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Detecting SAM-induced conformational change of a histone methyltransferase using a homogenous time-resolved fluorescence-based binding assay

Lin, Ying and Fan, Hong and Frederiksen, Mathias and Zhao, Kehao and Jiang, Lei and Wang, Zhaofu and Zhou, Shaolian and Guo, Weihui and Li, Shu and Harrington, Edmund and Meier, Peter and Scheufler, Clemens and Xu, Yao-Chang and Atadja, Peter and Lu, Chris and 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

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