CN102885846B - 2-硫代-6-氮杂尿苷在制备抗hiv-1病毒的药物中的应用 - Google Patents
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Abstract
本发明涉及2-硫代-6-氮杂尿苷在制备抗HIV-1病毒的药物中的应用。所述的药物能够抑制HIV-1辅助蛋白Vpu降解宿主限制因子BST-2,提高细胞表面BST-2含量,从而抑制HIV-1病毒释放。
Description
技术领域
本发明属于医药生物领域,具体涉及2-硫代-6-氮杂尿苷在制备抗HIV-1病毒的药物中的应用。
背景技术
病毒的耐药性是目前艾滋病药物治疗中最亟需解决的问题。耐药是病毒的药物作用靶位蛋白变异的结果。因为耐药株经常对一组抗病毒药物耐药,并且同一类药物之间交叉耐药非常常见。HIV-1之所以能够迅速产生针对现有临床应用的抗病毒药物的耐药性,其主要根源在于这些药物所针对的靶点都是病毒本身。而病原病毒天然就具有高变异的特点,在药物的选择压力下,很容易引起病毒的迅速变异从而产生耐药。因此,解决HIV-1的耐药性问题的关键之一在于必须突破传统的“以病原病毒本身为靶”的药物发展模式。
在生物进化的漫长过程中,宿主细胞会形成针对不同病原病毒的防御体系,而病毒也会形成特异性的拮抗机制,以逃避宿主细胞的抑制作用。最近研究发现,宿主限制因子BST-2(也命名为Tetherin/HM1.24/CD317)可抑制包括HIV-1在内的多种包膜病毒的释放。BST-2属II型整合膜蛋白,整个蛋白为同源二聚体,异源糖基化,大小为30-36kDa。BST-2在多种细胞中均有表达,包括末端分化B细胞、骨髓基质细胞、激活的T细胞和树突状浆细胞、Hela细胞,并且干扰素诱导可显著增强表达水平。
BST-2将新生的成熟HIV-1病毒颗粒束缚在细胞膜表面,阻止其释放。束缚的病毒颗粒被内吞到细胞的CD63内体结构(CD63-positiveendosomal compartments)中降解。研究表明,BST-2不仅可以阻止游离病毒颗粒的释放,而且可以抑制HIV-1病毒颗粒的主要传播方式—细胞间扩散。
HIV-1通过其编码的辅助蛋白Vpu来拮抗BST-2的抑制作用。Vpu通过其跨膜区同BST-2跨膜区相互作用,下调膜表面BST-2,使BST-2远离其作用位点,并且通过溶酶体途径降解BST-2,从而提高病毒释放。如能阻止Vpu降解BST-2,提高细胞表面BST-2含量,则可以抑制病毒释放,从而达到抗病毒效果。
化合物2-硫代-6-氮杂尿苷(2-thio-6-azauridine,CAS号:27089-56-1)是一种核苷类似物,是乳清酸核苷单磷酸脱羧酶(orotidine monophosphate decarboxylase)的抑制剂。最先报道该化合物具有抗多种RNA病毒活性,如日本脑炎病毒(Japanese encephalitisvirus)、黄热病毒(yellow fever virus)、白蛉热病毒(sandfly fevervirus)、西尼罗病毒(West Nile virus)、副流感病毒(parainfluenzavirus)。也有文献报道其具有抗肿瘤的活性,对小鼠L1210白血病体内体外实验均有活性,其抗肿瘤机制是通过抑制嘧啶核苷酸的从头合成途径。该化合物细胞水平的毒性较小,半数抑制浓度(IC50)大于1000μg/ml。
现有技术中,未见化合物2-硫代-6-氮杂尿苷在制备抗HIV-1病毒的药物方面的报道。
发明内容
为了解决上述技术问题,本发明的目的是提供2-硫代-6-氮杂尿苷在制备抗HIV-1病毒的药物方面的新用途。
本发明提供2-硫代-6-氮杂尿苷在制备抗HIV-1病毒的药物中的应用。
优选地,所述的药物为HIV-1的辅助蛋白Vpu降解宿主限制因子BST-2的抑制剂。
优选地,所述的药物为提高细胞表面BST-2含量的药物。
更优选地,所述的药物为抑制HIV-1病毒释放的药物。
由于2-硫代-6-氮杂尿苷能够抵抗HIV-1病毒,因此能够将2-硫代-6-氮杂尿苷作为有效成分制备抗HIV-1病毒的药物。
本发明还公开2-硫代-6-氮杂尿苷作为HIV-1的辅助蛋白Vpu降解宿主限制因子BST-2的抑制剂的应用。本发明人经研究意外地发现,2-硫代-6-氮杂尿苷能抑制HIV-1的辅助蛋白Vpu对宿主限制因子BST-2的降解,因此2-硫代-6-氮杂尿苷能作为HIV-1的辅助蛋白Vpu降解宿主限制因子BST-2的抑制剂。
本发明还公开2-硫代-6-氮杂尿苷在抑制HIV-1的辅助蛋白Vpu降解宿主限制因子BST-2中的应用。2-硫代-6-氮杂尿苷能抑制HIV-1的辅助蛋白Vpu对宿主限制因子BST-2的降解。
本发明还公开2-硫代-6-氮杂尿苷在提高细胞表面BST-2含量中的应用。2-硫代-6-氮杂尿苷能提高细胞表面BST-2含量。
本发明还公开2-硫代-6-氮杂尿苷在抑制HIV-1病毒释放中的应用。2-硫代-6-氮杂尿苷能抑制HIV-1病毒释放。
本发明人发现了化合物2-硫代-6-氮杂尿苷的新活性。2-硫代-6-氮杂尿苷能够抑制HIV-1的辅助蛋白Vpu降解宿主限制因子BST-2,提高细胞表面宿主限制因子BST-2的含量,从而抑制HIV-1病毒释放,达到抗HIV-1病毒的效果。因此该化合物作为HIV-1的辅助蛋白Vpu降解宿主限制因子BST-2的抑制剂,能用于抗HIV-1病毒。
本发明突破了传统的“以病原病毒本身为靶”的药物发展模式,解决了HIV-1的耐药性问题。
附图说明
图1示出Cell-ELISA方法检测2-硫代-6-氮杂尿苷对细胞膜表面BST-2表达水平的影响;
图2示出流式细胞术检测2-硫代-6-氮杂尿苷对细胞膜表面BST-2表达水平的影响;
图3示出化合物2-硫代-6-氮杂尿苷对BST-2mRNA转录的影响;
图4示出化合物2-硫代-6-氮杂尿苷对Vpu表达水平的影响;
图5示出化合物2-硫代-6-氮杂尿苷对HIV病毒颗粒释放的影响;
图6示出化合物2-硫代-6-氮杂尿苷对病毒感染性的测试结果;
图7示出化合物2-硫代-6-氮杂尿苷对IFNα诱导的IFNAR1降解的影响;
图8示出化合物2-硫代-6-氮杂尿苷对于Vpu、BST-2和β-TrCP2三者复合物相互作用的影响;
图9示出化合物2-硫代-6-氮杂尿苷对于Vpu诱导的BST-2泛素化的影响;
图10示出化合物2-硫代-6-氮杂尿苷抗HIV-1活性的IC50。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1化合物2-硫代-6-氮杂尿苷抑制Vpu下调细胞表面的BST-2
稳定表达Vpu蛋白的Hela-Vpu细胞为岑山等(岑山等。筛选拮抗Vpu降解BST-2活性的抗病毒药物的方法。专利申请号:201010123002.1)构建筛选得到,其表面BST-2被Vpu下调,较正常Hela低。
将Hela细胞和Hela-Vpu细胞接种于96孔板中,每孔细胞为1.0×104个。在37℃、5%CO2孵箱中培养24h后,分别加入二甲基亚砜(DMSO)、2-硫代-6-氮杂尿苷处理,继续培养24h后,采用Cell-ELISA的方法检测细胞膜表面BST-2的表达水平。检测过程为:吸去旧培养基,PBS洗两次,每次2min。每孔加入100μl4%多聚甲醛,室温固定20min。弃去,加入150μl PBS洗三次,每次1min。每孔加入50μl2%BSA配制的BST-2抗体,浓度为1:5000,37℃培养1h。PBS洗四次,每次1-2min。每孔加入50μl2%BSA配制的HRP驴抗兔(donkey anti-rabbit)抗体,浓度为1:6000,室温培养40min。PBS洗四次,每次1-2min。每孔加入100μl TMB显色液(现配现用),避光反应30min。每孔加入50μl0.5MH2SO4或者1M HCl终止反应,立即于450nm处测定吸光值。结果如图1所示,5μM2-硫代-6-氮杂尿苷可以提高50%Hela-Vpu细胞表面BST-2水平,而对正常Hela细胞表面BST-2水平没有明显的提高。
本发明人采用流式细胞术检测方法,进一步确证化合物2-硫代-6-氮杂尿苷对Hela-Vpu细胞表面BST-2水平的影响。流式细胞术检测方法如下:将Hela-Vpu细胞接种于6孔板中,每孔细胞为2.0×105个。在37℃、5%CO2孵箱中培养24h后,加入二甲基亚砜、2-硫代-6-氮杂尿苷处理,继续培养24h后,将培养好的细胞消化,用含10%FBS的PBS冲洗下来,收集到EP管中,于4℃下300g离心5min,弃上清,加入1mL预冷含3%FBS的PBS清洗细胞,4℃300g离心5min,弃上清,共三次。每管加入100μl含2%BSA的PBS配制的BST-2抗体(1:800),4℃冰浴60min。于4℃下300g离心5min,弃上清,加入1mL预冷含3%FBS的PBS清洗细胞,4℃300g离心5min,弃上清,共三次。每管加入100μl含2%BSA的PBS配制的FITC标记羊抗兔(1:200),冰浴40min。后续操作需要避光。于4℃下300g离心5min,弃上清,加入1mL预冷含3%FBS的PBS清洗细胞,4℃300g离心5min,弃上清,共三次。1mL PBS重悬细胞,流式细胞仪检测。结果与Cell-ELISA检测结果相同,如图2所示。
实施例2化合物2-硫代-6-氮杂尿苷对BST-2mRNA的转录的影响
为了测定化合物2-硫代-6-氮杂尿苷对BST-2mRNA转录的影响,将Hela-Vpu细胞接种于6孔板中,每孔细胞为2.0×105个。37℃、5%CO2孵箱中培养24h后,加入二甲基亚砜、2-硫代-6-氮杂尿苷处理,继续培养24h后,根据Invitrogen公司的TRIZOL使用说明,提取细胞总RNA。测定浓度后,取2μg总RNA,使用M-MLV逆转录酶和随机引物将RNA逆转录为cDNA。
cDNA扩增反应体系:SsoFast EvaGreen Supermix10μl,正向引物(10μM)1μl,反向引物(10μM)1μl,模板cDNA0.5μl,无RNA酶水7.5μl。反应条件为95℃,30sec;然后是40次循环,每次循环条件为95℃,5sec;60℃,20sec。熔解曲线为65℃-95℃,连续。
以管家基因gapdh为内对照,用2-ΔΔC T法分析bst-2mRNA转录水平的变化,正向引物和反向引物的序列见表1。
表1引物序列
结果如图3所示,5μM2-硫代-6-氮杂尿苷不影响BST-2mRNA的转录,说明2-硫代-6-氮杂尿苷对BST-2表达水平的提高是作用在转录后。
实施例3化合物2-硫代-6-氮杂尿苷的Vpu表达水平的影响
将Hela-Vpu细胞接种于6孔板中,每孔细胞为2.0×105个。37℃、5%CO2孵箱中培养24h后,加入二甲基亚砜、刀豆素A、2-硫代-6-氮杂尿苷处理,继续培养24h。刮取6孔板中细胞,1ml预冷PBS重悬,2×103g离心5min收集细胞,去上清,加入50μl预冷PBS重悬,再加入50μl2×上样缓冲液裂解细胞,沸水浴10min,每隔3min涡旋振荡。SDS-PAGE分离蛋白样品。转PVDF膜后,先后与一抗和二抗孵育,然后ECL显色。一抗使用如下浓度的兔源多克隆BST-2抗体(1:5000),兔源多克隆Vpu抗体(1:1000),鼠源单克隆β-actin抗体(1:2000);辣根过氧化物酶标记的二抗使用如下浓度的山羊抗小鼠(1:2000),山羊抗兔(1:5000)。
结果如图4所示,用化合物2-硫代-6-氮杂尿苷处理过的Hela-Vpu细胞的BST-2的含量上升,但是Vpu的表达没有改变。刀豆素A(ConcanamycinA)是溶酶体降解途径抑制剂,在本实验中作为阳性对照化合物。刀豆素A能抑制Vpu降解BST-2,所以BST-2含量上升。
实施例4化合物2-硫代-6-氮杂尿苷抑制HIV-1的释放与细胞中的BST-2相关
将293T细胞接种于六孔板,每孔细胞数为3.0×105个。37℃、5%CO2孵箱中培养24h后,用env缺失的HIV-1全基因组质粒pNL-Luc-E-0.2μg和表达水泡性口炎病毒包膜的质粒pHCMV-G0.14μg共转染细胞,同时转染BST-2质粒和等量空载体作为对照,并且加二甲基亚砜和2-硫代-6-氮杂尿苷处理24h后收集上清中的病毒,0.22μm滤膜过滤。根据使用说明,采用p24抗原测定ELISA试剂盒(HIV-1Ag购自Biomerieux公司)测出上清释放的病毒产量。
病毒HIV释放:结果如图5所示,在有BST-2存在的情况下2-硫代-6-氮杂尿苷能够明显抑制HIV的释放。在同时加化合物2-硫代-6-氮杂尿苷处理后,病毒的p24量,对照组(空载体)是实验组(BST-2)的四倍。
释放病毒感染性测定:96孔板每孔接种1×105SupT1细胞,取上述方法收到的10μl上清病毒液感染SupT1,在37℃、5%CO2孵箱中培养培养48h后,裂解细胞,采用荧光素酶活性测定试剂盒LuciferaseAssay System(Promega)测定裂解液的荧光素酶活性。
结果如图6所示,在有BST-2存在的情况下2-硫代-6-氮杂尿苷可以明显抑制HIV的释放。在同时加化合物2-硫代-6-氮杂尿苷处理后,病毒的感染性对照组(空载体)是实验组(BST-2)的四倍。
实施例5化合物2-硫代-6-氮杂尿苷对IFNα诱导的IFNAR1降解的影响
将Hela细胞接种在六孔板上,每孔细胞数为2.0×105个。24小时后,在Hela细胞中转染带有flag标签的IFNR1质粒,转染40h后,用IFN-处理来诱导IFNAR1降解,同时用放线菌酮处理来阻断IFNAR1的新合成,再加入二甲基亚砜和2-硫代-6-氮杂尿苷处理细胞。最后收取细胞,用Western Blot分析细胞内IFNAR1的含量。结果如图7所示,2-硫代-6-氮杂尿苷对于IFNα诱导的IFNAR1降解没有抑制作用,因为IFNα诱导的IFNAR1降解与Vpu诱导的BST-2的降解过程类似,所以初步证明2-硫代-6-氮杂尿苷特异的针对Vpu降解BST-2。
实施例6化合物2-硫代-6-氮杂尿苷对于Vpu、BST-2和β-TrCP2三者复合物相互作用的影响
将293T细胞接种于六孔板,每孔细胞数为3.0×105个。37℃、5%CO2孵箱中培养24h后,在293T细胞中共转染表达N端融合蛋白Rluc的BST-2和C端融合EYFP的Vpu。转染24h后,用2-硫代-6-氮杂尿苷处理293T细胞24小时,测定BRET比值。当BST-2与Vpu相互作用时,融合的Rluc和EYFP相互靠近,Rluc催化底物腔肠素产生的光波可以发生能量转移,激发受体EYFP,产生激发光。如果化合物影响BST-2和Vpu的相互作用,BRET比值应该随之而改变,如果增强两者相互作用,BRET比值应该升高,如果抑制两者相互作用BRET比值应相应降低。结果如图8所示,2-硫代-6-氮杂尿苷对BST-2和Vpu的相互作用增强。随后,Co-IP实验结果也证实了2-硫代-6-氮杂尿苷增强了Vpu和BST-2的相互作用。
实施例7化合物2-硫代-6-氮杂尿苷对于Vpu诱导的BST-2泛素化的影响
Vpu通过与BST-2相互作用来介导BST-2泛素化,从而下调BST-2。我们进一步研究了化合物2-硫代-6-氮杂尿苷对Vpu介导的BST-2泛素化的影响。结果如图9所示,Vpu的存在确实增强了BST-2的泛素化,而IMB-AZ处理后,泛素化BST-2的量明显减少,说明2-硫代-6-氮杂尿苷引起的BST-2和Vpu相互作用的增强并不对应于BST-2泛素化的增强。
实施例8化合物2-硫代-6-氮杂尿苷抗HIV-1活性的IC50测定
96孔板每孔接种1×105SupT1细胞,取一定量的清病毒液感染SupT1,并且加不同浓度的化合物2-硫代-6-氮杂尿苷处理细胞。37℃、5%CO2孵箱中培养培养48h后,裂解细胞,采用荧光素酶活性测定试剂盒Luciferase Assay System(Promega)测定裂解液的荧光素酶活性。如图10所示,结果表明该化合物的IC50为1.5μM。
Claims (4)
1.2-硫代-6-氮杂尿苷在制备抗HIV-1病毒的药物中的应用。
2.根据权利要求1所述的应用,其中所述药物为HIV-1的辅助蛋白Vpu降解宿主限制因子BST-2的抑制剂。
3.根据权利要求1所述的应用,其中所述药物为提高细胞表面BST-2含量的药物。
4.根据权利要求1所述的应用,其中所述药物为抑制HIV-1病毒释放的药物。
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