Work

High-Throughput Activity Reprogramming of Proteases (HARP)

Year: 2025

Type: article

Abstract: 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 K I TEVp uncompetitive inhibitor. Structural modeling and deep sequencing all provide insights into the molecular determinants of inhibitors and reinforce HARP’s foundational findings. Overall, HARP is a premier platform for discovering modulatory macromolecules from various synthetic scaffolds against enzyme targets. Graphical Abstract Workflow of HARP A yeast-based reporter of the interaction between a protease, its canonical substrate, and a modulator library within the yeast cell. Quantifying this interaction occurs by fluorescently labeling the displayed substrate cassettes on the surface of the cells, where the desired function (correlating with phenotype) can be selected using fluorescent-activated cell sorting (FACS). Isolated populations are sequenced and purified in preparation for secondary characterization to determine modulator effects and interaction strengths between the modulator and the protease target.

Source: ACS Chemical Biology

DOI: 10.1021/acschembio.5c00230