| Assay Method Information | |
| | KAT6A Biochemical Assay |
| Description: | TR-FRET based methods were used for assaying compounds of the present invention for KAT6A enzyme inhibitory activity. TR-FRET is a homogeneous proximity assay where Europium-labelled anti-acetyl lysine antibody binds to the acetylated substrate labelled with biotin, which in turn binds to streptavidin-labelled APC fluorescence acceptor. Europium can transfer energy to APC in the complex and the interaction of two dye-labelled binding partners is detected by the energy transfer between a donor and an acceptor dye and the subsequent light emission by the acceptor dye. KAT6A transfer an acetyl group from acetyl CoA to lysine amino acids of histones/target proteins. Typically, 5 μL of human-KAT6A (MYST domain 507-778 aa) in assay buffer (100 mM Tris HCl (pH 7.8), 15 mM NaCl, 1 mM EDTA, 0.01% Tween-20, 0.02% BSA, 1 mM DTT) is added to 384-well plate containing 5 μL of selected test compound in final 1% DMSO, serially diluted in 1:3 in an 8-10-point titration. The selected compound of the present invention and enzyme are incubated for 30 min on plate shaker at 300 rpm at 25° C. Next, 5 μL of substrate mix containing histone H4 peptide and acetyl-CoA in assay buffer is added to the plate. The final concentrations of H4 peptide and acetyl-CoA are 200 nM and 600 nM, respectively. Following 60 min reaction at 25° C. on plate shaker at 300 rpm, 5 μL of detection mix containing Europium-labelled anti-acetyl antibody and streptavidin-APC is added to the reaction wells. The plate is further incubated at 25° C. on shaker at 300 rpm and is read in TR-FRET mode (Ex:340 nm; Em: 615 nm and 665 nm) on a plate reader to achieve ˜5-7-fold activity. The percent inhibition was calculated from the ratio of the fluorescence (FL) intensities [(F665/F615)×10000) using the formula (Control FL ratio−(Sample FL ratio/Control FL ratio))×100. |
| Affinity data for this assay | |
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