| Assay Method Information | |
| | EZH2 LC-MS Assay |
| Description: | Representative compounds of the invention were serially and separately diluted 3-fold in DMSO to obtain twelve concentrations. Then the test compounds at each concentration (120 nL of each) were transferred by Mosquito into a 384-well Perkin Elmer ProxiPlate 384 plus plates. Solutions (6 μL) of 80 nM wild type PRC2 (wtPRC2) complex and 60 μM SAM in reaction buffer (20 mM Tris, pH 8.0, 0.1% BSA, 0.01% Triton, 0.5 mM DTT) were added to the wells that were then incubated with the test compound for 20 min. A 6 μL solution of 3 μM of the substrate peptide H3K27me1 (histone H3[21-44]-K27me1-biotin) and 6 μM regulatory peptide H3K27me3 (histone H3[21-44]-K27me3) in reaction buffer was added to initiate each reaction. The final components in the reaction solution include 40 nM wtPRC2 complex, 30 μM SAM, 1.5 μM H3K27me1 and 3 μM H3K27me3 peptides with varying concentration of the compounds. A positive control consisted of the enzyme at 40 nM, 30 μM SAM, 1.5 μM H3K27me1 and 3 μM H3K27me3 in the absence of the test compound, and a negative control consisted of 30 μM SAM, 1.5 μM H3K27me1 and 3 μM H3K27me3 only. Each reaction was incubated at room temperature for 120 min, then stopped by addition of 3 μL per of quench solution (2.5% TFA with 320 nM d4-SAH). The reaction mixture was centrifuged (Eppendorf centrifuge 5810, Rotor A-4-62) for 2 min at 2000 rpm and read on an API 4000 triple quadrupole mass spec with Turbulon Spray (Applied Biosystem) coupled with Prominence UFLC (Shimadzu). The levels of SAH production were normalized based on the values coming from the positive and negative controls to give percent enzyme activities. The data were fit to a dose response equation using the program Helios to get the IC50 values of the test compound. |
| Affinity data for this assay | |
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