UNC 2400

UNC 2400, a close analogue of UNC1999, is a negative control of UNC1999 that inhibits EZH2 and EZH1 [1].

Histone-lysine N-methyltransferase (EZH1 or EZH2) 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. Hypertrimethylation of H3K27 and overexpression of EZH2 are involved in cancers [1].

UNC 2400 is a negative control of UNC1999. UNC 2400 inhibited EZH2 with IC50 value of 13 µM and exhibited >1,000-fold less potency than UNC1999. Also, UNC 2400 inhibited EZH1 and EZH2 Y641F with IC50 values of 62 and >200 µM, respectively. In MCF10A cells, UNC 2400 exhibited little inhibition of H3K27me3 levels and had low cellular toxicity with EC50 value of 27.5 µM that was similar to UNC1999. In DB cells, UNC 2400 (3 µM) did not significantly inhibit DB cell proliferation and had no influence on H3K27me3 or EZH2 levels [1].

Reference:
[1].  Konze KD, Ma A, Li F, et al. An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem Biol, 2013, 8(6): 1324-1334.

Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia.[Pubmed:25395428]

Blood. 2015 Jan 8;125(2):346-57.

Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999's on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.

An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1.[Pubmed:23614352]

ACS Chem Biol. 2013;8(6):1324-34.

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 EZH2(Y641N) 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 analogue of UNC1999 with potency >1,000-fold lower than that of 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.