HTS for inhibitors of HIV-1 latency establishement

HIV-1 潜伏期抑制剂的 HTS 建立

• 批准号: 7494330
• 负责人: OLAF KUTSCH
• 金额: $ 53.37万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7494330
• 关键词: AIDS therapy; Adverse effects; Affect; Antiviral Agents; Biological; Biological Assay; Biological Testing; Calcineurin; Cell Density; Cell Line; Cells; Chemicals; Chemistry; Clinical; Condition; DNA Methylation; Data; Detection; Development; Diversity Library; Drug Delivery Systems; Drug Design; Drug toxicity; Event; Exhibits; FK506; Face; Failure; Flow Cytometry; Fluorescence; Genetic Transcription; Goals; HIV-1; Half-Life; Highly Active Antiretroviral Therapy; Histone Deacetylation; Histones; Infection; Intention; Laboratories; Lead; Light; Magic; Maintenance; Measures; Medical; Modification; Nature; Patients; Pattern; Performance; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacologic Substance; Play; Population; Preclinical Drug Evaluation; Process; Prograf; Public Health; Reporter; Research; Robotics; Role; Series; Standards of Weights and Measures; System; T-Lymphocyte; Tacrolimus; Techniques; Therapeutic; Time; Treatment Protocols; Viral; Viremia; antiretroviral therapy; base; design; feeding; inhibitor/antagonist; insight; latent infection; member; novel; prevent; programs; promoter; small molecule; therapeutic target

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.

描述（由申请人提供）：潜伏的HIV-1感染被认为是治愈性艾滋病治疗的主要障碍，因为它允许感染在抗逆转录病毒治疗中持续存在。鉴于正在进行的讨论是否潜伏库的内在稳定性是导致其长半衰期的唯一因素，或者低水平的病毒复制是否有助于通过持续补充潜伏库来测量稳定性，干扰潜伏建立可能是在治疗上干扰感染患者潜伏HIV-1库稳定性的另一种策略。为了确定治疗性干扰HIV-1潜伏期的潜在新靶点，我们开发了一种基于T细胞的实验系统，可以随时间跟踪细胞群中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，我们发现没有迹象表明这些对潜伏期维持很重要的过程在潜伏感染事件的建立中起作用。我们发现FK506 （Tacrolimus, Prograf.）是一种免疫抑制化合物，通过干扰钙调神经磷酸酶抑制NFAT激活，消除潜伏期建立而不影响活动性感染，这表明潜伏期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复用技术，使我们能够使用流式细胞术对潜伏期建立抑制剂进行高通量药物筛选。HTS分析使用荧光标记（荧光条形码），允许在不同浓度下同时分析药物效应（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。在此应用中，该分析将与一系列验证分析相结合，以确定化合物对甲基化，潜伏和活动性感染的浓度依赖性影响，从而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检测将用于筛选5万种化合物，以抑制潜伏期的建立。hit将接受药物化学计划，以开发先导化合物，中期目标是识别和开发HIV-1潜伏期建立的抑制剂，这些抑制剂比他克莫司具有更少的全身副作用，更有可能用于标准的HIV-1治疗团，目的是根除病毒。