Targeting Nucleic Acids with an Integrated Vitural and Actual Screen

描述（由申请人提供）：尽管最近的研究表明，不寻常的核酸结构在基因表达和DNA复制中起着深远的作用，但核酸是小分子治疗剂中代表性不足的分子靶点。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剂。我们的方法将实际的实验筛选分析与虚拟筛选分析相结合。前者是我们发明的一种独特的竞争透析测定，用于发现靶向特定核酸序列或结构的小分子。在竞争性透析分析中，一系列设计的核酸结构和序列对共同的测试配体溶液进行透析。在平衡状态下，更多的配体在透析细胞中积累，这些配体含有首选的结构或序列。该分析是严格的热力学，并允许定量测量配体结合自由能。在我们的综合方法中，将实施一种计算机虚拟筛选分析，该分析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。虚拟分析可以很容易地筛选数以百万计的化合物选择性结合特定的核酸结构的功能意义。来自虚拟屏幕的“命中”将被传递到实际的竞争透析分析进行验证。随着提出的改进，竞争透析可以快速筛选数百种化合物。我们的策略将是用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”。开发竞争透析分析的具体计划包括：i)以96孔板形式实施分析（促进自动化），ii)减少样本量，iii)扩大具有生物学意义的核酸阵列，iv)减少通量时间。虚拟和实际筛选将通过迭代反馈回路完全集成，以完善评分算法，使用从第一代透析分析中获得的实际数据，其中研究了126种化合物与13种核酸结构的相互作用。我们的方法与路线图计划的目标相匹配，它采用跨学科的方法，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.