2023 AIChE Annual Meeting

Discovering Darpin Inhibitors Against Disease-Relevant Targets for Potential Therapeutic Development.

Proteases are involved in several biological processes and, upon dysregulation, often contribute to disease onset and progression, making them critical therapeutic targets. Strategies for targeting protease therapeutics using small molecule inhibitors often rely on active site binding. Due to active sites being conserved across protease families, this strategy can lead to off-target toxicity, resulting in unintended consequences. Protein binders have been found to modulate proteases. Thanks to the large surface area of interaction, protein-based binders can modulate proteases in ways small molecules cannot. These include highly specific and selective inhibitors, activators, enhancers, and substrate-selective reprogrammers. However, isolating such modalities remains challenging because they are often discovered from high-throughput binding assays. To address this, our lab has 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 the activity of a protease. HARP provides us with the ability to engineer and discover protein binders that can reprogram the activity of proteases. These discoveries will enable us to understand protease mechanisms, establish their roles in disease, and help develop new novel targeted therapeutics.

We have applied our approach to discover tight binding proteases inhibitory Designed Ankyrin Repeat Proteins (DARPins) from randomly generated libraries. DARPins are genetically engineered protein binders with exceptionally high binding affinity and specificity. DARPins against a protein target are typically isolated using high-throughput screens such as ribosome display, which could potentially lead to discarding strong protease inhibitors. We show that we can isolate TEV inhibitory DARPins from randomized libraries and perform affinity maturation of weak inhibitors via error-prone PCR followed by fluorescence-activated cell sorting (FACS). Using this approach, we will be able to identify DARPins that are substrate-selective against disease-relevant proteases to potentially create new therapeutics. Our approach can fully enlist the potential of DARPins as protease modulators and provide the necessary foundation for modulator design.