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

描述（由申请人提供）： HIV-1潜伏期是有效、终生控制HIV-1感染的主要障碍，并且一直是该疾病治疗策略的克星。值得注意的是，潜伏感染细胞的全身负荷估计仅在 106 个范围内，这个数字非常小，似乎代表了一个易于处理的目标，尽管如此，之前使用抗 CD3 单克隆抗体或白细胞介素 2 (II-2)) 消除这些潜伏感染细胞的尝试并未成功。在本申请中，我们提议阿拉巴马大学伯明翰分校和南方研究所 (SRI) 之间的联合研究工作，重点是发现和验证选择性激活淋巴细胞中 HIV-1 表达的新型小分子候选药物。拟议的研究建立在 SRI 研究人员和我们小组先前工作的基础上 (J Virol. 76:8776, 2002)，这些工作既证明了该项目的可行性，又提供了立即提升到化合物库高通量筛选 (HTS) 所需的关键的基于细胞的检测方法。具体目标是： (i) 将现有的潜在 HIV-1 感染报告细胞系转移到 SRI 的全自动 HTS 平台，该细胞系使用增强型绿色荧光蛋白作为 HIV-1 表达的定量标记，并且适合 384 孔板格式的 HTS； (ii) 对超过 1,000 种化合物进行测定验证和初步筛选，以了解诱导 HIV-1 表达的活性； (iii) 生成第二代 HIV-1 潜伏期检测，最大限度地减少假阴性或阳性命中； (iv) 制定方案，评估 HTS 鉴定的先导化合物在 HIV 感染患者的病毒感染 CD4 记忆 T 淋巴细胞中诱导 HIV-1 表达的能力。拟议的研究利用了 DAB 和 SRI 在分子病毒学和药物发现方面独特且经过验证的优势，并有望提供一个科学和技术平台，允许未来筛选大型化合物库，以发现和临床前开发 HIV-1 重新激活剂。将 HIV-1 重新激活剂整合到现有的 HIV-1 治疗方案中，以消除潜在 HIV-1 的细胞库，将是制定 HIV-1 感染治疗策略的第一个重要步骤。

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.