Development and characterization of endolysosomal trafficking targeting chimera degraders of α-adrenergic receptor.

Introduction: Despite the booming targeted protein degradation technologies, degrading cell membrane proteins remains an enormous challenge. In particular, only a limited approach is appropriate for the degradation of the G protein-coupled receptor (GPCR) superfamily. It is encouraging that accelerating GPCRs' endocytosis and switching their post-endocytic fate from recycling to lysosomal degradation would represent a promising strategy for developing chemical degraders of GPCRs.

Objectives: This study aimed to elucidate the mechanism underlying post-endocytic sorting of internalized α-adrenergic receptor (α-AR) upon agonist stimulation and put forward a unique strategy for designing chemical degraders of GPCRs utilizing α-AR as an exemplary target.

Methods: The protein-protein interaction (PPI) of GASP1, Beclin 2, and α-AR was investigated by co-immunoprecipitation and GST pull-down, and the regulatory mechanism was explored using immunofluorescence imaging and biotin protection degradation assay. By conjugating the agonistic phenylephrine moiety and a Beclin 2-recruiting moiety, ML246 with linkers, the Endolysosomal Trafficking TArgeting Chimera (ETTAC) molecules were constructed as GPCR degraders for proof-of-concept studies.

Results: Mechanistically, the binding of Beclin 2 to GASP1 is crucial to the endolysosomal sorting and degradation of α-ARs. Recruiting Beclin 2 to enhance the Beclin 2-GASP1 binding, the ETTAC molecular proved to be highly efficient in reducing recycling and facilitating the degradation of α-AR. Furthermore, the representative ETTAC, PMA-37, effectively induces the α-ARs degradation in transfected and cancerous cells at the nanomole range in a GASP1 and Beclin 2-dependant manner and thus exhibits significant therapeutic effects against prostate tumor and benign prostatic hyperplasia.

Conclusions: Proof-of-concept studies of the ETTAC degraders for GPCR successfully elucidate the roles of post-endocytic sorting proteins and applied to directing the lysosomal degradation of α-ARs. Consequently, the ETTAC strategy represents a promising approach for the selective degradation of GPCRs and paves the way for future drug development.