Objective

Even successful PPI modulators (PPIM) have been discovered by cell free methods, thier validation with cell based assay is essential because; (1) Pseudo PPI modulating compounds such as surfactant-like or assay specific ones should be removed, (2) Penetration of PPIMs including peptides or macrocyclic compounds into living cells is often hampered by the plasma membrane and (3) PPIMs should have activity in the complicated intracellular environment where various substances are concentrated at high density. To provide a solution, we developed a very robust functional readout method called Fluoppi (Fluorescent Protein-Protein Interaction-visualization). The method allows visualization of the modulating effect of compounds on target PPIs inside living cells in single cell resolution. Fluoppi utilizes a simple output, where the distribution of fluorescence signal is either diffused (no PPI) or punctum patterned (PPI present). We have discovered that puncta derived from Fluoppi behave as liquid phase droplets which are known to be non-membrane intracellular structures undertaking fundamental functions for the cells and are partially caused by disordered proteins. Results of visualizing the effect of existing PPIMs for several targets such as KRAS(G12C)-Raf, cMyc-MAX, Keap1-p62(SQSTM1), Bcl family-BH3, p53-MDM2 and p53-MDM4 will be presented. In addition to them, recently we have visualized the effect of targeted protein degradation compounds such as PROTACs using Fluoppi. Assays of those compounds will be also presented in our presentation.