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

研究概述HIV-1病毒蛋白R(VPR)是一种多方面的蛋白质，通过一些独特的方式调节宿主细胞，促进HIV-1感染和病毒致病。VPR是HIV-1在单核细胞、巨噬细胞和不完全激活的T淋巴细胞等未分裂细胞中感染所必需的，因为它能够促进病毒前整合复合体(PIC)的核运输进入细胞核。VPR诱导细胞周期G2/M期停滞，从而优化病毒复制和逃避宿主免疫反应。此外，VPR引起的细胞凋亡被认为是导致CD4T细胞耗竭的原因之一。病毒复制激活和CD4T细胞耗尽被认为是导致艾滋病的标志。感染VPR缺陷病毒的恒河猴、黑猩猩或人类受试者与疾病进展缓慢相关的发现表明，抗VPR药物可能在对抗HIV-1感染的抗逆转录病毒治疗中有用。由于VPR具有阻断宿主细胞周期G2-M转换和诱导细胞凋亡的独特而有效的能力，针对这些VPR活性的新的分子探针的识别也可能在抗肿瘤新药设计中具有潜在的应用价值。这项建议的目标是使我们实验室建立的基于裂解酵母细胞的多重分析方法用于自动化高通量分子筛选(HTS)，以针对三种特定的VPR活性，即细胞凋亡、细胞周期G2/M调节和核运输，寻找新的分子探针。每个VPR活性将首先通过一次检测进行测量，并通过二次检测进行确认。相应的哺乳动物化验也将用于验证酵母筛选的结果。这些分析中的每一种都将根据96孔格式进行调整和验证，如果可能的话，还会进一步小型化。最后，所有这些分析都将被配置为HTS格式，并将通过使用一个小的ChemDiv40000化合物库进行中试运行。在原始HTS中确定的“HITS”的重要性将在哺乳动物细胞系统中进一步验证，无论是单独使用VPR还是在HIV-1感染的背景下。提出了两个具体的目标，以适应和配置基于分裂酵母细胞的分析到HTS格式，以筛选VPR介导的活性的小分子探针。在第一个目标中，我们将把我们实验室在过去11年中开发的三套基于分裂酵母的分析方法调整为96孔微滴定板格式，并对其进行优化，以便于操作和自动化。相应的哺乳动物方法也将用于验证酵母筛选中发现的小分子。完成这一具体目标将使我们能够展示所述每一种分析的健壮性质和重现性，此外，还将简化读数，使其更易于进行自动化分析；第二个具体目标将在马里兰大学巴尔的摩分校(UMB)的HTS核心设施进行，以进一步配置我们的HTS分析方法，并通过使用小型ChemDiv40000化合物库进行试运行。在适应和验证之后，这些基于分裂酵母细胞的分析应该准备好用于HTS，并参加分子文库筛选中心网络(MLSCN)，以针对每个特定的VPR活性识别额外的分子探针。总之，拟议研究的成功完成将使我们能够将基于裂解酵母的分析方法调整为HTS的格式，首次针对VPR进行初级药物筛选，并为我们提供参与NIH分子图书馆路线图倡议和分子图书馆筛选中心网络(MLSCN)的机会。已确定的小分子应该提供具有重要功能的先导化合物，以帮助我们在未来设计更多的抗艾滋病毒和癌症药物。

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.