| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | Assay 1: Compounds were tested for inhibition of methyltransferase activity in a radioisotope filter binding assay, similar to previously described in A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models. Chan-Penebre et al., Nat Chem Biol. (2015) 11(6):432-7. In the standard PRMT5/MEP50 enzyme inhibition assay, compounds were tested in a 10-dose IC50 mode with 3- or 5-fold serial dilution, in singlet, starting at 1, 10, or 100 μM. Control compound, SAH (S-(5′-Adenosyl)-L-homocysteine), was tested in 10-dose IC50 mode with 3-fold serial dilution starting at 100 μM. Reactions were carried out at 1 μM 3H-SAM (PerkinElmer) and 5 μM histone H2A as substrates for methyl transfer. Following a 60 min incubation at 30° C., the reaction was stopped with 20% TCA. Each reaction was spotted on a filter plate (Multiscreen FB Filter plate, Millipore) and washed 5 times in PBS, after which scintillation fluid was added and signal was detected in a scintillation counter. 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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