Ultra-potent HIV capsid inhibitors

摘要 抗逆转录病毒疗法（ART）已经将曾经致命的艾滋病毒/艾滋病疾病转变为可控制的， 慢性感染然而，目前的抗逆转录病毒疗法仍然存在一些紧迫的问题，包括 每天服用HIV-1药物的必要性，次优的治疗依从性，以及 耐药病毒表型的出现。因此，需要开发长效 靶向临床上未开发的病毒蛋白的抗逆转录病毒剂以减轻上述问题。HIV-1 衣壳蛋白是一个新的、有吸引力的靶点，因为它在病毒的生命周期中起着多种重要作用。 GS-6207（Lenacapavir，吉利德科学公司（Gilead Sciences））是一种最近发现的、一流的、长效的和超效的抗肿瘤药物。 有效的HIV-1衣壳抑制剂。最近完成的1期临床试验（NCT 03739866）表明， GS-6207进入2/3期临床试验（NCT 04143594/NCT 04150068），为期6个月， 给药间隔。我们的研究目标是阐明一种高效的 GS-6207的抗病毒活性，并利用所获得的知识开发第二代抑制剂。 为此，我们合成并检测了GS-6207的抗病毒活性。符合 由于衣壳在HIV-1生物学中的多方面作用，该抑制剂可有效（EC 50约为55 pM）削弱传入病毒 并且在病毒排出期间表现出第二种略微降低的（约314 pM的EC 50）抗病毒活性。模态 GS-6207的作用研究表明，该抑制剂通过稳定和 从而防止感染细胞的细胞质中衣壳壳的功能性分解。此外，本发明还提供了一种方法， GS-6207干扰衣壳与细胞HIV-1辅因子Nup 153和CPSF 6的结合， 病毒核输入和直接整合到染色质的基因丰富区域。我们的X射线晶体学 低温电子显微镜和氢氘交换实验表明，GS-6207 紧密结合两个相邻的衣壳亚单位，并促进远端六聚体内和六聚体间的相互作用， 显著地稳定了弯曲的衣壳晶格此外，我们的GS-6207的高分辨率X射线结构 与衣壳六聚体结合，使我们能够绘制与GS-6207非常接近的耐药变体 结合位点这些信息对于合理设计第二代抑制剂至关重要。我们提出 扩展这些研究，以更好地了解GS的多模式，特别有效的抗病毒活性， 6207在HIV-1复制的早期和晚期阶段。为此，我们将追求以下三个目标 具体目标：目标1将阐明抑制HIV进入后步骤的结构和机制基础， 目的2将剖析抑制病毒产生的潜在机制， 目的3将研究病毒对GS-6207耐药性的结构基础 合理开发耐药屏障增强的第二代抑制剂。综合起来看， 拟议的研究将剖析GS-6207的多模式抗病毒作用机制， 深入了解衣壳的病毒生物学，确定关键的衣壳相互作用并促进优化 这类化合物的临床应用。

Abstract Antiretroviral therapies (ART) have transformed the once deadly HIV/AIDS disease into a manageable, chronic infection. Yet, there are still a number of pressing problems associated with current ARTs, including the necessity of daily administration of HIV-1 medications, suboptimal treatment adherence, and the emergence of drug-resistant viral phenotypes. Therefore, there is a need for developing long-acting antiretroviral agents targeting clinically unexploited viral proteins to mitigate the above problems. HIV-1 capsid protein is a novel, attractive target as its plays multiple essential roles during the virus life cycle. GS-6207 (Lenacapavir, Gilead Sciences) is a recently discovered, first-in-class, long-acting, and ultra- potent HIV-1 capsid inhibitor. Recently completed phase 1 clinical trials (NCT03739866) have suggested advancement of GS-6207 into phase 2/3 clinical trials (NCT04143594/NCT04150068) with a six-month dosing interval. Our research objective is to elucidate structural and mechanistic bases for a highly potent antiviral activity of GS-6207 and exploit the knowledge obtained to develop second-generation inhibitors. For this, we have synthesized and examined the antiviral activities of GS-6207. Consistent with the multifaceted role of capsid in HIV-1 biology, the inhibitor potently (EC50 of ~55 pM) impaired incoming virus and exhibited a second, slightly reduced (EC50 of ~314 pM) antiviral activity during virus egress. Mode-of- action studies of GS-6207 revealed that the inhibitor blocks post-entry steps of infection by stabilizing and thereby preventing functional disassembly of the capsid shell in the cytoplasm of infected cells. In addition, GS-6207 interfered with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. Our x-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments have revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that strikingly stabilize the curved capsid lattice. Furthermore, our high-resolution x-ray structure of GS-6207 bound to a capsid hexamer enabled us to map drug-resistant variants in close proximity to the GS-6207 binding site. This information will be critical for rational design of second-generation inhibitors. We propose to extend these studies to better understand the multimodal, exceptionally potent antiviral activity of GS- 6207 during both early and late steps of HIV-1 replication. For this, we will pursue the following three specific aims: Aim 1 will elucidate structural and mechanistic bases for inhibition of post-entry steps of HIV- 1 infection by GS-6207; Aim 2 will dissect underlying mechanisms of inhibition of virus production and maturation by GS-6207; and Aim 3 will investigate the structural basis for viral drug-resistance to GS-6207 and rationally develop second-generation inhibitors with an enhanced barrier to resistance. Taken together, the proposed studies will dissect the multimodal antiviral mechanism of action of GS-6207, provide new insights into the viral biology of capsid, identify key inhibitor-capsid interactions and facilitate optimization of this class of compounds for their clinical use.