Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein

Maayan Gal, Biochemistry, Migal, Kiryat Shmona, Israel
Itai Bloch, Biochemistry, Migal, Kiryat Shmona, Israel

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. Shown here is a unique secondary screening technique by isothermal titration calorimetry, a label-free method capable of observing direct interactions, as an efficient tool for finding such an inhibitor. In this study we applied our strategy towards the search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. Following a virtual screening of a library of 15M small molecules, we tested by ITC 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.