MDM2 affinity measurements by Time Resolved Fluorescence Energy Transfer (TR-FRET) assay

To measure the ability of Mdm2i (NMI801) to inhibit p53 binding to MDM2 we utilized a competitive binding assay with a FRET readout. The measurements were performed in white 1536-well microtiterplates (Greiner Bio-One GmbH) in a total volume of 3.1 ul combining 100 nl of compounds diluted in 90% DMSO/10% H2O (3.2% final DMSO concentration) with 2 μl Europium-labeled streptavidin (final concentration 2.5 nM) in reaction buffer (PBS, 125 mM NaC1, 0.001% Novexin (Novexin Ltd.), Gelatin 0.01%, 0.2% Pluronic (BASF), 1 mM DTT), followed by the addition of MDM2-Bio diluted in assay buffer (final concentration 10 nM). The solution was pre-incubated for 15 min at room temperature, followed by addition of 0.5 ul Cy5-p53 peptide in assay buffer (final concentration 20 nM). The sample was incubated at room temperature for 10 min prior to reading the plate. Europium-Cy5 FRET was measured by an Analyst GT multimode microplate reader (Molecular Devices) with the following settings: dichroic mirror 380 nm, excitation 330 nm, emission donor 615 nm, and emission acceptor 665 nm. To validate the TR-FRET assay, a reference compound was added in each run. Only if the reference compound scored in the expected potency range, the data was considered reliable. IC50 values are calculated by curve fitting using XLfit. If not specified, reagents are purchased from Sigma Chemical Co, St. Louis, MO, USA.