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In-Cell Approach to Evaluate E3 Ligases for Use in Targeted Protein Degradation.

Yunan Zheng¹, Anamika Singh², Zeqi Niu¹

¹Technology & Therapeutic Platforms, AbbVie Inc., North Chicago, Illinois 60064, United States.

Journal of the American Chemical Society

|June 10, 2025

Summary

This summary is machine-generated.

This study introduces a novel method using genetic code expansion to create E3 ligase-binder constructs for targeted protein degradation. This platform enables the exploration and development of new therapeutic strategies for previously undruggable targets.

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In Vitro Analysis of E3 Ubiquitin Ligase Function

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Area of Science:

Biochemistry
Molecular Biology
Chemical Biology

Background:

Evaluating E3 ligases for targeted protein degradation (TPD) is hindered by the lack of specific binders.
Developing novel methods to create functional E3 ligase-binder constructs is crucial for advancing TPD therapeutics.

Purpose of the Study:

To develop a versatile platform for creating covalent E3 ligase-binder constructs using genetic code expansion and click chemistry.
To demonstrate the utility of this platform for mapping E3 ligase surfaces and enabling TPD of neosubstrates, including for ligases without known ligands.

Main Methods:

Utilized genetic code expansion to incorporate a tetrazine-containing noncanonical amino acid (Tet-ncAA) into E3 ligases expressed in living cells.
Employed click chemistry to conjugate the Tet-ncAA with a strained trans-cyclooctene (sTCO)-tethered neosubstrate binder.
Applied the developed E3-ligand-free degrader (ELF degrader) platform to cereblon (CRBN) and speckle-type POZ protein (SPOP) E3 ligases.

Main Results:

Demonstrated successful TPD of neosubstrates by engineered CRBN ligases, with efficiency dependent on Tet-ncAA placement and linker length.
Showcased the ability of the platform to map functional E3 surfaces and identify optimal TPD interfaces.
Successfully applied the strategy to SPOP, an E3 ligase lacking known specific ligands, revealing its potential for TPD applications and PROTAC-like development.

Conclusions:

The E3-ligand-free degrader (ELF degrader) platform provides a versatile approach to define functional degron sites and guide degrader design.
This method enables the interrogation of any E3 ligase surface in live cells and is applicable to a broad range of E3 ligases.
The ELF degrader platform unlocks new E3 ligases, including those without known ligands, for therapeutic applications in targeted protein degradation.