Lysine methyltransferases (KMTs) are involved in epigenetic gene regulation by covalent modification of histones. Domainex has addressed key technical challenges associated with KMTs, including generating a number of crystal structures, assays and a KMT chemical library. For instance, an EZH2 screening assay was developed using a peptide substrate and a more physiologically relevant substrate H3/H4 tetramer (Figure 1a&b). The biochemical assay was then utilised to screen a KMT library identified using LeadBuilder virtual screening focusing on both the SAM and peptide binding sites (Figure 2). KMT specific chemotypes were resolved for EZH2, SMYD2, SMYD3, G9a and NSD2. Binding conformations of chemotypes were illustrated against G9a using MicroScale Thermophoresis (MST) technology, and binding dependency of SAM evaluated (Figure 3a&b). Finally, EZH2 is known to methylate histone H3K27(me3) and we have demonstrated cellular efficacy of tool compounds via both FACS and Western Blotting techniques (Figure 4).
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Figure 1a: Development of an EZH2 AlphaScreen assay. Purified recombinant EZH2 complex was tested for its ability to methylate histone based substrates and resolve SAM ATP Kmapp. Rate of reaction was elucidated from a serial dilution of SAM in the presence of fixed concentrations of biotinylated peptide and EZH2 complex. Methylation was detected using alpha donor streptavidin and alpha acceptor anti- H3K27me3 beads.
Figure 1b: Effect of EPZ6438 on EZH2 enzyme activity. EPZ6438, a known EZH2 inhibitor, was titrated and pre-incubated with EZH2 complex plus SAM. Enzymatic reaction was initiated by the addition of 100nM biotinylated substrate. Assay was performed at 10 times SAM Km. Assay was stopped and IC50 values generated using four parameter fit.
Figure 2: KMT small molecule inhibition profile. A KMT library was identified through a LeadBuilder virtual screen. From this 1130 hit compounds were tested at 100μM for EZH2 inhibitory activity. In addition, selectivity assays were screened including SMYD3, SMYD2, NSD2 and G9a (green denotes >75% inhibition). Both selective and non-selective inhibitors were identified, along with other profiles.
Figure 3a: Binding of Domainex proprietary G9a inhibitor in the presence of SAM. Purified recombinant G9a was fluorescently labelled via lysine linkage. Low nano molar concentration of G9a was incubated with a titration of SAM and loaded onto the Nanotemper pico Monolith instrument. Using MST technology, changes in mobility of G9a were recorded and Kd resolved for SAM binding at 55μM.
Figure 3b: Effect of DMX compound on G9a mobility in the presence and absence of SAM. A titration of Domainex proprietary compound was incubated with fluorescently labelled G9a in the presence of; 10 times Kd SAM (blue line), Kd SAM (green line) and no SAM (orange lines). Compound binding is dependent on SAM binding.
Figure 4: Quantification of H3K27me3 in SU-DHL-6 cell lines. H3K27(me3) levels were measured in the SU-DHL-6 cell line either by FACS or Western blotting using an antibody to the methylation mark. Secondary antibodies were either labelled with Alexa Fluor-657 or AP respectively. The standard compound, EPZ6438 was tested at 20μM using FACS or titrated using Western blotting techniques. Decrease in methylation was observed in the presence of the EZH2 inhibitor.