In vitro activity: S63845 is a small molecule MCL1 inhibitor with Ki < 1.2 nM. S63845 is effective against haematological cancer-derived cell lines. S63845 potently killed MCL1-dependent H929 multiple myeloma cells. S63845 disrupted binding of BAK and BAX to MCL1 in HeLa cells. Treatment with S63845 increased MCL1 protein levels in the HCT-116 colon carcinoma cell line. In the tested myeloma cell lines, some were highly sensitive to S63845 (IC50< 0.1 μM), six lines were moderately sensitive (0.1 μM < IC50 < 1 μM) and two lines were insensitive (IC50> 1 μM). In a panel of human lymphomas and chronic myeloid leukaemia 11 cell lines: five lines were highly sensitive to S63845 (IC50 < 0.1 μM), three were moderately sensitive (0.1 μM < IC50< 1 μM) and three were insensitive to S63845 (IC50>1 μM). In a panel of eight AML cell lines: all lines were sensitive to S63845 (IC50 4–233 nM).

Kinase Assay: 10 mM HEPES pH 7.4, 175 mM NaCl, 25 μM EDTA, 1 mM TCEP, 0.01% P20 and 1% DMSO is used as a running buffer. The ligand surface is generated using double His-tagged proteins. Serial dilutions of the compound in buffer are injected over the protein surface. All sample measurements are performed at a flow rate of 30 μL per min (injection time 120 s, dissociation time 360 s). The sensor surface is regenerated by consecutive injections of 0.35 M EDTA pH 8.0 with 0.1 mg/mL trypsin, 0.5 M imidazole and 45% DMSO (60 s, 15 μL per min).

Cell Assay: S63845 is a highly selective and potent MCL1 inhibitor. S63845 bound human MCL1 with KD value of 0.19 nM. S63845 was approximately 1,000-fold more potent in killing MCL1-dependent H929 multiple myeloma cells than MCL1 inhibitor A-1210477. S63845 also induced caspase-dependent phosphatidyl-serine exposure, PARP cleavage and cytochrome c release from mitochondria. In HeLa cells, S63845 disrupted binding of BAK and BAX to MCL1. S63845 killed cancer cells through activation of the BAX/BAK-dependent mitochondrial apoptotic pathway by direct inhibition of MCL1.