| Assay Method Information | |
| | Binding Affinity Assay |
| Description: | Binding affinity of compounds having formula (I) to SMYD2 is indicia of their inhibition of the activity of this protein lysine methyltransferase. To determine the binding affinity of representative compounds having formula (I) to SMYD2, example compounds having Formula (I) were diluted in assay buffer (as detailed below) to concentrations of 0.0008 micromolar, 0.002 micromolar, 0.007 micromolar, 0.02 micromolar, 0.07 micromolar, 0.2 micromolar, 0.6 micromolar, 1.9 micromolar, 5 micromolar, 17.5 micromolar, and 50 micromolar and added (Echo Liquid Handler by Labcyte) to the wells of an assay plate (PerkinElmer Proxy Plate-384, Cat #6008289). 5 microliters of a 2×SMYD2 enzyme solution (20 nM, 1.8 mg/mL in assay buffer) were added to each well. To the control wells was also added 5 microliters of assay buffer. The assay buffer was comprised of Tris Cl (50 mMolar, obtained from Sigma, Cat # T23139 1 L), Tween 20 (0.01%, obtained from BioRad, Cat #170-6531), BSA (0.005%, obtained from Sigma, Cat# A-3059-50G), and DTT (1 mM, dithiothreitol (DTT)). The wells were incubated at room temperature for 30 minutes. The reaction was initiated by adding 2×p53 peptide (2.5 micromolar, GenMed, Cat #62529)+SAM (2 micromolar, S-adenosyl methionine, AK Scientific).Into the wells of the counter screen plate was added 5 microliters 2×SAH (300 nM, S-adenosyl homocysteine (SAH)), except the control wells. The counter screen plates are run to ensure that any compound activity observed are not artifacts. To the control wells was added 5 microliters of assay buffer, which comprised Tris Cl (50 mMolar, obtained from Sigma, Cat # T23139 1 L), Tween 20 (0.01%, obtained from BioRad, Cat #170-6531), BSA (0.005%, obtained from Sigma, Cat# A-3059-50G), and DTT (1 mM, dithiothreitol). |
| Affinity data for this assay | |
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