HTS for inhibitors of HIV-1 latency establishement

描述(申请人提供)：潜伏的HIV-1感染被认为是根治艾滋病治疗的主要障碍，因为它允许感染在接受抗逆转录病毒治疗的情况下持续存在。鉴于目前正在进行的讨论是潜伏库的内在稳定性是导致潜伏库半衰期较长的唯一因素，还是低水平的病毒复制有助于通过不断补充潜伏库来测量到的稳定性，干扰潜伏期的建立可能是在治疗上扰乱感染患者的潜伏性HIV-1库稳定性的一种替代策略。为了确定潜在的治疗干预HIV-1潜伏期的新靶点，我们开发了一个基于T细胞的实验系统，在该系统中，可以随着时间的推移跟踪HIV-1潜伏期在细胞群体中的建立和维持，并在单细胞水平上进行定量评估。我们证明潜伏期的建立是转录沉默病毒整合的结果。使用DNA甲基化或组蛋白去乙酰化的药物抑制剂，我们没有发现任何迹象表明，这些过程对潜伏期维持很重要，对建立潜伏感染事件起作用。我们的发现是，FK506(他克莫司，Prograf.)是一种免疫抑制化合物，通过干扰钙调神经磷酸酶来抑制NFAT的激活，在不影响活动性感染的情况下消除了潜伏期的建立，表明潜伏期的建立确实可以作为一种新的药物靶点。基于我们的分析，我们开发了一种基于细胞的多路技术，使我们能够使用流式细胞术对潜伏期建立的抑制剂进行高通量药物筛选。HTS检测使用荧光信号(荧光条形码)来同时分析不同浓度的药物效应(定量HTS(QHTS))；荧光模式和群体细胞密度的变化被用来量化特定的药物效应和可能的药物毒性。在这一应用中，该分析将与一系列验证分析相结合，这些验证分析定义了化合物对甲基化、潜伏和活跃感染的浓度依赖影响，以全面了解化合物对所有HIV-1转录状态的影响。一旦转移到我们的机器人平台，qHTS测试将被用于筛选50,000种化合物，寻找潜伏期建立的抑制剂。HITS将接受一项药物化学计划，以开发先导化合物，中期目标是确定和开发艾滋病毒-1潜伏期建立的抑制剂，这些药物具有比他克莫司更少的系统性副作用，更有可能用于旨在根除病毒的标准艾滋病毒-1治疗方案。 公共卫生相关性：潜伏的艾滋病毒-1感染被认为是艾滋病根治治疗的主要障碍，因为它允许在接受抗逆转录病毒治疗的情况下感染持续存在，然而，以前通过重新启动潜伏感染事件来根除艾滋病毒-1宿主的尝试失败了。在这里，我们证明潜伏感染的建立是可以预防的，潜伏感染的建立可以作为一种新的药物靶点。在这项应用中，我们建议对潜伏期形成的抑制剂进行药物筛选，中长期目标是开发用于HIV-1治疗的药物，目的是根除病毒。

DESCRIPTION (provided by applicant): Latent HIV-1 infection is believed to represent the principal obstacle to curative AIDS therapy, as it allows the infection to persist in the face of antiretroviral therapy. In light of the ongoing discussion whether the intrinsic stability of the latent reservoir is the only contributing factor to the long half-life of the reservoir, or whether low-level viral replication contributes to the measured stability by continuous replenishment of the latent reservoir, interference with latency establishment may be an alternative strategy to therapeutically perturb the stability of the latent HIV-1 reservoir in infected patients. To identify potential new targets for therapeutic interference with HIV-1 latency, we have developed a T cell based experimental system in which HIV-1 latency establishment and maintenance in a cell population can be followed over time and quantitatively assessed at the single cell level. We demonstrate that latency establishment is the result of transcriptionally silent viral integration. Using pharmacological inhibitors of DNA methylation or histone deacetylation, we find no indication that these processes, which have been shown important for latency maintenance, play a role for the establishment of latent infection events. Our finding that FK506 (Tacrolimus, Prograf.), an immuno-suppressive compound that inhibits NFAT activation by interference with calcineurin, abolishes latency establishment without affecting active infection, suggests that latency establishment could indeed serve as a novel drug target. Based on our assay, we have developed a cell-based multiplexing technique that allows us to perform high throughput drug screening for inhibitors of latency establishment using flow cytometry. The HTS assay uses fluorescence signatures (fluorescent barcoding) to allow for the simultaneous analysis of drug effects at various concentrations (quantitative HTS (qHTS)); changes in fluorescent patterns and population cell densities are used to quantify specific drug effects and possible drug toxicities. In this application, the assay will be combined with a series of verification assays that define the concentration-dependent influence of the compounds on methylated, latent and active infection to gain comprehensive insight on the compound effect on all HIV-1 transcription states. Once transferred to our robotic platform, the qHTS assay will be used to screen 50,000 compounds for inhibitors of latency establishment. Hits will be subjected to a medicinal chemistry program towards the development of lead compounds with the mid-term goal to identify and develop inhibitors of HIV-1 latency establishment that have less systemic side effects than tacrolimus and are more likely to be used in a standard HIV-1 therapeutic regiment with the intention of viral eradication. PUBLIC HEALTH RELEVANCE: Latent HIV-1 infection is believed to represent the principal obstacle to curative AIDS therapy, as it allows the infection to persist in the face of antiretroviral therapy, however, previous attempts to eradicate the HIV-1 reservoirs by reactivating latent infection events failed. Here, we demonstrate that the establishment of a latent infection can be prevented and that latency establishment can serve as a novel drug target. In this application, we propose to perform drug screening for inhibitors of latency formation with the mid- to long-tem goal of developing drugs to be used in HIV-1 therapy with the intention of viral eradication.