In-cell proximity target validation methods for heterobifunctional molecules with CRBN- or VHL-binder using AirID

Heterobifunctional molecules, such as proteolysis-targeting and autophagy-targeting chimera, represent new drug concepts. They are composed of two protein binders that can induce proximity interactions between two proteins and protein catalysis. Currently, cereblon (CRBN)- and von Hippel-Lindau (VHL)-binders with thalidomide- and VH032-backbones are widely used as E3 ligase binders. Here, we developed a method to validate proteins that interact with heterobifunctional molecules in cells using AirID, a proximity biotinylation enzyme. Interactome of target proteins was validated for six heterobifunctional molecules. ThBD-AirID, a fusion of the thalidomide-binding domain (ThBD) of CRBN and AirID, effectively biotinylated the target proteins. AirID fused to full-length VHL also exhibited highly effective biotinylation. Heterobifunctional molecules with the same target binder but different E3 binders showed different proximity interactome profiles in cells. Analysis using ThBD-AirID revealed a nuclear interaction between androgen receptor and ARV-110. AirID-fused ThBD and VHL could be useful for validating the heterobifunctional molecular interactome in cells. The development of AirID-based tools (ThBD–AirID, VHL–AirID) to validate protein interactomes of heterobifunctional molecules in cells reveals E3 binder–dependent proximity proteins of a protein of interest.