title: Long residence time inhibition of EZH2 in activated polycomb repressive complex 2.
abstract: EZH2/PRC2 catalyzes transcriptionally repressive methylation at lysine 27 of histone H3 and has been associated with numerous cancer types. Point mutations in EZH2 at Tyr641 and Ala677 identified in non-Hodgkin lymphomas alter substrate specificity and result in increased trimethylation at histone H3K27. Interestingly, EZH2/PRC2 is activated by binding H3K27me3 marks on histones, and this activation is proposed as a mechanism for self-propagation of gene silencing. Recent work has identified GSK126 as a potent, selective, SAM-competitive inhibitor of EZH2 capable of globally decreasing H3K27 trimethylation in cells. Here we show that activation of PRC2 by an H3 peptide trimethylated at K27 is primarily an effect on the rate-limiting step (kcat) with no effect on substrate binding (Km). Additionally, GSK126 is shown to have a significantly longer residence time of inhibition on the activated form of EZH2/PRC2 as compared to unactivated EZH2/PRC2. Overall inhibition constant (Ki*) values for GSK126 were determined to be as low as 93 pM and appear to be driven by slow dissociation of inhibitor from the activated enzyme. The data suggest that activation of EZH2 allows the enzyme to adopt a conformation that possesses greater affinity for GSK126. The long residence time of GSK126 may be beneficial in vivo and may result in durable target inhibition after drug systemic clearance.
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D and ‡Platform Technology and Sciences, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, Pennsylvania 19426, United States.
authors.forename: Glenn S
authors.initials: GS
authors.lastname: Van Aller
authors.forename: Melissa Baker
authors.initials: MB
authors.lastname: Pappalardi
authors.forename: Heidi M
authors.initials: HM
authors.lastname: Ott
authors.forename: Elsie
authors.initials: E
authors.lastname: Diaz
authors.forename: Martin
authors.initials: M
authors.lastname: Brandt
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Schwartz
authors.forename: William H
authors.initials: WH
authors.lastname: Miller
authors.forename: Dashyant
authors.initials: D
authors.lastname: Dhanak
authors.forename: Michael T
authors.initials: MT
authors.lastname: McCabe
authors.forename: Sharad K
authors.initials: SK
authors.lastname: Verma
authors.forename: Caretha L
authors.initials: CL
authors.lastname: Creasy
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tummino
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Kruger
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D000071221:Enhancer of Zeste Homolog 2 Protein; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D008745:Methylation; D009707:Nucleosomes / Q000187:drug effects / Q000201:enzymology; D017354:Point Mutation; D063151:Polycomb Repressive Complex 2 / Q000037:antagonists & inhibitors* / Q000235:genetics; D011485:Protein Binding; D011728:Pyridones / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; C577920:GSK-2816126; D007211:Indoles; D009707:Nucleosomes; D011728:Pyridones; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2