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

项目总结 卡波西肉瘤相关疱疹病毒(KSHV)是伽玛疱疹病毒亚家族的成员之一，已被 被证明是卡波西氏肉瘤、原发性渗出性淋巴瘤和多中心Castleman病的病原体。 疾病。卡波西肉瘤是与人类感染有关的最常见的恶性肿瘤 免疫缺陷病毒(HIV)，在约20%的获得性免疫缺陷综合征(AIDS)中发展 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的发生直接相关， 但病毒的裂解复制也直接或间接地促进了肿瘤的发生。因此，重要的是要 开发特定的裂解复制抑制物来控制KSHV相关癌症。目前，大多数 抗疱疹病毒感染的现有药物是针对病毒DNA合成的核酸类似物。 然而，这些核酸药物表现出广泛的不良反应，包括频繁的药物诱导 耐药突变病毒和各种毒性。此外，没有专门针对KSHV感染的药物 是可用的。因此，有必要开发抗KSHV的药物。我们认为除了 病毒DNA合成，病毒衣壳组装，这是产生后代病毒粒子的关键步骤，可以 作为一种新颖而有效的药物靶点。KSHV衣壳由955个衣壳亚基组成。这些亚基形成一种 耐压网络，可承受其~150 kb冷凝产生的数十个大气压的压力 DsDNA基因组。我们假设，如果一个抑制剂与955个衣壳单位中的任何一个结合，它将要么 防止衣壳的形成或导致不稳定的衣壳在高压下破裂。这个 关键的一点是，955个单位中只有一个单位必须成为目标，从而将药物与目标的比率降低约 一千倍。潜在地，这种方法可以通过~3-Log减少药物的剂量，显著地最小化其 可能的毒性和副作用。换句话说，较高的内部压力和大量的亚基使得 疱疹病毒衣壳组装容易中断，因此可靶向引导结构 抗病毒药物的开发。 最近，我们用电子计数冷冻法解析了KSHV衣壳的原子分辨结构。 电子显微镜(低温电子显微镜)。在低温电子显微镜结构和功能分析结果的指导下，我们鉴定出一种 主要衣壳蛋白(MCP)上结构域上的疏水凹槽，是很有希望的可用药部位。 此R21应用程序的目标是识别针对该可用药部位的化学抑制剂 具体目标如下：1)通过两次互补的高通量筛选来鉴定化学抑制剂， 2)通过功能鉴定鉴定先导化合物。中断疱疹病毒的先导化合物 衣壳组装，不仅将是科学研究的有用工具，而且有可能成为第一个- 抗疱疹病毒感染类药物。

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.