A GFP-based HTS Assay for HIV-1 Reactivating Agents

基于 GFP 的 HIV-1 重新激活剂 HTS 测定

• 批准号: 7225977
• 负责人: OLAF KUTSCH
• 金额: $ 31.04万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7225977
• 关键词: Advanced Development; Alabama; Allogenic; Biological Assay; Body Burden; Cell Line; Cell Survival; Cells; Characteristics; Clinical; Development; Disease; Drug Design; Evaluation; Fluorescence; Future; Generations; Genes; Goals; HIV-1; Human; In Vitro; Infection; Institution; Interleukin-2; Introns; Joints; Kinetics; Lead; Libraries; Lymphoid Cell; Molecular Target; Molecular Virology; Monoclonal Antibodies; Monoclonal Antibody HuM291; Muromonab-CD3; Numbers; Patients; Pharmaceutical Preparations; Preclinical Drug Evaluation; Process; Proteins; Protocols documentation; Proviruses; Ramp; Range; Reader; Reading; Reporter; Reproducibility; Research; Research Institute; Research Personnel; Rest; Robotics; Screening procedure; Site; System; T memory cell; T-Cell Activation; T-Lymphocyte; Testing; Therapeutic; Therapeutic Agents; Treatment Protocols; Universities; Validation; Viral; Work; base; drug discovery; enhanced green fluorescent protein; high throughput screening; in vivo; memory CD4 T lymphocyte; novel; pre-clinical; programs; scale up; small molecule; small molecule libraries

DESCRIPTION (provided by applicant): HIV-1 latency is a major obstacle to effective, lifelong control of HIV-1 infection and has been the nemesis of curative strategies for the disease. Remarkably, estimates of the total body burden of latently-infected cells are in the range of only 106, a notably small number that would seem to represent a tractable target, nonetheless, previous attempts to eliminate these latently-infected cells using anti-CD3 monoclonal antibodies or Interleukin 2 (II-2)) have been unsuccessful. In this application, we propose a joint research effort between the University of Alabama at Birmingham and the Southern Research Institute (SRI) focusing on the discovery and validation of novel small molecule drug candidates that selectively activate HIV-1 expression in lymphoid cells. The proposed research builds on a body of previous work by SRI investigators and by our group (J Virol. 76:8776, 2002) that both demonstrates feasibility of the project and provides the critical cell-based assays needed for immediate ramp-up to high throughput screening (HTS) of compound libraries. Specific aims are: (i) to transfer an existing latently HIV-1 infected reporter cell line that uses enhanced green fluorescent protein as a quantitative marker of HIV-1 expression and is amenable to HTS in a 384-well plate format to the fully automated HTS platform at the SRI; (ii) to perform assay validation and initial screens of >1,000 compounds for activity in inducing HIV-1 expression; (iii) to generate second generation HIV-1 latency assays that will minimize false negative or positive hits; (iv) to develop protocols that allow for the evaluation of the ability of lead compounds identified by HTS to induce HIV-1 expression in virally-infected CD4+ memory T lymphocytes from HIV-infected patients. The proposed research capitalizes on unique and proven strengths at DAB and SRI in molecular virology and drug discovery and promises to provide a scientific and technical platform that will allow for the future screening of large compound libraries with the objective to discover and preclinically develop HIV-1 reactivating agents. Integration of HIV-1 reactivating agents into the existing HIV-1 treatment schedule with the goal to deplete the cellular reservoirs of latent HIV-1 will be a first essential step towards the development of a therapeutic treatment strategy for HIV-1 infection.

描述（由申请人提供）： HIV-1潜伏期是对HIV-1感染有效控制的有效控制的主要障碍，并且一直是该疾病治愈策略的二聚体。值得注意的是，对潜在感染细胞的总体负担的估计仅在106范围内，这一点似乎代表了一个可拖动的靶标，尽管如此，先前尝试使用抗CD3单克隆抗体或使用静脉内抗体或金属蛋白素2（II-2）来消除这些潜在受感染的细胞的尝试均未成立。在此应用程序中，我们提出了阿拉巴马大学伯明翰大学与南方研究所（SRI）之间的共同研究工作，重点是发现和验证新型的小分子药物候选者，这些候选物有选择地激活淋巴样细胞中的HIV-1表达。拟议的研究基于SRI研究人员和我们的小组（JVirol。76：8776，2002）的一系列作品，这些研究都证明了该项目的可行性，并提供了立即将基于基于细胞的关键测定方法逐步升级到高吞吐量筛选（HTS）的化合物图书馆。具体目的是：（i）将现有的潜在HIV-1感染的记者细胞系传递，该细胞系使用增强的绿色荧光蛋白作为HIV-1表达的定量标记，并且可以在384-WELL板格式的HTS中转移到SRI的全自动HTS平台中的HTS； （ii）在诱导HIV-1表达的活动中执行测定验证和> 1,000种化合物的初始筛选； （iii）生成第二代HIV-1潜伏期测定，以最大程度地减少假阴性或正命中； （iv）开发方案，以评估HTS在病毒感染的CD4+记忆T淋巴细胞中诱导HTS诱导HIV-1表达的铅化合物的能力。拟议的研究利用了分子病毒学和药物发现中DAB和SRI的独特和可靠的优势，并有望提供一个科学和技术平台，该平台将允许将来筛选大型化合物库，以发现并明确开发HIV-1重新激活剂。将HIV-1重新激活的剂整合到现有的HIV-1治疗时间表中，其目标是耗尽潜在的HIV-1的细胞储层，这将是朝着制定HIV-1感染治疗策略的第一步。