An Orally Bioavailable Chemical Probe of the Lysine Methyltransferases EZH2 and EZH1

Abstract

EZH2 or EZH1 is the catalytic subunit of the polycomb repressive complex 2 that catalyzes methylation of histone H3 lysine 27 (H3K27). The trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive post-translational modification. Overexpression of EZH2 and hypertrimethylation of H3K27 have been implicated in a number of cancers. Several selective inhibitors of EZH2 have been reported recently. Herein we disclose UNC1999, the first orally bioavailable inhibitor that has high in vitro potency for wild-type and mutant EZH2 as well as EZH1, a closely related H3K27 methyltransferase that shares 96% sequence identity with EZH2 in their respective catalytic domains. UNC1999 was highly selective for EZH2 and EZH1 over a broad range of epigenetic and non-epigenetic targets, competitive with the cofactor SAM, and non-competitive with the peptide substrate. This inhibitor potently reduced H3K27me3 levels in cells and selectively killed diffused large B cell lymphoma cell lines harboring the EZH2Y641N mutant. Importantly, UNC1999 was orally bioavailable in mice, making this inhibitor a valuable tool for investigating the role of EZH2 and EZH1 in chronic animal studies. We also designed and synthesized UNC2400, a close analog of UNC1999 with >1,000-fold lower potency than UNC1999 as a negative control for cell-based studies. Finally, we created a biotin-tagged UNC1999 (UNC2399) which enriched EZH2 in pull-down studies, and a UNC1999 – dye conjugate (UNC2239) for co-localization studies with EZH2 in live cells. Taken together, these compounds represent a set of useful tools for the biomedical community to investigate the role of EZH2 and EZH1 in health and disease.

Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity

“…Ezh2 lies within the PRC1/2 complex and confers gene silencing by trimethylating histone H3 at lysine 27. To directly analyze the contribution of Ezh2 methyltransferase activity to regulation of NK cell development, we used small-molecule inhibitors UNC1999 and EPZ005687 (23,24) to examine the effect of inhibitors on mouse NK cell development by treatment of HSPCs at the start of cellular differentiation (Fig. 3A).…”

A Role for Widely Interspaced Zinc Finger (WIZ) in Retention of the G9a Methyltransferase on Chromatin

“…Western Blotting and Densitometry-Western blotting was performed using a Typhoon imager (GE Healthcare) as described elsewhere (31) or using a LI-COR Odyssey as described elsewhere (32). Densitometry was performed using ImageJ software or LI-COR Image Studio software.…”

EZH2 Inhibitor GSK126: Metabolism, drug transporter and rat pharmacokinetic studies

“…However, our research needs and financial constraints extended to multiple research groups; it required multi-gram quantities of the drug substance and it was thus more economical to prepare 4 ourselves than to merely procure it. As there are some scientific publications which provide various synthetic procedural information to prepare molecules such as 2-7 (Brackley et al, 2011;Knutson et al, 2012;McCabe et al, 2012;Qia et al, 2012;Verma et al, 2012;Konze et al, 2013), we prepared 4 (Scheme 1) by brominating 2-methyl-3-nitro benzoic acid to give bromo-nitro-benzoic acid 8. Compound 8 was then subjected to addition & cyclization reactions to yield bromoindole methyl ester 9.…”