Targeting Nucleic Acids with an Integrated Vitural and Actual Screen

描述(申请人提供)：尽管最近的研究表明，不寻常的核酸结构在基因表达和DNA复制中发挥着深远的作用，但核酸是小分子治疗剂的代表不足的分子靶标。我们提出了一种创新的、跨学科的、高通量的筛选方法，用于发现新的核酸靶向治疗药物。我们的方法将实际的实验筛选试验与虚拟筛选试验相结合。前者是我们发明的一种独特的竞争透析法，用于发现靶向特定核酸序列或结构的小分子。在竞争透析法中，一系列设计的核酸结构和序列被针对共同的测试配基溶液进行透析。在平衡时，更多的配基积累在包含首选结构或序列的透析室中。该分析是热力学严格的，并允许定量测量配体结合自由能。在我们的综合方法中，将实施一种电子虚拟筛选分析，它将包含与竞争透析分析中使用的实际核酸阵列相同的受体阵列。这种虚拟测试可以很容易地筛选出数百万种化合物，以确定它们与具有功能意义的特定核酸结构的选择性结合。来自虚拟屏幕的“命中”将被传递到实际的竞争透析分析以进行验证。通过建议的改进，竞争透析法可以快速筛选数百种化合物。我们的策略将是通过虚拟屏幕识别选择性识别特定结构的化合物库，然后通过高通量竞争透析严格验证这些“命中”。竞争透析法开发的具体计划包括：i)以96孔板的形式实施该分析(便于自动化)，ii)减少样品体积，iii)扩大具有生物意义的核酸阵列，以及iv)缩短吞吐时间。虚拟屏幕和实际屏幕将通过迭代反馈循环完全整合，以改进评分算法，使用从第一代透析实验获得的实际数据，在该实验中，研究了126种化合物与13种核酸结构的相互作用。我们的方法符合路线图倡议的目标，因为它是跨学科的方法，并强调扩大可用于发现新疗法的工具箱。

DESCRIPTION (provided by applicant): Nucleic acids are under-represented molecular targets for small molecule therapeutic agents, even though recent research shows that unusual nucleic acid structures play a profound role in gene expression and DNA replication. We propose an innovative, interdiscplinary, high-throughput screening approach for the discovery of new nucleic acid-targeted therapeutic agents. Our approach integrates an actual experimental screening assay with a virtual screening assay. The former is a unique competition dialysis assay that we invented for the discovery of small molecules that target specific nucleic acid sequences or structures. In the competiton dialysis assay, an array of designed nucleic acid structures and sequences are dialyzed against a common test ligand solution. At equilibirum, more ligand accumulates in the dialysis cell that contains the preferred structure or sequence. The assay is thermodynamically rigorous, and allows for quantitative measurement of ligand binding free energies. In our integrated approach, an in silico virtual screening assay will be implemented that will contain an array of receptors that is identical to the actual array of nucleic acids used in the competiton dialysis assay. The virtual assay can easily screen millions of compounds for their selective binding to particular nucleic acid structures of functional significance. "Hits" from the virtual screen will be passed to the actual competition dialysis assay for validation. With proposed improvements, the competiton dialysis can rapidly screen hundreds of compounds. Our strategy will be to identify libraries of compounds that selectively recognize particular structures with the virtual screen, then to rigorously verify these "hits" by high-throughput competition dialysis. Specific plans for the development of the competition dialysis assay include : i) implementation of the assay in a 96-well plate format (facilitating automation), ii) reduction of sample volumes, iii) expansion of the biologically significant nucleic acid array, and iv) reduction of throughput time. The virtual and actual screens will be fully integrated by an iterative feedback loop to refine the scoring algorithm, using actual data obtained from the first generation dialysis assay in which the interaction of 126 compounds with 13 nucleic acids structures was studied. Our approach matches the Road Map Initiative goals by its interdisciplinary approach and by its emphasis on expanding the toolbox available for the discovery of new therapeutics.