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）：据信潜在的HIV-1感染代表了治疗艾滋病疗法的主要障碍，因为它使感染能够在面对抗逆转录病毒疗法的情况下持续存在。鉴于正在进行的讨论是否是潜在储层的固有稳定性是导致储层长期寿命的唯一因素，还是低级病毒复制是否有助于通过连续补充潜在储层，干扰，有助于测量的稳定性通过潜伏期建立，可能是治疗策略，可以在受感染的患者中扰动潜在的HIV-1储层的稳定性。为了确定对HIV-1潜伏期治疗干扰的潜在新靶标，我们开发了一种基于T细胞的实验系统，在该系统中，可以随时间遵循HIV-1潜伏期的建立和维护，并在单个细胞水平上进行定量评估。我们证明了潜伏期的建立是转录静音病毒整合的结果。使用DNA甲基化或组蛋白脱乙酰基化的药理抑制剂，我们没有发现这些过程对维持潜伏期很重要，这对于建立潜在感染事件起着作用。我们的发现，一种免疫抑制性化合物FK506（他克莫司（artarlimus，prograf）。通过干扰钙调神经蛋白而抑制NFAT激活，废除了不影响活跃感染的潜伏期，这表明潜伏期的建立确实可以用作新的药物目标。根据我们的测定，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仪进行高通量药物筛查，以对潜伏期的抑制剂进行抑制作用。 HTS分析使用荧光特征（荧光条形码），以同时分析各种浓度（定量HTS（QHTS））的药物效应；荧光模式和种群细胞密度的变化用于量化特定的药物作用和可能的药物毒性。在此应用中，该测定法将与一系列验证测定法相结合，这些测定定义化合物对甲基化，潜在和活性感染的浓度依赖性影响，以获得对所有HIV-1转录状态的化合物效应的全面见解。一旦转移到我们的机器人平台，QHTS分析将用于筛选50,000种延迟抑制剂的化合物。 HITS将受到一项药物化学计划，以开发具有中期目标的铅化合物，以识别和开发HIV-1潜伏期抑制剂的系统性副作用，该抑制剂的系统性副作用比他克莫司群较少，并且更有可能用于标准HIV-1治疗团以消除病毒。 公共卫生相关性：据信潜在的HIV-1感染代表了治疗艾滋病治疗的主要障碍，因为它使感染能够在抗逆转录病毒疗法面前持续存在，但是，先前试图通过反应潜在感染来消除HIV-1储量事件失败。在这里，我们证明可以预防潜在感染的建立，并且潜伏期的建立可以作为一种新型的药物靶标。在本应用中，我们建议对潜伏形成的抑制剂进行药物筛查，并以中高为中心的目的，即开发用于HIV-1治疗的药物，以消除病毒。