Lin, W., Li, Y., Yang, L., & Chen, T. (2020). ACS omega, 6(1), 680-695.
BODIPY FL VH032 is a novel, small-molecule fluorescent probe designed for the highly sensitive detection of von Hippel-Lindau (VHL) E3 ligase ligands. This probe was used to develop the first BODIPY FL VH032-mediated time-resolved fluorescence resonance energy transfer (TR-FRET) assay for the VHL-ElonginC-ElonginB (VCB) protein complex. It binds VCB with high affinity (Kd = 3.01 nM), enabling efficient ligand displacement analysis through TR-FRET signal modulation.
In the TR-FRET assay, terbium-labeled anti-GST antibody binds the GST-tagged VCB complex, acting as the donor fluorophore, while BODIPY FL VH032 serves as the acceptor. When a competing VHL ligand displaces BODIPY FL VH032 from its binding site, the FRET signal at 520 nm diminishes, allowing real-time monitoring of ligand binding events.
Compared to fluorescence polarization (FP) assays, BODIPY FL VH032-based TR-FRET exhibits superior sensitivity and lower protein consumption, with reduced assay interference. It reliably distinguishes between VHL ligands and unrelated molecules, confirming high specificity. With an average Z' value of 0.91, this assay is suitable for high-throughput screening.
Overall, BODIPY FL VH032 is a powerful tool for the identification and characterization of VHL ligands, facilitating drug discovery efforts targeting the ubiquitin-proteasome system via PROTAC and molecular glue strategies.
