Pooled endogenous protein tagging and recruitment for systematic profiling of protein function

Area of Science:

• Biochemistry and Molecular Biology
• Chemical Biology
• Drug Discovery and Development

Background:

• Induced proximity therapeutics, a rapidly advancing field, aim to bring effector and target proteins together, often to induce target degradation.
• Progress in this field is limited by the absence of scalable and unbiased tools for exploring effector-target protein interactions.

Purpose of the Study:

• To develop and apply a novel screening methodology for identifying new effector proteins that can induce target degradation.
• To expand the toolkit for induced proximity therapeutics, including protein degradation and other proximity-dependent mechanisms.

Main Methods:

• Combined pooled endogenous gene tagging with a ligand-binding domain and generic small-molecule-based recruitment to screen for induced protein proximity.
• Employed two orthogonal screening strategies: fluorescence-based monitoring of target protein levels and a cellular growth assay dependent on essential protein degradation.
• Utilized a platform for pooled induction of endogenous protein-protein interactions.

Main Results:

• Identified novel effector proteins capable of inducing target degradation, including previously validated examples.
• Screens converged on members of the C-terminal-to-LisH (CTLH) complex as key effectors.
• Demonstrated the efficacy of the developed platform in identifying new effectors for protein degradation.

Conclusions:

• The developed platform provides a scalable and unbiased approach to explore effector-target interactions for induced proximity therapeutics.
• This methodology significantly expands the repertoire of effector proteins available for targeted protein degradation and other induced proximity applications.
• The findings pave the way for the discovery of new therapeutic strategies based on induced proximity.