Molecules that induce interactions between proteins, often referred to as "molecular glues", are increasingly recognized as important therapeutic modalities and as entry points for rewiring cellular signaling networks. Here, we report a new PACE-based method to rapidly select and evolve molecules that mediate interactions between otherwise noninteracting proteins: rapid evolution of protein-protein interaction glues (rePPI-G). By leveraging proximity-dependent split RNA polymerase-based biosensors, we developed E. coli-based detection and selection systems that drive gene expression outputs only when interactions between target proteins are induced. We then validated the system using engineered bivalent molecular glues, showing that rePPI-G robustly selects for molecules that induce the target interaction. Proof-of-concept evolutions demonstrated that rePPI-G reduces the "hook effect" of the engineered molecular glues, due at least in part to tuning the interaction affinities of each individual component of the bifunctional molecule. Altogether, this work validates rePPI-G as a continuous, phage-based evolutionary technology for optimizing molecular glues, providing a strategy for developing molecules that reprogram protein-protein interactions.
Keywords: PACE; continuous directed evolution; molecular glue; protein engineering; protein interaction stabilizers; protein−protein interactions (PPIs).