Structure-guided development of chemical inhibitors against Kaposi’s sarcoma-associated herpesvirus (KSHV)

项目摘要 卡波西肉瘤相关疱疹病毒（KSHV）是γ-疱疹病毒亚科的一员， 被证明是卡波西肉瘤、原发性渗出性淋巴瘤和多中心Castleman's的病原体 疾病卡波西肉瘤是人类最常见的恶性肿瘤， 免疫缺陷病毒（HIV），约20%的获得性免疫缺陷综合征（AIDS） 没有接受抗逆转录病毒治疗的患者。KSHV的病毒潜伏期被认为与肿瘤发生直接相关， 但病毒裂解性复制也直接或间接地促进肿瘤发生。因此， 开发特异性裂解性复制抑制剂来控制KSHV相关的癌症。目前，大多数 可用的抗疱疹病毒感染的药物是靶向病毒DNA合成的核酸类似物。 然而，这些核酸药物显示出广泛的不良反应，包括频繁的药物诱导， 抗性突变病毒和各种毒性。此外，没有专门针对KSHV感染的药物， 服务器租用-美国服务租用因此，需要开发抗KSHV的药物。我们认为，除了 病毒DNA合成、病毒衣壳组装是产生子代病毒体的关键步骤， 作为一种新的有效的药物靶点。KSHV衣壳含有955个衣壳亚基。这些亚基形成一个 抗压网络，以抵御其冷凝~150-kb产生的数十个大气压的压力 dsDNA基因组。我们假设，如果抑制剂与955个衣壳单位中的任何一个结合， 阻止衣壳的形成或导致在高压下破裂的不稳定衣壳。的 关键是955个单位中只有一个必须被靶向，从而将药物与靶向的比率降低约 一千倍潜在地，这种方法可以将药物的剂量减少~ 3-Log，显著地最小化其剂量。 可能的毒性和副作用换句话说，高的内部压力和大量的亚基使得 疱疹病毒衣壳装配容易中断，因此可以靶向结构指导的 抗病毒剂的发展。 最近，我们解决了原子分辨率结构的KSHV衣壳采用电子计数冷冻， 电子显微镜（cryoEM）。根据cryoEM结构和功能分析结果，我们确定了一个 主要衣壳蛋白（MCP）的上结构域上的疏水沟是有希望的可药用位点。 该R21申请的目的是鉴定靶向该可药用位点的化学抑制剂， 以下具体目的：1）通过两种互补的高通量筛选鉴定化学抑制剂， 2）通过功能评价鉴定先导化合物。阻断疱疹病毒的先导化合物 衣壳组装，不仅将成为科学研究的有用工具，而且有可能成为第一个- 治疗疱疹病毒感染的同类药物。

PROJECT SUMMARY Kaposi's sarcoma-associated herpesvirus (KSHV), a member of the gamma-herpesvirus subfamily, has been shown to be an etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Kaposi's sarcoma is the most common malignancy associated with infection of human immunodeficiency virus (HIV) and develops in about 20% of acquired immunodeficiency syndrome (AIDS) patients without antiretroviral therapy. Viral latency of KSHV is thought to be directly linked to tumorigenesis, but viral lytic replication also contributes to tumorigenesis, directly and indirectly. Therefore, it is important to develop specific lytic replication inhibitors to control KSHV-associated cancers. Currently, the majority of the available drugs fighting herpesvirus infection are nucleic acid analogues targeting viral DNA synthesis. However, these nucleic acid drugs show extensive adverse effects including the frequent induction of drug resistant mutant viruses and various kinds of toxicity. Moreover, no drugs specifically targeting KSHV infection is available. Therefore, there is a need for the development of anti-KSHV drugs. We believe that aside from viral DNA synthesis, virus capsid assembly, which is a critical step for the production of progeny virions, can serve as a novel and potent drug target. KSHV capsids contain 955 capsid subunits. These subunits form a pressure-resistant network to withhold tens of atmospheres of pressure generated by its condensed ~150-kb dsDNA genome. We hypothesize that if an inhibitor binds to any one of the 955 capsid units, it will either prevent the formation of the capsid or result in an unstable capsid which bursts under the high pressure. The key point is that only one of the 955 units has to be targeted, thereby reducing the drug-to-target ratio by about a thousand-fold. Potentially, this method can reduce a drug's dose by ~ 3-Log, significantly minimizing its possible toxicity and side effects. In other words, the high internal pressure and large number of subunits make herpesvirus capsid assembly prone to interruption and can therefore be targeted for the structure-guided development of antiviral agents. Recently, we resolved the atomic-resolution structure of the KSHV capsid by employing electron-counting cryo- electron microscopy (cryoEM). Guided by the cryoEM structure and functional analysis results, we identified a promising druggable site, which is a hydrophobic groove on the upper-domain of Major Capsid Protein (MCP). The objective of this R21 application is to identify chemical inhibitors targeting this druggable site with the following specific aims: 1) to identify chemical inhibitors by two complementary high-throughput screenings, and 2) to identify lead compounds by functional evaluation. The lead compounds, which interrupt herpesvirus capsid assembly, will not only be a useful tool for scientific research but also have the potential to be the first- in-class drugs against herpesvirus infection.