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

描述（由申请人提供）：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）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相互作用介导了基因组识别、二聚化和包装过程中的关键功能，构成了开发新治疗策略的理想靶点。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立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。拟议的检测将需要简单地混合预期配体与预先形成的蛋白质- rna和RNA-RNA组装体。重新建立结合平衡后，溶液中不同复合物的独特分子质量和同位素组成特征将允许通过直接ESI-FTICR分析对其进行明确的识别，从而提供检测输出的即时读数。所选择的分析平台将能够探索最广泛的化学空间，而不需要存在发色团或放射性标签。没有衍生化和分离程序不仅可以加快分析速度，还可以最大限度地减少人工结果的风险。试点研究将确定完成检测的条件，并将评估其在广泛配体文库的高通量初步筛选中的潜力。预计该方案将为检测NIH分子文库小分子库（MLSMR）中包含的化合物提供快速和敏感的方法，以确定基于细胞的二次筛选的可能线索。1 .耐药菌株日益增长的威胁大大增加了寻找新的治疗策略以控制艾滋病毒大流行的紧迫性。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在病毒生命周期中具有关键功能，是目前尚未开发的理想干预靶点。NC和不同茎环之间相互作用的高度特异性提供了在不影响正常宿主过程的情况下抑制这些功能的机会。提出的分析将为探索化学空间寻找能够破坏这些相互作用的小分子配体提供一个强大的工具。所选择的分析平台提供的灵活性将能够检测可能与目标组装结合而不诱导解离的物种，从而扩大从分析中可获得的信息范围。应用于大型化合物文库，这些检测有望识别小分子，这些小分子可能为开发针对HIV-1基因组识别、二聚化和包装过程的抑制剂和诊断工具提供线索。1

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