A High-Throughput Screening Platform to Discover RNA Methylation Inhibitors

发现 RNA 甲基化抑制剂的高通量筛选平台

基本信息

批准号：
10705980
负责人：
Kathryn D Meyer
金额：
$ 37.28万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10705980
关键词：
Address Adenosine Antibodies Benchmarking Biological Assay Biology Cancer Biology Cell Differentiation process Cell Line Cell physiology Cells Chemicals Complex Consumption Couples Data Deposition Development Drug Targeting Fluorescence Gametogenesis Gene Expression Goals Human In Vitro Investigation Learning Life Cycle Stages Link Malignant Neoplasms Mass Spectrum Analysis Measurement Measures Memory Messenger RNA Methods Methylation Methyltransferase Modification Molecular Neurons Oncogenes Performance Pharmaceutical Preparations Physiological Processes Play Predisposition Protein Methyltransferases Proteins RNA RNA methylation Reader Regulation Role Structure System Technology Time Tumor Suppressor Proteins Virus cell type cost cost effective cross reactivity design detection method drug candidate drug discovery genetic manipulation genome wide methylation high throughput screening human disease immunoregulation improved in silico in vivo inhibitor neurodevelopment novel novel therapeutics screening sensor small molecule stem cell proliferation therapeutic target tool tumor progression

项目摘要

Summary Cellular mRNAs are susceptible to a variety of chemical modifications that play important roles in regulating gene expression and cellular function. The most abundant internal mRNA modification is m6A, which occurs when adenosine residues become methylated. m6A influences nearly every aspect of the mRNA life cycle and is critical for a variety of physiological processes, such as gametogenesis, neurodevelopment, learning and memory, immune regulation, and stem cell proliferation and differentiation. Additionally, m6A has emerged as an important RNA regulatory mechanism during cancer progression: altered levels of m6A readers, writers, and erasers are observed in several human cancers, and methylation of oncogenes and tumor suppressors has been shown to impact their expression and promote cancer development. Thus, there is a great need both for understanding how m6A is regulated in cells as well as for developing drugs that target the m6A methyltransferase machinery. However, a major limitation has been the lack of simple, cost-effective methods for detecting changes in m6A methylation in cells in a manner that is compatible with high-throughput screening (HTS). Here, we overcome this barrier by developing a novel genetically encoded m6A sensor which provides a simple fluorescent readout for m6A methylation in living cells. Aim 1 will optimize the m6A sensor system and generate cellular tools to facilitate using the system for diverse applications. Aim 2 will develop the m6A sensor into a HTS-compatible system and demonstrate its utility for drug discovery efforts by performing a pilot HTS designed to identify m6A methyltransferase inhibitors. Aim 3 will create molecular tools incorporating the system and measure the performance of the m6A sensor in vivo. Altogether, these studies will provide a much-needed tool for detecting m6A dynamics in cells and will develop an optimized system for HTS-based studies of m6A methylation. Our technology is likely to have an immediate impact both for basic investigations of m6A biology in cancer as well as drug discovery efforts aimed at identifying novel m6A methyltransferase inhibitors.

概括细胞 mRNA 容易受到各种化学修饰的影响，这些修饰在基因调节中发挥着重要作用表达和细胞功能。最丰富的内部 mRNA 修饰是 m6A，它发生在腺苷残基被甲基化。 m6A 几乎影响 mRNA 生命周期的各个方面，并且至关重要对于各种生理过程，如配子发生、神经发育、学习和记忆，免疫调节、干细胞增殖和分化。此外，m6A 已成为重要的癌症进展过程中的 RNA 调控机制：m6A 读取器、写入器和擦除器水平的改变在几种人类癌症中观察到，癌基因和肿瘤抑制基因的甲基化已被证明影响它们的表达并促进癌症的发展。因此，非常需要了解 m6A 在细胞中的调节方式以及开发针对 m6A 甲基转移酶机制的药物。然而，一个主要的限制是缺乏简单、经济有效的方法来检测 m6A 的变化以与高通量筛选 (HTS) 兼容的方式在细胞中进行甲基化。在这里，我们克服了通过开发一种新型基因编码 m6A 传感器来克服这一障碍，该传感器提供简单的荧光读数用于活细胞中的 m6A 甲基化。目标 1 将优化 m6A 传感器系统并生成蜂窝工具方便将该系统用于各种应用。 Aim 2 将 m6A 传感器开发成兼容 HTS 的传感器系统并通过执行旨在识别 m6A 的试点 HTS 来展示其在药物发现工作中的实用性甲基转移酶抑制剂。目标 3 将创建结合该系统的分子工具并测量 m6A传感器的体内性能。总而言之，这些研究将为检测提供急需的工具细胞中的 m6A 动力学，并将开发一个优化系统，用于基于 HTS 的 m6A 甲基化研究。我们的该技术可能会对癌症中 m6A 生物学的基础研究产生直接影响旨在识别新型 m6A 甲基转移酶抑制剂的药物发现工作。