Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
High Throughput Screening Laboratory, Shankel Structural Biology Center, University of Kansasgrid.412016.0grid.266515.3grid.412016.0grid.266515.3, Lawrence, Kansas, USA.
J Virol. 2022 Jan 26;96(2):e0132621. doi: 10.1128/JVI.01326-21. Epub 2021 Oct 20.
Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe () that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using -expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% inhibition of B19V infection at 10 μM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC]/half-maximal effective concentration [EC] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC). A purine derivative (P7) demonstrated an antiviral effect (EC = 1.46 μM) without prominent cytotoxicity (CC = 71.8 μM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 μM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site () and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC]/half-maximal effective concentration [EC] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 μM (two times the EC). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.
细小病毒 B19（B19V）感染可导致怀孕期间血液系统疾病和胎儿水肿。目前，尚无治疗或预防 B19V 感染的抗病毒药物或疫苗。为了寻找针对 B19V 复制的新型小分子抗病毒药物，我们开发了一种高通量筛选（HTS）测定法，该测定法基于使用重组病毒大非结构蛋白（NS1N）的氨基末端氨基酸 1 至 176 进行缺口测定和荧光标记的 DNA 探针（），该探针跨越病毒 DNA 复制起点的缺口位点。我们总共筛选了 17040 种化合物，发现了 2178 种（12.78％）具有> 10％抑制 NS1 缺口活性的化合物，其中 84 种化合物被证实以剂量依赖性方式抑制缺口。使用 B19V 感染的扩增的原代人红细胞祖细胞（EPC），我们验证了 24 种在 10 μM 时可抑制 B19V 感染> 50％的化合物，这些化合物可分为 7 种结构支架。根据 EPC 中的治疗指数（半最大细胞毒性浓度[CC]/半最大有效浓度[EC]比），选择了前 4 种化合物，以在 EPC 中在 90％最大有效浓度（EC）的两倍时检查它们对 B19V 感染的抑制作用。嘌呤衍生物（P7）在 EPC 中表现出抗病毒作用（EC = 1.46μM），而没有明显的细胞毒性（CC = 71.8μM），在 3.32μM 时可抑制 B19V 感染 92％，可作为未来治疗 B19V 感染引起的血液系统疾病的先导化合物。 B19V 编码一种大的非结构蛋白，NS1。其由氨基酸 1 至 176 组成的 N 末端结构域（NS1N）与病毒 DNA 结合，并作为内切核酸酶对病毒 DNA 复制起点进行缺口，这是滚环依赖性 B19V DNA 复制的关键步骤。为了进行抗 B19V 抗病毒药物的高通量筛选（HTS），我们将基于荧光的缺口测定法（该测定法使用跨越末端分辨率位点（）和 NS1N 蛋白的荧光标记探针）微型化为 384 孔板格式。 HTS 测定法在筛选 17040 种化合物方面表现出很高的可靠性和能力。根据 EPC 中的治疗指数（半最大细胞毒性浓度[CC]/半最大有效浓度[EC]比），嘌呤衍生物在 EPC 中对 B19V 感染的抑制作用为 92％，EC 为 3.32μM（EC 的两倍）。我们的研究证明了一种强大的 HTS 测定法，可用于筛选针对 B19V 感染的抗病毒药物。
