| Kinase Assay |
To determine the inhibition of Moz activity by the test compounds, assay reactions are conducted in a volume of 8 μL in 384-well low volume assay plates. The reactions are performed in assay buffer (100 mM Tris-HCl, pH 7.8, 15 mM NaCl, 1 mM EDTA, 0.01% Tween-20, 1 mM Dithiothreitol, and 0.02% m/v chicken egg white albumin). Reactions are set up with 0.4 μM Acetyl coenzyme A (AcCoA), 50 nM N-terminal histone H4 peptide (sequence SGRGKGGKGLGKGGAKRHRKV-GGK-biotin), 10 nM MOZ enzyme, and an acetyl-lysine specific antibody (final dilution 1: 10000). 11-point dilution series of the compounds (WM-8014) of the invention are prepared in DMSO; a volume of 100 nL is transferred using a pin tool into assay plates containing substrates, before adding enzyme to start the reaction. Positive (no compound) and negative (AcCoA omitted) control reactions are included on the same plates and receive the same amount of DMSO as the compound treated wells. After adding all reagents, the plates are sealed with adhesive seals and incubated for 90 minutes at room temperature[1].
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