Abstract:
Difficulty in preventing crops from plant viruses urges to discover novel efficient antiviral chemicals, which is sped up by precise screening methods. Fluorescence-based methods have recently been applied as innovative and rapid tools for visually monitoring the replication of viruses and screening of antivirals, whereas the quantification of fluorescence signals mainly depends on manually calculating the fluorescent spots, which is time-consuming and imprecise. In the present work, the fluorescence spots were automatically identified, and the fluorescence area was directly quantified by a program developed in our group, which avoided subjective errors from the operators. We further employed this digital and visual screening assay to identify antivirals using the tobacco mosaic virus-green fluorescence protein (TMV-GFP) construct, in which the expression of GFP intuitively reflected the efficacy of antivirals. The accuracy of this assay was validated by quantifying the activities of the commercial antiviral inhibitors ribavirin and ningnanmycin and then was applied to evaluate the subtle activity differences of a series of newly synthesized carbazole and β-carboline alkaloid derivatives. Among them, compounds 5 (76%) and 11 (63%) exhibited anti-TMV activities comparable to that of ningnanmycin (65%) at 50 μM, and they delayed the multiplication of TMV in the early stage of infection without phytotoxicity. Taken together, these findings demonstrated that the digital and visual TMV-GFP screening method was competent to test the antiviral activities of compounds with subtle modifications and facilitated the discovery of novel antivirals.
摘要:
在防止作物从植物病毒的困难促使发现新的有效的抗病毒化学物质,这是通过精确的筛选方法加速的。基于荧光的方法最近被用作创新和快速的工具,用于视觉监测病毒的复制和抗病毒药物的筛选。而荧光信号的定量主要取决于手动计算荧光点,这是耗时且不精确的。在目前的工作中,荧光斑点被自动识别,荧光面积由我们小组开发的程序直接量化,避免了操作人员的主观错误。我们进一步利用这种数字和视觉筛选试验来鉴定使用烟草花叶病毒绿色荧光蛋白(TMV-GFP)构建体的抗病毒药物,其中GFP的表达直观地反映了抗病毒药物的功效。通过定量商业抗病毒抑制剂利巴韦林和宁南霉素的活性来验证该测定的准确性,然后将其用于评估一系列新合成的咔唑和β-咔啉生物碱衍生物的细微活性差异。其中,化合物5(76%)和11(63%)在50μM时表现出与宁南霉素(65%)相当的抗TMV活性,它们在没有植物毒性的感染早期延迟了TMV的增殖。一起来看,这些发现表明,数字和视觉TMV-GFP筛选方法能够测试具有细微修饰的化合物的抗病毒活性,并促进了新型抗病毒药物的发现。