Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation.
Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation.
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DOI：
10.1016/j.dib.2022.108206
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发表时间：
2022-06
期刊：
影响因子：
1.2
通讯作者：
Patel, Nikesh
中科院分区：
文献类型：
--
作者：
Abulwerdi, Fardokht;Fatehi, Farzad;Manfield, Iain W.;Le Grice, Stuart F. J.;Schneekloth, John S.;Twarock, Reidun;Stockley, Peter G.;Patel, Nikesh
关键词：
感染细菌、植物或人类的多种ssRNA病毒在组装过程中使用RNA包装信号（PS）介导的调节来忠实有效地包装其基因组。PS通常包含短核苷酸识别基序，最常存在于RNA茎环的未配对区域中，并且通常以纳摩尔亲和力结合其同源外壳蛋白（CP）。迄今为止鉴定的PS在面对其病毒编码的聚合酶的典型易错复制时具有弹性，使其成为潜在的药物靶标。针对荧光标记的寡核苷酸淘选小分子量药物样化合物的固定阵列，所述寡核苷酸包含最保守的B肝炎病毒（HBV）PS、PS1，已知其是核衣壳形成中的主要决定因素。这鉴定了在阵列中独特地结合PS1的> 70种化合物。测试了其中66种市售药物对HBV核帽样颗粒（NCP）体外组装的潜在影响，确定了有效的组装抑制剂。在这里，我们描述了一个高通量的屏幕上使用荧光各向异性在96孔微孔板格式的这种效果。用Alexa Fluor 488染料5′标记HBV基因组RNA（gRNA）和包含PS1的短寡核苷酸。使用Biomek 4000液体处理机器人，将过量的（相对于化学计量T = 4 NCP形成）HBV核心蛋白（Cp）二聚体逐步自动滴定到含有这些RNA中的每一种的溶液中。使用POLARstar酶标仪监测这些混合物的各向异性值。用RNase A挑战NCP样结构以鉴定不导致完全NCP形成的反应。结果表明，约50%的化合物阻止了完全的NCP形成，突出了PS介导的组装和PS结合化合物作为具有新分子靶标的潜在直接作用的抗病毒药物。重要的是，该方法允许高通量体外筛选这种主要人类病原体中的组装抑制剂。
Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets. An immobilised array of small molecular weight, drug-like compounds was panned against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1, known to be a major determinant in nucleocapsid formation. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly in vitro, which identified potent assembly inhibitors. Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5′ labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T = 4 NCP formation) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation. The results imply that ∼50% of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.
影响因子：
28.3
作者：
Patel N;White SJ;Thompson RF;Bingham R;Weiß EU;Maskell DP;Zlotnick A;Dykeman E;Tuma R;Twarock R;Ranson NA;Stockley PG
通讯作者：
Stockley PG
影响因子：
5.4
作者：
Porterfield, J. Zachary;Dhason, Mary Savari;Zlotnick, Adam
通讯作者：
Zlotnick, Adam
影响因子：
5.6
作者：
Patel N;Abulwerdi F;Fatehi F;Manfield IW;Le Grice S;Schneekloth JS Jr;Twarock R;Stockley PG
通讯作者：
Stockley PG