title: Detecting S-adenosyl-L-methionine-induced conformational change of a histone methyltransferase using a homogeneous time-resolved fluorescence-based binding assay.
abstract: A homogeneous 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 the 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 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-CJP702 binding 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 change induced by compound binding with an HTRF assay.
authors.affiliation: China Novartis Institute for Biomedical Research, Pudong New Area, Shanghai 201203, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Lin
authors.forename: Hong
authors.initials: H
authors.lastname: Fan
authors.forename: Mathias
authors.initials: M
authors.lastname: Frederiksen
authors.forename: Kehao
authors.initials: K
authors.lastname: Zhao
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.forename: Zhaofu
authors.initials: Z
authors.lastname: Wang
authors.forename: Shaolian
authors.initials: S
authors.lastname: Zhou
authors.forename: Weihui
authors.initials: W
authors.lastname: Guo
authors.forename: Jun
authors.initials: J
authors.lastname: Gao
authors.forename: Shu
authors.initials: S
authors.lastname: Li
authors.forename: Edmund
authors.initials: E
authors.lastname: Harrington
authors.forename: Peter
authors.initials: P
authors.lastname: Meier
authors.forename: Clemens
authors.initials: C
authors.lastname: Scheufler
authors.forename: Yao-Chang
authors.initials: YC
authors.lastname: Xu
authors.forename: Peter
authors.initials: P
authors.lastname: Atadja
authors.forename: Chris
authors.initials: C
authors.lastname: Lu
authors.forename: En
authors.initials: E
authors.lastname: Li
authors.forename: X Justin
authors.initials: XJ
authors.lastname: Gu
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D001381:Azepines / Q000737:chemistry; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011799:Quinazolines / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D001381:Azepines; C518299:BIX 01294; D005456:Fluorescent Dyes; D011799:Quinazolines; D011994:Recombinant Proteins; D012435:S-Adenosylhomocysteine; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D000241:Adenosine; C006235:sinefungin