ITC was used to measure the dissociation constant (KD) of MDM2 ligands Nutlin-3a and AM-7209. All titrations (10 µM proteins in the cell and 100 µM Nutlin-3a or 150 µM AM-7209 in the syringe) were performed using a MicroCal Auto-iTC200 isothermal titration calorimeter from Malvern Panalytical, assuming one site of binding. The data were analyzed using the MicroCal PEAQ-ITC Analysis Software version 1.1.0.1262.13. For some constructs, measuring the binding affinity directly through titration proved difficult, and hence a competitive titration was used in these cases.