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

Neomorphic protein-protein interactions (neoPPIs) 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 to 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 assay to characterize KRAS-CYPA neoPPI-inducing molecular glues. We show that the developed time-resolved fluorescence resonance energy transfer 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. Molecular glues offer a promising strategy to target “undruggable” proteins by inducing neomorphic protein-protein interactions. This study develops a high-throughput, cell lysate–based time-resolved fluorescence resonance energy transfer assay to detect molecular glue–induced KRAS–cyclophilin A neomorphic protein-protein interaction in a physiologically relevant environment, enabling the discovery of small molecules that could expand therapeutic options for KRAS-driven cancers.