| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | Assay 2: Compounds were tested for inhibition of methyltransferase activity in 384-well plate assay format using mass spectrometry technology. In this enzyme inhibition assay, compounds were tested in a 11-dose IC50 mode with 3-serial dilution, in duplicate, starting at 1, 10, or 100 μM. Reactions were carried out at 1 μM SAM and 0.1 μM histone H4 1-21 peptide as substrates for methyl transfer. Following an 18-hour incubation at rt, the reaction was stopped with 0.5% formic acid. Products of the reaction were captured while unreacted substrates were washed away, prior to MALDI mass spectrometry detection and analysis. Percent enzyme activity was calculated based on no inhibitor DMSO control as 100% activity. EC50 values were determined in GraphPad Prism 8 using the [inhibitor] vs. response Variable slope (four parameters) function. |
| Affinity data for this assay | |
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