High-throughput Activity Reprogramming of Proteases (HARP).

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Developing specific protease inhibitors is challenging due to the need for extensive characterization.
• Existing platforms prioritize high-affinity binders, often missing rare, potent inhibitors.

Purpose of the Study:

• To develop a novel platform for efficient discovery of protease inhibitors.
• To isolate low-nanomolar inhibitors against specific proteases using a functional screen.

Main Methods:

• Developed the High-throughput Activity Reprogramming of Proteases (HARP) platform, a yeast-based functional screen.
• Coupled protease inhibition to a selectable cell-surface phenotype for high-throughput screening.
• Utilized structural modeling and deep sequencing for inhibitor analysis.

Main Results:

• Successfully isolated low-nanomolar inhibitory nanobodies against tobacco etch virus protease (TEVp) and human kallikrein 6.
• Identified a rare 7.6 nM K_i uncompetitive inhibitor for TEVp.
• HARP demonstrated high dynamic range and resolution in inhibitor discovery.

Conclusions:

• HARP is a premier platform for discovering modulatory macromolecules against enzyme targets.
• The platform facilitates the isolation of potent and selective protease inhibitors.
• Provides insights into molecular determinants of inhibitor efficacy.