以EV71 3Cpro为靶标的抗病毒药物筛选模型的建立及抗3Cpro化合物筛选

建立一种以EV71 3C蛋白酶为靶标的抗肠病毒药物筛选模型,并应用于小分子化合物库筛选具有抗EV71活性的化合物.从临床手足口病例标本中分离肠道病毒进行PCR鉴定及基因组测序.通过插入突变在黄色荧光YFP编码框合适位点处引入EV71 3C酶切位点,构建对3C蛋白酶敏感的报告质粒pc DNA3-m YFP,然后将其与表达3C的质粒共转293A细胞,在3C抑制剂Rupintrivir存在与否的情况下通过荧光显微镜和酶标仪检测Ex(500nm)/Em(535nm)荧光信号的变化,判断建模是否成功;利用建好的筛选模型在高通量药物筛选平台对小分子化合物库进行初筛和复筛;再利用空斑分析检测筛选出的活性化合物是否对临床分离的EV71毒株具有抑制作用.m YFP在293A细胞中表达良好,3C的表达使荧光信号下降80%,Rupintrivir的存在则几乎不影响荧光表达,说明以3C为靶位的筛选模型构建成功.经过高通量初筛和复筛从26 000多种小分子化合物中获得26种能够显著回复m YFP表达的活性化合物;空斑分析显示其中2种化合物具有较为明显的抑制EV71复制的活性.因此,我们所构建的3C-m YFP共表达系统是一种简便有效的、可用于高通量筛选抗EV71 3C~(pro)药物的筛选模型.

Establishment and Application of High-throughput Screening Model for Antiviral Agents Targeting EV71 3Cpro

Enterovirus 71 (EV71) is the main causing agent of hand-foot-mouth disease (HFMD), however, the specific antiviral agents are still not commercially available. In order to find antiviral agents against EV71, a high throughput drug-screening model targeting EV71 3C^pro was established and a small-molecular compound library was screened. The virus EV71-MZ was isolated from an HFMD patient, and identified by PCR. A 3C^pro recognition site was inserted into the middle region of YFP open reading frame to generate the mYFP by insertion mutation. The full length mYFP proteins were observed by fluorescence microscope and the protein level was measured by using microplate reader in Ex(500 nm)/Em(535 nm). Change of the fluorescence value reflected the degree of the inhibition on 3C^pro activity. A small-molecular compound was screened by using the established screening model in the high throughput drug screening system, then the antiviral activity of the active compounds was further evaluated by plaque assay. As a result, mYFP expressed well in 293A cell; the expression of 3C^pro reduced the fluorescence signal remarkably, however, the signal was recovered by adding Rupintrivir, an inhibitor of 3C^pro. These results indicated that the screening model targeting 3C^pro was established successfully. 26 of 26 000 compounds significantly reverted the fluorescence signal of the mYFP in the presence of 3C^pro; noticeably, two of the 26 compounds, i.e. numbering 3 and 8, exhibited strong antiviral activity by plaque assay. All together, 3C-mYFP co-expression system is an optimized and effective screening method for high-throughput screening of anti-EV71 3C^{pro drugs.}