Programmable peptide-guided protein degradation

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Summary Over 600 human proteins have been recently prioritized as key cancer targets, with nearly half being considered ‘intractable’ by standard small-molecule inhibition approaches, due to target instability and active site accessibility constraints. By redirecting the ubiquitin-proteasomal pathway (UPS) for targeted protein degradation, the proteolysis-targeting chimera (PROTAC) technology provides a potential solution, enabling rapid and continuous target consumption as well as the stronger pharmacological effects than small molecule inhibition. Nonetheless, PROTACs suffer from similar developmental hurdles as small molecules and cannot be easily designed for motif or post-translational modification-specific targeting. To address these hurdles, research efforts have shifted toward gene therapy approaches by introducing the concept of protein-mediated protein degradation. Here, E3 ubiquitin ligases are redirected by replacing their natural substrate binding domains with “off-the-shelf” binding domains, including nanobodies, antibodies, and DARPins, to generate target-specific ubiquibodies. To augment this platform, we recently exploited natural protein-protein interaction information to develop algorithmic pipelines that prioritize target-selective peptides which can be fused to the E3 ubiquitin ligase conjugation domains to induce target protein degradation. In this project, we will augment our current methods to enable the development of these ubiquibodies (uAbs) for any protein, including those deemed ‘intractable’ by small molecule-based means. To do this, we will automate a bipartite algorithmic pipeline that leverages recent advancements in protein language modeling as well as existing co-complex databases to design peptide binders to diverse protein targets, including those with solved co-crystals as well as those with minimal structural information. Specifically, our pipeline will take user-specified target proteins as inputs, and generate prioritized lists of candidate peptide binders as outputs, enabling subsequent generation of uAbs for target degradation. Through library-on-library fluorescence-based assays in human cells and subsequent encapsulation of uAb mRNA in lipid nanoparticles (LNPs), we will develop a scalable method to test and translate our degraders for downstream in vivo validation. In total, this work will generate a robust peptide design tool that will enhance targeted protein degradation efforts and lay the foundation for programmable proteome editing.