Split-luciferase Epigenetic Assays for Drug Discovery

项目摘要 表观遗传控制对于维持转录完整性是必不可少的。动态调节 表观基因组是通过DNA和组蛋白上的一系列翻译后修饰介导的 尾组蛋白修饰是由酶介导的，这些酶可以广泛地分为“作家”和“编辑者”。 催化化学修饰的添加，去除修饰的“橡皮擦”， “阅读器”可以通过特定的蛋白质结构域识别化学修饰。写作 酶消除组蛋白标记是一个动态过程，当失调时， 与癌症、炎症和神经系统疾病有关。 在本应用中，我们重点关注两类组蛋白修饰酶，即甲基赖氨酸 转移酶（KMT）和赖氨酸脱甲基酶（KDM），旨在开发和验证细胞- 基于靶向KMT和KDM的测定。这些试验，基于三杂交分裂 荧光素酶系统是可逆的，允许抑制剂结合的剂量依赖性定量。 这些努力既重要又创新，因为它们可以报告药物的直接结合 靶蛋白在其预定的作用位点，从而促进铅的产生 用于研究组蛋白生物学的治疗候选物和化学探针的鉴定， 途径。

Project Summary Epigenetic control is essential for maintaining transcriptional integrity. Regulation of the dynamic epigenome is mediated through a range of post translational modifications on DNA and histone tails. Histone modifications are mediated by enzymes which can be broadly classified into “writers” that catalyze the addition of chemical modifications, “erasers” that remove the modifications, and “readers” that can recognize chemical modifications through specific protein domains. The writing and erasing of histone marks by enzymes is a dynamic process, and when dysregulated is associated with cancer, inflammatory and neurological diseases. In this application we focus on the two classes of histone modifying enzymes, lysine methyl transferases (KMTs) and lysine demethylases (KDMs), and aim to develop and validate cell- based assays targeting both KMTs and KDMs. These assays, based on a three-hybrid split luciferase system, are reversible allowing dose dependent quantification of inhibitor binding. These efforts are both significant and innovative as they can report on the direct binding of a drug to the target protein at its intended site of action, thereby facilitating the generation of lead therapeutic candidates and identification of chemical probes for studying histone biology and pathways.