NVP-BAG956 is an ATP-competitive PI3K inhibitor (IC50s: 34/56/112/444 nM for PI3Kδ/PI3Kα/PI3Kγ/PI3Kβ).

PI3Kγ:117 nM, PDK1:240 nM, PI3Kα:56 nM, PI3Kβ:446 nM, PI3Kδ:35 nM, VEGFR1:2.56 μM

NVP-BAG956 also inhibits PDK1 (IC50: 240 nM) and VEGFR1 (IC50: 2.56 μM). NVP-BAG956 blocks phosphorylation of PKB/Akt in A2058 cells (IC50: 67 nM). Inhibition of PKB/Akt phosphorylation correlated with loss of A2058 cell proliferation for NVP-BAG956 (IC50: 290 nM). In the presence of NVP-BAG956, A2058 cells are only able to exit G2-M and then remain in G1. The p27Kip1 expression is clearly induced by NVP-BAG956 in A2058 cells but not in C32 cells.

One day after plating (7×10^3 cells/cm2), melanoma cells (A2058, B16F1, B16F10, C32, HBL, Malme, Malme3M, NA8, SKMel2, SKMel23, A375, Hs294T, WM35, and 1205lu cells) are exposed to LY294002 (25 μM), Wortmannin (500 nM), NVP-BAG956 (1 μM), NVP-BBD130 (1 μM), NVP-BEZ235 (1 μM), and ZSTK474 (1 μM), and Rapamycin (100 nM). Compound concentrations are set 2 log units above the IC50 in vitro to ensure full PI3K inhibition, except for the μM inhibitor LY294002. Cells are trypsinized and counted, and the volume is quantified using a Casy Counter and Analyser. To determine the nuclear volume, cells are resuspended in CASYton containing 0.5% Triton X-100, followed by repetitive pipetting (8×), before volume measurements.