In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types. Additionally, somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 occur in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The Y641 residue is the most frequently mutated residue, with up to 22% of germinal centre B-cell DLBCL and follicular lymphoma harbouring mutations at this site. These lymphomas have increased H3K27 tri-methylation (H3K27me3) owing to altered substrate preferences of the mutant enzymes. However, it is unknown whether specific, direct inhibition of EZH2 methyltransferase activity will be effective in treating EZH2 mutant lymphomas. Here we demonstrate that GSK126, a potent, highly selective, S-adenosyl-methionine-competitive, small-molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.

A central challenge in structure-based ligand design is the accurate prediction of binding free energies. Here we apply alchemical free energy calculations in explicit solvent to predict ligand binding in a model cavity in T4 lysozyme. Even in this simple site, there are challenges. We made systematic improvements, beginning with single poses from docking, then including multiple poses, additional protein conformational changes, and using an improved charge model. Computed absolute binding free energies had an RMS error of 1.9 kcal/mol relative to previously determined experimental values. In blind prospective tests, the methods correctly discriminated between several true ligands and decoys in a set of putative binders identified by docking. In these prospective tests, the RMS error in predicted binding free energies relative to those subsequently determined experimentally was only 0.6 kcal/mol. X-ray crystal structures of the new ligands bound in the cavity corresponded closely to predictions from the free energy calculations, but sometimes differed from those predicted by docking. Finally, we examined the impact of holding the protein rigid, as in docking, with a view to learning how approximations made in docking affect accuracy and how they may be improved.

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k cat /K m ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k cat /K m ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k cat /K m ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.

We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed relative binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 (phenol) and 1.1 (catechol) kcal/mol. To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0Å (diverse set), 1.8Å (phenol derived predictions) and 1.2Å (catechol derived predictions). We found that to predict accurate affinities and rank-orderings required nearnative starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe these results can help guide future improvements in physics-based absolute binding free energy methods.