Fission Yeast as a HTS Platform for New Molecular Probes of HIV-1 VPR-Medicated A

裂变酵母作为 HIV-1 VPR 药物 A 新分子探针的高温超导平台

• 批准号: 7556254
• 负责人: RICHARD YUQI ZHAO
• 金额: $ 15万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7556254
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Affect; Agar; Anti-Retroviral Agents; Antiviral Agents; Apoptosis; Automation; Baltimore; Biological Assay; CD4 Positive T Lymphocytes; Cancerous; Cell Cycle; Cell Cycle Arrest; Cell Death; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Complex; Core Facility; Defective Viruses; Development; Disease; Disease Progression; Drug Design; Engineering; Factor Analysis; Fission Yeast; Fluorescence; Future; G2/M Arrest; G2/M Transition; Goals; HIV; HIV-1; Immune response; Infection; Interphase Cell; Laboratories; Lead; Libraries; Life; Link; Liquid substance; Macaca mulatta; Malignant Neoplasms; Mammalian Cell; Maryland; Measures; Mediating; Methods; Molecular; Molecular Bank; Molecular Probes; Monitor; Nature; Nuclear Import; Numbers; Pan Genus; Pan troglodytes; Patients; Performance; Pharmaceutical Preparations; Phase; Preclinical Drug Evaluation; Property; Proteins; Public Health; Quality Control; Reaction; Regulation; Reproducibility; Research; Running; Screening procedure; Signal Transduction; System; T-Cell Depletion; T-Lymphocyte; Testing; Time; United States National Institutes of Health; Universities; Validation; Viral; Viral Pathogenesis; Viral Proteins; Yeasts; assay development; base; design; high throughput screening; human subject; inhibitor/antagonist; macrophage; miniaturize; monocyte; nucleocytoplasmic transport; small molecule; software systems

DESCRIPTION (provided by applicant): Research Summary HIV-1 viral protein R (Vpr) is a multifaceted protein that modulates host cells through a number unique ways to facilitate HIV-1 infection and viral pathogenesis. Vpr is required for HIV-1 infection in non-dividing cells such as monocytes, macrophages and incomplete activated T-lymphocytes because of its ability to facilitate nuclear transport of the viral pre-integration complex (PIC) into the nucleus. Vpr induces cell cycle G2/M arrest that is shown to optimize viral replication and escape of host immune responses. In addition, Vpr causes apoptosis that is believed to contribute to depletion of CD4 T-cell depletion. Activation of viral replication and depletion of CD4 T-cell depletion are considered as hallmark of causing AIDS. The findings that rhesus monkeys, chimpanzees or human subjects infected with Vpr defective viruses are associated with slow disease progression indicate that anti-Vpr agents could be useful in anti-retroviral therapies against HIV-1 infection. Because of the unique and efficient ability of Vpr to interrupt host cell cycle G2-M transition and to induce apoptosis, identification of new molecular probes against these Vpr activities could also have potential utility in new drug designs against cancerous cells. The goal of this proposal is to adapt multiple fission yeast cell-based assays, established in our laboratory, for the use in automated high throughput molecular screening (HTS) for new molecular probes against three specific Vpr activities, i.e., apoptosis, cell cycle G2/M regulation and nuclear transport. Each Vpr activity will be first measured by a primary assay and confirmed by a secondary assay. Corresponding mammalian assays will also be used to validate the findings from yeast screenings. Each of these assays will be adapted and validated for 96-well formats, further miniaturized if possible. Finally all of these assays will be configured into HTS format and pilot testing will be carried out through test runs with a small ChemDiv40000 compound library. Significance of the "hits" identified in the primary HTS will be further verified in mammalian cell systems either with Vpr alone or in the context of HIV-1 infection. Two specific aims are proposed to adapt and configure the fission yeast cell-based assays into the HTS format to screen for small molecule probes of Vpr-mediated activities. In the first aim, we will adapt three sets of the fission yeast-based assays, which were developed in our laboratory over the last 11 years, into 96-well microtiter plate format by optimizing them for easy handling and automation. Corresponding mammalian methods will also be used to verify the small molecules identified in the yeast screenings. Completion of this specific aim would allow us to demonstrate the robust nature and reproducibility of each of the described assays, furthermore, to simplify the readouts and to make them more amenable to automated analyses; the second Specific Aim will be carried out at the HTS Core Facility of the University of Maryland at Baltimore (UMB) to further configure our assays for HTS and to conduct a pilot run by using a small ChemDiv40000 compound library. After adaptation and validation, these fission yeast cell-based assays should be ready for HTS and to participate in the Molecular Libraries Screening Centers Network (MLSCN) to identify additional molecular probes against each of the specific Vpr activities. All together, successful completion of the proposed studies will enable us to adapt the fission yeast-based assays into the formats for HTS, to conduct primary drug screening against Vpr for the first time and to provide opportunities for us to participate in the NIH Molecular Libraries Roadmap Initiative and the Molecular Libraries Screening Centers Network (MLSCN). The identified small molecules should provide functionally significant lead compounds to help us in the future design of additional drugs against HIV and cancers. Public Health Relevance Statement HIV/AIDS is one of the most devastating diseases in the world with approx. 33 million people living with HIV in 2007 and approx. 2.5 million new infections in the same year. Activities of HIV-1 viral protein R (Vpr) has been linked to activation of viral replication and depletion of CD4 T-cell depletion, both of which are hallmark of AIDS. In addition, rhesus monkeys, chimpanzees or human subjects infected with HIV carrying defective Vpr typically show slow disease progressions. Thus finding drugs that could limit the Vpr's activities could potentially alleviate the detrimental effects of Vpr on HIV-infected patients. At present, there is no antiviral drug or HTS system that is designed to screening drugs against Vpr. In this proposal, multiple fission yeast cell-based assays, which have been developed in our laboratory over the last 11 years to measure specific activities of Vpr such as nuclear transport, cell cycle arrest and apoptosis, will be adapted to the high throughput platform to screen potential inhibitors of Vpr. The proposed assays could additionally identify compounds that affect cell proliferation and apoptosis, two cellular processes that are involved in the development of cancers.

描述（由申请人提供）：研究概述HIV-1病毒蛋白R（Vpr）是一种多方面的蛋白质，通过许多独特的方式调节宿主细胞，以促进HIV-1感染和病毒发病机制。Vpr是HIV-1在非分裂细胞如单核细胞、巨噬细胞和不完全活化的T淋巴细胞中感染所必需的，因为它能够促进病毒整合前复合物（PIC）的核转运进入细胞核。Vpr诱导细胞周期G2/M停滞，其显示出优化病毒复制和宿主免疫应答的逃逸。此外，Vpr引起细胞凋亡，据信这有助于CD 4 T细胞耗竭的耗竭。病毒复制的激活和CD 4 T细胞的耗竭被认为是导致AIDS的标志。感染Vpr缺陷型病毒的恒河猴、黑猩猩或人类受试者与缓慢的疾病进展相关的发现表明，抗Vpr剂可用于抗HIV-1感染的抗逆转录病毒疗法。由于Vpr独特而有效地中断宿主细胞周期G2-M转换并诱导细胞凋亡的能力，鉴定针对这些Vpr活性的新分子探针在针对癌细胞的新药设计中也具有潜在的实用性。本提案的目标是调整我们实验室建立的基于多个分裂酵母细胞的测定，用于针对三种特异性Vpr活性的新分子探针的自动化高通量分子筛选（HTS），即，细胞凋亡、细胞周期G2/M调控和核转运。首先通过一次试验测量各Vpr活性，并通过二次试验确认。相应的哺乳动物试验也将用于验证酵母筛选的结果。这些试验中的每一种都将针对96孔格式进行调整和验证，如果可能，进一步小型化。最后，将所有这些检测试剂盒配置为HTS格式，并通过使用小型ChemDiv 40000化合物库进行测试运行进行中试测试。将在哺乳动物细胞系统中单独使用Vpr或在HIV-1感染的情况下进一步验证在原代HTS中鉴定的“命中”的意义。提出了两个具体的目标，以适应和配置的裂变酵母细胞为基础的测定到HTS格式筛选小分子探针的Vpr介导的活动。在第一个目标中，我们将通过优化将我们实验室在过去11年中开发的三套基于裂变酵母的测定方法改造成96孔微量滴定板格式，以易于操作和自动化。相应的哺乳动物方法也将用于验证酵母筛选中鉴定的小分子。这一特定目标的完成将使我们能够证明每种所述测定的稳健性和再现性，此外，简化读数并使其更适合于自动化分析;第二个“特定目标”计划将在位于巴尔的摩的马里兰州大学的高温超导核心设施（HTS Core Facility）内实施进一步配置我们的HTS分析，并使用小型ChemDiv 40000化合物库进行试验运行。经过调整和验证后，这些基于裂变酵母细胞的检测试剂盒应准备好用于HTS，并参与分子库筛选中心网络（MLSCN），以识别针对每种特定Vpr活性的其他分子探针。总而言之，成功完成拟议的研究将使我们能够将基于裂变酵母的测定方法应用于HTS的形式，首次针对Vpr进行主要药物筛选，并为我们提供参与NIH分子库路线图倡议和分子库筛选中心网络（MLSCN）的机会。这些小分子应该能提供功能上重要的先导化合物，以帮助我们在未来设计更多的抗艾滋病毒和癌症药物。 艾滋病毒/艾滋病是世界上最具破坏性的疾病之一，每年约有2007年有3300万人感染艾滋病毒，2.5同年新增感染者100万人。HIV-1病毒蛋白R（Vpr）的活性与病毒复制的激活和CD 4 T细胞的耗竭有关，这两者都是AIDS的标志。此外，感染携带缺陷Vpr的HIV的恒河猴、黑猩猩或人类受试者通常显示缓慢的疾病进展。因此，找到可以限制Vpr活性的药物可能会减轻Vpr对HIV感染患者的有害影响。目前，还没有设计用于筛选抗Vpr药物的抗病毒药物或HTS系统。在该提案中，我们实验室在过去11年中开发的用于测量Vpr特定活性（如核转运、细胞周期阻滞和细胞凋亡）的多个基于裂变酵母细胞的测定将适用于高通量平台，以筛选Vpr的潜在抑制剂。所提出的检测方法还可以识别影响细胞增殖和凋亡的化合物，这两个细胞过程参与癌症的发展。