CN117618429A - 去泛素化酶usp8抑制剂在制备治疗乙型脑炎药物中的应用 - Google Patents
去泛素化酶usp8抑制剂在制备治疗乙型脑炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及医药技术领域,具体是去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用。本发明以人神经母细胞瘤细胞(SH‑SY5Y)作为靶细胞,利用泛素化化合物库中的抑制剂靶向泛素化通路中的关键酶进行抑制,来寻找与乙型脑炎病毒(JEV)感染SH‑SY5Y细胞相关的宿主因子,有助于认识JEV侵入中枢神经系统引起神经元细胞损伤的机制,同时也为JEV引起的中枢神经系统感染和致病的治疗药物提供新的作用靶点。本发明通过实验发现去泛素化酶USP8抑制剂具有抑制JEV感染SH‑SY5Y细胞的特性。本发明提供了去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用,为JEV的预防和治疗提供了新的靶点和治疗方案。
Description
技术领域
本发明涉及医药技术领域,具体地说,是去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用。
背景技术
乙型脑炎(Japanese encephalitis,JE)是由蚊子传播的乙型脑炎病毒(Japaneseencephalitis virus,JEV)引起的,JEV属于黄病毒科,黄病毒属,为单股正链RNA病毒。JEV具有神经致病性,可致以脑实质炎症为主要病变的乙型脑炎,简称乙脑。重症乙脑患者主要表现为高热、头痛、昏迷等症状,出现疾病症状者的病死率可高达30%,约30%-50%的重症存活者伴有瘫痪、智力障碍等神经系统后遗症(Ashraf U,Ding Z,Deng S,etal.Pathogenicity and virulence of Japanese encephalitis virus:Neuroinflammation and neuronal cell damage.Virulence.2021;12(1):968-980.)。乙型脑炎主要流行于东亚、东南亚以及大洋洲的部分地区,目前主要的控制手段为疫苗预防以及蚊虫的消除。虽然疫苗的使用大大降低了乙型脑炎的发病率,但乙型脑炎的发病率仍在增加。近年来有关抗乙脑药物的研发是国内外医学及生物学研究领域的热点,经过科研人员的不懈努力,已经发现多种具有抗乙脑活性的药物。按照药物性质大体可分为两类:一是人工合成的抗病毒药,其中包括核酸靶向治疗剂、核酸类似物、细胞因子类、黄酮类化合物、抗生素类等;二是天然药物中获得的抗病毒药,其中包括酚类化合物、牛蒡子甙元以及一些中药成分的提取物等。但是这些药物作用效果评估主要停留在动物模型、细胞和分子水平上,能否投入临床应用还有待深入研究。
泛素特异性蛋白酶8(Ubiquitin-specific protease 8,USP8)是去泛素化酶(deubiquitinating enzyme,DUB)超家族中的一类半胱氨酸酶,属于泛素特异性蛋白酶USP家族成员,大小约130kDa。DUB家族蛋白是一种专门水解泛素与目标蛋白之间或泛素分子之间异肽键的蛋白酶,因此USP8也被称为泛素异肽酶Y(ubiquitin isopeptidase Y,UBPY)。USP8与许多其他DUB一样,其特征在于自身结构的多结构域。除催化结构域外,还存在特定的蛋白质-蛋白质相互作用结构域,这些结构域有助于USP8底物的募集,调控和靶向不同的蛋白质复合物(Dufner A,Knobeloch KP.Ubiquitin-specific protease 8(USP8/UBPy):aprototypic multidomain deubiquitinating enzyme with pleiotropicfunctions.Biochem Soc Trans.2019;47(6):1867-1879.)。研究表明,USP8在生命体中发挥多种作用,而USP8的功能异常与多种疾病包括癌症,神经退行性疾病等密切相关。USP8还可以直接调节表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)的降解。近些年研究也发现USP8参与了多种病毒的感染与致病过程。如寨卡病毒(Zika virus)可以借助USP8的功能来感染细胞(Zheng Y,Liu Q,Wu Y,et al.Zika virus elicitsinflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1axis.EMBO J.2018;37(18):e99347.)。
(E)-9-(乙氧基亚氨基)-9H-茚并[1,2-b]吡嗪-2,3-二甲腈((E)-9-(Ethoxyimino)-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile)又名去泛素化酶抑制剂2(deubiquitinase inhibitor 2,DUB-IN-2),是一种茚并[1,2-b]吡嗪类化合物(化学结构式如式I所示),能够靶向抑制USP8的活性。USP8在肿瘤细胞中常呈过度活化,激活的USP8参与调控下游信号转导途径,与肿瘤细胞的恶性行为密切相关。研究表明垂体促肾上腺皮质激素腺瘤中USP8的突变率为35%-60%。DUB-IN-2已被证明在小鼠垂体瘤细胞(AtT-20)中发挥作用(Kageyama K,Asari Y,Sugimoto Y,et al.Ubiquitin-specific protease8inhibitor suppresses adrenocorticotropic hormone production and corticotrophtumor cell proliferation.Endocr J.2020;67(2):177-184.)。熊等报道,通过使用DUB-IN-2可以重塑发炎的肿瘤微环境(TME),显著增强了抗PD-1/PD-L1免疫疗法的功效(XiongW,Gao X,Zhang T,et al.USP8 inhibition reshapes an inflamed tumormicroenvironment that potentiates the immunotherapy.Nat Commun.2022;13(1):1700.)。
但是关于USP8抑制剂在乙型脑炎中的治疗作用目前还未见报道。
发明内容
本发明的目的在于提供去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用。
本发明的第一方面,提供去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用。
进一步的,所述的去泛素化酶USP8抑制剂为(E)-9-(乙氧基亚氨基)-9H-茚并[1,2-b]吡嗪-2,3-二甲腈((E)-9-(Ethoxyimino)-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile)又名去泛素化酶抑制剂2(deubiquitinase inhibitor 2,DUB-IN-2),是一种茚并[1,2-b]吡嗪类化合物(结构式如式I所示),能够靶向抑制USP8的活性。
进一步的,所述的去泛素化酶USP8抑制剂在制备抑制乙型脑炎病毒感染和细胞毒性的药物中的应用。
本发明的第二方面,提供一种治疗乙型脑炎的药物,其包含:
(A)有效量的去泛素化酶USP8抑制剂;以及
(B)药学上或免疫学上可接受的载体或赋形剂。
进一步的,所述的去泛素化酶USP8抑制剂为去泛素化酶抑制剂2(deubiquitinaseinhibitor 2,DUB-IN-2)。
本发明优点在于:
1、本发明以人神经母细胞瘤细胞(SH-SY5Y)作为靶细胞,利用泛素化化合物库中的抑制剂靶向泛素化通路中的关键酶进行抑制,来寻找与乙型脑炎病毒(JEV)感染SH-SY5Y细胞相关的宿主因子,有助于认识JEV侵入中枢神经系统引起神经元细胞损伤的机制,同时,也为JEV引起的中枢神经系统感染和致病的治疗药物提供新的作用靶点。本发明通过实验发现去泛素化酶USP8抑制剂同时具有抑制乙型脑炎病毒感染SH-SY5Y细胞的特性。
2、本发明提供了去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用,为乙型脑炎病毒的预防和治疗提供了新的药物,具有较好的市场价值和临床应用前景。
附图说明
图1中A为利用JEV体外细胞培养系统,通过对泛素化化合物库进行筛选的部分免疫荧光检测结果图,B为相应的病毒感染的抑制率统计图;
Control:不添加任何抑制剂的JEV感染SH-SY5Y细胞组(空白对照组);
Chloroquine:添加浓度为100μM氯喹的JEV感染SH-SY5Y细胞组(阳性药对照组);
Inhibitor:添加抑制剂浓度为5μM靶向泛素-蛋白酶体系统中不同关键酶的JEV感染SH-SY5Y细胞组(实验组)。
图2为USP8抑制剂DUB-IN-2对病毒感染的抑制作用图,其中A为使用不同浓度的DUB-IN-2作用于靶细胞后抑制JEV感染及细胞毒性检测的示意图,图中主纵坐标轴表示JEV病毒量,次纵坐标轴表示对细胞毒性的影响,B为不同浓度的DUB-IN-2作用于靶细胞后对病毒感染性影响的免疫荧光检测图。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1
一、实验材料
泛素化化合物库-Ubiquitination Compound Library(Cat.No.:HY-L050),购自MCE公司。
DUB-IN-2为泛素特异性蛋白酶8抑制剂(Cat.No.:HY-50737A),购自MCE公司。
人神经母细胞瘤系SH-SY5Y,购自ATCC,保藏号:ATCC CRL-2266。
二、实验方法
1.siRNA干扰
1.1RNA转染
转染步骤参照Lipofectamine 2000说明书
1)提前12-16h将SH-SY5Y细胞(购自ATCC,保藏号:ATCC CRL-2266)铺在24孔细胞培养板上培养,使得转染时细胞密度为80%-90%。
2)取2μL Lipofectamine 2000加入50μL opti-MEM中并轻柔混匀,室温孵育5min;另取5μL浓度为5μM的干扰RNA和50μL opti-MEM混合。孵育结束后,将稀释的Lipofectamine2000转染试剂加入稀释的RNA中,并轻柔吹吸混匀。室温孵育20min后,加入SH-SY5Y细胞中,补加400μL opti-MEM,使得RNA终浓度为50nM。
3)转染后6-8h更换含有双抗的新鲜培养基。
2.实时荧光定量PCR(RT-PCR)检测JEV病毒量
1)TRIzol提取对照组与处理组细胞的总RNA,具体步骤如下:
靶细胞处理后去培养上清,在细胞中加入1ml TRIzol,充分混合室温裂解细胞3-5min。加入1/5体积的氯仿,手动剧烈混合15s。于4℃、12,000r/min离心15min。取上层水相并转移到新的EP管中,加入等体积异丙醇,充分混合,室温沉淀10min。于4℃、12,000r/min离心10min。弃上清,加入1ml预冷的75%乙醇。于4℃、12,000r/min离心5min。充分弃上清,室温晾干RNA沉淀,加入DEPC处理水溶解沉淀,得到总RNA。
2)利用takara反转录试剂盒获取对照组与干扰组细胞的cDNA,具体步骤如下:
在PCR管中加入如下反应体系,
轻柔混合混匀,置于37℃反应15min,然后置于85℃加热5s灭活逆转录酶。
3)荧光定量RT-PCR检测
利用takara的SYBR Premix Ex Taq试剂盒进行反应,反应体系如下,
利用Rotor Gene 3000A仪器进行两步法扩增,95℃预变性2min,进行40个PCR循环,95℃5s,60℃30s。
3.JEV病毒感染SH-SY5Y细胞
3.1SH-SY5Y细胞的JEV病毒感染实验
SH-SY5Y细胞转染RNA后48h,进行JEV病毒感染实验。将培养上清吸出,用预温PBS润洗2次,以MOI=0.5的病毒量接种JEV,37℃孵育2h后弃去病毒液,并用预温PBS润洗3次,加入新鲜培养基继续培养。
3.2免疫荧光染色检测JEV抗原表达
SH-SY5Y细胞感染病毒后继续培养48h,采用免疫荧光法检测病毒抗原的表达,具体步骤如下:
1)细胞固定:将96孔板中的培养液移去,加入PBS清洗细胞2次,每孔加入100μl预冷甲醇,于-20℃条件下固定20min,用预冷的PBS清洗细胞3次。
2)透膜:固定后的细胞每孔加入100μl 0.1% TritonX-100,室温孵育15min,用预冷PBS洗涤3次。
3)封闭:每孔加入100μl 3% BSA,于室温下孵育1h。
4)一抗孵育:每孔加入JEV特异性兔源单抗GTX125868(1:1000稀释)100μl,室温孵育1h,用预冷的PBS洗涤3次。
5)二抗孵育:每孔加入AF 488荧光标记抗兔IgG(1:1000稀释)100μl,室温避光孵育1h,用预冷的PBS避光洗涤2次。
6)标记细胞核:每孔加入细胞核荧光染料DAPI(1:10000,PBS稀释),室温避光孵育15min,用预冷的PBS避光洗涤3次。
7)荧光显微镜下检测并计算绿色AF 488阳性细胞克隆数。
4.蛋白免疫印迹
1)用蛋白裂解液分别提取不同处理组SH-SY5Y细胞的总蛋白。
2)蛋白质定量后分别将30ug蛋白加到12.5%浓度的聚丙烯酰胺凝胶中电泳,并截取相应条带用电转仪转到PVDF膜上。
3)蛋白的非特异性位点用5%的脱脂牛奶封闭,然后用USP8或EGFR抗体封闭,4℃过夜,用TBST缓冲液洗三遍,洗去一抗。
4)然后用HRP标记的二抗室温孵育2h,继而用TBST缓冲液洗三遍。
5)最后,利用显色液显色并拍照分析。
5.对泛素化化合物库进行筛选
1)分组:实验分为空白对照组、阳性药对照组和实验组,每一组每个药物均设置3个复孔,实验独立重复三次。
Control:不添加任何抑制剂的JEV感染SH-SY5Y细胞组(空白对照组);
Chloroquine:添加浓度为100μM氯喹的JEV感染SH-SY5Y细胞组(阳性药对照组);
Inhibitor:添加抑制剂浓度为5μM靶向泛素-蛋白酶体系统中不同关键酶的JEV感染SH-SY5Y细胞组(实验组)
2)提前12-16h将SH-SY5Y细胞铺在96孔细胞培养板上培养,使得处理时细胞密度为80%-90%。吸出培养上清,用37℃预温的PBS润洗2次,每孔分别加入不同抑制剂100μl,于37℃孵育6h,阳性对照组加入100μl浓度为100μM氯喹的全培液,空白对照组加入等量全培液。
弃去抑制剂溶液,每孔加入等量的JEV(MOI=0.5),37℃孵育48h后弃去病毒液,PBS润洗3次。免疫荧光检测方法同步骤3.2。
6.抑制剂(DUB-IN-2)作用
1)分组:实验分为空白对照组、阳性药对照组和抑制剂干扰组,每一组每个药物浓度均设置3个复孔,实验独立重复三次。
2)提前12-16h将SH-SY5Y细胞铺在24孔细胞培养板上培养,使得处理时细胞密度为80%-90%。吸出培养上清,用37℃预温的PBS润洗2次,每孔分别加入不同浓度梯度的药物500μl,于37℃孵育6h,阳性对照组加入浓度为100uM氯喹的全培液,空白对照组加入等量全培液。
弃去化学药物溶液,每孔加入等量的JEV(MOI=0.5),37℃孵育2h后弃去病毒液,PBS润洗3次,加入蛋白酶K溶液(1mg/ml)去除结合于靶细胞表面的病毒颗粒。JEV感染靶细胞后续培养和检测方法同步骤3.2。
7.细胞毒性实验
采用CCK-8方法检测使用DUB-IN-2后对细胞增殖的影响,具体步骤如下:
收集对数生长期细胞,以每孔3000个的密度接种于96孔板。待细胞过夜贴壁后,添加DUB-IN-2,培养48h后检测细胞增殖情况。弃去原有培养基,每孔加入含10μL CCK-8的新鲜培养基110μL,培养3h后用多功能酶标仪在450nm波长检测各孔吸光度值。实验独立重复3次,计算平均值。
三、实验结果
利用JEV体外细胞培养系统,通过对泛素化化合物库进行筛选发现,去泛素化酶USP8抑制剂DUB-IN-2具有较好的抗JEV活性,其能够抑制JEV感染SH-SY5Y细胞(图1A)。同时我们设置了空白对照组(未添加药物,Control)、阳性药对照组(100μM Chloroquine)和抑制剂处理组(5μM),在药物处理6h后感染JEV48 h,然后通过间接免疫荧光法检测病毒抗原,统计感染率并均标准化到Control对照,以对JEV的相对抑制率>50%(即感染率<50%,图中虚线为感染率=50%)且抑制效果优于Chloroquine作为候选药物纳入标准。其中去泛素化酶USP8抑制剂DUB-IN-2处理后对JEV有较为明显的抑制效果(图1B)。
进一步,采用不同浓度梯度的去泛素化酶USP8抑制剂DUB-IN-2作用后,检测对病毒感染的影响。结果发现随着抑制剂浓度的提高,JEV的病毒量逐渐降低,而不同浓度抑制剂对细胞活性并没有明显的影响(图2A),间接免疫荧光法检测病毒抗原得到相一致的结果(图2B),这些结果表明去泛素化酶USP8抑制剂DUB-IN-2能够抑制JEV感染且抗病毒能力与药物浓度成正相关性。
为明确去泛素化酶USP8抑制剂DUB-IN-2的抑制靶点USP8对JEV感染的影响,在转染USP8分子siRNA后,通过免疫印迹法检测USP8蛋白分子的表达。结果显示,转染USP8分子siRNA后,能够明显抑制USP8蛋白分子的表达。免疫荧光法检测病毒抗原表明USP8下调后,明显降低了JEV对SH-SY5Y细胞的感染。这些结果表明,与对照细胞相比,USP8下调后JEV对SH-SY5Y细胞的感染能力明显下降,病毒量减少。因此,去泛素化酶USP8可作为抑制JEV对SH-SY5Y细胞感染的新的药物靶点。
通过以上实验结果证明:本发明发现去泛素化酶USP8抑制剂可明显抑制JEV在SH-SY5Y细胞中的感染,进一步发现抑制去泛素化酶USP8蛋白分子的表达可以降低JEV的感染,起到抗病毒的作用。本发明为JEV引起的乙型脑炎的预防和治疗提供了新的治疗药物和作用靶点,具有较好的市场价值和临床应用前景。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (5)
1.去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用。
2.根据权利要求1所述的去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用,其特征在于,所述的去泛素化酶USP8抑制剂为(E)-9-(乙氧基亚氨基)-9H-茚并[1,2-b]吡嗪-2,3-二甲腈,结构式如式I所示:
3.根据权利要求1所述的去泛素化酶USP8抑制剂在制备治疗乙型脑炎药物中的应用,其特征在于,所述的去泛素化酶USP8抑制剂在制备抑制乙型脑炎病毒感染和细胞毒性的药物中的应用。
4.一种治疗乙型脑炎的药物,其特征在于,包含:
(A)有效量的去泛素化酶USP8抑制剂;以及
(B)药学上或免疫学上可接受的载体或赋形剂。
5.根据权利要求4所述的治疗乙型脑炎的药物,其特征在于,所述的去泛素化酶USP8抑制剂为(E)-9-(乙氧基亚氨基)-9H-茚并[1,2-b]吡嗪-2,3-二甲腈,结构式如式I所示:
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