NVP-BAG956 (BAG956) is a potent PI3K/PDK inhibitor that inhibits PI3Kδ, PI3Kα, PI3Kγ, and PI3Kβ, shows strong cytotoxicity against T-ALL cell lines, and can be used to study melanoma.

A2058 cells:290 nM, PI3Kγ:117 nM, PI3Kδ:35 nM, PI3Kβ:446 nM, VEGFR1:2.56 μM, PI3Kα:56 nM, PDK1:240 nM, pAkt:67 nM

NVP-BAG956 inhibited PDK1 (IC50: 240 nM) and VEGFR1 (IC50: 2.56 μM). It also blocked the phosphorylation of PKB/Akt in A2058 cells with an IC50 of 67 nM.The inhibition of PKB/Akt phosphorylation was closely correlated with the reduction of A2058 cell proliferation, with an IC50 of 290 nM for NVP-BAG956.In the presence of NVP-BAG956, the A2058 cells were only able to exit from the G2-M phase, and thereafter stayed in the G1 phase. In addition, NVP-BAG956 significantly induced the expression of p27Kip1 in A2058 cells, but not in C32 cells. [1]

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.