Kinase Assay: The reaction mixture consisted of the following components in 10 μL assay buffer (50 mM Hepes pH 7.5, 10 mM MgCl2, 3 mM MnCl2, 1 mM EGTA, 2 mM DTT, 0.01%Tween-20): 0.10 μg/mL of in-house generated mTOR enzyme, 0.05 μM ULight-eIF4E-binding protein 1 (Thr37/46) peptide and 10 μM ATP. The mixture was incubated for 60 mins at room temperature. 10 μL of Detection mixture consisted of 16 mM EDTA, 0.004 mM Eu-W1024-labeled Anti-Phospho-eIF4E-binding protein 1-(Thr37/46) antibody and 1 × LANCE? Detection Buffer was then added and incubated for 60 mins.

Cell Assay: VS-5584 showed high anti-proliferative activity in a broad spectrum of cancer cells, with H929 cells (multiple myeloma) exhibiting the highest sensitivity in our panel (IC50 = 48 nM).

VS-5584 is an ATP-competitive inhibitor which selectively inhibits PI3K/mTOR signaling with equivalent low nanomolar potency against all human Class I PI3K isoforms and mTOR kinase. VS-5584 is approximately 10-fold selective for cancer stem cells with an EC50 of 15 nM in HMLE breast cancer cells. VS-5584 preferentially decreases CD44Hi/CD24Lo cells in an HMLER immortalized mammary cancer cell line. In SUM159 cells, VS-5584 effectively eliminates the cancer stem cell side population. A large human cancer cell line panel screen (436 lines) reveals broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. In the FLT3-ITD harboring MV4-11 cells, VS-5584 blocks pAkt (S473) and pAkt (T308) with IC50 of 12 and 13 nM, respectively. The IC50 of VS-5584 for pS6 (S240/244), pAkt (S473), and pAkt (T308) are 20, 23, and 15 nM, respectively

[1] Hart S, et al. Mol Cancer Ther, 2013, 12(2), 151-161.