Evaluation of molecular glue-induced neomorphic protein-protein interaction with a cell lysate-based time-resolved fluorescence resonance energy transfer assay.

Abstract

Neo-protein-protein interactions (neoPPI) induced by mutated amino acid residues present a promising class of therapeutic targets for proximity pharmacology-enabled drug discovery. Small molecule compounds, such as cyclosporin and FK506, have the capability to mimic the mutational effect to create new interaction interfaces to enhance the interaction of a protein with an unrelated, or low affinity protein. Such small molecules can function as molecular glues to purposefully generate neoPPIs for physiological reprogramming and therapeutic intervention. Thus, an unbiased approach for the systematic discovery of neoPPI-inducing compounds is much needed to accelerate the proximity pharmacology field. With the demonstrated small molecule induced neoPPI of oncogenic KRAS with cyclophilin A (CYPA), here we report the development of a robust cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay to characterize KRAS-CYPA neoPPI inducing molecular glues. We show that the developed TR-FRET assay can monitor the induced interaction of CYPA with oncogenic KRAS variants, KRAS-G12C, G12D and G12V. The assay was optimized to achieve a robust performance in a miniaturized format, which was utilized to demonstrate the potent effect of RMC-7977, a known molecular glue, in inducing the KRAS-CYPA interaction. This validated assay platform allows for the characterization of molecular glue-induced KRAS-CYPA neoPPI, which is readily applicable to explore the vast chemical space for the discovery of novel KRAS-CYPA neoPPI inducers and can be generally applicable for unbiased discovery of proximity-inducing molecular glues.