Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with...

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions but challenging to target with inhibitors.
• Developing inhibitors for PPIs requires understanding complex molecular systems and physical interaction principles.

Purpose of the Study:

• To identify a novel small molecule inhibitor capable of disrupting the interaction between tumor suppressor p53 and E3-ligase MDM2.
• To establish an efficient strategy combining virtual screening and isothermal titration calorimetry for PPI inhibitor discovery.

Main Methods:

• Utilized virtual screening to filter a library of 15 million small molecules, identifying 80 virtual hits.
• Employed isothermal titration calorimetry (ITC) as a label-free secondary screening method to detect direct molecular interactions.
• Applied 2D NMR spectroscopy and fluorescence polarization to characterize the binding and inhibitory mechanism of the identified compound.

Main Results:

• Identified a specific small molecule, (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide, that binds MDM2 with a dissociation constant of approximately 2 µM.
• Confirmed the molecule's ability to inhibit the p53-MDM2 interaction through spectroscopic analyses.
• Demonstrated the efficacy of the combined virtual screening and ITC approach for PPI inhibitor discovery.

Conclusions:

• The developed strategy combining virtual screening and ITC is highly effective for identifying novel PPI inhibitors.
• The identified small molecule represents a promising lead compound for targeting the p53-MDM2 interaction, with potential therapeutic implications.
• Further characterization confirmed the molecule's binding site and competitive inhibition mechanism against p53.