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，潜伏感染细胞的总身体负荷的估计值仅在106的范围内，这是一个非常小的数字，似乎代表了一个易处理的靶标，尽管如此，先前使用抗CD 3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不成功。在这项申请中，我们提出了一个联合研究工作之间的亚拉巴马大学伯明翰和南方研究所（SRI）的重点是发现和验证新的小分子候选药物，选择性地激活HIV-1在淋巴细胞的表达。拟议的研究建立在SRI调查人员和我们小组以前工作的基础上（J Virol. 76：8776，2002），其既证明了该项目的可行性，又提供了化合物文库的高通量筛选（HTS）所需的立即斜升所需的关键的基于细胞的测定。具体目标是：（i）将现有的潜伏性HIV-1感染的报告细胞系转移到SRI的全自动HTS平台，所述报告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，并且在384孔板格式中适合HTS;（ii）进行测定验证和对> 1，000种化合物进行诱导HIV-1表达的活性的初始筛选;（iii）产生第二代HIV-1潜伏期测定，其将使假阴性或阳性命中最小化;（iv）开发允许评价通过HTS鉴定的先导化合物在来自HIV感染患者的病毒感染的CD 4+记忆T淋巴细胞中诱导HIV-1表达的能力的方案。拟议的研究利用了DAB和SRI在分子病毒学和药物发现方面的独特和成熟的优势，并有望提供一个科学和技术平台，以便将来筛选大型化合物库，目的是发现和临床前开发HIV-1再活化剂。将HIV-1再活化剂整合到现有的HIV-1治疗方案中，以耗尽潜伏HIV-1的细胞库，将是开发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.