(175o) Discovering Novel Ankyrin Protein Modulators through an Optimized Yeast Surface Display System

Proteases are involved in regulating several biological processes. Thus, protease dysregulation often contributes to disease, making them critical therapeutic targets. A major objective in biomedicine has been to develop selective protease modulators. As part of this objective, there have been increased efforts to discover and engineer protein binders such as endogenous inhibitors and antibodies that inhibit proteases. These protein-based modulators are ideal modulators because they have high affinity, specificity and can be engineered. Among these proteins are Designed Ankyrin Repeat Proteins (DARPins). DARPins are synthetic antibody mimics that are much smaller (14-18 kDa) than antibodies (150 kDa), more thermostable, and can be produced in bacteria while maintaining the very strong affinity and specificity of antibodies. The current challenge with DARPin discovery methods is that they are often screened using ribosome display. Ribosome display is very good at identifying strong binders but gives no information about the potential of these binders to inhibit their target. Moreover, affinity binding high-throughput screens will often miss strong inhibitors with moderate binding affinities. This work aims to validate a new discovery method for protease inhibitory DARPins. To discover these DARPins, we have developed a High-throughput Activity Screen for the functional Reprogramming of Proteases (HARP). HARP is a yeast surface display system that allows us to measure protease activity through interactions between a protease and its substrates within the Endoplasmic Reticulum (ER). The outcomes of protease catalytic turnovers are then secreted and displayed on the surface. These outcomes are then measured using flow cytometry after staining epitope tags with fluorescent antibodies. Using HARP, we can screen protein-based modulator libraries to measure the inhibitory potential of individual variants. Thus, rather than screening a library purely based on binding affinity, we screen for library variants that can inhibit a protease. By leveraging HARP, we were able to discover novel ankyrin protein sequences to modulate protease activity paving the way for breakthroughs in the field of protease modulation for therapeutic use.