In vitro activity: NVP-AEW541 also inhibits InsR, Tek, Flt1 and Flt3 with IC50 of 140 nM, 530 nM, 600 nM and 420 nM in purified kinases/recombinant kinase domains assay. NVP-AEW541 is more selective and shows 27-fold more potent than InsR at the cellular level. NVP-AEW541 suppresses the IGF-I-mediated survival, soft agar and proliferation of MCF-7 cells with IC50 of 0.162 μM, 0.105 μM and 1.64 μM, respectively. NVP-AEW541 also reduces the level of phospho-IGF-1R and phospho-PKB in NWT-21 cells. NVP-AEW541 shows growth inhibitory effect on TC-71 musculoskeletal sarcoma cells in low-serum medium as well as in 10% FBS–containing medium. NVP-AEW541 inhibits cell cycle progression and induces specific G1 arrest in sarcoma cell lines (TC-71, SK-N-MC, SaoS-2, RD/18 and RH4). NVP-AEW541 could inhibit the growth of human neuroblastoma cells with IC50 of 0.4-6.8 μM. An increase in the hypodiploid fraction and the depletion of the S and G2-M compartments could be detected in these cell lines. NVP-AEW541-driven inhibition of IGF-1R causes a reduction of phosphorylation of Akt, but not of Erk1 and Erk2 in neuroblastoma cells. NVP-AEW541 inhibits glioma cell growth and disrupts the autocrine loop initiated by HIF1α stabilization. A recent study shows that NVP-AEW541 suppresses the proliferation and viability of PC3, DU145, and 22Rv1 prostate cancer cells, without necessarity of associated cell death. NVP-AEW541 decreases phospho-Akt levels in 22Rv1 and DU415 cells but not PC3 cells, without affecting total Akt levels, which shows that PTEN status could determine the effectiveness of NVP-AEW541 with essential Akt. NVP-AEW541-induced radiosensization is dependent on Akt activation status. NVP-AEW541 could increase the H2AX phosphorylation (a measure of DSBs) in PC3, DU145, and 22Rv1 cells.

Kinase Assay: NVP-AEW541 is dissolved in DMSO (10 mM) and stored at -20 °C. Dilutions are freshly made in DMSO/water 1:1. The final concentration of DMSO in the enzyme assays is<0.5 %. The protein kinase assays are carried out in 96-well plates at RT and terminated by the addition of 20 μL of 125 mM EDTA. Subsequently, 30 μL (c-Abl, c-Src, IGF-1R) of the reaction mixture are transferred onto Immobilon-PVDF presoaked for 5 min with methanol, rinsed with water, then soaked for 5 min with 0.5 % H3PO4 and mounted on vacuum manifold. After spotting all samples, vacuum is connected and each well rinsed with 200 μL 0.5 % H3PO4. Membranes are removed and washed 4× on a shaker with 1.0 % H3PO4, once with ethanol. After drying, mounting in Packard TopCount 96-well frame, and adding of 10 μL/well of Microscint, membranes are counted. IC50 values are calculated by linear regression analysis of the percentage inhibition of NVP-AEW541 in duplicate, at four concentrations (usually 0.01, 0.1, 1, and 10 μM). One unit of protein kinase activity is defined as 1 nmol of 33P transferred from [γ33P]ATP to the substrate protein per minute per mg of protein at 37 °C.

Cell Assay: Between 3 × 103 and 6 × 103 cells/well are seeded in 96-well plates with a total media volume of 100 μL/well. Increasing concentrations of NVP-AEW541 is added 24 hours thereafter in quadruplicate. 72 hours later, cells are fixed by addition of 25 μL/well Glutaraldehyde (20%) and incubation for 10 min at RT. Cells are then washed 2× with 200 μL/well H2O and 100 μL Methylene Blue (0.05%) is added. After incubation for 10 min at RT, cells are washed 3× with 200 μL/well H2O. 200 μL/well HCl (3%) is added, and following incubation for 30 min at RT on a plate shaker, absorbance is measured at 650 nm.

Cancer Cell. 2004 Mar;5(3):231-9; Cancer Res. 2005 May 1;65(9):3868-76; Clin Cancer Res. 2006 Nov 15;12(22):6772-80.