Protein-protein interactions (PPI) play a critical role in regulating many cellular processes.
Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great
challenge, mainly due to the complexity involved in characterizing multi-molecular systems and
limited understanding of the physical principles governing PPIs. Here we show that the combination of
virtual screening techniques, which are capable of filtering a large library of potential small molecule
inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method
capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study
we applied this strategy in a search for a small molecule capable of interfering with the interaction of
the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that
were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures
of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of
the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8
tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the
identification of this single bioactive compound, spectroscopic measurements were used to further characterize the
interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the
small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.