Dysregulation of Hepatitis B Virus Nucleocapsid Assembly in vitro by RNA-binding Small Ligands.
Dysregulation of Hepatitis B Virus Nucleocapsid Assembly in vitro by RNA-binding Small Ligands.
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DOI：
10.1016/j.jmb.2022.167557
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发表时间：
2022-05-30
影响因子：
5.6
通讯作者：
Stockley PG
中科院分区：
文献类型：
--
作者：
Patel N;Abulwerdi F;Fatehi F;Manfield IW;Le Grice S;Schneekloth JS Jr;Twarock R;Stockley PG
分布在B型肝炎病毒基因组中的RNA序列/基序在体外以不依赖于RNA聚合酶/聚合酶的方式调节核心蛋白（Cp）形成核衣壳样颗粒（NCP）。这些多个RNA包装信号（PS）可以各自形成茎-环，在其环中包含Cp-识别基序-RGAG-。通过用荧光标记的RNA寡核苷酸筛选包含该序列的固定化配体文库，鉴定了以纳摩尔亲和力结合这些PS位点中最重要的用于NCP组装调节的药物样分子。这些“命中”，与亲和力范围从低纳摩尔到高微摩尔的66，购买非固定化版本。它们对PS的亲和力和对NCP组装的影响通过表面等离子体共振在体外测定。高亲和力配体结合依赖于PS环内-RGAG-基序的存在，这与PS位点之间的配体交叉结合一致。简单的结构-活性关系表明，它也依赖于这些配体中特定官能团的存在。一些化合物在纳摩尔浓度下是体外NCP组装的有效抑制剂。尽管有适当的logP值，但这些配体在细胞培养中不抑制HBV复制。然而，建模证实了使用PS结合配体靶向NCP组装作为一种新的抗病毒策略的潜力。这也允许针对体内不同分子靶标的抗病毒配体之间的潜在协同效应的计算探索。新型小分子RNA结合配体可以使HBV PS调节的组装失调，从而为开发针对该主要病原体的直接作用的抗病毒药物开辟了新的靶点。
RNA sequences/motifs dispersed across the genome of Hepatitis B Virus regulate formation of nucleocapsid-like particles (NCPs) by core protein (Cp) in vitro, in an epsilon/polymerase-independent fashion. These multiple RNA Packaging Signals (PSs) can each form stem-loops encompassing a Cp-recognition motif, -RGAG-, in their loops. Drug-like molecules that bind the most important of these PS sites for NCP assembly regulation with nanomolar affinities, were identified by screening an immobilized ligand library with a fluorescently-labelled, RNA oligonucleotide encompassing this sequence. Sixty-six of these "hits", with affinities ranging from low nanomolar to high micromolar, were purchased as non-immobilized versions. Their affinities for PSs and effects on NCP assembly were determined in vitro by Surface Plasmon Resonance. High-affinity ligand binding is dependent on the presence of an -RGAG-motif within the loop of the PS, consistent with ligand cross-binding between PS sites. Simple structure−activity relationships show that it is also dependent on the presence of specific functional groups in these ligands. Some compounds are potent inhibitors of in vitro NCP assembly at nanomolar concentrations. Despite appropriate logP values, these ligands do not inhibit HBV replication in cell culture. However, modelling confirms the potential of using PS-binding ligands to target NCP assembly as a novel antiviral strategy. This also allows for computational exploration of potential synergic effects between antiviral ligands directed at distinct molecular targets in vivo. HBV PS-regulated assembly can be dysregulated by novel small molecule RNA-binding ligands opening a novel target for developing directly-acting anti-virals against this major pathogen.
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影响因子：
5.9
作者：
Cihlar, Tomas;Fordyce, Marshall
通讯作者：
Fordyce, Marshall
影响因子：
--
作者：
Bradner, James E.;McPherson, Olivia M.;Koehler, Angela N.
通讯作者：
Koehler, Angela N.
影响因子：
16.6
作者：
Brown, Rebecca S.;Anastasakis, Dimitrios G.;Kielian, Margaret
通讯作者：
Kielian, Margaret
影响因子：
56.9
作者：
Deres, K;Schröder, CH;Rübsamen-Waigmann, H
通讯作者：
Rübsamen-Waigmann, H
DOI：
10.3390/v2061279
发表时间：
2010-06
期刊：
Viruses
影响因子：
--
作者：
De Clercq E;Férir G;Kaptein S;Neyts J
通讯作者：
Neyts J