Kinase Assay: the potency of compounds against Akt enzymes is measured. Since GSK2110183 and GSK2141795 are highly potent inhibitors of the 3 isoforms of Akt, the true potency (Ki) of the inhibitors is initially determined at low enzyme concentrations (0.1 nM Akt1, 0.7 nM Akt2, and 0.2 nM Akt3) using a filter binding assay and then confirmed with progress curve analysis. In the filter binding assay, a pre-mix of enzyme plus inhibitor is incubated for 1 h and then added to a GSKα peptide (Ac-KKGGRARTSSFAEPG-amide) and [γ33P] ATP. Reactions are terminated after 2 h and the radio labeled Akt peptide product is captured in a phospho-cellulose filter plate. Progress curve analysis utilized continuous real-time fluorescence detection of product formation using the Sox-Akt-tide substrate (Ac-ARKRERAYSF-d-Pro-Sox-Gly-NH2).

Cell Assay: A 3-day proliferation assay using CellTiter-Glo is performed to measure the growth inhibition by the compounds at 0-30 μM. Cell growth is determined relative to untreated (DMSO) controls. EC50’s are calculated from inhibition curves using a 4- or 6-parameter fitting algorithm in the Assay Client application. Cells used: Hematological cell lines and solid tumor cell lines

Afuresertib inhibits the kinase activity of the E17K AKT1 mutant protein with EC50 of 0.2 nM. Afuresertib shows a concentration-dependent effect on multiple AKT substrate phosphorylation levels, including GSK3b, PRAS40, FOXO and Caspase 9. Overall 65% of the hematological cell lines are sensitive to afuresertib (EC50 < 1 μM). Among tested solid tumor cell lines, 21% have EC50 < 1 μM in response to afuresertib.