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.

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design.

Understanding protein conformational variability remains a challenge in drug discovery. The issue arises in protein kinases, whose multiple conformational states can affect the binding of small-molecule inhibitors. To overcome this challenge, we propose a comprehensive computational framework based on Markov state models (MSMs). Our framework integrates the information from explicit-solvent molecular dynamics simulations to accurately rank-order the accessible conformational variants of a target protein. We tested the methodology using Abl kinase with a reference and blind-test set. Only half of the Abl conformational variants discovered by our approach are present in the disclosed X-ray structures. The approach successfully identified a protein conformational state not previously observed in public structures but evident in a retrospective analysis of Lilly in-house structures: the X-ray structure of Abl with WHI-P154. Using a MSM-derived model, the free energy landscape and kinetic profile of Abl was analyzed in detail highlighting opportunities for targeting the unique metastable states.

Objective-Vascular calcification significantly increases cardiovascular morbidity and mortality. We recently reported that the deficiency of cartilage oligomeric matrix protein (COMP) leads to vascular mineralization. We characterized the COMP-degrading metalloproteinase, a disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7). Here, we tested whether ADAMTS-7 facilitates vascular calcification. Methods and Results-ADAMTS-7 expression was markedly upregulated in calcifying rat vascular smooth muscle cells (VSMCs) in vitro, calcified arteries of rats with chronic renal failure in vivo, and radial arteries of uraemic patients.Silencing of ADAMTS-7 markedly reduced COMP degradation and ameliorated VSMC calcification, whereas ectopic expression of ADAMTS-7 greatly enhanced COMP degradation and exacerbated mineralization. The transcriptional activity of ADAMTS-7 promoter was not altered by high phosphate. We used bioinformatics and quantitative polymerase chain reaction analysis to demonstrate that high-phosphate upregulated ADAMTS-7 mRNA and protein via miR-29a/b repression, which directly targeted the 3′ untranslated region of ADAMTS-7 in VSMCs. MicroRNA (MiR)-29a/b mimic markedly inhibited but miR-29a/b inhibitor greatly enhanced high-phosphate-induced ADAMTS-7 expression, COMP degradation, and subsequent VSMC calcification. ADAMTS-7 silencing significantly diminished miR-29a/b repressionexaggerated VSMC calcification. Conclusion-Our data reveal a novel mechanism by which ADAMTS-7 upregulation by miR-29a/b repression mediates vascular calcification, which may shed light on preventing cardiovascular morbidity and mortality.