Potency assay (cells):
In H29 cells, A-1210477 competed with BIM for MCL1 binding in co-IP assays (tested at 10 uM) and increased MCL1 protein but not RNA expression. In a coupled co-IP-ELISA assay, A-1210477 dose-dependently decreased BIM co-IP with and IC50 in the low uM range and disrupted NOXA-MCL1 interaction in a mammalian yeast two hybrid assay (IC50 ~1 uM). In cellular fluorescence localization assays, A-1210477 redistributed BIM labeled with mCherry from the mitochondria to the cytosol. In MCL1-dependent cells (H929 cells), A-1210477 induced apoptosis (IC50 <10 uM).
Target engagement assay (cells):
Indirect, coupled co-IP ELISA assay