Detecting S-adenosyl-l-methionine-induced conformational change of a histone methyltransferase using a HTRF-based binding assay

Literature Life Science

Development of an HTRF assay to measure the binding of the HMT to its inhibitors

Abstract

A homogeneous time-resolvedfluorescence (HTRF)-based bindingassay has been established to measure the binding of the histonemethyltransferase (HMT) G9a to its inhibitor CJP702 (a biotin analog of the known peptide-pocket inhibitor, BIX-01294). This assay was used to characterize G9a inhibitors. As expected, the peptide-pocket inhibitors decreased the G9a-CJP702binding signal in a concentration-dependent manner. In contrast, the S-adenosyl-L-methionine (SAM)-pocket compounds, SAM and sinefungin, significantly increased the G9a-CJP702 binding signal, whereas S-adenosyl-L-homocysteine (SAH) showed minimal effect. Enzyme kinetic 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 an increased E:SAM or E:sinefungin concentration. Thus, the G9a-CJP702binding assay not only can be used to characterize the peptide-pocket inhibitors but also can 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 changeinduced by compound binding with an HTRF assay.

Details

Anal Biochem. 2012;423(1):171-7.

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