CN114939166A - 钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途 - Google Patents
钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途 Download PDFInfo
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Abstract
本发明属于药物制备技术领域，涉及钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途。本发明中首次发现钙离子通道抑制剂具有高效的抗汉坦病毒活性，可以高效抑制汉坦病毒感染细胞，降低病毒载量，为开发抗汉坦病毒新药提供依据和可能，为保障人类健康提供一种新的安全可靠的技术手段。
Description
技术领域
本发明属于药物制备技术领域，涉及钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途。
背景技术
汉坦病毒(Hantaviruses,HTV)属于布尼亚病毒科，为单股负链RNA病毒，基因组分三个节段。目前发现汉坦病毒有二十余种血清/基因型，主要包括汉滩病毒(Hantaanvirus,HTNV)、汉城病毒(Seoul Virus，SEOV)、普马拉病毒(Puumah Virus,PUUV)、多布拉伐-贝尔格莱德病毒(Dobrava-Belgrade virus，DOBV)、辛诺柏病毒(Sin Nombre Virus,SNV)、安第斯病毒(Andes virus，ANDV)等病毒。汉坦病毒广泛分布于世界各地，可引起严重程度不等的人类疾病，是重要的人兽共患病病原体。汉坦病毒在自然界主要保持在特定的自然宿主中，并不引起明显症状。但感染人类后，主要引起两种急性发热性疾病，分别是肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)和汉坦病毒心肺综合征(hantavirus cardiopulmonary syndrome,HCPS)。汉坦病毒感染后病死率高，是全球关注的公共卫生问题。
汉坦病毒感染尚无特效治疗药物，发病早期可采用利巴韦林进行抗病毒治疗。在HFRS病程早期应用利巴韦林后，可以提高HFRS患者的存活率，但在汉坦病毒肺综合征患者中使用利巴韦林并不能降低病死率。因此，需要不断寻找新的抗病毒药物。
发明内容
本发明所要解决的技术问题在于针对现有技术的不足，提供了钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途，所述钙离子通道抑制剂具有高效的抗病毒活性，可以高效抑制病毒感染细胞，降低病毒载量。
本发明提供了钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途。
进一步地，所述钙离子通道抑制剂为盐酸贝尼地平、西尼地平、非洛地平、马尼地平、盐酸尼卡地平和尼索地平中的任意一种。
进一步地，所述钙离子通道抑制剂能够用于制备抑制RNA水平病毒复制的药物。
进一步地，所述钙离子通道抑制剂能够用于制备抑制病毒NP蛋白表达的药物。
进一步地，所述钙离子通道抑制剂能够用于制备抑制靶病毒S基因表达的药物。
进一步地，所述汉坦病毒为汉滩病毒、汉城病毒、普玛拉病毒、多布拉伐-贝尔格莱德病毒、安第斯病毒和辛诺柏病毒中的任意一种。
与现有技术相比，本发明的有益效果在于：
1、本发明提供了盐酸贝尼地平及其他钙离子通道抑制剂(西尼地平、非洛地平、马尼地平、盐酸尼卡地平和尼索地平)在制备抗汉坦病毒感染药物中的用途，本发明首次发现包括盐酸贝尼地平的钙离子通道抑制剂具有高效的抗病毒活性，可以高效抑制病毒感染细胞，降低病毒载量。本发明为开发抗病毒新药提供依据和可能，为保障人类健康提供一种新的安全可靠的技术手段。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明中盐酸贝尼地平在RNA水平对病毒复制的抑制作用结果图；
其中，图A表示Vero E6细胞中盐酸贝尼地平在RNA水平对病毒复制的抑制作用；
图B表示A549细胞中盐酸贝尼地平在RNA水平对病毒复制的抑制作用；
图C表示Huh7细胞中盐酸贝尼地平在RNA水平对病毒复制的抑制作用；
图2为本发明中盐酸贝尼地平在蛋白水平对NP蛋白的抑制作用结果图；
其中，图A表示Huh7细胞中盐酸贝尼地平在蛋白水平对NP蛋白的抑制作用；
图B表示A549细胞中盐酸贝尼地平在蛋白水平对NP蛋白的抑制作用；图3为本发明中盐酸贝尼地平在荧光水平对病毒NP蛋白的抑制作用结果图；
图4为本发明中不同浓度盐酸贝尼地平对细胞活力的影响结果图；
其中，图A表示不同浓度盐酸贝尼地平对Vero E6细胞活力的影响；
图B表示不同浓度盐酸贝尼地平对A549细胞活力的影响；
图5为本发明中盐酸贝尼地平对汉滩病毒(HTNV)、汉城病毒(SEOV)、普马拉病毒(PUUV)、多布拉伐-贝尔格莱德病毒(DOBV)、辛诺柏病毒(SNV)、安第斯病毒(ANDV)等病毒抑制感染结果图。
图6为本发明中钙离子通道抑制剂的西尼地平(Cilnidipine)、非洛地平(Felodipine)、马尼地平(Manidipine)、盐酸尼卡地平(Nicardipine HCl)和尼索地平(Nisoldipine)对汉滩病毒(HTNV)抑制感染结果图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
本实施例提供盐酸贝尼地平在制备抗汉坦病毒感染药物中的用途。
本发明基于体外细胞水平筛选，利用多种实验技术如Quantitative Real-timePCR(qPCR)、Western blot(WB)、Immunofluorescence(IF)等，发现盐酸贝尼地平在体外可高效抑制病毒感染细胞，降低病毒载量；本发明利用CCK8实验检测了盐酸贝尼地平(Benidipine HCl)对细胞活力的影响，发现盐酸贝尼地平的使用浓度对细胞活力几乎没有影响，表明使用浓度在安全范围内。
一、盐酸贝尼地平抗病毒活性评价
1、盐酸贝尼地平在mRNA水平抗病毒活性评价
(1)细胞接种及药物处理
将人肝癌细胞(Huh7)、肺癌细胞(A549)、非洲绿猴肾细胞(Vero E6)以1×105个细胞接种到12孔板中，第二天将盐酸贝尼地平用含2％FBS的DMEM培养基稀释至终浓度为10μM，每孔加入1mL含有盐酸贝尼地平的DMEM培养基，药物处理1h后，弃掉培养基，用DPBS洗三遍，加入HTNV感染(Moi＝0.1)2h，每15min摇晃一次。2h后，弃掉病毒液，用DPBS洗三遍，加入含有2％FBS的DMEM培养基。
(2)RNA提取
RNA提取说明书提取RNA，所得RNA后冻于-80℃冰箱保存。
(3)qPCR检测靶基因S基因mRNA的表达
利用反转录试剂盒对RNA进行反转录，反转录产物冻于-20℃，具体过程如下：
a：gDNA消化
在RNase-free离心管中配制如下混合液，用移液器轻轻吹打混匀。
表1 Real time qPCR gDNA消化反应体系
b：逆转录反应体系配制(20μL体系)
表2 Real time qPCR cDNA逆转录反应体系
取上述已反转录好的cDNA，稀释5倍，以GAPDH为内参，利用qPCR方法检测细胞内靶基因S基因的表达量，qPCR反应程序如下：95℃，2min；95℃，5s，60℃，30s，39个循环；95℃，5s；65℃，5s；95℃50s。所使用的引物序列如表3所示：
表3 qPCR引物
图1是本实施例中利用qPCR技术检测盐酸贝尼地平在RNA水平对病毒复制的抑制作用，采用Students’t检验，*P<0.05，**P<0.01，**P<0.001，****P<0.0001)。与阴性对照组DMSO(二甲基亚砜)组相比，盐酸贝尼地平(Benidipine HCl)能够显著抑制靶病毒S基因的表达，表明盐酸贝尼地平具有显著的抗病毒效果。
2、盐酸贝尼地平在蛋白水平抗病毒活性评价
2.1WB
(1)细胞接种及药物处理
将人肝癌细胞(Huh7)、肺癌细胞(A549)以2.5×105个细胞接种到6孔板中，第二天将盐酸贝尼地平用含2％FBS的DMEM培养基稀释至终浓度为10μM，每孔加入1mL含有盐酸贝尼地平的DMEM培养基，药物处理1h后，弃掉培养基，用DPBS洗三遍，加入HTNV感染(Moi＝0.1)2h，每15min摇晃一次。2h后，弃掉病毒液，用DPBS洗三遍，加入含有2％FBS的DMEM培养基放置细胞培养箱培养。
(2)蛋白提取
48h后，弃掉6孔板中的培养基，DPBS洗一遍，加入含有磷酸酶和蛋白酶抑制剂的RIPA裂解液在冰上裂解30min，用细胞刮将细胞刮下来加入到1.5mL EP管中，在日立离心机上离心(12000rpm,15min,4°)，将上清转移至新准备的EP管中，利用BCA蛋白定量试剂盒进行蛋白定量。将蛋白定量加入loading buffer煮沸10min后，在10％的分离胶的蛋白胶中每个泳道加入30μg蛋白进行电泳(70V，30min；120V，1.5h)、转膜(200mA,2h)。将含有蛋白条带的PVDF膜转移到3％BSA(TBST)封闭液中封闭1h，随后移除3％BSA，加入NP蛋白一抗1A8(原液，本科室自己制备)4°摇床过夜，第二天回收1A8一抗，TBST洗三遍，加入内参GAPDH一抗(TBST稀释，1：10000，CST)4℃摇床过夜，第二天回收GAPDH一抗，TBST洗三遍，加入含有二抗(TBST稀释，1：10000，CST)室温摇床孵育1h，TBST洗三遍，利用奥德赛成像仪进行扫膜(如图2)。
图2是本实施例中利用Western blot技术检测盐酸贝尼地平对HTNV病毒NP蛋白表达影响。如图所示，与阴性对照组DMSO相比，盐酸贝尼地平能够显著降低NP蛋白的表达，表明盐酸贝尼地平具有显著的抗病毒活性。
2.2IF
(1)细胞接种及药物处理
肺癌细胞(A549)以5×104个细胞接种到12孔板中，第二天将盐酸贝尼地平用含2％FBS的DMEM培养基稀释至终浓度为10μM，每孔加入1mL含有盐酸贝尼地平的DMEM培养基，药物处理1h后，弃掉培养基，用DPBS洗三遍，加入HTNV感染(Moi＝0.1)2h，每15min摇晃一次。2h后，弃掉病毒液，用DPBS洗三遍，加入含有2％FBS的DMEM培养基。
(2)免疫荧光染色
48h后，弃掉12孔板中的培养基，DPBS洗2遍，加入4％多聚甲醛固定30min，DPBS洗2遍，加入0.5％Triton-X100室温破膜10min，DPBS洗2遍，加入3％BSA封闭30min(现配现用)，DPBS洗2遍，加入一抗1A8(1：100，本科室自己制备，具体记载于文献《An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential AntiviralTherapeutic Against Hantavirus Infection》)4°过夜，第二天DPBS洗5遍，加入cy5标记的鼠源二抗(1：500，CST)室温避光孵育1h，弃去二抗，DPBS洗5遍，加入细胞核染料DAPI(1：10000)室温孵育5min，弃去染料，DPBS洗5遍，利用荧光倒置显微镜进行观察拍照(如图3)。
如图3所示，与阴性对照组DMSO组相比，盐酸贝尼地平药物处理组1A8荧光数量显著减少，表明盐酸贝尼地平具有显著的抗病毒活性。
二、盐酸贝尼地平对细胞毒性的影响
1、CCK8
(1)细胞接种及药物处理
将人肝癌细胞(Huh7)、肺癌细胞(A549)、非洲绿猴肾细胞(Vero E6)以1×104个细胞接种到96孔板中，第二天将盐酸贝尼地平用含2％FBS的DMEM培养基稀释至不同浓度，每组加入0.1mL含有不同浓度盐酸贝尼地平的DMEM培养基。
(2)CCK8检测
药物处理24h后，每孔加入100μL含有10％CCK8的DMEM培养基，4h后，利用epoch酶标仪在450nm检测吸光度(如图4)。
如图4所示，与阴性对照DMSO相比，盐酸贝尼地平在10μM浓度下没有显著差异性，表明盐酸贝尼地平使用浓度在安全范围内。
三、盐酸贝尼地平抗汉滩病毒(HTNV)、汉城病毒(SEOV)、普马拉病毒(PUUV)、Dobrava病毒(DOBV)、辛诺柏病毒(SNV)、安第斯病毒(ANDV)等假病毒感染细胞评价
1、细胞接种及药物处理
(1)细胞接种及药物处理
肺癌细胞(A549)以1×104个细胞接种到96孔板中，第二天将盐酸贝尼地平用含2％FBS的DMEM培养基稀释至终浓度为10μM，每孔加入0.1mL含有盐酸贝尼地平的DMEM培养基，药物处理1h后，弃掉培养基，用DPBS洗三遍，加入感染汉滩病毒(HTNV)、汉城病毒(SEOV)、普马拉病毒(PUUV)、Dobrava病毒(DOBV)、辛诺柏病毒(SNV)、安第斯病毒(ANDV)等假病毒2h，每15min摇晃一次。2h后，弃掉病毒液，用DPBS洗三遍，加入含有2％FBS的DMEM培养基。
2、荧光拍照
24h后，利用上述倒置荧光显微镜对GFP进行拍照(如图5)；
如图5所示，与阴性对照组DMSO组相比，盐酸贝尼地平药物处理组荧光数量显著减少，表明盐酸贝尼地平能够抑制汉滩病毒(HTNV)、汉城病毒(SEOV)、普马拉病毒(PUUV)、多布拉伐-贝尔格莱德病毒(DOBV)、辛诺柏病毒(SNV)、安第斯病毒(ANDV)等假病毒感染细胞。
四、西尼地平、非洛地平、马尼地平、盐酸尼卡地平和尼索地平等钙离子通道抑制剂抗汉滩病毒(HTNV)感染细胞评价(具体过程与盐酸贝尼地平抗病毒活性评价中的步骤一致)
1、细胞接种及药物处理
将人肺癌细胞(A549)以1×105个细胞接种到12孔板中，第二天将西尼地平、非洛地平、马尼地平、盐酸尼卡地平和尼索地平分别用含2％FBS的DMEM培养基稀释至终浓度为10μM，每孔加入1mL含有西尼地平、非洛地平、马尼地平、盐酸尼卡地平或尼索地平的DMEM培养基，药物处理1h后，弃掉培养基，用DPBS洗三遍，加入HTNV感染(Moi＝0.1)2h，每15min摇晃一次。2h后，弃掉病毒液，用DPBS洗三遍，加入含有2％FBS的DMEM培养基。
2)汉滩病毒靶基因S基因检测
24h后按RNA提取说明书提取RNA，所得RNA后冻于-80℃冰箱保存。利用反转录试剂盒对RNA进行反转录，反转录产物冻于-20℃，具体过程如前述盐酸贝尼地平抗病毒活性评价中的步骤一致。
图6是本实施例中利用qPCR技术检测西尼地平、非洛地平、马尼地平、盐酸尼卡地平和尼索地平钙离子通道抑制剂等在RNA水平对病毒复制的抑制作用，采用Students’t检验(*P<0.05，**P<0.01，**P<0.001，****P<0.0001)。与阴性对照组DMSO(二甲基亚砜)组相比，西尼地平、非洛地平、马尼地平、盐酸尼卡地平和尼索地平能够显著抑制靶病毒S基因的表达，表明具有显著的抗病毒效果。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
<110> 中国人民解放军空军军医大学
<120> 钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途
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Claims (6)
1.钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途。
2.根据权利要求1所述的钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途，其特征在于，所述钙离子通道抑制剂为盐酸贝尼地平、西尼地平、非洛地平、马尼地平、盐酸尼卡地平和尼索地平中的任意一种。
3.根据权利要求2所述的钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途，其特征在于，所述钙离子通道抑制剂能够用于制备抑制RNA水平病毒复制的药物。
4.根据权利要求3所述的钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途，其特征在于，所述钙离子通道抑制剂能够用于制备抑制病毒NP蛋白表达的药物。
5.根据权利要求4所述的钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途，其特征在于，所述钙离子通道抑制剂能够用于制备抑制靶病毒S基因表达的药物。
6.根据权利要求5所述的钙离子通道抑制剂在制备抗汉坦病毒感染药物中的用途，其特征在于，所述汉坦病毒为汉滩病毒、汉城病毒、普玛拉病毒、多布拉伐-贝尔格莱德病毒、安第斯病毒和辛诺柏病毒中的任意一种。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5614193A (en) * | 1991-11-14 | 1997-03-25 | The United States Of America As Represented By The Secretary Of The Army | Hantavirus vaccine |
| WO2005046676A1 (en) * | 2003-11-12 | 2005-05-26 | University Of South Carolina | Treatment or prevention of cardiovascular and respiratory disorders with novel substituted cyclic amp-specific phosphodiesterase inhibitors |
| US20130053301A1 (en) * | 2010-01-22 | 2013-02-28 | Ascendis Pharma A/S | Dipeptide-based prodrug linkers for aliphatic amine-containing drugs |
| CN110743015A (zh) * | 2019-11-27 | 2020-02-04 | 中国科学院武汉病毒研究所 | 钙离子通道抑制剂的新应用 |
-
2022
- 2022-05-11 CN CN202210509795.3A patent/CN114939166B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5614193A (en) * | 1991-11-14 | 1997-03-25 | The United States Of America As Represented By The Secretary Of The Army | Hantavirus vaccine |
| WO2005046676A1 (en) * | 2003-11-12 | 2005-05-26 | University Of South Carolina | Treatment or prevention of cardiovascular and respiratory disorders with novel substituted cyclic amp-specific phosphodiesterase inhibitors |
| US20130053301A1 (en) * | 2010-01-22 | 2013-02-28 | Ascendis Pharma A/S | Dipeptide-based prodrug linkers for aliphatic amine-containing drugs |
| CN110743015A (zh) * | 2019-11-27 | 2020-02-04 | 中国科学院武汉病毒研究所 | 钙离子通道抑制剂的新应用 |
Non-Patent Citations (3)
| Title |
|---|
| WANG ET AL.: "Dihydropyridine-derived calcium channel blocker as a promising anti-hantavirus entry inhibitor", 《FRONTIERS IN PHARMACOLOGY》 * |
| 刘玉柱;陈可彬;吴玮;: "缬沙坦氨氯地平治疗肾综合征出血热急性肾衰竭效果研究", 中国实用医药 * |
| 蒋伟: "NLRP3炎症小体在汉滩病毒感染THP-1细胞中的作用", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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