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

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.