Lin, Ying, Fan, Hong, Frederiksen, Mathias, Zhao, Kehao, Jiang, Lei, Wang, Zhaofu, Zhou, Shaolian, Guo, Weihui, Li, Shu, Harrington, Edmund, Meier, Peter, Scheufler, Clemens, Xu, Yao-Chang, Atadja, Peter, Lu, Chris, Li, En and Gu, Justin (2012) Detecting SAM-induced conformational change of a histone methyltransferase using a homogenous time-resolved fluorescence-based binding assay. Analytical Biochemistry, 423. pp. 171-177.

Abstract

A homogenous time-resolved fluorescence (HTRF)-based binding assay has been established to measure the binding of the histone methyltransferase (HMT) G9a to its inhibitor CJP702 (a biotin analog of a known peptide-pocket inhibitor, BIX-01294). This assay was used to characterize G9a inhibitors. As expected, the peptide-pocket inhibitors decreased the G9a-CJP702 binding signal in a concentration-dependent manner. In contrast, the SAM-pocket compounds, SAM and sinefungin significantly increased the G9a-CJP702 binding signal; whereas SAH showed minimal effect. Enzyme kinetics studies showed that CJP702 is an uncompetitive inhibitor (vs SAM) that has a strong preference for the E:SAM form of the enzyme. Other data presented suggest that the SAM/sinefungin-induced increase in the HTRF signal is secondary to the increased E:SAM or E:sinefungin level. Thus the G9a-CJP702 binding assay can not only be used to characterize the peptide-pocket inhibitors; it can also detect the subtle conformational differences induced by the binding of different SAM-pocket compounds. To our knowledge, this is the first demonstration of using an uncompetitive inhibitor as a probe to monitor the conformational change induced by compound-binding with a HTRF assay.

Item Type:	Article
Date Deposited:	13 Oct 2015 13:15
Last Modified:	13 Oct 2015 13:15
URI:	https://oak.novartis.com/id/eprint/5231