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

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.