Kinase experiment:

The effect of UNC3866 on the methyltransferase activity of G9a, EHMT1, SUV39H1, SUV39H2, SETDB1, SETD8, SUV420H1, SUV420H2, SETD7, MLL1 trimeric complex, MLL3 tetrameric complex, EZH2 trimeric complex, PRMT1, PRMT3, PRMT5-MEP50 complex, PRMT6, PRMT7, PRMT8, PRDM9, SETD2, SMYD2, SMYD3, BCDIN3D and DNMT1 is assessed by monitoring the incorporation of tritium-labeled methyl group to lysine or arginine residues of peptide substrates using Scintillation Proximity Assay (SPA). Assays are performed in a 20 μL reaction mixture containing 3H-SAM at substrate concentrations close to the Km values for each enzyme. Three concentrations (1 μM, 10 μM, and 50 μM) of UNC3866 are used in all selectivity assays. To stop the enzymatic reactions, 7.5 M Guanidine hydrochloride is added, followed by 180 μL of buffer (20 mM Tris, pH 8.0). The reactions are mixed and then transferred to a 96-well FlashPlate. The reaction mixtures in Flash plates are incubated for 1 hour and the CPM are measured using a TopCount plate reader. The CPM counts in the absence of compound for each data set are defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set are defined as background (0%)[1].

Cell experiment:

PC3 cells are seeded at 200 cells/well into 24-well plates. Cells are allowed to adhere overnight. The media (DMEM supplemented with 10 % FBS) is then exchanged with fresh media containing DMSO, UNC3866 or UNC4219. On day three, the media are exchanged with fresh media containing DMSO, UNC3866 or UNC4219. For dose-response studies, the EC50 is derived from a six-point titration ranging from 100 μM to 0.4 μM of UNC3866 or UNC4219. At day 0, 3 or 6, cells are fixed with ice-cold methanol for 30 sec. and rehydrated with PBS. Nuclei of the cells are stained with DAPI (0.05 μg/mL) and numerated using High Content Microscopy. For dose-response studies, the cell count of UNC3866- or UNC4219-treated cells is normalized to the average cell count of DMSO-treated cells. The EC50 is calculated using the “log[inhibitor] vs. the normalized response-Variable slope” equation in GraphPad Prism 5[1].

UNC3866 is a potent antagonist of CBX4 and CBX7 chromodomains with a Kd of ~100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains. UNC3866 antagonizes the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. [1]

UNC3866 is the most potent ligand reported for CBX7, with a Kd of 97 ± 2.4 nM. UNC3866 antagonizes the CBX7-H3 interaction, assayed by AlphaScreen (IC50 = 66 ± 1.2 nM). Affinity of UNC3866 for CBX2, CBX4, CBX6 and CBX8 is surprisingly well associated with the percent sequence identity of each chromodomain relative to that of CBX7. UNC3866 is equally potent for CBX4, which is most similar to CBX7, whereas it is 18-, 6- and 12-fold selective for CBX4 and CBX7 over CBX2, CBX6 and CBX8, respectively. UNC3866 is 65-fold selective for CBX4 and CBX7 over CDY1 and 9-fold selective for CBX4 and CBX7 over CDYL1b and CDYL2. Each backbone amide of UNC3866 engages in at least one hydrogen bond with the backbone of CBX7, while the N-terminal tert-butylbenzoyl cap of UNC3866 primarily connects the side chains of D50, R52 and L53. X-ray crystallography exhibited that interactions of UNC3866 and the CBX chromodomains closely mock those of the methylated H3 tail. UNC3866 suppresses PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage. [1]

Reference:

1.A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1. Nat Chem Biol. 2016 Mar;12(3):180-7.