而有利于NRF2在該細胞區室中的泛素化和降解(Yu等,2011)。 E3連接酶適配子β-TrCP標記IκBα(Winston等人,1999)和NRF2(Rada等人,2011,2012; Cuadrado,2015)用于蛋白酶體降解,因此它可以導致增加的NF-kB活性。
     
     


     
    圖7. NF-κB和NRF2之間的串擾發生在不同的水平。(A)已在NFE2L2的啟動子區域中鑒定出響應元件。(B)NRF2和NF-κB轉錄因子都競爭結合轉錄共激活因子CREB結合蛋白(CBP / p300)。(C)NF-κB活化激酶IKKb含有ETGE基序,其允許KEAP1結合和隨后的泛素蛋白-蛋白酶體降解。(D)據報道NF-κB結合并將KEAP1轉移至細胞核,從而促進NRF2降解。(E)炎癥過程中產生的ROS激活NF-κB和NRF2; 最后,NRF2減弱ROS并因此減弱NF-κB活性。(F)不同的促炎信號激活Rho GTP酶RAC1,導致NF-κB和NRF2活化。 然后NRF2抑制RAC1介導的NF-kB活化。
    NRF2的抗炎活性被認為僅依賴于氧化還原代謝的調節或與NF-κB的串擾。然而,NRF2還可以在暴露于LPS后直接阻斷巨噬細胞中促炎基因白細胞介素(IL)-6和IL-1β的轉錄(Kobayashi等,2016)。LPS暴露或NRF2的藥理學活化導致其與這些促炎基因的近端啟動子結合并阻斷RNA pol II的募集。該機制似乎與NRF2與其成熟的ARE增強子的結合無關。在其他研究中,NRF2可以直接調節其他幾種巨噬細胞特異性基因的表達,例如具有膠原結構的巨噬細胞受體,細菌吞噬作用所需的受體,或CD36,氧化低密度脂蛋白的清道夫受體(Harvey等,2011;Ishii和Mann,2014)。類似地,編碼促炎細胞因子IL-17D的基因含有AREs,并且該NRF2-T輔助因子(Th)17軸似乎賦予針對腫瘤發生和病毒感染的保護作用(Saddawi-Konefka等,2016)。
    慢性炎癥過程涉及白細胞粘附到血管內皮并滲入受損組織。兩種過程似乎都被NRF2與其編碼血紅素加氧酶-1(HO-1)的至少一種靶基因HMOX1一起調節。NRF2 / HO-1軸通過調節幾種細胞粘附分子如血管細胞粘附分子1的表達來抑制炎性細胞與內皮的粘附(Banning和Brigelius-Flohe,2005; Wenzel等,2015)。另外,NRF2 / HO-1抑制巨噬細胞中的金屬蛋白酶-9,這是組織內免疫細胞遷移所必需的(Bourdonnay等,2009)。
    許多臨床前研究報道,天然化合物(Satoh等,2013)或通過破壞其負調節因子KEAP1激活NRF2導致髓樣白細胞(Kong等,2011)和巨噬細胞(Lin等, 2008)的強效抗炎作用。 在觀察性研究中,NFE2L2中的多態性與轉錄活性降低相關,與炎癥性腸?。ˋrisawa等,2008b)和慢性胃炎(Arisawa等,2007)的風險增加相關。NRF2的免疫調節作用的一個例子是在中樞神經系統。受損神經元釋放出趨化因子,一種特異性激活小膠質細胞中磷脂酰肌醇3激酶/ AKT(PI3K / AKT)途徑的趨化因子,導致GSK-3β的抑制和NRF2的上調(LastresBecker等,2014)。在這項研究中,來自AD和進行性核上性麻痹患者的尸檢顯示出趨化因子水平的補償性增加以及上調的NRF2蛋白,表明該途徑有助于限制病變大腦中的炎癥反應。

    B. NRF2在自身免疫性疾病中的作用

    在NRF2疾病群的外圍,我們發現了幾種自身免疫疾病表型,如白癜風,哮喘,多發性硬化癥(MS)和系統性紅斑狼瘡(SLE)。實際上,在實驗性自身免疫性腦脊髓炎(EAE)和類風濕性關節炎(RA)的動物模型中的大量工作,以及MS和牛皮癬中的臨床證據進一步指出NRF2在自身免疫疾病中的作用。氧化組織損傷和細胞凋亡可以增加自身抗原的產生,導致T細胞的活化和B細胞產生自身抗體,例如,如對3-硝基酪氨酸陽性蛋白所觀察到的那樣(Thomson等,2012)。此外,II相解毒酶的丟失,其中許多由NRF2轉錄調節,導致活性中間體的產生增加,這有助于形成半抗原或受損的大分子,有時變得具有免疫原性,從而增加引發自身免疫反應的自身抗原庫。因為NRF2調節的酶在許多化學物質的解毒中起關鍵作用,因此可以認為NRF2可能是一種保護機制,可以抵抗環境對自身免疫發病機制的作用(Ma, 2013)。NRF2介導的自身免疫調節的潛在機制還涉及抑制促炎性Th1和Th17應答以及免疫抑制性T調節(Treg)和Th2應激的激活。還有越來越多的證據表明NRF2可以控制參與抗原呈遞和適應性免疫應答調節的樹突細胞(DC)和巨噬細胞的分化和功能。事實上,NRF2缺陷表明,通過增加共刺激分子的表達并因此增加抗原特異性T細胞反應性來改變DCs的功能和表型(Al-Huseini等,2013)。
    MS是一種慢性炎性疾病,其特征在于自身反應性免疫細胞浸潤到中樞神經系統中。NRF2的缺失加劇了EAE的發展,EAE是MS的小鼠模型(Johnson等人,2010)。與NRF2缺乏相關的部分影響可能與HO-1水平降低有關。因此,具有骨髓特異性HO-1缺陷的小鼠表現出較高的病變發生率,伴隨著抗原呈遞細胞的活化和炎性Th17和髓鞘特異性T細胞的浸潤(Tzima等人,2009)。敲除KEAP1(Kobayashi等,2016)或用各種激活NRF2的小分子治療(Buendia等,2016)抑制了疾病的發展和嚴重程度。NRF2在活躍的MS病變中強烈上調,并且NRF2應答基因的表達主要在初始髓鞘破壞的區域中被發現(Licht-Mayer等,2015)。在MS腦中,NRF2及其靶標NADPH:醌氧化還原酶(NQO1)和HO-1主要在浸潤性巨噬細胞中表達,在較小程度上在星形膠質細胞中表達,最有可能作為對病理性ROS形成的代償性反應。相反,在少突膠質細胞中缺乏NRF2和抗氧化基因表達,這可能是它們在MS中的損傷和損失的基礎(van Horssen等,2010)。由于免疫和氧化還原穩態的改變,MS患者外周血單核細胞中HO-1表達降低,并且在疾病惡化期間下調(Fagone等,2013)。值得注意的是,干擾素-β治療患者的基因表達譜分析鑒定出NRF2是長期抗氧化反應和神經元保存的潛在介質(Croze等,2013)。
    SLE由高氧化環境、失調的細胞死亡和去除死細胞的缺陷而突出,這導致細胞壞死作為自身抗原的來源。NRF2缺陷的雌性小鼠隨年齡增長而發展為類似于SLE的多器官自身免疫性疾病,其特征為DNA氧化增加,脂質過氧化,脾細胞凋亡,抗雙鏈DNA抗體和史密斯抗原的存在,以及伴隨重要的組織損傷(血管炎,腎小球腎炎,肝炎和心肌炎)(Li等,2004)。只有雌性小鼠出現SLE進展的事實表明,雌性特異性因子可能有助于打破對自身抗原的免疫耐受(Li等,2004)。NRF2缺乏還導致CD4 + T細胞的增殖反應增強、CD4 + / CD8 +比率改變、以及SLE中促炎性Th17的促進(Ma等人,2006; Zhao等人,2016)。事實上,NRF2耗竭與狼瘡性腎炎發展過程中的Th17分化和功能有關,這似乎是通過調節細胞因子信號傳導抑制因子3 /磷酸化信號轉導和轉錄激活因子(STAT)3途徑和IL-1b信號來調節的(Zhao 等,2016)。此外,Nrf2-/ -小鼠的唾液腺顯示出強烈的淋巴細胞浸潤,使人想起Sjögren綜合征,這通常與SLE有關(Ma等,2006)。SLE患者表現出氧化性DNA損傷修復機制的改變(Evans等,2000),高血清氧化蛋白水平,載脂蛋白C3(Morgan等,2007),氧化磷脂和抗氧化修飾脂蛋白的自身抗體(Frostegard等,2005)。NRF2多態性尚未與SLE易感性相關,盡管SNP rs35652124與墨西哥女性兒童期發病腎炎風險增加有關(Cordova等,2010)。
    類風濕關節炎(RA)是一種全身炎癥性疾病,具有復雜但仍難以捉摸的自身免疫特性,在炎癥關節中,中性粒細胞、巨噬細胞和淋巴細胞被積極募集和激活。這導致促炎介質如ROS / RNS,類二十烷酸,細胞因子(IL-17,TNF-α,干擾素-?,IL-6和IL-1β)和分解代謝酶的分泌增加,這些促炎介質引發滑膜成纖維細胞的過度增殖, 關節腫脹,軟骨和骨的逐漸破壞(Roberts等,2015)。NRF2基因的缺失增加了實驗性RA模型中關節改變的易感性。例如,在表達T細胞受體KRN和主要組織相容性復合體II類分子A(g7) (K/BN關節炎模型)的小鼠中,以及在抗體誘導的關節炎中,NRF2缺乏加速了發病率并加重了疾病進程( Maicas等,2011; Wu等,2016b)。NRF2缺乏顯著上調炎癥細胞的遷移、環加氧酶-2和誘導型一氧化氮合酶的表達、ROS和RNS的產生以及促炎細胞因子和趨化因子的釋放。此外,NRF2可能是關節炎中骨代謝的保護因子(Maicas等,2011),NRF2 / HO-1活化在RA動物模型和人滑膜成纖維細胞中發揮抗炎和抗氧化作用(Wu等,2016b)。有趣的是,抗風濕金(I)化合物通過激活NRF2和上調HO-1和GCLC來刺激抗氧化反應,證明其對RA的臨床療效(Kobayashi 等, 2016)。此外,NRF2 / HO-1激活介導了滑膜細胞凋亡的誘導和西洛他唑對促炎細胞因子產生的抑制(Park等,2010)以及H2S和相關化合物的抗炎作用,它們能通過對KEAP1的半胱氨酸殘基進行硫化物修飾(Wu等,2016b)。其他誘導NRF2和HO-1信號傳導的藥物,如瑞巴派特,可以轉導人和小鼠CD4+ T細胞向免疫抑制性Treg表型的分化,并通過特異性抑制STAT3來抑制TCD4+細胞向炎性Th17細胞的分化(Moon等,2014)。發炎的滑膜內過量的ROS產生似乎有助于RA的發病機制,因為患者的ROS形成,脂質過氧化,蛋白質氧化,DNA損傷和抗氧化防御機制活性降低顯著增加,所有這些都促成了組織損傷和疾病進展(Datta等,2014)。響應于病理性ROS的形成,NRF2途徑在RA患者的滑膜細胞和抗體誘導的關節炎小鼠的關節中被激活,但是該反應顯然不足以抵消疾病進展(Wu等,2016b)。
    白癜風是一種皮膚炎癥性疾病,其特征在于表皮中ROS的積累,其參與黑素細胞的死亡。這些分子修飾DNA和黑素體蛋白,形成自身抗原并激活針對黑素細胞的自身免疫應答(Xie等,2016)。 遺傳學研究發現NRF2啟動子SNPs與白癜風易感性有關,如-650位點的SNP (Guan等, 2008),而rs35652124的C等位基因在漢族人群中被證明具有保護作用(Song等, 2016)。NRF2及其下游解毒靶基因NQO1,GCLC和GCLM在白癜風患者的表皮中上調,表明這種防御機制的激活不足(Natarajan等,2010)。

    C. NRF2在慢性呼吸系統疾病中的作用

    NRF2在呼吸系統疾病中的相關性在2010年得到了回顧(Cho和Kleeberger,2010),在這項工作中,我們將僅重點介紹最相關的發現(圖8)。香煙煙霧是慢性阻塞性肺?。–OPD)的主要危險因素。COPD患者具有功能失調的肺泡巨噬細胞,其導致不受控制的ROS產生,促炎介質,吞噬作用缺陷和一系列參與組織損傷的金屬蛋白酶。慢性阻塞性肺病(COPD)患者的肺泡巨噬細胞功能失調,導致ROS生成失控、促炎介質、有缺陷的吞噬作用以及一系列參與組織損傷的金屬蛋白酶。事實上,肺氣腫肺組織顯示實質中肺泡巨噬細胞密度與肺破壞嚴重程度之間存在直接關系(Finkelstein等,1995)。肺泡巨噬細胞的吞噬活性受損是引起COPD急性加重的細菌和病毒復發的主要原因,并且是發病率和死亡率的主要來源。Nrf2-/- 小鼠對香煙煙霧誘發的肺氣腫表現出增強的易感性(Rangasamy等,2004)。重要的是,用異硫氰酸酯蘿卜硫素(SFN)激活NRF2可恢復對細菌的識別和吞噬作用,增強肺泡巨噬細胞對肺細菌的清除,并減少野生型小鼠的炎癥,但對于暴露于接觸香煙煙霧的Nrf2-/-小鼠不會發生(Harvey等,2011)。在人類中,與吸煙和非吸煙的非肺氣腫患者相比,吸煙相關性肺氣腫患者肺泡巨噬細胞中NRF2的轉錄特征降低(Goven等, 2008)。NRF2表達降低與脂質過氧化產物4-羥基壬烯醛巨噬細胞表達增加有關。在NFE2L2啟動子中,由三個SNPs和一個三聯體重復構成的功能性單倍型,產生低至中等NRF2表達的多態性(Yamamoto等,2004),與發生COPD的風險增加有關(Hua等,2010)。低表達單倍型是發展呼吸衰竭的重要預測因子。因此,SNP rs6721961的-617A等位基因發生急性肺損傷的風險顯著較高(Marzec等,2007)。
    病理性ROS形成可能在慢性肺纖維化的發病機制中起作用。早期研究表明,博來霉素誘導的肺纖維化在Nrf2-/- 中比在野生型小鼠中更嚴重(Cho等,2004)。事實上,野生型小鼠通過上調NRF2誘導抗氧化和抗炎反應,而這在Nrf2-/-小鼠中無法實現。后來證實,患有特發性肺纖維化或慢性結節病/過敏性肺炎的患者表現出NRF2的表達增加,并且支氣管肺泡灌洗液中的低摩爾.wt.抗氧化劑水平增加,如尿酸,抗壞血酸,視黃醇和α-生育酚 ,表明對ROS挑戰的適應性反應不成功(Markart等,2009)。機制地,NRF2缺乏增加肌成纖維細胞分化,而用SFN藥理誘導NRF2導致肌成纖維細胞數量減少和轉化生長因子-β(TGF-β)的促纖維化作用減弱(Artaud-Macari等人,2013)。
     
      
     
    圖8. NRF2在慢性病的常見機制和病理表型中的作用。該圖提供了從圖5的NRF2疾病組中提取的一些實例。這些疾病的常見病理機制包括異常高的ROS水平和參與組織損傷的低度慢性炎癥。NRF2通過調節許多細胞保護基因的表達,提供針對這些和其他組織特異性改變的細胞保護特征。

    D. 消化系統中的NRF2

    NRF2疾病組中消化系統病理表型的顯著地位突出了NRF2的轉錄特征作為一種對異生素引發的慢性氧化損傷和炎癥應激的有效適應機制的相關性。在胃腸道(GI)中,長期接觸異生素可引發腸腔微生物群與免疫系統之間功能失調的相互作用(Aviello和Knaus,2017)。這可導致胃腸道的慢性疾病,如包括克羅恩病和潰瘍性結腸炎的炎性腸?。↖BD)表型,其中存在激活保護性NRF2應答的證據。例如,在來自IBD患者的結腸組織中,結腸上皮細胞通過與蛋白酶體蛋白表達增加相關的NRF2依賴性適應對炎癥信號起反應(Kruse等,2016)。來自IBD患者的單核細胞衍生的巨噬細胞證明了特異性NRF2依賴性基因表達譜,其響應于LPS而加重,進一步表明這是一種減輕炎癥反應的嘗試(Baillie等, 2017)。在遺傳水平上,NFE2L2基因的特定基因型(-686-684)與日本隊列研究中的潰瘍性結腸炎的發展相關,尤其是在女性中(Arisawa等,2008a)。事實上,GI中的病理過程高度依賴于宿主的遺傳背景,與腸腔微生物群和免疫系統之間的功能失調相互作用有關(Aviello和Knaus,2017)。
    GI的微生物群的共生作用之一是釋放適量的ROS,其引起由NRF2介導的上皮細胞和浸潤的免疫細胞中的細胞保護反應(Jones等,2015)。此外,在真核生物中受NRF2轉錄控制的細胞保護分子也可以由共生細菌產生。例如,微生物群中的HO-1同源物可能對胃腸道內穩態有很大的貢獻,這可以用于治療一氧化碳在腸道的局部遞送(Onyiah等, 2014)。
    NRF2在維持胃腸道穩態方面的作用已被證實,使NRF2這一轉錄因子成為IBD中有希望的治療靶點。因此,幾種化合物和膳食補充劑可能表現出有益效果,如褪黑激素,3-(3-吡啶基亞甲基)-2-吲哚滿酮,丁酸鹽,干酪乳桿菌,左旋肉堿,4-乙烯基-2,6-二甲氧基苯酚(卡諾爾), 乳枸杞(脫脂牛奶中枸杞的配方產品)等(Orena等,2015)。因此,明確NRF2在消化道慢性和急性疾病中的參與情況,對于更好地指導NRF2通路的調節治療方法至關重要。
    肝臟也是抵抗食物異生素的第一道防線。因此,NRF2疾病組強調該轉錄因子在與肝損傷相關的病理表型中的相關性也就不足為奇了。早期使用Nrf2-/-小鼠模型證明其對對乙酰氨基酚誘導的肝細胞損傷、苯并[a]芘誘導的腫瘤形成以及Fas-和TNF-α介導的肝細胞凋亡具有保護作用(Aleksunes和Manautou,2007)。Nrf2-/-小鼠對化學毒性的較高敏感性與解毒酶的基礎表達和誘導表達降低相關。在人類中,導致NRF2表達降低的三種NRF2啟動子SNPs的功能性單倍型與胃粘膜炎癥的發展顯著相關,無論是獨立地還是通過與幽門螺桿菌感染相互作用(Arisawa等,2007)。對原發性膽汁性膽管炎患者NRF2轉錄特征的分析表明,這些患者表現出NRF2表達降低以及HO-1和GCLC蛋白水平低,這些損傷在肝硬化患者中更為嚴重(Wasik等,2017)。
    病理性ROS形成是非酒精性脂肪性肝炎(NASH)患者肝細胞損傷和疾病進展的關鍵機制(圖8)。該疾病分兩個階段發展,一個是肝細胞中脂肪酸的逐漸積累,另一個是肝損傷和炎性病理性ROS的形成(Wang等,2018)。因此,喂食高脂肪飲食(HFD)的小鼠出現了單純的脂肪變性,其特征是肝臟脂肪沉積增加而沒有炎癥或纖維化,但Nrf2-/-小鼠出現加劇的肝臟脂肪變性和大量炎癥,與NASH一致(Reccia等,2017)。然而有趣的是,肝細胞特異性KEAP1的缺失在減少肝臟脂肪變性的同時,并沒有改變NASH發展過程中的炎癥反應,提示其具有代償機制(Ramadori等, 2016)。至少在NASH的大鼠模型中,飲食NRF2激活劑減弱了肝纖維化的進展(Shimozono等,2013)。在NASH患者的肝臟活組織檢查中病理性ROS形成的標志物增加,并且NRF2特征增加,表明其試圖減少氧化劑和炎癥負荷(Takahashi等人,2014)。

    E. 心血管系統中的NRF2

    NRF2疾病群指出心血管系統對細胞氧化還原平衡的變化以及眾所周知的動脈粥樣硬化、高血壓和糖尿病等共病的發展具有高度敏感性(Griendling and FitzGerald, 2003a,b;Harrison等,2003;Jay等,2006)(圖8)。NRF2在預防這些病理表型方面的作用已經在NRF2 -/-小鼠中得到證實,NRF2 -/-小鼠在慢性耐力運動中表現出心臟結構受損(更多的重塑事件)和功能受損(較少縮短分數)(Shanmugam等, 2017a)。它們也更容易在心肌梗塞后發生心力衰竭(Strom和Chen,2017)。相反,NRF2的組成型活化產生還原狀態,其特征書是心臟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)。在人類中,Tako-Tsubo心肌病的微陣列分析表明,在這種收縮功能障礙的急性期,病理性ROS水平增加,NRF2代償性上調(Nef等,2008)。最近,血液透析患者的全身性炎癥和病理性ROS形成與NRF2的下調相關(Pedruzzi等,2015),并且兩種啟動子多態性(rs35652124和rs6721961)與這些患者的死亡風險增加相關(Shimoyama等,2014)。
    NRF2在內皮穩態中最相關的靶點之一是HO-1,其通常與鐵蛋白的上調相平行,因此降低游離鐵水平,并阻止芬頓型反應。由HO-1和膽綠素還原酶的聯合活性產生的膽紅素是清除ROS / RNS的最強大的內源性抗氧化劑之一(Jansen等,2010),并且在體外預防脂質過氧化方面非常有效( Stocker等,1987)。Hmox1-/-小鼠顯示出慢性缺氧引起的肺動脈高壓升高(Christou等,2000),藥理學HO-1誘導可改善糖尿病并發癥(Kruger等,2006),以及硝酸甘油誘導的血管功能障礙( 硝酸鹽耐受性)(Wenzel等,2007)。最近,Hmox1-/-小鼠在血管緊張素II響應中顯示NADPH氧化酶-2上調、血管病理ROS形成、炎癥標志物、內皮功能障礙和血壓升高(Wenzel et al., 2015)。事實上,高血清膽紅素水平與冠狀動脈疾病的發病率呈負相關(Hopkins等,1996)。膽紅素阻止血管NADPH氧化酶的活化(Kwak等,1991),其參與心血管疾病的發展(Griendling和FitzGerald,2003a,b; Harrison等,2003; Jay等,2006)。外周動脈疾病是動脈粥樣硬化的常見表現,患有外周動脈疾病的患者HO-1水平降低(Signorelli等, 2016)。

    F. 代謝性疾病中的NRF2

    2型糖尿?。═2DM)是最常見的慢性代謝疾病之一,并且在NRF2疾病組中尤為突出。胰島素敏感組織以及胰腺中的病理性ROS形成已在T2DM患者中發現,導致胰腺β細胞分泌胰島素和外周組織胰島素作用嚴重受損(Uruno等,2015)(圖 8)。同樣,由于蛋白質的非酶促糖化,病理性ROS水平也有助于糖尿病并發癥的發病機理。這已在糖尿病腎病中得到證實,其中腎小球表現出病理性ROS水平和NRF2的代償性升高(Jiang等人,2010)。自2007年以來,一些研究利用動物模型和細胞系研究了NRF2在T2DM及其并發癥中的作用。體外對人體細胞的研究表明,NRF2的激活是在急性高糖環境下實現的,而較長時間的培育時間或振蕩的葡萄糖濃度均不能激活NRF2 (Ungvari等, 2011;Liu等, 2014)。因此,這些研究指出NRF2活化依賴于葡萄糖濃度和動力學。相反,NRF2在前驅糖尿病和糖尿病患者的外周血單核細胞中下調,表明NRF2可能是重要的治療靶點(Jimenez-Osorio等,2014)。
    NRF2缺乏對高血糖的影響首先在Nrf2-/-小鼠中顯示,其中氧化和亞硝化改變增強并導致早期腎損傷(Yoh等,2008)。 在隨后的研究中,鏈脲佐菌素誘導的糖尿病Nrf2-/-小鼠表現出加劇的腎小球損傷,同時ROS產生增加和促纖維化標志物TGF-β和纖連蛋白表達的增加(Jiang等人,2010)。在這個糖尿病模型中,NRF2對血視網膜屏障功能障礙和糖尿病視網膜病變的進展具有保護作用(Xu等, 2014)。同樣,HFD誘導的血管ROS水平的增加在Nrf2-/-小鼠中顯著加劇,并伴有嚴重的內皮功能障礙,表現為乙酰膽堿誘導的主動脈松弛減弱和細胞間粘附分子-1和TNF-α表達增加( Ungvari等,2011)。
    NRF2在組織特異性胰島素抵抗中起復雜作用。因此,與野生型小鼠相比,HFD喂養的Nrf2-/-小鼠由于肝臟和骨骼肌中胰島素信號的增強而表現出更好的胰島素敏感性,但相反,這些小鼠由于與病理ROS形成相關的肝脂毒性過高而產生嚴重的NASH (Meakin等, 2014)。因此,該研究將肝胰島素抵抗與NASH的發展分離。根據這些數據,隨后的研究表明,由于CYP2A5酶的表達減弱,HFD喂養的Nrf22-/-小鼠的肝臟由于GSH的顯著消耗而表現出更高的病理性ROS形成(Cui等人,2013)。NRF2在肝細胞中的敲低增強了棕櫚酸誘導的細胞凋亡,棕櫚酸是一種在胰島素抵抗性肥胖患者中高度升高的脂肪酸。這種效應與病理性ROS的產生增加相關,再次強化了NRF2在NASH進展中的關鍵作用(Pilar Valdecantos等,2015)。
    為了進一步研究NRF2在代謝綜合征中的作用,我們在瘦素缺乏(ob/ob)小鼠模型中去除NRF2,該模型具有極好的正能量平衡(Xue等, 2013)。有趣的是,全身ob / ob / Nrf2-/-小鼠或脂肪細胞特異性ob/ob/ Nrf2-/-小鼠顯示白色脂肪量減少,顯示NRF2是脂肪生成的關鍵參與者。這些小鼠具有更嚴重的代謝綜合征,其特征在于高脂血癥,加重的胰島素抵抗和高血糖,表明代謝綜合征與病理性ROS形成之間的機制聯系。
    另一組研究評估了持續誘導NRF2在葡萄糖代謝中的作用。NRF2基因誘導利用Keap1的一個亞型等位基因(Keap1flox/- 突變體)降低肥胖糖尿病db/db小鼠的血糖,是通過抑制肝細胞中cAMP-CREB信號傳導以及其他糖異生基因(例如過氧化物酶體增殖激活受體輔激活因子-1α)來抑制肝葡萄糖6磷酸酶(Uruno等,2013)。此外,Keap11敲低小鼠中NRF2活性的增強增加了肝臟中AMP活化蛋白激酶(AMPK)的磷酸化,以及骨骼肌中的胰島素信號傳導,導致葡萄糖耐量的顯著改善(Xu等,2013)。由于NRF2在T2DM背景下的多效活性,所有這些和其他研究的結果證明需要設計多種遺傳和藥理學策略來闡明參與全身葡萄糖穩態控制的組織中的全部NRF2功能。
    除了年齡,體重和血糖等糖尿病因素外,與NRF2相關的遺傳因素在人類中研究較少。在中國人群中,SNP rs6721961與病理性ROS形成和新診斷的T2DM風險有關,并且還可能導致胰島素分泌能力受損和胰島素抵抗增加(Wang等,2015)。在墨西哥混血男性中,相同的SNP與糖尿病相關(Jimenez-Osorio等,2017)。在一項漢族志愿者參與的病例對照研究中,發現T2DM患者無論有無并發癥,包括周圍神經病變、腎病、視網膜病變、足潰瘍和微血管病變,NFE2L2基因4個SNPs的基因型和等位頻率存在顯著差異(Xu等, 2016b)。

    G. 神經退行性疾病中的NRF2

    NRF2疾病組提供NRF2參與幾種神經退行性疾病的證據,包括AD和PD,其代表老年人最普遍的認知和運動障礙。在神經退行性疾病中,低級病理性ROS形成與蛋白質穩態之間的聯系尤為重要,因為大多數這些病理表型的特征在于特定蛋白質的異常聚集(圖8)。在細胞和動物模型中提供了指向蛋白病變中病理ROS形成以及NRF2作為蛋白酶體和自噬調節因子的證據(Pajares等, 2017)。最初,據報道自噬貨物蛋白質sequestosome 1(SQSTM1)與NRF2競爭結合KEAP1。最初,有報道稱自噬貨物蛋白sequestosome 1 (SQSTM1)與NRF2競爭與KEAP1的結合。SQSTM1將KEAP1置于自噬體降解途徑,因此上調NRF2(Komatsu等,2010)。SQSTM1將KEAP1置于自噬體降解途徑,因此上調NRF2(Komatsu等,2010)。最近,發現NRF2調節參與自噬起始、貨物識別、延伸和自溶酶體清除的自噬基因的表達(Pajares等,2016)。在本研究中,Nrf2-/-小鼠中,轉基因過表達人突變淀粉樣前體蛋白和tau蛋白導致淀粉樣病變和tau蛋白病加重。NRF2缺乏癥和神經退行性變之間的聯系被越來越多的動物模型證據所支持(Johnson and Johnson, 2015)。一般的觀點是受損的神經元試圖激活NRF2依賴性轉錄,可能是為了增加它們自身的存活率。此外,星形膠質細胞中NRF2的上調參與代謝補償,包括增加GSH的供應以增強其增殖能力(Bolanos,2016),而小膠質細胞中的NRF2上調使該免疫細胞恢復到靜止狀態(Rojo等,2014a)。
    Ramsey等人(2007)證明了NRF2在PD患者的多巴胺能神經元中的核定位。其他研究發現淀粉樣蛋白前體蛋白和tau損傷神經元表達NRF2及其靶SQSTM1水平升高,可能是通過自噬清除這些毒性蛋白的補償機制(Lastres-Becker等,2014; Pajares等,2016)。與這些結果一致,AD和PD腦中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,與NRF2表達相關的細胞保護蛋白,如NQO1和SQSTM1,在路易體中被部分隔離,表明PD患者中NRF2特征的神經保護能力受損(Lastres-Becker等,2016)。這種差異的一種可能解釋是NRF2及其靶基因水平可能在衰老和疾病進展過程中發生變化。
    NFE2L2的一些SNP單倍型與ALS,AD或PD的風險降低或延遲發作相關。在關于先前與高基因表達相關的三種功能性啟動子SNPs的兩項研究中分析了ALS的發病。有趣的是,這種單倍型與ALS發病延遲4年有關(Bergstrom等,2014),但另一項研究未發現明確的相關性(LoGerfo等,2014)。關于AD,一個單倍型等位基因與AD的2年早期發病相關,表明NFE2L2基因的變體可能影響AD進展(von Otter等,2010b)。已經更詳細地分析了NFE2L2與PD的遺傳關聯。NFE2L2啟動子中的三個SNPs(rs6721961,rs6706649和rs35652124)在病例對照研究中被證明為保護性單倍型(von Otter等,2010a)。這種單倍型在瑞典隊列中延遲了疾病的發作,甚至降低了波蘭隊列中PD的風險。這些結果得到了四項新的獨立的歐洲病例對照研究的支持(von Otter等,2014),但未在臺灣人群中復制(Chen等,2013),這表明種族和環境因素存在差異。另一種方法是將來自嗅黏膜的PD細胞暴露于煙提物或殺蟲劑中,以評估基因-環境的相互作用,并且鑒定了幾種影響對這些毒素的易感性的SNPs(Todorovic等,2015)??偠灾?,NRF2的輕微激活(例如對于NFE2L2基因的某些功能性單倍型所發現的)應該足以觸發大腦中的保護機制。

    IV. 癌癥中Keap1的悖論

    NRF2在腫瘤發生和進一步腫瘤進展中的作用似乎存在明顯的二分法。一方面,通過激活生物轉化反應,NRF2可以防止化學誘導的癌癥發生。臨床前研究表明NRF2在大鼠體內經藥理激活后,對黃曲霉毒素B(1)誘導的肝癌具有完全的保護作用(Johnson等, 2014)。相反,NRF2引發的保護性反應在確定的癌癥中提供了生長優勢,這將是本節的重點。
    不斷增加的ROS水平可以通過改變基因組穩定性來維持腫瘤發生,同時激活特定的氧化還原信號轉導和促進腫瘤細胞存活和增殖的炎癥過程(Reuter等,2010)。因此,NRF2的上調代表癌細胞適應的機制,以耐受推動腫瘤進展的高ROS水平(Schumacker,2006)以及維持導致腫瘤復發和遠處轉移形成的癌癥干細胞(Ryoo等,2016)。例如,來自人類結腸直腸腫瘤的癌癥干細胞中的NRF2特征指出了由高水平的GCLC,谷胱甘肽過氧化物酶和硫氧還蛋白還原酶-1介導的保護機制,這是這些細胞抵抗應激物和化學治療劑的能力的基礎(Emmink等,2013)。從這個角度來看,NRF2在癌細胞中表現得像一個致癌基因,通過誘導ARE介導的細胞保護反應的慢性激活,可以幫助其適應其氧化環境(Panieri和Santoro,2016)。已經報道了NRF2的惡性激活的幾種機制,包括體細胞突變,表觀遺傳學和致癌信號傳導改變。
    據報道,沿著NFE2L2的編碼序列在癌癥中接近600個體細胞突變(Gao等人,2017)。在圖9中,我們顯示了來自10,000名癌癥患者的數據集的結果(Zehir等人,2017)。在大多數情況下,這些突變改變了NRF2的DLG和ETGE基序與KEAP1的相互作用,從而誘導NRF2在幾種實體瘤(包括食道癌,皮膚癌,肺癌和喉癌)中的過度活化(Kim等,2010b; Taguchi和 Yamamoto,2017)。例如,在晚期食管鱗狀細胞癌中,NRF2功能獲得性突變與腫瘤復發和預后不良相關,其原因包括增殖增加、獨立依附生存以及對化療和放療的抵抗(Shibata 等, 2011)。KEAP1基因中的功能喪失突變在一些實體腫瘤例如肺癌中也是常見的(Singh等人,2006)?;谶@一通路調控β-TrCP/NRF2的有力證據,奇怪的是在NRF2與β-TrCP的交界面未發現干擾體突變。這一事實表明,由于未知的原因,這些突變是不可生存的,或者β-TrCP逃逸導致NRF2水平的升高不足以驅動致癌性。
    盡管如此,體細胞突變僅在一小部分癌癥患者中引起長期/慢性NRF2激活。在基因表達水平上,有趣的是位于人NRF2基因的ARE增強子的SNP rs6721961(-617C>A)的等位基因消除了NRF2的自身誘導,這與這些癌癥患者的顯著存活相關(Okano等,2013)。已經在肺腫瘤中描述了由于KEAP1的三個CpG位點的啟動子高甲基化引起的表觀遺傳變化,導致隨后的NRF2活化,其可以通過5-氮雜-2’-脫氧胞苷處理逆轉NRF2的活化(Wang等,2008)。最近已經回顧了miRNAs在NRF2水平的轉錄后調節中的作用(Kurinna和Werner,2015)。簡而言之,miR200a靶向人乳腺癌細胞中的KEAP1 mRNA,導致其降解并隨后激活NRF2(Eades等,2011)。反過來,miR28促進NRF2 mRNA的降解(Yang等人,2011)。
    致癌基因或突變的腫瘤抑制因子可以增強癌癥中NRF2的活化。KRAS,BRAF或c-MYC的內源性致癌等位基因上調NRF2,可能是通過致癌基因介導的ROS和隨后的KEAP1慢性失活(DeNicola等,2011)。腫瘤抑制因子p53的突變形式,通過增強營養攝取和構建塊的合成來維持癌細胞的生長,也可以通過與結合NRF2啟動子的Sp1轉錄因子的串擾來上調NRF2(Tung等,2015)。
    各種蛋白激酶對NRF2(磷酸-NRF2)的磷酸化是107種肝細胞癌中潛在的激活機制。增加的磷酸化NRF2水平與表現出這種獨特表型的患者的KEAP1表達降低和5年總體存活率差有關(Chen等,2016)。此外,磷酸酶和張力蛋白同源物(PTEN)腫瘤抑制因子的突變維持過度活躍的和致癌的磷脂酰肌醇-3-激酶(PI3K)-AKT信號傳導,并由于為NRF2蛋白酶體降解的PTEN / GSK-3 / β-TrCP途徑的下調而導致NRF2活性增加。(Rada等,2011,2012; Cuadrado,2015)。針對PTEN / GSK-3 / β-TrCP途徑的治療干預措施應考慮到GSK-3既可作為腫瘤抑制因子又可作為腫瘤啟動子,并且還與癌癥干細胞的產生有關(McCubrey等,2014)。
    一些應激誘導的蛋白質與KEAP1相互作用,因此在癌細胞中,這些蛋白與KEAP1結合的NRF2競爭。因此,NRF2逃脫了KEAP1介導的降解。NRF2與KEAP1結合的競爭對手之一是自噬銜接蛋白SQSTM1的磷酸化形式,該蛋白發生在癌細胞用于維持自身生長的選擇性自噬過程中(Shimizu等, 2016)。還證明了促進癌癥干細胞中細胞周期停滯的細胞周期蛋白依賴性激酶抑制劑p21通過其KRR基序與NRF2中的DLG和ETGE基序的相互作用抑制NRF2與KEAP1的結合(Chen等人,2009)。最近,顯示具有ETGE基序的二肽基肽酶3可以與NRF2競爭結合KEAP1(Hast等人,2013)??赡苡裳趸€原狀態的長期改變誘導的二肽基肽酶3的過表達與ARE基因的表達增加和預后不良相關,特別是在雌激素受體陽性乳腺癌中(Lu等人,2017)。
    NRF2誘導導致癌癥進展的代謝變化。例如,一項多平臺非靶向代謝組學研究確定了乳腺腫瘤樣本中代謝物變化的模式(Tang等,2014)。他們發現GSH和3-(4-羥基苯基)乳酸通過與NRF2的相互作用與BRCA1參與氧化還原穩態呈正相關。代謝組學研究還表明,NRF2可以增加癌細胞中的有氧糖酵解,以支持其高能量需求。這通過NRF2介導的錳-超氧化物歧化酶表達的誘導發生,導致線粒體過氧化氫的產生升高和AMPK的活化。該過程由caveolin-1調節,其直接結合NRF2和KEAP1,并阻礙NRF2活化并因此阻礙糖酵解轉變。這顯然解釋了為什么通常更具侵略性的糖酵解腫瘤具有caveolin-1低/ Mn超氧化物歧化酶高的表型(Hart等,2016)。NRF2還可以驅動葡萄糖和谷氨酰胺向腫瘤細胞增殖所需的合成代謝途徑(Mitsuishi等,2012)。在PI3K-AKT信號通路活躍和KEAP1活性缺失的情況下,NRF2被證明可以誘導癌癥中葡萄糖代謝從糖酵解向合成代謝途徑(嘌呤合成)的轉移(Mitsuishi等, 2012;Xu等, 2016a)。以上這一點被強調通過NRF2介導的參與磷酸戊糖途徑的基因轉錄和NADPH(葡萄糖6磷酸脫氫酶;磷酸葡萄糖酸脫氫酶,蘋果酸酶1,異檸檬酸脫氫酶1,轉酮醇酶和轉醛酶)的產生以及涉及嘌呤核苷酸合成(磷酸核糖焦磷酸酰胺轉移酶,亞甲基四氫葉酸脫氫酶2)的相關基因。
    NRF2的激活賦予癌細胞生長優勢的事實可能會爭辯說,在這項工作中提到的慢性疾病NRF2的藥理學活化可能意味著患癌癥的高風險。然而,必須考慮到NRF2的致癌活性需要其基因或KEAP1發生突變,從而導致NRF2信號的持續高水平誘導。但在藥理學治療中并非如此,在藥理學治療中,調節藥物劑量和NRF2活性是可能的。此外,經驗證據表明,參與NRF2激活劑臨床試驗的受試者沒有顯示出更高的癌癥風險。自從2013年獲得監管機構批準以來,已經服用NRF2活化劑富馬酸二甲酯數年的MS患者就是最好的例證。相反,在癌癥患者中使用NRF2抑制劑可能導致NRF2疾病組中描述的病理表型的表現。當NRF2抑制劑到達臨床時,這種可能性需要進一步研究。

    V. NRF2藥物組

      本節試圖開發一種NRF2藥物組,該藥物組可用于未來的臨床指導,以NRF2疾病組的病理表型為中心靶向NRF2進行治療。如圖2B中所述,通過靶向KEAP1來正在尋求NRF2的藥理學活化以增加其穩定性。這些策略基于發現改變KEAP1結構的親電化合物或阻止NRF2與KEAP1對接的小分子。雖然尚未經驗證實,但GSK-3抑制劑應阻止b-TrCP識別NRF2,并且可發現一些化合物以防止NRF2與b-TrCP的結合。用靶向bZip二聚化結構域的化合物分析NRF2的抑制作用,以防止形成活性NRF2 / MAF異二聚體。通過與其他轉錄因子比較,可能發現阻礙NRF2 / MAF異二聚體與ARE結合的小分子(圖9C)。本節總結了新藥物發現和重新調整藥物用途的轉化觀點中最重要的發現。

     
     
    圖9. 在MSK IMPACT臨床測序組(MSKCC)研究(Zehir等,2017)的腫瘤中發現的體細胞突變和抑制NRF2的藥理學策略。(A)具有NRF2突變的腫瘤的百分比。 (B)沿NRF2多肽的突變分布。(C)NRM2 / MAFF異二聚體與ARE元件之間相互作用的PyMOL表示。 藍色:NRF2; 粉紅色:MAFF。 紅色箭頭表示可能通過小分子抑制NRF2的機制,這些小分子可以靶向NRF2和MAF蛋白之間相互作用的bZip結構域(PPI抑制劑)或NRF2-MAF異二聚體與ARE相互作用的界面[DNA-蛋白質相互作用(DPI)]

    A. 親電NRF2誘導劑

    大多數已知的生理學或藥理學NRF2誘導劑是親電分子,其通過氧化或烷基化共價修飾富含硫醇的KEAP1蛋白中存在的半胱氨酸殘基(Hur和Gray,2011; Satoh等,2013)。KEAP1是最適合作為親電子或氧化還原傳感器的蛋白質之一,因為它在人體中含有27個半胱氨酸殘基,并起到親電子陷阱的作用。KEAP1的半胱氨酸C151,C273和C288似乎是最容易發生親電子反應的(圖2B),盡管存在一些特異性(Yamamoto等,2008; Saito等,2015)。親電子加合物以兩種不同的方式抑制KEAP1。 一種是誘導KEAP1的構象變化,這將導致其對NRF2的結合能力喪失。另一個是阻斷KEAP1和CUL3 / RBX1之間的相互作用,導致KEAP1與NRF2的隔離以及新合成的NRF2的進一步穩定化(Rachakonda等,2008; Baird和Dinkova-Kostova,2013; Cleasby等, 2014; Saito等,2015)。
    NRF2調制劑的至少30項近期專利在世界國際產權組織中被收錄。這些專利正在保護查爾酮衍生物,新型酰胺三萜衍生物,氘取代的富馬酸衍生物,3-烷基氨基-1H-吲哚丙烯酸酯衍生物,甲醇酰胺,含有活化乙烯基的芐基衍生物,穿心蓮內酯或[S]+阿撲嗎啡,以及倍半萜內酯衍生物(Sun等,2017)。盡管從臨床前概念驗證的角度來看,大多數這些化合物在某種程度上被證明是有用的,但它們的臨床價值迄今通常非常有限。其中只有少數藥物進入了臨床試驗,而食品和藥物管理局(Food and Drug Administration)或歐洲藥品管理局(European Medicines Agency)等監管機構批準的藥物更少。我們在這項研究中討論了轉化管道中最發達的NRF2激活劑。
    富馬酸酯是KEAP1改性劑的最突出的例子,并且富馬酸二甲酯(DMF)是迄今為止唯一的食品和藥物管理局和歐洲藥品管理局批準的藥物,其注冊為NRF2活化劑。單酯形式的DMF,富馬酸單甲酯(MMF)被描述為其活性代謝物。DMF和MMF是邁克爾受體,其直接與KEAP1中存在的半胱氨酸殘基反應(Lin等,2011)。
    從NRF2的功能尚不清楚開始,DMF和其他富馬酸酯已經用于治療牛皮癬超過50年。該化合物在歐洲以商品名Fumaderm獲得許可。臨床試驗顯示,在DMF治療12-16周后,銀屑病面積和嚴重程度指數降低至50%-80%(Altmeyer等,1994; Mrowietz等,1998)。最近,在III期試驗(BRIDGE)中,DMF已證明其在治療患有中度至重度慢性斑塊狀銀屑病的成人中的功效(Mrowietz等,2017)。富馬酸鹽在銀屑病病變緩解中的作用機制包括外周T細胞數量的減少以及從Th1向Th2免疫應答的轉變(Ghoreschi等,2011; Tahvili等,2015)。在另一種自身免疫性疾病SLE中,富馬酸酯已被用作治療嚴重、廣泛和難治性皮膚表現的全身聯合療法(Saracino和Orteu,2017)。
    2013年,DMF以商品名Tecfidera被批準用于治療MS(Xu等,2015)。在MS患者中使用DMF是通過在EAE的MS小鼠模型中獲得的陽性結果推動的。對疾病進程和組織學的顯著治療效果與脊髓中巨噬細胞介導的炎癥顯著減少有關。血液中的多重細胞因子分析證明DMF處理的動物中抗炎細胞因子IL-10的增加(Schilling等人,2006)。此外,DMF還改善了野生型中髓鞘,軸突和神經元的保存,但在Nrf2-/-小鼠中沒有(Ellrichmann等,2011)。在人類中,DMF顯示MS病變和年復發率顯著降低(Schimrigk等,2006)。兩項III期臨床試驗DEFINE和CONFIRM證實了這些結果(Fox等,2012; Gold等,2012)。因此,DMF目前被用作復發緩解型MS的第一線治療,其不能通過傳統療法治療。新的DMF配方正在測試和申請專利,以提高藥物的生物利用度和功效(Sun等,2017)。例如,MMF已被用于開發第二代NRF2誘導物作為前藥(Zeidan等,2014)。鉛化合物ALKS-8700,一種MMF的2-(2,5-二氧代-1-吡咯烷基)乙酯衍生物,在體內迅速轉化為MMF,因此提高了其生物利用度并減少了胃腸道副作用。ALKS-8700目前正在進行III期臨床試驗(EVOLVE MS)。
    DMF和MMF調節免疫應答。例如,它們通過減少炎性細胞因子的釋放并因此減少DCs處理抗原的能力來抑制DCs的成熟。此外,DMF和MMF激活天然殺傷細胞以裂解DCs并增強DCs和T細胞的凋亡(Ghoreschi等,2011; Al-Jaderi和Maghazachi,2015)。因此,DMF和MMF阻礙T細胞介導的自身反應。一些研究表明,DMF還通過觸發GSH耗竭誘導II型DCs,這導致HO-1活性增強和STAT1磷酸化抑制。這些經典的II型DCs抑制Th1和Th17介導的反應,支持Th2的反應。此外,通過DCs增加IL-10的產生有利于CD4+T細胞向抑制性Treg表型的分化(Ockenfels等,1998; Ghoreschi等,2011)。DMF還抑制NF-κB的核轉位(Peng等,2012),從而在小膠質細胞和星形膠質細胞(Brennan等,2017)以及外周血單核細胞(Eminel等,2017)中產生炎癥介質,如TNF-α,IL-1β,IL-6,趨化因子,粘附分子和一氧化氮。此外,DMF發揮抗血管生成作用,其依賴于內皮細胞中血管內皮生長因子受體-2表達的下調(Meissner等,2011)。最近的研究結果表明,DMF減少了CD4+,CD8+,Th1和Th17細胞的數量,而CD4+/CD8+比例和Th2亞群在這些患者的血液中增加。有趣的是,DMF/MMF對T細胞活化的抑制作用主要局限于記憶T細胞(Wu等, 2017)。DMF或MMF的這些免疫調節活性對于保護少突膠質細胞免受ROS誘導的細胞毒性具有重要意義(Scannevin等,2012)。
    其他機制可以解釋NF-κB的抑制,而與NRF2活化無關。因此,DMF可能與幾種調節NF-kB信號傳導的蛋白質中的半胱氨酸殘基相互作用(Blewett等,2016)。此外,DMF可以抑制泛素偶聯酶,從而防止NF-κB的IkB抑制因子在IL-1β或Toll樣受體激動劑的作用下降解(McGuire等, 2016)。此外,DMF直接結合蛋白激酶C-θ中的特定半胱氨酸殘基,這是參與T細胞受體信號傳導的關鍵激酶(Blewett等,2016)。此外,MMF和DMF激活羥基羧酸受體-2,導致NF-κB的抑制和促炎細胞因子和粘附分子的下調(Chen等,2014; Gillard等,2015)并導致中性粒細胞浸潤減少 (Chen等,2014)。盡管這些NRF2非依賴性作用與EAE的急性炎癥期相關,但DMF在慢性自身免疫性脫髓鞘中的神經保護功效取決于NRF2活化(Linker等,2011)。在Nrf2-/-和野生型小鼠中DMF治療的臨床益處與炎性Th1和Th17細胞的減少以及抗炎M2單核細胞的誘導相關。同時,在野生型中觀察到CD80和CD86共刺激分子的表達降低,但在Nrf2-/-小鼠中未觀察到,表明至少這些作用是NRF2依賴性的(Schulze-Topphoff等,2016)。
    DMF用于治療自身免疫疾病的成功表明,具有由慢性,低級炎癥和病理性ROS形成強調的共同病理機制的其他疾病可能受益于該藥物的重新定位。在亨廷頓舞蹈病的小鼠模型中,DMF治療保留了存活率、肌肉功能和體重,這與完整神經元數量的增加有關(Ellrichmann等,2011)。此外,在最近的PD臨床前研究中,使用該疾病的α-突觸核蛋白病模型,由于自噬誘導受損,DMF在野生型中具有神經保護作用,但在Nrf2-/-小鼠中則不具有神經保護作用(Lastres-Becker等,2016)。
    DMF被證明可預防糖尿病小鼠的內皮功能障礙和心血管病理性ROS形成和炎癥(Sharma等,2017),并且在注射鏈脲佐菌素后載脂蛋白E缺陷小鼠中動脈粥樣硬化、腎功能不全和其他糖尿病并發癥減少(Tan等,2014)。此外,一些研究表明,DMF可能通過抑制NF-κB途徑發揮抗腫瘤活性,因此在治療侵襲性癌癥中增加了治療價值(Kastrati等,2016)。DMF是網絡藥理學方法中老藥新用概念的相關實例。
    合成三萜類化合物是2-氰基-3,12-二氧代 - 油酸-1,9(11) - 二烯-28-酸鹽(CDDO; 巴多索隆,RTA401)的衍生物,其類似于天然產物齊墩果酸。它們通過其α-β不飽和支架表現出邁克爾受體活性,并且代表了最有效的NRF2誘導劑(Sun等,2017)。它們與KEAP1的C151相互作用并阻礙其與CUL3的相互作用,從而導致NRF2激活(Cleasby等,2014)。原理證明研究強烈支持合成的三萜類化合物用于退行性疾病,并且正在成為Reata / Abbott作為炎癥抗氧化劑調節劑的深入研究的焦點。例如,CDDO-咪唑(CDDO-Im RTA403)在野生型而非Nrf2-/-小鼠的腹膜中性粒細胞中誘導各種抗氧化基因(Hmox1,Gclc,Gclm和Nqo1)的表達,并減弱LPS誘導的ROS產生和產生和促炎細胞因子的產生,從而降低死亡率(Thimmulappa等,2006b)。CDDO-乙基酰胺(RTA405)和CDDO-CDDO-三氟乙基酰胺(RTA 404)在毒素誘導的PD模型(1-甲基-4-苯基-1,2,3,6-四氫吡啶)中測量的所有終點均具有顯著作用 (Kaidery等,2013)。在MS的EAE模型中,CDDOCDDO-三氟乙基酰胺抑制炎癥、病理性ROS形成和髓鞘變性(Pareek等,2011)。
    CDDO-甲酯(CDDO-Me,RTA 402)是在用于治療糖尿病腎病的臨床試驗中達到的第一個CDDO(Pergola等,2011)。雖然第二階段的結果非常令人鼓舞,但由于心血管安全問題(Zhang,2013),CDDO-Me后來在第三階段(BEACON試驗)被撤回,這與NRF2無關,但最有可能是脫靶改變內皮素信號傳導(de Zeeuw等,2013; Chin等,2014)。目前,CDDO-Me正在進行臨床研究,作為Alport綜合征和肺動脈高壓的潛在治療方法(表2)。為了改善其安全性,進一步的研究導致了CDDO-二氟丙酰胺(RTA-408, omaveloxone)的開發,該藥物目前正在進行二期試驗,用于治療弗里德雷希的共濟失調、眼部炎癥和眼部手術后疼痛。
    奧替普拉是一種有機硫化合物,用作抗血吸蟲病藥物,目前正在進行III期試驗,用于治療非酒精性脂肪性肝炎。用于治療亨廷頓氏病的高級臨床試驗正在開發中,米諾環素是一種抗生素,由于NRF2活化而具有神經保護作用(Kuang等,2009)。用于治療急性腎病的I期臨床研究中的另一種NRF2誘導劑是CXA-10,一種通過激活NRF2具有抗炎特性的硝基脂肪酸(Batthyany和Lopez,2015)。近年來,許多具有相同作用機制的NRF2誘導劑已被描述(Buendia等, 2015a,b, 2016),其中一些還處于臨床前研究階段,如化合物VEDA-1209,它是一種查爾酮衍生物,具有良好的抗炎作用,可用于潰瘍性結腸炎的治療。
    SFN是由有機硫化合物蘿卜硫苷的酶促裂解產生的異硫氰酸酯,其存在于西蘭花、卷心菜和其他十字花科植物的芽苗中。催化反應由植物中發現的黑芥子酶和胃腸道的微生物群驅動(Kensler等,2013)。催化反應由植物中發現的黑芥子酶和胃腸道的微生物群驅動(Kensler等,2013)。最近,SFN已經通過化學合成獲得(Kim等,2015)。通過向患有T2DM的患者施用含有SFN的花椰菜芽粉,實現了SFN向臨床的轉化(Bahadoran等,2012)。西蘭花粉降低血漿丙二醛和氧化低密度脂蛋白(LDL),提高總抗氧化能力。心血管危險因素如血清甘油三酯,氧化LDL/LDL比率和血漿致動脈粥樣硬化指數(甘油三酯對數/高密度脂蛋白比率)也降低。此外,促炎標志物如C-反應蛋白和IL-6減少。在最近的一項研究中,SFN作為濃縮西蘭花芽提取物使用,通過NRF2的核轉位抑制肝細胞葡萄糖的生成,并降低參與糖異生的關鍵酶的表達。此外,SFN降低了患有T2DM的肥胖患者的空腹血糖和糖化血紅蛋白(Axelsson等,2017)。SFN誘導的NRF2活化通過降低ROS負荷和抑制NF-κB和TGF-β1信號傳導途徑來保護腎細胞免于狼瘡性腎炎(Jiang等,2014a)。
    關于神經退行性疾病,已經表明SFN穿過血腦屏障并提供足夠的腦生物利用度來激活NRF2特征并減少LPS誘發的神經炎癥,這反映在促炎性標志物(誘導型一氧化氮合酶、IL-6、TNF-α)的減少中和海馬中的小神經膠質細胞增生(Innamorato等,2008)。SFN還保護多巴胺能神經元免受帕金森病毒1-甲基-4-苯基-1,2,3,6-四氫吡啶的影響,并減弱星形膠質細胞增生和小神經膠質細胞增生(Jazwa等,2011)。與這些發現一致,SFN降低了磷酸化tau水平并增加了Beclin-1和LC3-II,表明NRF2活化可能通過大腦中的自噬促進這種毒性蛋白的降解(Jo等,2014)。SFN處理的脊髓損傷大鼠炎癥因子水平明顯降低,挫傷體積減小,協調性改善(Wang等, 2012)。該藥還通過保留血腦屏障和減少病理性ROS形成和炎癥細胞數量來改善EAE(Li等,2013)。SFN迄今已用于至少32項針對慢性疾病的臨床研究,如癌癥、哮喘、慢性腎病、T2DM、囊性纖維化、自閉癥和精神分裂癥(Duran等,2016; Houghton等,2016)(表2)。
     
    表2. 選擇的NRF2誘導劑作為KEAP1的親電子修飾劑
    該引用對應于ClinicalTrials.gov中的代碼。
     
     
     
     
     
     

     
    總之,這些觀察結果為其他SFN衍生化合物的開發鋪平了道路,這些化合物表現出更好的藥代動力學特征。SFN是一種在親水性介質中穩定性較差的油性物質。其物理化學特征促使Evgen Pharma(Wilmslow,Cheshire,England)開發出一種環糊精復合物制劑Sulforadex,該制劑正在進行用于治療蛛網膜下腔出血的II期臨床試驗。SFN還與褪黑激素雜交以產生ITH12674,該化合物被設計為具有用于治療腦缺血的雙重藥物-前藥作用機制(Egea等人,2015)。

    姜黃素是姜黃中發現的主要類姜黃素,已被用于治療肥胖癥、代謝綜合征和前驅糖尿病。采用氣相色譜-電子沖擊質譜法對姜黃素對大鼠肝臟的影響進行了非靶向代謝組學研究。間斷攝入姜黃素可上調NRF2,并在抗肝損傷方面發揮抗氧化和抗炎作用(Qiu等,2016)??诜S素可有效降低血清甘油三酯、IL-1β、IL-4和血管內皮生長因子,以及增加血液中脂聯素水平。在T2DM患者中,姜黃素降低空腹血糖,糖化血紅蛋白,血清游離脂肪酸,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)。

    白藜蘆醇是一種保護植物免受真菌感染的多酚,存在于葡萄皮、紅葡萄酒、漿果和許多其他植物中。白藜蘆醇通過下調KEAP1表達和激活蛋白去乙?;竤irtuin-1來激活NRF2信號通路,從而發揮抗氧化作用(Ungvari等, 2010)。在健康受試者中,白藜蘆醇的飲食施用防止了血漿中膽固醇、內毒素、促氧化劑和炎性標記物(p47phox,KEAP1,IL-1β和TNF-α)的升高。這些事件與NRF2活性的升高相關,NRF2活性升高是由NRF2靶標NQO1和谷胱甘肽S-轉移酶的表達增強所確定的(Ghanim等,2011)。在T2DM患者中,治療4周后胰島素敏感性得到改善,這通過AKT增強胰島素信號傳導,病理性ROS形成減少和糖化血紅蛋白水平降低來確定(Brasnyo等,2011; Bhatt等,2012)??偟膩碚f,白藜蘆醇在動物模型和患者中可預防高血壓、高膽固醇血癥、動脈粥樣硬化、缺血性心臟病、糖尿病和代謝綜合征等主要心血管、炎癥、氧化和代謝并發癥(Xia等, 2017)。

    經常被忽視的問題是親電KEAP1抑制劑缺乏選擇性。親電試劑與細胞中不同的親核試劑發生反應,從而表現出非靶向和非預期的副作用。例如,CDDO-Im可與500多種不同的目標相互作用(Yore等,2011)。通常,幾種蛋白磷酸酶在其催化中心含有對氧化還原敏感的半胱氨酸,并且一些KEAP1抑制劑可以修飾和滅活這些磷酸酶,因此擾亂信號傳導網絡。這些磷酸酶之一是PTEN(Lee等,2002; Kitagishi和Matsuda,2013; Han等,2015)。PTEN的催化C124殘基可以通過與強親電試劑例如CDDO-Im(Pitha-Rowe等,2009)和叔丁基氫醌(Rojo等,2014b)形成加合物來修飾。然后,PI3K/AKT通路的激活增加涉及GSK-3的抑制和NRF2隨后的穩定(圖2C) (Rada等, 2011, 2012)。此外,KEAP1與其他同樣含有高親和力結合基序ETGE的蛋白相互作用(Hast等, 2013),如Bcl-2和IKKβ (Kim等, 2010a;Cazanave等,2014)。因此,從KEAP1缺陷細胞獲得的一些結果可能不一定與NRF2活化有關。

    B. 用于NRF2激活的蛋白質-蛋白質相互作用抑制劑

    為了克服選擇性的缺陷,出現了一類阻止NRF2與KEAP1對接的新型NRF2誘導劑(Richardson等,2015)。通過先前闡明KEAP1(Padmanabhan等人,2006)的X射線晶體結構與含有NRF2的高親和力結合ETGE基序的肽結合,已經實現了PPI抑制劑的使用(Lo等,2006)。KEAP1包含六葉β螺旋槳,具有特定的疏水和親水殘基,參與采用β-發夾結構的EGGE基序的對接。對接主要受KEAP1的幾個精氨酸與ETGE基序中的兩個谷氨酸之間的靜電相互作用的支持(Lo等人,2006; Padmanabhan等人,2006)。NRF2的低親和力DLG基序與KEAP1的對接也被表征(Tong等, 2007)?;谶@些相互作用,擬肽化合物是PPI抑制劑的第一個例子,其對親電試劑的選擇性顯著提高(Hu等,2013; Marcotte等,2013; Winkel等,2015)。這些抑制劑在細胞中表現出較弱的活性,現在已經報道了一種新的基于循環肽的激發策略。這些肽之一顯示出對KEAP1的高結合親和力和NRF2的激活,并在小鼠巨噬細胞中引發抗炎作用(Lu等,2018)。

    最近對KEAP1/NRF2相互作用的新多肽和小分子抑制劑的發現進行了綜述(Abed等, 2015;Jiang等, 2016)。簡而言之,最初使用表面等離振子共振和熒光偏振測定評估一系列截短的NRF2肽作為PPI的直接抑制劑(Hu等,2013)。抑制能力最小的肽序列為LDE-ETGE-FL的9-mer序列(Chen等, 2011;Inoyama等,2012)。與此同時,Wells和合作者(Hancock等,2013)使用噬菌體展示文庫結合高通量熒光偏振測定法搜索新推定的肽配體。他們發現,與單獨的天然肽相比,基于NRF2和SQSTM1的ETGE基序的雜合肽對KEAP1具有更高的結合活性。為了促進細胞攝取,設計一種肽,其中ETGE基序與HIV-Tat蛋白的細胞轉導結構域和鈣蛋白酶的切割序列(DEETGE-Cal-Tat)融合。該肽在腦缺血的小鼠模型中顯示出神經保護和認知保護作用(Tu等,2015)。

    已經描述了五個PPI抑制劑家族:四氫異喹啉(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匯總了最近針對這些小分子的專利。 盡管這些化合物非常有前途,但仍需要證明它們對KEAP1/NRF2相互作用具有選擇性,因為KEAP1也至少靶向Bcl2和IKK(Kim等,2010a; Hast等,2013; Cazanave等,2014)。
    從可用文庫中索引的大量化合物中,化合物LH601,苯磺?;奏ね?,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非常適合的候選者(Hu等,2013; Jnoff等,2014; Bertrand等,2015; Wen等,2015; Nasiri等,2016)。這些研究詳細描述了與KEAP1的原子相互作用、親和力和結合的熱力學參數。這些化合物的治療功效將在未來的工作中進行分析,其中應該解決安全性,效力和血腦屏障滲透性。
     
    表3. 選擇的NRF2誘導劑充當NRF2-KEAP1蛋白質 - 蛋白質相互作用抑制劑

     
     
     

    C. 用于NRF2激活的Keap1以外的藥物靶

    蛋白激酶GSK-3磷酸化NRF2的DSGIS序列中的兩個絲氨酸殘基以產生一個磷酸化依賴性降解基序或磷酸化二核(圖2)。該磷酸二核被E3連接酶適配子β-TrCP識別,導致NRF2的泛素依賴性蛋白酶體降解。因此,GSK-3抑制劑應通過阻止這種磷酸二核的產生來阻止NRF2降解。GSK-3是AD和其他病理表型中的重要激酶。它使細胞骨架蛋白tau磷酸化,促進神經原纖維纏結的形成,神經原纖維纏結是病理性細胞內聚集體,其干擾軸突運輸并導致神經元死亡(Silva等,2014)。因此,推測GSK-3抑制可能具有防止神經原纖維纏結形成和NRF2降解的雙重益處。不幸的是,大多數用于開發GSK-3抑制劑的管道由于無效而已經停止,盡管在大多數情況下沒有很好的證據表明靶標調節(Palomo和Martinez,2017)。

    從概念上講,β-TrCP-磷酸化NRF2相互作用的抑制劑也應該導致NRF2活化,因為它們應該破壞NRF2降解的這一分支(圖2)。β-TrCP的β-螺旋和含有NRF2磷酸二核的肽之間的分子相互作用已經通過NMR解決(Rada等,2012)。正如KEAP1/EGTE所發生的那樣,最相關的氨基酸似乎是β-TrCP的幾個精氨酸殘基,其與DpSGIpS基序的兩個磷酸絲氨酸相互作用。然而,能夠抑制β-TrCP-磷酸化NRF2相互作用的小分子的發現仍在進行中。
    已經開發了其他策略來抑制NRF2抑制因子BACH1,這是一種bZip蛋白,可與MAF蛋白形成異二聚體并阻斷ARE基因的表達。已經在體外描述了HPP-4382化合物對BACH1的有效抑制(Attucks等,2014),但是,在完整的臨床試驗之前,必須在體內證明HPP-4382的安全性和功效特征??紤]到其他途徑也可能影響NRF2的活性,我們有理由推測,組合方法將是激活該轉錄因子的最佳途徑。

    D. NRF2抑制劑

    NRF2在構成性和高度過表達時具有與其致癌活性相關的“陰暗面”。因此,已提出NRF2抑制作為使癌細胞對化學治療藥物或放射療法敏感的機制(Milkovic等,2017)??梢栽O想兩種策略用小分子抑制NRF2:破壞NRF2和MAFs之間的bZip相互作用的PPI抑制劑,以及阻斷NRF2-MAF與ARE結合的DNA-蛋白質相互作用抑制劑(圖9C)。這兩種策略都受到了阻礙,因為這類藥物需要克服蛋白質-蛋白質之間以及蛋白質-DNA界面(在較小程度上)之間的大量自由能。 然而,已發現其他bZip轉錄因子如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是一種促氧化合物,通過靶向NRF2克服了白血病抗性(Manna等,2015)??箟难?維生素C),一種著名的ROS清除劑,被發現通過降低NRF2/ARE復合物水平,降低GCLC基因的表達,降低GSH水平,來增強伊馬替尼耐藥癌細胞的敏感性(Tarumoto等,2004)。全反式維甲酸是NRF2抑制劑的另一個實例,其在體外和體內顯著降低有效的親電NRF2誘導物對NRF2的活化。它激活視黃酸受體α,其與NRF2形成復合物,因此阻礙轉錄因子與ARE基因的結合(Wang等,2007)。

    天然產物如鴉膽子苦醇(Ren等,2011; Olayanju等,2015),赭曲霉毒素A(Tarumoto等,2004; Limonciel和Jennings,2014)和葫蘆巴堿(Arlt等,2013)也有被發現抑制NRF2。然而,他們的作用機制還沒有完全明白。事實上,與現有化合物相關的一個重要問題是它們可能具有的深遠的脫靶效應。例如,最近發現鴉膽子苦醇對蛋白質合成具有普遍和非特異性抑制作用,導致NRF2水平下降,而且許多其他快速轉換蛋白下降,因此最近不鼓勵使用鴉膽子苦醇(Harder et al。,2017))。類似地,獸醫實踐中使用的抗原蟲劑鹵蟲酮通過抑制NRF2積累來增強癌細胞的化學敏感性,但這種效應似乎是間接的,它通過抑制核糖基轉移RNA的合成,這是NRF2以及許多其他含脯氨酸的蛋白質的核糖體轉化所強烈需要的(Tsuchida等,2017)。

    最近報道了一種識別選擇性NRF2抑制劑的新方法,即使用小分子抑制劑的高通量定量篩選(Singh等, 2016)。作者確定了一種名為ML385的一流化合物,它最有可能阻止NRF2與其他bZip共激活因子的結合。該化合物阻斷NRF2轉錄活性并使KEAP1缺陷細胞對卡鉑和其他化學治療藥物敏感。需要進一步的研究來確認ML385是否對NRF2具有選擇性,或者它是否也抑制其他bZip轉錄因子。

    鑒于NRF2在多種腫瘤病理表型中具有良好的全身效應,用小分子抑制劑特異性靶向NRF2似乎提供了一種良好的臨床途徑。然而,有必要確定用NRF2抑制劑治療癌癥是否會增加NRF2疾病組中的其他病理表型的風險。


    E. 藥物新用代替新藥物的發現與開發

    如前所述,許多化合物正在開發中,為與NRF2疾病組相關的病理表型提供益處。另一種方法是給已經在臨床中用于某種病理機制的藥物一個新的用途,用于治療與NRF2有關的其他病理機制。本節根據一些常用藥物在NRF2調控中的作用,為其重新定位提供依據。

    二甲雙胍是T2DM的一線單藥治療。根據圖6,它為與葡萄糖代謝相關的病理表型的NRF2亞群提供治療益處。事實上,SFN可降低肝臟葡萄糖的產生,并改善T2DM患者的血糖控制(Axelsson等,2017)。有趣的是,一些證據表明,二甲雙胍可能有效預防NRF2疾病組中的其他非糖尿病病理表型,包括心血管疾?。∟esti和Natali,2017),呼吸系統疾?。⊿ato等,2016),消化系統(Bauer和Duca,2016),神經退行性疾?。∕arkowicz-Piasecka等,2017),自身免疫(Schuiveling等,2017)和腫瘤(Heckman-Stoddard等,2017)疾病。二甲雙胍的作用機制尚不完全清楚,但它涉及抑制線粒體復合物I,從而增加AMP/ATP比值(El-Mir等,2000; Owen等,2000)并導致能量傳感器AMPK的激活(Hardie,2004; Rena等,2017)。重要的是,AMPK激活NRF2(Wang等,2017a; Zhao等,2017),并且該軸的藥理學靶向可減輕中風后的炎癥(Wang等,2017c)或內毒素暴露(Ci等,2017; Lv等,2017)。事實上,二甲雙胍以AMPK依賴的方式激活NRF2,從而抑制臨床前嚙齒動物短暫性全腦缺血模型的炎癥反應(Ashabi等, 2015;Kaisar等,2017)。葡萄糖代謝和炎癥可能不是二甲雙胍/NRF2作用的唯一病理機制。實際上,已經描述了氧化還原的其他有益效果(Kocer等,2014; Kelleni等,2015)和蛋白質穩態(Tsai等,2017)。

    他汀類藥物可預防和減少心血管病變表型。除了降脂作用外,他汀類藥物似乎可以預防與NRF2網絡相關的病理機制,如炎癥(Pantan等,2016; Wu等,2016a; Hwang等,2017)和病理性ROS形成( Abdanipour等,2014)。它們是3-羥基-3-甲基-戊二酰-CoA還原酶的競爭性抑制劑,其催化膽固醇合成中的限速反應。其他多效性影響包括轉錄因子Krüppel樣因子2的上調,其在肝硬化進展期間早期誘導,并減輕肝血管功能障礙的發展(Marrone等,2015)。最近的證據表明,至少一些他汀類藥物會激活NRF2。在分離的肝細胞中進行的一項研究中,高濃度的辛伐他汀激活了NRF2,可能作為一種防御機制(Cho等,2013)。用洛伐他汀預處理神經干細胞激活NRF2途徑并引發針對過氧化氫誘導的細胞死亡的保護(Abdanipour等,2014)。在肝硬化中,辛伐他汀激活由Krüppel樣因子2和NRF2形成的軸,以減少星狀細胞的氧化負荷和炎癥反應,改善肝纖維化,內皮功能障礙和門靜脈高壓。辛伐他汀激活NRF2的機制尚不完全清楚,但似乎涉及NRF2相互作用組中發現的元素,如絲裂原活化蛋白激酶、PI3K/AKT途徑(Jang等,2016)和GSK-3(Lin等,2016)。

    圖4的NRF2相互作用組可以推斷出藥物新用的其他病例,特別是信號激酶。如圖2C所示,GSK-3磷酸化NRF2的Neh6結構域,導致β-TrCP的識別和進一步泛素依賴性蛋白酶體降解。GSK-3在沒有刺激的情況下是活躍的,而在激活AKT和其他激酶的信號級聯導致GSK-3在其N端偽底物域磷酸化時是不活躍的。因此,已知靶向信號激酶的藥物可用于上調(GSK-3抑制劑)或下調(PI3K/AKT抑制劑)NRF2特征。

    GSK-3參與NRF2疾病組中發現的至少一些病理表型,例如糖尿病和神經退行性疾?。˙eurel等,2015; Maqbool和Hoda,2017)。從天然和合成來源發現了廣泛的GSK-3抑制劑(Khan等,2017),但將GSK-3抑制劑重新利用來增加NRF2活性的最佳證據可能源于臨床使用鋰作為情緒穩定劑(Chiu等,2013)。雖然此時在NRF2疾病組中未發現雙相情感障礙和抑郁,但很明顯它們表現出神經炎癥和退行性病變表型,至少在小鼠模型中暗示NRF2失調(Martin-de-Saavedra等,2013; Freitas等,2016; Yao等,2016)。

    NRF2相互作用組也為阻斷信號激酶從而激活GSK-3的癌癥藥物抑制NRF2提供了一個理由。例如,表皮生長因子受體抑制劑厄洛替尼導致NRF2抑制,參與非小細胞肺癌中的腫瘤細胞感覺(Xiaobo等,2016)。用于治療肝細胞癌的激酶級聯抑制劑索拉非尼也導致NRF2及其下游靶標金屬硫蛋白-1(Houessinon等,2016)和亞甲基四氫葉酸脫氫酶1的抑制(Lee等人,2017)。

    最后,在兩項相關研究中,迄今為止一直在尋找可能影響NRF2調節的再利用藥物。使用基于熒光相關光譜的篩選系統,1633種藥物中的兩種顯著增加HepG2細胞中的NRF2蛋白水平:葉綠酸和bonaphton(Yoshizaki等,2017)。在另一項研究中,分析了連接圖數據庫,其包含用1309種試劑處理的人細胞系的基因表達譜(Lamb等,2006; Iorio等,2010)(Zhang等,2017),通過激活NRF2以尋找有潛力的氧化還原調節劑(Xiong等,2014)。該研究發現阿司咪唑是一種有效的抗組胺藥,用于過敏性疾病,是一種新型的NRF2激活劑。
     

    VI. 生物標志物作為NRF2特征參與監測目標

    由于ROS的半衰期短,在毫秒或納秒的范圍內,對患者或群體研究中的氧化還原狀態的評估受到阻礙(Ghezzi等,2017b)。因此,病理性ROS形成的生物標志物基于測量ROS留下的痕跡,ROS通常是細胞分子的末端氧化產物,其中許多是非特異性的(Frijhoff等,2015)。相反,NRF2的激活和其靶基因的后續表達是對生物體對病理性ROS形成的總暴露的間接但可靠的估計。由于NRF2激活是對環境應激源的一種成熟的細胞反應,所以它被認為是暴露于異生素的生物標志物。在肺部,通過對多個轉錄研究的數據挖掘,并結合獨創性通路分析,報道了NRF2信號在健康吸煙者中上調,因此表明NRF2調控的抗氧化基因在預防煙草煙霧的毒性作用中發揮核心作用(Comandini等, 2010)。類似地,NRF2調控的一種酶NQO1在過量服用對乙酰氨基酚患者的肝組織中含量是正常水平的15倍(Aleksunes等, 2006)。疾病與營養之間的關聯通?;诓豢煽康淖晕覉蟾妫ˋrcher等,2015)。通過激活NRF2來測量對營養素響應的生物標志物,據稱具有有益效果,這可以提供一種可靠的方法來驗證營養研究。但是,這種可能性仍未得到探索。
    與NRF2轉錄相關的變化可用作監測藥物功效的生物標志物,所述藥物旨在通過黃嘌呤氧化酶和NADPH氧化酶抑制劑減少病理性ROS形成。同樣,可以通過定義全局蛋白和基因表達譜來檢測和監測環境化學品的暴露情況(Ghezzi等,2017a)。這種方法類似于第一階段藥物代謝酶的使用,其中細胞色素P450是各種異種生物通過Ah受體誘導的,可以作為海洋污染的指標(Cajaraville等, 2000)。12周內每日口服富馬酸酯與銀屑病患者皮膚中NRF2靶基因表達增加有關(Onderdijk等,2014)。同樣,在接受每日劑量CDDO-Me治療3周的癌癥患者外周血單核細胞中,NQO1的mRNA水平增加了5倍(Hong等, 2012)。

    使用NRF2的轉錄特征作為生物標記需要很好地了解ARE基因活化所涉及的機制,因為大多數NRF2靶標受另外的轉錄因子調節。因此,分析幾種ARE基因的表達很重要。例如,一項使用NRF2作為肺鱗癌治療反應預測因子的研究提出使用28個基因來定義NRF2激活譜(Cescon等, 2015)。
     

    VII. 結論

    系統醫學和網絡藥理學共同強調了NRF2在慢性疾病中起基礎性作用的一組病理表型。這些疾病具有共同的機制,包括氧化,炎癥和代謝改變。 本研究中提出的NRF2相互作用組,NRF2疾病組和NRF2藥物組仍處于早期發展階段,但它們代表了將NRF2構建為一種常見的治療和系統醫學方法的首次嘗試。 即將完善的現有數據庫和即將公布的臨床結果數據將進一步提高這一新方法的準確性,這一新方法是以藥理學和機制為基礎的藥物新用。本文為藥物發現激活或抑制NRF2的綜合策略提供了路線圖,并強調需要為新藥的開發或針對NRF2作為慢性病常見成分的藥物的重新利用的轉化而努力。
     

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