Allali-Hassani Abdellah, Wasney Gregory A, Siarheyeva Alena, Hajian Taraneh, Arrowsmith Cheryl H, Vedadi Masoud
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
J Biomol Screen. 2012 Jan;17(1):71-84. doi: 10.1177/1087057111422256. Epub 2011 Oct 4.
The histone methyltransferase (HMT) family of proteins consists of enzymes that methylate lysine or arginine residues on histone tails as well as other proteins. Such modifications affect chromatin structure and play a significant regulatory role in gene expression. Many HMTs have been implicated in tumorigenesis and progression of multiple malignancies and play essential roles in embryonic development and stem cell renewal. Overexpression of some HMTs has been observed and is correlated positively with various types of cancer. Here the authors report development of a continuous fluorescence-based methyltransferase assay in a 384-well format and its application in determining kinetic parameters for EHMT1, G9a, PRMT3, SETD7, and SUV39H2 as well as for screening against libraries of small molecules to identify enzyme inhibitors. They also report the development of a peptide displacement assay using fluorescence polarization in a 384-well format to assay and screen protein peptide interactions such as those of WDR5 and EED, components of MLL and EZH2 methyltransferase complexes. Using these high-throughput screening methods, the authors have identified potent inhibitors and ligands for some of these proteins.
组蛋白甲基转移酶（HMT）蛋白家族由能够使组蛋白尾部的赖氨酸或精氨酸残基以及其他蛋白质发生甲基化的酶组成。此类修饰会影响染色质结构，并在基因表达中发挥重要的调节作用。许多HMT与多种恶性肿瘤的发生和进展有关，在胚胎发育和干细胞更新中发挥着重要作用。已观察到某些HMT的过表达，且与多种类型的癌症呈正相关。在此，作者报告了一种基于荧光的384孔板格式的连续甲基转移酶测定方法的开发，及其在确定EHMT1、G9a、PRMT3、SETD7和SUV39H2的动力学参数以及针对小分子文库进行筛选以鉴定酶抑制剂方面的应用。他们还报告了一种基于荧光偏振的384孔板格式的肽置换测定方法的开发，用于测定和筛选蛋白质 - 肽相互作用，如MLL和EZH2甲基转移酶复合物的组成部分WDR5和EED之间的相互作用。使用这些高通量筛选方法，作者已经为其中一些蛋白质鉴定出了有效的抑制剂和配体。
