CN104922111A - 一种小分子化合物在抗流感病毒药物中的应用 - Google Patents
一种小分子化合物在抗流感病毒药物中的应用 Download PDFInfo
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Abstract
本发明请求保护一种小分子化合物在抗H1N1型流感病毒药物中的应用。本发明提供的化合物均具有较好的抗流感病毒作用，为进一步的抗流感病毒药物研发提供了强有力的理论基础和实践基础，具有重要的研发价值和开发意义。
Description
技术领域
本发明涉及一种新的化合物，更具体地，涉及一种抗流感病毒化合物及其应用。
背景技术
流行性感冒是流感病毒引起的急性呼吸到感染的传染病，特别是由甲型流感病毒引起的流行性感冒是严重危害家养畜禽及人类健康的一种人畜共患病。其对人类致病性高，曾多次引起了世界性的大流行，给许多国家和地区造成了巨大的经济损失和社会恐慌。
目前市面上生成的针对流感的抗病毒药物主要是针对流感病毒的血凝素（HA）和神经氨酸酶（NA）为靶标的药物，如扎那米韦和达菲。但是与抗生素不同，抗病毒的药物抗病毒谱比较窄，而HA和NA突变率很高，所以不断研究和开发新型的抗流感药物是一项艰巨而又需要不断坚持的重要工作。
发明内容
本发明提供一种抗病毒化合物在抗流感病毒药物中的应用，所述的抗病毒化合物的结构式如式I所示：
式I
本发明具有以下优点：
该化合物利用流感病毒聚合酶复合体活性体系验证其作用机制，该化合物针对的靶点为聚合酶复合体，具体相对广泛抗流感病毒的活性。
附图说明
图1：抗病毒化合物对H1N1亚型流感的抑制效果的IC50。
图2：抗病毒化合物对细胞的毒性实验（MTS）（CC50）。
图3：抗病毒化合物对H1N1亚型流感病毒的聚合酶复合体的复制活性的抑制效果。
具体实施方式
为了更好地理解本发明的实质，下面结合实验和结果来说明这种抗病毒化合物对抗H1N1亚型流感的应用。
实验一：抗病毒化合物的抗病毒效果的空斑实验
1）准备24孔板的MDCK细胞，待细胞密度到80%-90%左右，可以开始感染实验；
2）去掉24孔细胞的上清培养基，用PBS洗1-2遍，加入稀释好的病毒感染液；
3）配好细胞培养液， 感染1 h后，去掉病毒感染液，用PBS洗1遍，加入含有梯度稀释浓度的药物的细胞培养基。
4）感染24 h后，收取感染的细胞上清进行空斑实验。准备好密度为90% 以上的12孔MDCK细胞。取感染的细胞上清进行稀释，一般稀释倍数有104倍、105倍、106倍，即-4、-5、-6。
5）去掉MDCK细胞上清培养基，用PBS洗1-2遍，加入稀释好的感染上清，每孔300 ul，孵育1-2h；
6）把2*琼脂糖胶放入微波炉中低火3min至完全溶解，在放入水浴锅温浴；
7）待1*琼脂糖培养基温度为42°C左右，去掉孵育的上清，用PBS洗1遍，加入1*琼脂糖胶培养基1 ml/12孔；放入4度冰箱5-10 min左右，待琼脂糖胶凝固后，倒扣放入37度培养箱。
8）48小时后观察噬斑。
本实验证明该抗病毒化合物具有良好的抗H1N1亚型流感病毒的作用。
实验二：细胞毒性和凋亡活性检测
分别将0.5uM, 1uM, 10uM, 50uM 的抗病毒化合物与MDCK细胞共同孵育，48小时后用MTS法检测细胞活力。
本实验证明该抗病毒化合物不具有诱导细胞凋亡的能力。
实验三：抗病毒化合物对流感病毒聚合酶复合体活性的抑制实验
1）构建真核表达的H1N1亚型流感病毒的聚合酶复合体蛋白的质粒：pcDNA3.1-PB1，pcDNA3.1-PB2，pcDNA3.1-PA和pcDNA3.1-NP。
2) 取生长良好的人肾上细胞株239T细胞，接种于48孔细胞培养板中，每孔1×105细胞。使用的培养基是完全培养基：高糖DMEM，10%胎牛血清以及1%双抗，培养条件是5%二氧化碳、37℃；
3) 24 h贴壁后，共转染流感聚合酶质粒系统。转染采用脂质体包裹转染，试剂使用lipo2000，转染液50μl。
3) 转染12 h后，加入化合物，每孔加入化合物终浓度为0.5uM, 1uM, 10uM, 50uM。
4) 培养24h后，利用双报告系统检测荧光素酶luciferase与renilla表达的比值。
实验结果如图3所示，从实验结果可以看出，抗病毒化合物均具有抑制H1N1亚型流感病毒聚合酶复合体活性的作用。
本实验说明该抗病毒化合物能够抑制H1N1亚型流感病毒的复合酶活性。
Claims (1)
1.一种小分子化合物在抗流感病毒药物中的应用，其特征在于，所述的抗病毒化合物的结构式如式I所示：
式I。
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| WO1996039144A1 (en) * | 1995-06-06 | 1996-12-12 | Procyte Corporation | Stable copper(i) complexes as active therapeutic substances |
| WO1997001559A1 (en) * | 1995-06-29 | 1997-01-16 | Procyte Corporation | Zinc(ii) complexes and methods related thereto |
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| WO1996039144A1 (en) * | 1995-06-06 | 1996-12-12 | Procyte Corporation | Stable copper(i) complexes as active therapeutic substances |
| WO1997001559A1 (en) * | 1995-06-29 | 1997-01-16 | Procyte Corporation | Zinc(ii) complexes and methods related thereto |
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