High-Throughput Protease Reprogramming Powered By a Suite of Integrative Vectors

Proteases make up approximately 2% of the human proteome and are involved in several biological processes, including apoptosis, intracellular signaling, blood coagulation, digestion, inflammation, and disease. Proteases are tightly regulated; when dysregulated, they are a hallmark of disease, making them critical therapeutic targets. Protease modulators, particularly inhibitors, are often discovered using high-throughput screening platforms. However, most existing high-throughput screening technologies identify molecules that bind to a protease, with little information on how binding relates to modulation. Moreover, protease inhibitors generally target the enzyme’s active site. This mechanism leads to off-target toxicity since related proteases have similar active sites and may begin the progression of on-target side effects because proteases have multiple physiological substrates. These substrates and pathways are necessary for cellular health. Therefore, there is a need to modulate proteases beyond catalytic inhibition. In particular, methods to discover or engineer molecules that selectively alter protease activity would enable new therapeutic opportunities. Such discoveries would also deepen our knowledge of protease modulatory landscapes.

Here we present a high-throughput platform to isolate, characterize, and discover protease modulatory nanobodies that can reprogram a protease. We have developed a Suite of Integrative Vectors (SIVs) to streamline this technology. The High-throughput Activity screen for functional Reprogramming of Proteases (HARP) is a functional screen that utilizes yeast surface display to measure protease activity. When coupled with Fluorescent Activated Cell Sorting (FACS), HARP can isolate protease variants with desired activities, protease substrates that are highly active, and protein molecules that can reprogram a protease. Introducing SIVs into this system allows us to integrate the transcriptional cassettes for protease, modulator, and substrate expression into the yeast chromosome. This approach leads to a high signal-to-noise ratio, increased dynamic range, and streamlines molecular cloning. Plasmid-based systems can suffer from inconsistent plasmid copies and nonuniform induction, which results in reproducibility issues. By enabling efficient integration of gene cassettes in the yeast chromosome, the integrative plasmids will allow us to reprogram protease activity on multiple substrates and to find substrate selective protein-based reprogrammers.