Development of the Gag/Lysyl tRNA synthetase interaction as a target for anti-HIV

开发 Gag/赖氨酰 tRNA 合成酶相互作用作为抗 HIV 靶点

基本信息

批准号：
7641008
负责人：
LAWRENCE KLEIMAN
金额：
$ 61.23万
依托单位：
SIR MORTIMER B. DAVIS JEWISH GEN HOSP
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7641008
关键词：
Alanine Anisotropy Anti-HIV Therapy Antiviral Agents Binding Binding Proteins Binding Sites Biological Assay C-terminal Capsid Cell Culture Techniques Cells Co-Immunoprecipitations Collaborations Complex Computer Simulation DNA Data Development Dimerization Enzymes Fluorescence Anisotropy Gagging Genome HIV-1 Human In Vitro Label Libraries Life Cycle Stages Lysine-Specific tRNA Lysine-tRNA Ligase Mammalian Cell Maps Measures Minnesota Monitor Mutagenesis Mutate Mutation N Domain NMR Spectroscopy Ohio Peptides Protein Footprinting Proteins RNA RNA-Directed DNA Polymerase Reaction Reporting Resolution Reverse Transcription Scanning Screening procedure Site Small Interfering RNA Techniques Testing Tracer Transfer RNA Universities Viral Viral Proteins Viral Reverse Transcription Virion Virus Work X-Ray Crystallography Yang analog aptamer design drug development gag Gene Products high throughput screening improved in vivo inhibitor/antagonist mutant novel particle prevent small molecule viral DNA viral RNA

项目摘要

DESCRIPTION (provided by applicant): The genome of HIV-1 is composed of RNA, and after this RNA enters a cell, it must be converted to viral DNA, which can then insert into the cell's DNA, and be used to produce new viral protein and viruses. The RNA is converted into DNA by the viral enzyme reverse transcriptase. This enzyme requires a primer to start reverse transcription. The primer for reverse transcription in HIV-1 is human tRNALys3, which is selectively packaged into the virion during viral assembly in the cell, and binds (anneals) to a region in the viral RNA genome termed the primer binding site (PBS). It is from this bound tRNALys3 that reverse transcription begins. The selective packaging of tRNALys3 occurs because the viral protein Gag specifically interacts with a major tRNALys3-binding protein in the cell, lysyl-tRNA synthetase (LysRS), so that both tRNALys3 and LysRS are packaged into the assembling virus. Disrupting this reaction through mutations in either Gag or LysRS prevent tRNALys3 from being packaged into the virus, and greatly reduces reverse transcription and viral infectivity. The Gag/LysRS interaction, therefore, represents a novel target for anti-HIV-1 therapy. In this project, we are seeking to identify small molecule inhibitors of this interaction. This will be done using high throughput screening (HTS) of libraries of compounds to find molecules that inhibit the Gag/LysRS interaction. The interaction of LysRS with either Gag or the smaller capsid (CA) molecule will be measured using fluorescent anisotropy (FA), using fluorescently-labeled CA as the tracer in the HTS of the LysRS/CA interaction. The ability of small peptides or RNA molecules to inhibit this interaction will also be investigated, using peptides from Gag or LysRS that are known to be involved in the Gag/LysRS interaction, or SELEX to isolate RNA molecules that specifically bind to CA. Project Narrative Many reports have now demonstrated that the selective packaging into HIV-1 of the primer for reverse transcription, tRNALys3, is essential for the viral life cycle. This selective incorporation of tRNALys3 requires a specific interaction between Gag and LysRS, making the Gag/LysRS interaction an attractive target for antiviral drug development. The feasibility of using a fluorescence anisotropy assay to detect the binding interaction has already been demonstrated, and will be used in this project facilitate the development of rapid screening techniques to search for inhibitors of this novel target, which will then be tested for their ability to specifically inhibit HIV-1 replication.

描述（申请人提供）：HIV-1的基因组由RNA组成，该RNA进入细胞后，必须转化为病毒DNA，然后病毒DNA才能插入细胞的DNA中，用于产生新的病毒蛋白质和病毒。 RNA通过病毒逆转录酶转化为DNA。该酶需要引物来启动逆转录。 HIV-1 中逆转录的引物是人 tRNALys3，它在细胞中的病毒组装过程中被选择性包装到病毒粒子中，并与病毒 RNA 基因组中称为引物结合位点 (PBS) 的区域结合（退火）。逆转录正是从结合的 tRNALys3 开始。 tRNALys3 的选择性包装之所以发生，是因为病毒蛋白 Gag 与细胞中主要的 tRNALys3 结合蛋白赖氨酰-tRNA 合成酶 (LysRS) 特异性相互作用，从而将 tRNALys3 和 LysRS 都包装到组装病毒中。通过 Gag 或 LysRS 突变破坏这一反应，可以防止 tRNALys3 被包装到病毒中，并大大降低逆转录和病毒感染性。因此，Gag/LysRS 相互作用代表了抗 HIV-1 治疗的新靶标。在这个项目中，我们正在寻求识别这种相互作用的小分子抑制剂。这将通过对化合物库进行高通量筛选 (HTS) 来找到抑制 Gag/LysRS 相互作用的分子来完成。 LysRS 与 Gag 或较小的衣壳 (CA) 分子的相互作用将使用荧光各向异性 (FA) 进行测量，使用荧光标记的 CA 作为 LysRS/CA 相互作用的 HTS 中的示踪剂。还将研究小肽或 RNA 分子抑制这种相互作用的能力，使用已知参与 Gag/LysRS 相互作用的 Gag 或 LysRS 肽，或使用 SELEX 来分离特异性结合 CA 的 RNA 分子。项目叙述现在许多报告表明，逆转录引物 tRNALys3 选择性包装到 HIV-1 中对于病毒生命周期至关重要。 tRNALys3 的这种选择性掺入需要 Gag 和 LysRS 之间的特定相互作用，从而使 Gag/LysRS 相互作用成为抗病毒药物开发的有吸引力的靶点。使用荧光各向异性测定来检测结合相互作用的可行性已经得到证明，并将在该项目中使用，以促进快速筛选技术的开发，以寻找该新靶标的抑制剂，然后将测试其能力特异性抑制 HIV-1 复制。