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、终身控制HIV-1感染的主要障碍，也是该疾病治疗策略的克星。值得注意的是，对潜伏感染细胞体内总负担的估计只有106个，这是一个非常小的数字，似乎是一个容易对付的目标，然而，以前使用抗CD3单抗或白细胞介素2(IL-2)消除这些潜伏感染细胞的尝试都没有成功。在这项申请中，我们建议阿拉巴马大学伯明翰分校和南方研究所(SRI)联合开展研究，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调查人员和我们小组(J Virol)先前工作的基础上。76：8776,2002)，既证明了项目的可行性，又提供了立即提高化合物文库高通量筛选(HTS)所需的关键的基于细胞的分析。具体目标是：(I)将现有的潜伏感染HIV-1的报告细胞系转移到SRI的全自动HTS平台上，该报告细胞系使用增强型绿色荧光蛋白作为HIV-1表达的定量标记物，并以384孔板的形式服从HTS；(Ii)对诱导HIV-1表达的&gt；1000种化合物进行分析验证和初步筛选；(Iii)产生第二代HIV-1潜伏期分析，将假阴性或阳性命中降至最低；(4)制定方案，评估HTS确定的先导化合物在HIV感染者的病毒感染的CD4记忆T淋巴细胞中诱导HIV-1表达的能力。这项拟议的研究利用了DAB和SRI在分子病毒学和药物发现方面独特和经过验证的优势，并承诺提供一个科学和技术平台，使未来能够对大型化合物库进行筛选，目的是发现和临床前开发HIV-1复活剂。将艾滋病毒-1复活剂纳入现有的艾滋病毒-1治疗计划，目的是耗尽潜伏的艾滋病毒-1的细胞储存库，这将是制定艾滋病毒-1感染治疗战略的第一个基本步骤。

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.