MS-based screening of candidate inhibitors of protein-RNA and RNA-RNA interaction

基于 MS 筛选蛋白质-RNA 和 RNA-RNA 相互作用的候选抑制剂

• 批准号: 7555867
• 负责人: Daniele Fabris
• 金额: $ 14.8万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 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 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7555867
• 关键词: Affect; Anti-Retroviral Agents; Antiviral Therapy; Area; Binding; Biological Assay; Cells; Characteristics; Chemicals; Complement; Complex; Condition; Cyclotrons; Detection; Development; Diagnostic; Dimerization; Dissociation; Drug resistance; Effectiveness; Electrospray Ionization; Endopeptidases; Equilibrium; Fourier Transform; Genome; Goals; HIV; HIV Infections; HIV-1; In Vitro; Individual; Integrase; Intervention; Ions; Label; Libraries; Life Cycle Stages; Ligands; Mass Spectrum Analysis; Mediating; Methods; Molecular Bank; Multi-Drug Resistance; Nature; Nucleocapsid; Nucleocapsid Proteins; Output; Peptide Hydrolases; Pharmaceutical Preparations; Pilot Projects; Pliability; Procedures; Process; Proteins; Protocols documentation; RNA; RNA-Directed DNA Polymerase; Radioactive; Reading; Resistance; Resolution; Risk; Screening procedure; Signal Transduction; Solutions; United States National Institutes of Health; Viral; Virus; base; chromophore; drug development; gag Gene Products; inhibitor/antagonist; molecular mass; novel therapeutics; pandemic disease; prospective; repository; small molecule; tool; transmission process

DESCRIPTION (provided by applicant): This project is aimed at the development of mass spectrometry-based assays for the identification of small- molecule ligands capable of disrupting the binding of HIV-1 nucleocapsid (NC) protein with stemloop domains of the genome packaging signal ((-RNA). These specific protein-RNA and RNA-RNA interactions mediate critical functions in the process of genome recognition, dimerization, and packaging, which constitute ideal targets for the development of new therapeutic strategies. With the goal of identifying small-molecule candidates that may disrupt such processes, we propose to employ electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry to determine the effects of potential ligands on these specific interactions. The proposed assays will require simple mixing of prospective ligand with preformed protein-RNA and RNA-RNA assemblies. After binding equilibria are re-established, the unique molecular masses and isotopic compositions characteristic of the different complexes in solution will allow for their unambiguous identification by direct ESI-FTICR analysis, thus providing an immediate reading of the assay output. The selected analytical platform will enable the exploration of the widest possible chemical space with no need for the presence of chromophores or radioactive labels. The absence of derivatization and separation procedures will not only expedite analysis, but will also minimize the risk of artifactual results. The pilot study will define the conditions for completing the assays and will evaluate their potential for high-throughput primary screening of extensive ligand libraries. It is expected that this protocol will provide a rapid and sensitive approach for examining compounds included in the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify possible leads for cell-based secondary screening. 1 The growing threat of drug resistant strains has greatly increased the urgency of the search for new therapeutic strategies to control the HIV pandemic. Due to their critical functions in the virus life cycle, the assemblies formed by the nucleocapsid (NC) protein with stemloop domains of the genome packaging signal ((-RNA) constitute ideal targets for intervention, which are still untapped. The highly specific nature of the interactions between NC and the different stemloops offers the opportunity of inhibiting such functions without affecting normal host processes. The proposed assays will provide a robust tool for exploring the chemical space in search of small-molecule ligands capable of disrupting these interactions. The flexibility afforded by the selected analytical platform will enable the detection of species that may bind to target assemblies without inducing dissociation, thus expanding the scope of the information attainable from the assays. Applied to large compound libraries, these assays are expected to enable the identification of small molecules that may serve as leads for the development of inhibitors and diagnostic tools targeting the processes of genome recognition, dimerization, and packaging in HIV-1. 1

描述（由申请方提供）：本项目旨在开发基于质谱的检测方法，用于鉴定能够破坏HIV-1核衣壳（NC）蛋白与基因组包装信号（（-RNA）的茎环结构域结合的小分子配体。这些特定的蛋白质-RNA和RNA-RNA相互作用介导基因组识别、二聚化和包装过程中的关键功能，这些功能构成了开发新治疗策略的理想靶标。为了确定可能破坏这些过程的小分子候选物，我们建议采用电喷雾电离傅里叶变换离子回旋共振（ESI-FTICR）质谱法来确定潜在配体对这些特定相互作用的影响。所提出的测定将需要将预期配体与预先形成的蛋白质-RNA和RNA-RNA组装体简单混合。在重新建立结合平衡后，溶液中不同复合物的独特分子量和同位素组成特征将允许通过直接ESI-FTICR分析对其进行明确鉴定，从而提供测定输出的即时阅读。选定的分析平台将能够探索尽可能广泛的化学空间，而不需要存在发色团或放射性标记。没有衍生化和分离程序不仅可以加快分析，而且还可以最大限度地减少人为结果的风险。试点研究将确定完成测定的条件，并将评估其用于广泛配体文库的高通量初步筛选的潜力。预计该方案将提供一种快速灵敏的方法，用于检查NIH分子库小分子库（MLSMR）中包含的化合物，以确定基于细胞的二级筛选的可能线索。1耐药菌株的威胁日益严重，这大大增加了寻找新的治疗策略以控制艾滋病毒流行的紧迫性。由于它们在病毒生命周期中的关键功能，由核衣壳（NC）蛋白与基因组包装信号（-RNA）的茎环结构域形成的组装体构成了理想的干预靶标，这仍然是未开发的。NC和不同茎环之间相互作用的高度特异性提供了抑制这些功能而不影响正常宿主过程的机会。拟议的测定将提供一个强大的工具，探索化学空间，寻找能够破坏这些相互作用的小分子配体。由所选分析平台提供的灵活性将使得能够检测可能与靶组装体结合而不诱导解离的物质，从而扩大可从测定获得的信息的范围。应用于大型化合物库，这些测定预计能够鉴定小分子，这些小分子可作为开发抑制剂和诊断工具的先导，靶向HIV-1中的基因组识别、二聚化和包装过程。1