High-throughput Activity Reprogramming of Proteases (HARP)

Developing potent and selective protease inhibitors remains a grueling, iterative, and often unsuccessful endeavor. Although macromolecular inhibitors can achieve single-enzyme specificity, platforms used for macromolecular inhibitor discovery are optimized for high-affinity binders, requiring extensive downstream biochemical characterization to isolate rare inhibitors. Here, we developed the High-throughput Activity Reprogramming of Proteases (HARP) platform, HARP is a yeast-based functional screen that isolates protease-inhibitory macromolecules from large libraries by coupling their inhibition of endoplasmic reticulum-resident proteases to a selectable phenotype on the cell surface. Endowed with high dynamic range and resolution, HARP enabled the isolation of low-nanomolar-range inhibitory nanobodies against tobacco etch virus protease and human kallikrein 6, including a rare 7.6 nM KI TEVp uncompetitive inhibitor. Structural modeling and deep sequencing all provide insights into the molecular determinants of inhibitors and reinforce foundational findings. Overall, HARP is a premier platform for discovering modulatory macromolecules from various synthetic scaffolds against enzyme targets.