HTS for inhibitors of HIV-1 latency establishement

描述（由申请人提供）：潜伏性HIV-1感染被认为是治愈性艾滋病治疗的主要障碍，因为它使感染在抗逆转录病毒治疗面前持续存在。根据正在进行的讨论，无论是潜伏性储库的内在稳定性是储库半衰期长的唯一影响因素，还是低水平病毒复制通过持续补充潜伏性储库而有助于测定的稳定性，干扰潜伏期建立可能是治疗性干扰感染患者中潜伏性HIV-1储库稳定性的替代策略。为了确定潜在的新靶点，用于治疗性干扰HIV-1潜伏期，我们开发了一种基于T细胞的实验系统，在该系统中，可以随着时间的推移跟踪细胞群中HIV-1潜伏期的建立和维持，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理学抑制剂，我们没有发现这些过程，这已被证明是重要的潜伏期的维护，发挥作用的建立潜伏感染事件。我们发现FK 506（他克莫司，NF.），一种通过干扰钙调磷酸酶抑制NFAT活化、消除潜伏期建立而不影响活动性感染的免疫抑制化合物表明潜伏期建立确实可以作为新的药物靶点。基于我们的测定，我们开发了一种基于细胞的多路复用技术，该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检测使用荧光特征（荧光条形码），允许同时分析不同浓度下的药物效应（定量HTS（qHTS））;荧光模式和细胞群密度的变化用于定量特定药物效应和可能的药物毒性。在本申请中，该试验将与一系列验证试验相结合，这些试验确定了化合物对甲基化、潜伏和活动性感染的浓度依赖性影响，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测定将用于筛选50，000种化合物的潜伏期建立抑制剂。命中将进行药物化学计划，以开发先导化合物，中期目标是鉴定和开发HIV-1潜伏期建立的抑制剂，其全身副作用比他克莫司少，更有可能用于标准HIV-1治疗方案，目的是根除病毒。 公共卫生相关性：潜伏的HIV-1感染被认为是治愈性艾滋病治疗的主要障碍，因为它使感染在抗逆转录病毒治疗面前持续存在，然而，以前通过重新激活潜伏感染事件来根除HIV-1宿主的尝试失败了。在这里，我们证明了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。在本申请中，我们建议进行药物筛选的潜伏期形成的抑制剂，中期至长期的目标是开发药物用于HIV-1治疗的目的是根除病毒。

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.