In vitro activity: NVP-CGM097 binding to MDM2 is species dependent. It was shown to be selective for the p53:MDM2 interaction compared to the p53:MDM4 interaction (1176-fold selectivity) and the Ras:Raf interaction (3000-fold selectivity). In addition, NVP-CGM097 showed no significant activity against Bcl-2:Bak, Bcl-2:Bad, Mcl-1:Bak, Mcl-1:NOXA, XIAP:BIR3, and c-IAP:BIR3 protein-protein interactions. NVP-CGM097 was able to significantly redistribute wild-type p53 into the cell nucleus with an IC50 of 0.224 μM, demonstrating its ability to inhibit the p53:MDM2 interaction in living cells. NVP-CGM097 treatment leads to p53 nuclear translocation that results in cell growth inhibition in a p53-dependent manner.

Kinase Assay: NVP-CGM097 binds to human MDM2 with an IC50 of 1.7 nM and shows high selectivity over MDM4 (IC50=2000 nM). NVP-CGM097 is about four times more potent than Nutlin-3a (IC50=8 nM). In addition, NVP-CGM097 shows no significant activity against Bcl-2:Bak, Bcl-2:Bad, Mcl-1:Bak, Mcl-1:NOXA, XIAP:BIR3, and c-IAP:BIR3 protein-protein interactions. NVP-CGM097 significantly inhibits the proliferation of cells expressing wild-type p53, while sparing the p53 null cells with a 35-58-fold difference. NVP-CGM097 is able to significantly redistribute wild-type p53 into the cell nucleus with an IC50 of 0.224 μM, demonstrating its ability to inhibit the p53:MDM2 interaction in living cells. In addition, NVP-CGM097 activity against the p53:MDM2 interaction is assessed in proliferation assays using either wild-type p53 or p53 null cells. NVP-CGM097 significantly inhibits the proliferation of cells expressing wild-type p53, while sparing the p53 null cells with a 35-58-fold difference. NVP-CGM097 inhibtis HCT116 (p53WT/WT) with IC50 of 454±136 nM.

Cell Assay: Cells (Bon1 cells, NCI-H727 cells, Got1 cells) were seeded in appropriate densities (Bon1 cells: 1500 cells/well, NCI-H727 cells: 2000 cells/well, Got1 cells: 50000 cells/well) into 96-well plates and grown for 24 hrs in complete medium containing serum/antibiotic. The next day, the cells were incubated with various concentrations of NVP-CGM097 (0.1 nM-2500 nM), 5-fluorouracil (100 nM-100 μM), streptozotocin (1 nM-100 μM), temozolomide (1 μM-1 mM), everolimus (10 nM) or octreotide (100 nM-10 μM) in 10 % FBS medium (antibiotic-free). After 48 hrs, 96 hrs, 144 hrs or 216 hrs the metabolic activity was measured with 'Cell Titer 96 Aqueous One Solution' cell proliferation assay. The measurement was performed at 492 nm with an ELISA plate reader.