In vitro activity: RG7388 (also known as Idasanutlin and RO5503781) is a novel, potent and highly selective antagonist of the p53-MDM2 protein-protein interaction with IC50 of 6 nM. Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein-protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7388 exerts its effect by binding to MDM2 and blocking the interaction between MDM2 protein and the tumor suppressor protein p53. By disrupting the MDM2-p53 interaction, p53 is not degraded enzymatically and the transcriptional activity of p53 is restored, which results in the p53-mediated induction of tumor cell apoptosis. MDM2 is a negative regulator of the p53 pathway and is often overexpressed in tumor cells. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical development for treating cancer.

Kinase Assay: The p53-MDM2 HTRF assay is performed in buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM DTT, 0.02 or 0.2 mg/mL BSA. Small-molecule inhibitors are stored in aliquots as 10 mM stock solutions in DMSO at 4°C in 96-deep-well plates. It is thawed and mixed immediately prior to testing. The compound is incubated with GST-MDM2 and a biotinylated p53 peptide for one hour at 37°C. Phycolink goat anti-GST (Type 1) allophycocyanin and Eu-8044-streptavidin are then added and followed by one hour incubation at room temperature. Plates are read using the Envision fluorescence reader. IC50 values are determined from inter-plate duplicate or triplicate sets of data.

Cell Assay: RG7388 (also known as Idasanutlin) inhibits cell proliferation with IC50 value of 30 nM, it induces a dose-dependent p53 stabilization, cell cycle arrest, as well as cell apoptosis in cancer cells expressing wild-type p53. RG7388 (300 nM or 1.8 μM) induces apoptosis in SJSA osteosarcoma cells. Cell proliferation is evaluated by the tetrazolium dye assay. The concentration at which 50% inhibition (IC50) or 90% inhibition (IC90) of cell proliferation is determined from the linear regression of a plot of the logarithm of the concentration versus percent inhibition. RG7388 was prepared at concentrations of 1 and 10 mmol/L in DMSO and stored in aliquots at –20°C. SJSA, RKO, HCT116, H460, A375, SK-MEL-5, SW480, MDA435, and HeLa cells were obtained from the ATCC. Cell lines were authenticated by short tandem repeat analysis through Promega authentication services. For in vitro studies, cells were cultured in their ATCC-designated media. Medium was supplemented with 10% FBS and 1% 200 nmol/L l-glutamine. To assess cell viability, cells were seeded at densities identified for best growth for a 5-day assay in 96-well plates in normal growth media. Serial dilutions of RG7388 (1–3 in fresh media) starting at 300 μmol/L were applied to wells (1–10) in triplicate for a final concentration range of 0.01 to 30 μmol/L and control wells were treated with 0.3% DMSO equivalent to DMSO at the highest RG7388 concentration. Cell respiration, as an indicator of cell viability, was measured by the reduction of MTT to formazan as previously described