Developing Reliable In Silico Methods to Design Small Molecules Targeting RNA

描述(申请人提供)：microRNAs(MiRNAs)是一种小的非编码RNAs，通过靶向mRNA降解或翻译抑制来调节基因表达。不足为奇的是，miRNA表达异常会导致疾病，特别是癌症。在此，我们建议进行一系列研究，以探索与乳腺癌相关的miRNA前体结合并抑制其在乳腺癌细胞中的生物发生的化合物的治疗潜力。这些化合物的开发是我们目前资助的R01基金(R01-GM097455)的直接结果，在该基金中，我们建议开发计算方法来设计渴望和选择性结合miRNA前体的小分子。这些研究有可能将新的抗癌疗法推向临床，并能够开发针对导致其他疾病的miRNAs的一般策略，如丙型肝炎感染、阿尔茨海默病和心脏病等。我们的母公司R01建议：(I)建立一种计算方法，使用RNA基序-小分子相互作用的数据库来识别所需RNA靶标的铅小分子，并从转录本中识别新的RNA靶标；以及(Ii)使用这些计算工具来识别与人miRNAs结合的铅小分子，并评估它们在哺乳动物细胞系中抑制生物发生的作用。事实上，这种方法被用来合理地设计一种小分子，它选择性地结合致癌的miRNA前体miR-96，并抑制其在乳腺癌细胞中的生物发生。重要的是，miRNA图谱研究表明，我们的小分子比Anagomir更具选择性，Anagomir是最先进的miRNA靶向方式！因此，这种小分子是推进癌症动物模型并测试小分子确实可以药物“无法下药”的RNA靶标的假设的典范案例。具体目标是：目的1：评价我们设计的Pri-miR-96小分子在三阴性和转移性乳腺癌模型中的治疗潜力。目的2：验证我们设计的化合物的体内靶点、化合物选择性和作用机制。此前，我们的团队已经证明，小分子可以被设计成与活细胞中的RNA靶标反应。这种共价方法显著提高了生物活性(2500倍)，并可用于验证细胞靶点 小分子。目的3：评估在pri-miR-96中同时结合DROSHA加工位点和邻近内环的二聚体小分子。我们设计了一种有效的二聚体小分子，在体外针对pri-miR-96(比我们的铅单体小分子强约500倍)。我们将使用原位异种移植模型，评估该化合物在MDA-MB-231乳腺癌细胞以及转移和耐药变异株中的治疗潜力。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting a mRNA for degradation or translational repression. Not surprisingly, aberrant miRNA expression can cause disease, in particular cancers. Herein, we propose a series of investigations to explore the therapeutic potential of compounds that bind a miRNA precursor associated with breast cancer and inhibit its biogenesis in breast cancer cells. These compounds were developed as a direct result of our currently funded R01 grant (R01- GM097455) in which we proposed to develop computational methods to design small molecules that avidly and selectively bind miRNA precursors. These studies have the potential to advance new anti-cancer therapies to the clinic and to enable development of general strategies to target miRNAs that cause other diseases such as Hepatitis C infections, Alzheimer's disease, and heart disease, among others. Our parent R01 grant proposes to: (i) establish a computational approach to identify lead small molecules for a desired RNA target using a database of RNA motif-small molecule interactions and to identify novel RNA targets from transcriptomes; and (ii) use these computational tools to identify lead small molecules that bind human miRNAs and evaluate them for inhibiting biogenesis in mammalian cell lines. Indeed, this approach was employed to rationally design a small molecule that selectively binds an oncogenic miRNA precursor, miR- 96, and inhibits its biogenesis in breast cancer cells. Importantly, miRNA profiling studies reveal that our small molecule is more selective than an antagomir, the state-of-the-art miRNA targeting modality! Thus, this small molecule is an exemplary case to push forward into animal models of cancer and test the hypothesis that small molecules can indeed drug "undruggable" RNA targets. The Specific Aims are: Aim 1: Evaluate the therapeutic potential of our designed pri-miR-96 small molecule in models of triple negative and metastatic breast cancer. Aim 2: Validate in vivo targets, compound selectivity and mechanism of action of our designer compounds. Previously, our group has shown that small molecules can be designed to react with RNA targets in live cells. This covalent approach significantly improves bioactivity (>2500-fold) and can be used to validate the cellular targets of small molecules. Aim 3: Evaluate a dimeric small molecule that simultaneously binds the Drosha processing site and an adjacent internal loop in pri-miR-96. We have designed a potent dimeric small molecule that targets pri-miR-96 in vitro (~500-fold more potent than our lead monomeric small molecule). We will evaluate the therapeutic potential of this compound in MDA-MB-231 breast cancer cells and metastatic and drug-resistant variants, using orthotopic xenograft models.