Abstract
The von Hippel-Lindau (VHL) tumor suppressor associates with transcription factors elongin-C and elongin-B to form the VHL-elongin-C-elongin-B protein complex and carry out its functions, such as degradation of hypoxia-inducible factors. VHL ligands are used not only to modulate hypoxia-signaling pathways and potentially treat chronic anemia or ischemia but also to form bivalent ligands as proteolysis-targeting chimeras to degrade proteins for potential therapeutic applications. Sensitive and selective VHL-based binding assays are critical for identifying and characterizing VHL ligands with high-throughput screening approaches. VHL ligand-binding assays, such as isothermal titration calorimetry, surface plasmon resonance, and fluorescence polarization assays, are reported but with limitations. Isothermal titration calorimetry requires higher protein concentrations with a lower throughput than fluorescence-based assays do. Surface plasmon resonance requires protein immobilization, which introduces variation and is not suitable for testing a large number of ligands. Fluorescence polarization can be sensitive with high-throughput capability but is susceptible to assay interference, and small-molecule-based fluorescent probes are not available. We developed the first small-molecule-based high-affinity VHL fluorescent probe BODIPY FL VH032 (5), with a K d of 3.01 nM, for a time-resolved fluorescence resonance energy-transfer assay. This new assay is sensitive, selective, resistant to assay interference, and capable of characterizing VHL ligands with a wide range of affinities. It is also suitable for VHL ligand identification and characterization with high-throughput screening.