cb4008748_si_001.pdf (544.6 kB)
Long Residence Time Inhibition of EZH2 in Activated Polycomb Repressive Complex 2
journal contribution
posted on 2014-03-21, 00:00 authored by Glenn S. Van Aller, Melissa Baker Pappalardi, Heidi M. Ott, Elsie Diaz, Martin Brandt, Benjamin J. Schwartz, William H. Miller, Dashyant Dhanak, Michael
T. McCabe, Sharad K. Verma, Caretha L. Creasy, Peter J. Tummino, Ryan
G. KrugerEZH2/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.