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In-cell proximity target validation methods for heterobifunctional molecules with CRBN- or VHL-binder using AirID.
Kohdai Yamada1, Satoshi Yamanaka2, Hiroyuki Yamakoshi3
1Division of Cell-Free Sciences, Proteo-Science Center, PIAS, Ehime University, Matsuyama, Ehime, Japan.
Communications Biology
|August 30, 2025
Summary
Researchers developed a new method using AirID to validate protein interactions with heterobifunctional molecules like proteolysis-targeting chimeras. This technique helps understand how these novel drugs engage with target proteins within cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Heterobifunctional molecules, including proteolysis-targeting chimeras (PROTACs) and autophagy-targeting chimeras (AUTOTACs), are emerging drug modalities.
- These molecules induce proximity between proteins, enabling targeted protein degradation or other catalytic functions.
- Cereblon (CRBN) and von Hippel-Lindau (VHL) are commonly used E3 ligase binders in current heterobifunctional molecule design.
Purpose of the Study:
- To develop and validate a novel method for identifying protein interactomes of heterobifunctional molecules in cellular environments.
- To assess the efficacy of AirID, a proximity biotinylation enzyme, in capturing these interactions.
- To compare the proximity interactome profiles generated by molecules with identical target binders but different E3 ligase binders.
Main Methods:
- Development of AirID-based proximity biotinylation tools fused to E3 ligase binding domains (ThBD of CRBN and full-length VHL).
- Application of these tools to validate the interactome of six different heterobifunctional molecules in cells.
- Comparative analysis of interactome data generated by molecules differing in their E3 ligase component.
Main Results:
- The ThBD-AirID fusion protein effectively biotinylated target proteins, validating its utility.
- AirID fused to full-length VHL also demonstrated high biotinylation efficiency.
- Significant differences in proximity interactome profiles were observed for molecules with the same target binder but distinct E3 binders.
- A specific nuclear interaction between androgen receptor and ARV-110 was identified using ThBD-AirID.
Conclusions:
- AirID-based proximity biotinylation is a robust method for validating cellular interactomes of heterobifunctional molecules.
- AirID-fused ThBD and VHL are valuable tools for studying the complex interactions of these novel drug candidates.
- Understanding these proximity interactomes is crucial for optimizing the design and efficacy of heterobifunctional therapeutics.
