CN107286044B - 一种能够抑制流感病毒pb2蛋白与rna帽结合的化合物 - Google Patents
一种能够抑制流感病毒pb2蛋白与rna帽结合的化合物 Download PDFInfo
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Abstract
本发明提供了一种化合物，所述化合物为式(Ⅰ)、式(Ⅱ)或式(Ⅲ)所示的化合物，所述化合物均能够抑制流感病毒PB2蛋白与RNA帽结合，具有抗流感病毒的功能。
Description
技术领域
本发明涉及一种化合物，具体涉及一种能够抑制流感病毒PB2蛋白与RNA帽结合的化合物。
背景技术
流行性感冒在世界范围内导致了很高的发病率和死亡率，被认为是对人类来说最为重要的病毒威胁之一。接种疫苗是控制流感病毒传播的主要途径。然而，由于流感疫苗的制备存在诸多局限，例如在新爆发病毒的鉴定到疫苗的临床应用大约要有6个月的滞后。所以，抗病毒药物是目前来说迫切需要的预防和治疗流感的方法。两类抗病毒药物已批准在临床治疗使用，即M2通道蛋白抑制剂(金刚烷胺和金刚乙胺)和神经氨酸酶(NA)抑制剂(奥司他韦和扎那米韦)。然而，耐药菌株的出现，如季节性H3N2，2009年甲型H1N1，H5N1和H7N9高致病性禽流感等，很大程度上削弱了这两类药物的临床使用价值。所以，新开发的能够在不同流感病毒亚型间提供交叉保护作用的抗病毒药物备受关注。流感病毒蛋白的生物学和结构功能学研究已在广泛地开展，这也为新药筛选提供了更多的靶点。需要特别指出的是，流感病毒RNA聚合酶中相关功能结构域的晶体结构已被成功解析。由于这些蛋白在不同流感亚型之间相当保守。因此，针对RNA聚合酶的功能结构域设计抑或是筛选的药物更有机会对流感产生广谱的抗病毒效果。
流感病毒RNA聚合酶由PA、PB1和PB2三个亚基组成，它们与核蛋白(NP)一起形成的核糖核蛋白(RNP)负责催化病毒RNA在宿主细胞核内的转录和复制。在一个被称为“偷帽”(cap-snatching)的过程中，病毒mRNA的转录首先取决于PB2蛋白捕获宿主mRNA中的帽状结构，紧随其后，PA蛋白能够利用其核酸内切酶活性剪切帽子结构下游的RNA序列。该序列则被当作起始病毒mRNA转录的引物。研究表明，PB2亚单位上的第318-483位氨基酸残基形成了RNA帽结合结构域(PB2cap-binding domain)。该功能结构域可以通过5'帽子结构识别并结合宿主mRNA。由于PB2帽结合结构域不同于宿主细胞中其他蛋白与mRNA帽的结合模式，这表明抑制PB2蛋白和帽子结构的结合的药物存在很小的可能会影响到宿主细胞对帽子结构的正常应用。所以，PB2帽结合结构域是潜在的新型抗病毒药物的筛选靶点。此外，相关研究已经表明替换PB2帽结合结构域中的关键氨基酸位点将显著减少PB2帽结合活性以及整个RNA聚合酶的活性。所以，由PB2帽结合结构域筛选而来的抗病毒药物所诱导产生耐药毒株的可能性会大大降低。
发明内容
本发明的目的在于克服现有技术存在的不足之处而提供了一种能够抑制流感病毒PB2蛋白与RNA帽结合的化合物。
为实现上述目的，所采取的技术方案：一种能够抑制流感病毒PB2蛋白与RNA帽结合的化合物，所述化合物为式(Ⅰ)、式(Ⅱ)或式(Ⅲ)所示的化合物，所述式(Ⅰ)所示的化合物的结构式如下：
所述式(Ⅱ)所示的化合物的结构式如下：
所述式(Ⅲ)所示的化合物的结构式如下：
将上述所述式(Ⅰ)所示的化合物命名为PB2-19，将上述所述式(Ⅱ)所示的化合物命名为PB2-35，将上述所述式(Ⅲ)所示的化合物命名为PB2-39。本发明所述式(Ⅰ)、(Ⅱ)和(Ⅲ)所示的化合物都能够干扰RNA帽与流感病毒PB2帽结合结构域结合。其中所述PB2-39是当中最有效的流感病毒抑制剂。PB2-39能够抑制多个亚型的流感病毒在细胞中的复制，包括H1N1、H3N2、H5N1、H7N7、H7N9和H9N2亚型。BALB/c小鼠在感染致命剂量的流感病毒后接受PB2-39鼻腔给药，能够显著提高的存活率并减少肺组织中的病毒量。
本发明提供了上述所述的化合物在制备抗流感病毒的药物中的用途。
优选地，所述流感病毒为甲型、乙型和丙型流感病毒中的至少一种。
本发明的有益效果在于：本发明提供了一种化合物，所述化合物为式(Ⅰ)、式(Ⅱ)或式(Ⅲ)所示的化合物，所述化合物均能够抑制流感病毒PB2蛋白与RNA帽结合，具有抗流感病毒的功能。
附图说明
图1为本发明实施例1中所述PB2-39的体外抗病毒效果；
图2为本发明实施例1中所述PB2-39的小鼠体内抗病毒疗效；
图3为本发明实施例1中所述PB2-19的小鼠体内抗病毒效果。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
实施例1
1.材料和方法
1.1细胞、病毒
犬肾细胞(MDCK)在包含有10％热灭活胎牛血清(FBS)，50单位/毫升青霉素和50微克/毫升链霉素(P/S)的MEM培养基中培养。MDCK在病毒感染后，在含有1微克/毫升的TPCK胰蛋白酶但不含有胎牛血清的MEM中培养。总共8株/6种亚型的流感病毒株，即A/HK/415742/09(H1N1)、A/Hong Kong/1/1968(H3N2)、A/Shenzhen/406H/2006(H5N1_SZ)、A/HongKong/156/97(H5N1_HK)、A/Vietnam/1194/2004(H5N1_VN)、A/Netherlands/219/2003(H7N7)、A/Anhui/1/2013(H7N9)和A/HK/1073/1999(H9N2)分别在MDCK细胞中增殖并在本研究中使用。病毒的滴度通过空斑试验(plaque assay)测定后，分装并保存在-80摄氏度。所有与活病毒相关试验是在生物安全2级或3级设施中进行的。
1.2选择性指数
选择性指数(selectivity index)是通过50％的细胞毒性浓度(CC50)除以50％的病毒抑制浓度(IC50)计算而来的，该指标数值越高则指示该药物有越好的临床应用前景。我们通过MTT法测定了本发明所述化合物的CC50，再通过空斑减数试验确定IC50值，最终得到了奔赴吗所述化合物的选择性指数。
1.3多周期病毒生长试验
MDCK细胞在接种了感染复数(MOI)为0.002的流感H1N1病毒后，在含有20μM的PB2-39以及1毫克/毫升TPCK胰蛋白酶的MEM培养基中培养。感染后0、6、21、25、32、47和54小时后，收集病毒上清液并使用空斑法测定病毒滴度。
1.4交叉保护力试验
本发明所述化合物针对多个亚型的流感病毒，包括甲型H1N1、H3N2、H5N1、H7N7、H7N9和H9N2的抗病毒效果也进行了测试。简单地说，MDCK细胞在接种0.002个MOI的流感病毒后1小时，用PBS洗净，并将培养基置换成新鲜的含有不同浓度(20、5、1.25、0.31μM)本发明所述化合物的MEM培养基。病毒接种24小时候后，收集细胞上清液，并采用反转录-实时荧光定量聚合酶链式反应(RT-qPCR)技术测定病毒的滴度。
1.5本发明所述化合物体内抗病毒疗效评估
本实验采用6-8周龄的BALB/c雌性小鼠作为实验动物。所有实验遵循生物安全3级动物设施标准作业程序并得到香港大学动物伦理委员会的批准。麻醉后,共42只小鼠(14只/组)经鼻腔接种一定剂量的流感病毒。药物在病毒接种6小时后由鼻腔途径给药，每日2次，共3天。第一组的小鼠接受20微升1毫克/毫升的PB2-39(即2.5毫克/千克体重)。第二组的小鼠经鼻腔接受20微升2.5毫克/毫升的扎那米韦作为阳性对照。最后一组小鼠经鼻腔注射以20微升PBS作为阴性对照。动物的生命体征总共监测21天或直至死亡。在病毒接种后的第4天，随机从每组小鼠中选取四只处死后收集肺部组织。一半的肺组织碾磨后取上清采用空斑法和RT-qPCR法测定病毒滴度，另一半肺组织立即被固定在10％福尔马林中进行的组织病理学分析。PB2-19在老鼠模型上的抗病毒效果评价采用与PB2-39完全相同的方法。不同的是，由于水溶解度的限制，PB2-19的用量为1毫克/千克体重。
2.结果
我们测定了本发明所述化合物的IC50和CC50，并通过CC50除以IC50计算选择性指标(selectivity index)。一般来说，该指标数值越高则指示该药物有越好的临床应用前景。因为PB2-19和PB2-39显示出的选择性指数高于100(表1)，所以我们在随后的研究中对该两个化合物详细进行了评价。
表1 本发明所述化合物的选择性指数结果
2.1 PB2-39提供广谱的抗甲型流感病毒作用
首先，我们测试了PB2-39在多周期病毒生长试验中的表现。如图1A所示，该化合物显示了明显的抗流感病毒作用。针对H1N1亚型流感病毒，使用20μM的PB2-39能够使上清液中的病毒滴度减少100-1000倍.由于PB2帽结合结构域的序列在不同亚型的流感病毒之间高度保守，我们进一步评估了PB2-39对H5N1、H7N7、H7N9、H9N2、H3N2亚型的毒株在细胞水平的抗病毒效果。如图1B所示，PB2-39能够以剂量依赖的方式抑制所有测试的流感毒株的复制。然而，不同亚型的病毒对PB2-39表现出不同的敏感性。
2.2 PB2-39能够在小鼠体内抑制流感病毒生长
紧接着，我们评估了PB2-39在小鼠体内的抗病毒效果。首先，我们测定了经不同亚型流感病毒感染的小鼠在经过PB2-39治疗后的存活率。如图2A所示，当小鼠接种5个LD50(半数致死量)的H1N1病毒后，经阴性对照PBS处理的小鼠全部死亡(存活率0％)，而经阳性对照(扎那米韦)或者PB2-39治疗的小鼠全部存活(存活率100％)；当小鼠接种1个LD50的H5N1流感病毒后，经阴性对照PBS处理的小鼠有一半死亡(存活率50％)，而经阳性对照(扎那米韦)或者PB2-39治疗的小鼠全部存活(存活率100％)；当小鼠接种1个LD80的H7N9病毒后，经阴性对照PBS处理的小鼠有80％死亡(存活率20％)，而经阳性对照(扎那米韦)或者PB2-39治疗的小鼠全部存活(存活率100％)。该试验结果显示，PB2-39能够针对不同流感病毒的感染显著提高小鼠的存活率。在感染过后的第4天，每个组中随机挑选4只小鼠取肺并通过空斑试验测定肺组织中的病毒含量。如图2B结果表明，PB2-39的治疗能够显著减少肺组织中的病毒量。此外，如图2C所示的组织病理学检查进一步表明，经过PB2-39治疗的小鼠，肺组织间质炎性细胞浸润及肺泡损伤亦得到改善。我们的结果表明PB2-39能有效抑制流感病毒在小鼠体内的复制。
2.3 PB2-19能够在小鼠体内抑制流感病毒生长
在此前的试验中，因为PB2-19也显示出较好的临床应用前景，即选择性指数为273(表1),所以我们也评价了PB2-19在小鼠体内的抗病毒效果。如图3所示，采用的试验手段与PB2-39基本相同，不同的是，我们只评价了PB2-19针对H1N1流感病毒的体内抗病毒效果。结果表明，当小鼠接种1个LD60的H1N1病毒后，经阴性对照PBS处理的小鼠有60％死亡(存活率40％)，而经阳性对照(扎那米韦)或者PB2-19治疗的小鼠全部存活(存活率100％)(图3A)。此外，PB2-19也能够有效得减少病毒在实验小鼠肺部的繁殖(图3B)。组织病理学检查的结果也支持PB2-19能够提供体内的抗流感病毒效果(图3C)。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (2)
1.式（Ⅰ）、式（Ⅱ）或式（Ⅲ）所示的化合物在制备抗流感病毒的药物中的用途，其特征在于，所述式（Ⅰ）所示的化合物的结构式如下：
（Ⅰ）；
所述式（Ⅱ）所示的化合物的结构式如下：
（Ⅱ）；
所述式（Ⅲ）所示的化合物的结构式如下：
（Ⅲ）。
2.根据权利要求1所述的用途，其特征在于，所述流感病毒为甲型、乙型和丙型流感病毒中的至少一种。
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