Development of BODIPY FL VH032 as a High-Affinity and Selective von Hippel–Lindau E3 Ligase Fluorescent Probe and Its Application in a Time-Resolved Fluorescence Resonance Energy-Transfer Assay

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.

General Stepwise Approach to Optimize a TR-FRET Assay for Characterizing the BRD/PROTAC/CRBN Ternary Complex

“…The panel of compounds included the pan-BRD ligands (+)-JQ1 and HJB97, CRBN ligands thalidomide and lenalidomide, and the irrelevant compounds RVX-208 [a BRD­(BD2) selective ligand] and VH032 (a VHL ligand; Figure ). , We included HJB97 and thalidomide because they are components of the bivalent PROTAC BET Degrader-1 that binds to BRD and CRBN, respectively. All chemicals were tested in 1–3 dilutions with respective concentration ranges of 0.56 nM to 100 μM for (+)-JQ1, RVX-208, and VH032 and 0.017 nM to 3 μM for HJB97, thalidomide, and lenalidomide.…”

“…DMSO is a commonly used solvent for chemicals, and we used DMSO to prepare all of our stock chemical solutions. At high concentrations, DMSO can affect TR-FRET assays. ,,− We used the TR-FRET assay for the BRD2­(BD1)/PROTAC BET Degrader-1/CRBN ternary complex as an example to evaluate the assay’s DMSO tolerance. We selected PROTAC BET Degrader-1 as a representative PROTAC because of its highest signal window (Figure ).…”

“…RVX-208 was purchased from MedKoo Biosciences, Inc. (Morrisville, NC). We purchased (+)-JQ1 from Cayman Chemical (Ann Arbor, MI), and VH032 was prepared in house . Triton X-100 and bovine serum albumin were purchased from Sigma (St. Louis, MO).…”

“…In our TR-FRET ternary complex formation assay, the formation of the BRD2­(BD1)/PROTAC BET Degrader-1/CRBN­(DDB1) ternary complex was blocked by either of the BRD2­(BD1) ligands (+)-JQ1 and HJB97 or the CRBN ligands thalidomide and lenalidomide in a dose-dependent manner but not by compounds irrelevant to the complex, such as the BRD­(BD2) selective ligand RVX-208 or the VHL ligand VH032 (Figure ), , further confirming the specificity of the assay. This indicates that the assay can be configured to characterize BRD or CRBN ligands for their specific inhibitory binding activities.…”

A protocol for high-throughput screening of histone lysine demethylase 4 inhibitors using TR-FRET assay

“… For compounds in powders, dissolve them in DMSO to prepare stock solutions at 10 mM. For primary single dose screen, compound stock solutions in DMSO are pre-arrayed in barcoded 384-well compound plates (Corning 3656) ( Cui et al., 2011 ; Lin et al., 2021 ) at 10 μL/well in areas of columns 3-to-12 and 15-to-24; wells in columns 1, 2, 13 and 14 are empty ( Figure 1 A).

Figure 1 Plate layouts for primary single dose screen and for dose response test (A) Plate layout for primary single dose screen.

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