Simard Jeffrey R, Plant Matthew, Emkey Renee, Yu Violeta
Amgen, Inc., Cambridge, Massachusetts 02142, USA.
Assay Drug Dev Technol. 2013 Apr;11(3):152-62. doi: 10.1089/adt.2012.481. Epub 2013 Feb 14.
The methylation state of lysine residues within histone H3 is a major determinant of active and inactive regions of the genome. Enhancer of Zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is part of the polycomb repressive complex 2 (PRC2). Elevated EZH2 expression levels have been linked to hypertrimethylation of histone H3 lysine 27 (H3K27), repression of tumor repressor genes, and the onset of several types of cancers. We used the AlphaLISA technology to develop a high-throughput assay for identifying small molecule inhibitors of EZH2. AlphaLISA Acceptor Beads coated with antibodies directed against methylated H3K27 provided a sensitive method of detecting EZH2 activity through measurement of K27 methylation of a biotinylated H3-based peptide substrate. Optimized assay conditions resulted in a robust assay (Z'>0.7) which was successfully implemented in a high-throughput screening campaign. Small molecule inhibitors identified by this method may serve as powerful tools to further elucidate the potential importance of EZH2 in the development and treatment of cancer.
组蛋白H3内赖氨酸残基的甲基化状态是基因组活性和非活性区域的主要决定因素。锌指增强子同源物2（EZH2）是一种组蛋白赖氨酸甲基转移酶，是多梳抑制复合物2（PRC2）的一部分。EZH2表达水平升高与组蛋白H3赖氨酸27（H3K27）的超甲基化、肿瘤抑制基因的抑制以及几种癌症的发生有关。我们使用AlphaLISA技术开发了一种高通量检测方法，用于鉴定EZH2的小分子抑制剂。涂有针对甲基化H3K27的抗体的AlphaLISA受体珠提供了一种通过测量生物素化的基于H3的肽底物的K27甲基化来检测EZH2活性的灵敏方法。优化的检测条件产生了一种稳健的检测方法（Z'>0.7），该方法已成功应用于高通量筛选活动。通过这种方法鉴定的小分子抑制剂可能成为进一步阐明EZH2在癌症发展和治疗中的潜在重要性的有力工具。
