Abstract
Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes. While several degraders that harness the proteasome or the lysosome have been developed, a technology that simultaneously degrades targets and accelerates cellular autophagic flux is still missing. In this study, we develop a general chemical tool and platform technology termed AUTOphagy-TArgeting Chimera (AUTOTAC), which employs bifunctional molecules composed of target-binding ligands linked to autophagy-targeting ligands. AUTOTACs bind the ZZ domain of the otherwise dormant autophagy receptor p62/Sequestosome-1/SQSTM1, which is activated into oligomeric bodies in complex with targets for their sequestration and degradation. We use AUTOTACs to degrade various oncoproteins and degradation-resistant aggregates in neurodegeneration at nanomolar DC50 values in vitro and in vivo. AUTOTAC provides a platform for selective proteolysis in basic research and drug development.
Highlights
Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes
Targeted protein degradation (TPD) is the latest of emerging modalities in drug discovery and development and typically employs heterobifunctional chimeric molecules comprised of a target binder linked to a degradation-inducing moiety, which offer an attractive therapeutic means to eradicate disease-associated proteins, especially those belonging to the once-considered undruggable proteome[3,4,5,6,7]
Degradation of ERβ upon PHTPP-1304 treatment with proteasomal inhibition in MCF7 cells exhibited subnanomolar DC50 and Dmax values (Supplementary Fig. 4g). This robust degradation persisted up to at least 8 h post-washout of PHTPP-1304 (Supplementary Fig. 4h), suggesting that AUTOphagy-TArgeting Chimera (AUTOTAC) may display sustained degradative efficacy and be recycled from the lysosome. These results suggest that AUTOTAC does not require ubiquitin-dependent and PPI-driven cooperativity for its sustained autophagic proteolysis
Summary
Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes. LYTAC is applicable to only extracellular proteins, ATTEC directly targets mutant Htt or lipid droplets to the autophagosome without prior sequestration, and AUTAC utilizes S-guanylation that is still dependent on ubiquitination of the target[14,15,16,17] These challenges of the existing TPD modalities necessitate further development of a generally applicable TPD platform independent of Ub and the proteasome. S-guanylation-inducing molecular tags (AUTAC) or targeting extracellular and secreted/membrane proteins directly to the lysosome via peptidic tags (LYTAC)—attempt to address the degradation of PROTAC-resistant cargoes, no degrader technology yet exists for directly sequestering and targeting proteins and their aggregates using autophagy cargo receptors (e.g., p62). Binding of the Nt-Arg residue through the p62-ZZ domain conformationally activates p62 into an autophagy-compatible form, accelerating its self-oligomerization, interaction with LC3, and autophagosome biogenesis, facilitating autophagic targeting of p62-cargo complexes in a multi-step manner[21,22,23]
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