Quantitative Degradation Rate Assessment of bioPROTACs Based on Peptide Degrons, E3 Domains, Adapters and Conjugated...

Protein-based bispecific degraders, known as bioPROTACs, have emerged as powerful tools for targeted protein degradation through the ubiquitin-proteasome system (UPS). However, the relative efficacy of various recruitment domains within these degraders remains poorly understood. To address this knowledge gap, we conducted a comprehensive comparison of recruitment domains in bioPROTACs, utilizing eGFP as a proof-of-principle degradation target and an eGFP-binding DARPin with known structure as an adapter. Our innovative approach combined microinjection and live-cell microscopy, enabling a detailed assessment of directly measured degradation rates as a single-cell kinetic readout, unaffected by uptake or biosynthesis rates of the degrader, and across the different chemical classes. We examined nine degron peptides, three E3 ligase domains or adapters, and two series of small-molecule binders, linked in various geometries. Our results revealed that bioPROTACs based on E3 or adapter protein domains and small molecules generally exhibited the highest degradation rates, while most degron peptides showed comparatively low efficacy. Notably, for VHL-ligand-1 and thalidomide, the placement of the coupling site and linker position significantly influenced performance. This study provides crucial insights into the design and optimization of bioPROTACs, paving the way for the development of more effective degraders for specific applications. Our findings contribute to the growing field of targeted protein degradation and offer valuable guidance for researchers seeking to enhance the efficacy of bioPROTAC-based therapeutic approaches.