Chemoselective Semisynthesis of Covalent Nanobody-Guided Protein Degraders in Neurons

The selective removal of pathological proteins represents a promising strategy for treating neurodegenerative diseases driven by protein aggregation and synaptic dysfunction. Here, we present a chemoselective semisynthesis platform that uses expressed protein ligation (EPL) to generate nanobody-guided degraders (nanodegraders, NDs) incorporating SuFEx (sulfur–fluoride exchange) covalent modules for proximity-enabled crosslinking, azido-lysine handles for E3-ligand conjugation, and a chaperone-mediated autophagy (CMA1) motif for lysosomal targeting. Subsequent attachment of a cell-penetrating peptide (CPP) enables intracellular delivery, yielding α-synuclein (α-Syn) NDs that engage lysosomal, proteasomal, or dual-proteolytic degradation pathways. Dual-proteolytic NDs revealed route competition rather than synergy, defining design limits for multi-route degradation in neurons. The optimized covalent lysosome-targeting ND efficiently internalized into human iPSC-derived A53T α-Syn neurons, cleared aggregated α-Syn, restored calcium alterations and synaptotoxic effects, and remained stable and selective in vivo. This work establishes a modular chemical strategy for engineering nanobody-based degraders that dismantle neurotoxic proteins through multiple proteolytic systems.