Developing Reliable In Silico Methods to Design Small Molecules Targeting RNA

描述（由申请人提供）：RNA具有基本和多样的生物学功能，包括催化、基因调控和细胞定位。功能异常可导致癌症、囊性纤维化和肌肉萎缩症等疾病。总之，RNA是小分子干预的重要治疗靶标。由于基因组测序的努力，新的RNA靶点被发现的速度正在增加。相比之下，设计或筛选用于结合和调节RNA功能的配体的方法远远落后。用于鉴定结合RNA的小分子的最常用方法是高通量筛选。不幸的是，RNA靶点的命中率远低于蛋白质。这部分是由于缺乏对小分子中使它们易于结合RNA的特征的理解，以及缺乏对可靶向的RNA空间的理解，这显著阻碍了RNA治疗学和RNA化学生物学领域的发展。拟议工作的目标是开发计算方法来识别基因组序列和二级结构中的可药用RNA靶标，并合理设计小分子配体来靶向它们。该提案的具体目标是：（1）建立一种计算方法来识别可以用小分子靶向的RNA。a.）构建一个可搜索的RNA基序-配体相互作用的数据库。数据库将使用开放源码MySQL关系引擎构建，并可通过命令行界面和网络访问; B.）开发搜索RNA二级结构以鉴定具有结合数据库中的配体的两个或更多个基序的RNA结构域的计算机程序; c.）更新可检索的数据库，因为在文献中可以获得关于靶向RNA的小分子的新信息。通过网络上的服务器提供对软件的开放访问。(2)使用这些计算工具搜索所有人类pri-microRNA和pre-microRNA的二级结构，以与目标1中描述的数据库重叠。基于这种重叠，配体将被设计为结合存在于感兴趣的microRNA中的两个或更多个基序，并且将使用模块化组装方法合成，以增强靶亲和力和特异性。(3)测试单体和模块化组装的配体，用于靶向它们被设计为使用生物化学技术结合的microRNA。在存在和不存在总细胞RNA的情况下测定配体结合位点，并使用双荧光素酶报告系统测定体内配体效价。项目描述第6页 公共卫生相关性：RNA是一种重要的生物分子，与疾病状态有关;然而，很少有药物靶向它。拟议研究的目标是汇编所有已知的RNA-小分子相互作用，并开发计算工具来设计与疾病相关的RNA结合并调节其功能的配体。公共卫生相关性声明第7页

DESCRIPTION (provided by applicant): RNA has essential and diverse biological functions including catalysis, gene regulation, and cellular localization. Aberrant function can lead to diseases such as cancer, cystic fibrosis, and muscular dystrophy. Taken together, RNA is an important therapeutic target for small molecule intervention. The rate at which new RNA targets are being discovered is increasing due to genomic sequencing efforts. In contrast, methods to design or screen ligands for binding to and modulating RNA function are lagging far behind. The most common method employed to identify small molecules that bind RNA is high throughput screening. Unfortunately, the hit rates for RNA targets are much lower than proteins. This is in part due to the lack of understanding about the features in small molecules that pre-dispose them for binding RNA and a lack of understanding about the RNA space that is targetable, significantly hampering developments in the fields of RNA therapeutics and RNA chemical biology. The goal of the proposed work is to develop computational methods to identify druggable RNA targets in genomic sequences and secondary structures and to rationally design small molecule ligands to target them. The specific aims of the proposal are: (1) Establish a computational approach to identify RNAs that can be targeted with a small molecule. a.) Construct a searchable database of RNA motif-ligand interactions that have been previously identified. The database will be constructed using the open-source MySQL relational engine and will be accessible via a command-line interface and the web; b.) Develop a computer program that searches RNA secondary structures to identify RNA domains that have two or more motifs that bind a ligand in the database; c.) Update the searchable database as new information on small molecules targeting RNA becomes available in the literature. Provide open access to the software via a server on the web. (2) Use these computational tools to search the secondary structures of all human pri- and pre- microRNAs for overlap with the database described in Aim 1. Based on this overlap, ligands will be designed that bind two or more motifs present in a microRNA of interest and will be synthesized using a modular assembly approach in order to enhance target affinity and specificity. (3) Test monomeric and modularly assembled ligands for targeting the microRNA to which they were designed to bind using biochemical techniques. Determine the ligand binding site in the presence and absence of total cellular RNA and ligand potency in vivo using a dual luciferase reporter system. Project Description Page 6 PUBLIC HEALTH RELEVANCE: RNA is an important biomolecule that is associated with disease states; however, very few drugs target it. The goal of the proposed research is to compile all known RNA-small molecule interactions and develop computational tools to design ligands that bind to an RNA associated with disease and modulate its function. Public Health Relevance Statement Page 7