An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New...

Area of Science:

• Biophysics
• Structural Biology
• Drug Discovery

Background:

• Integral membrane proteins (IMPs) are crucial in cellular functions and disease, necessitating efficient methods for studying their structure and interactions.
• Understanding IMPs and their interactions with small molecule ligands is vital for early-stage drug discovery.
• Robust, high-throughput biophysical methods are required for IMP analysis and ligand screening.

Purpose of the Study:

• To develop and validate a novel differential scanning fluorimetry (DSF) screening method for integral membrane proteins (IMPs).
• To assess the utility of a thiol-reactive BODIPY FL-cystine dye for monitoring IMP thermal unfolding.
• To evaluate the method's capability in detecting and characterizing low molecular weight (LMW) ligand-target interactions.

Main Methods:

• A differential scanning fluorimetry (DSF) assay was developed using a thiol-reactive BODIPY FL-cystine dye.
• The assay monitored the thermal unfolding of the G-protein-coupled receptor (GPCR), CXCR2.
• The method was validated using well-characterized antagonists and cross-validated with differential static light scattering (DSLS).

Main Results:

• The novel DSF assay reliably assessed the stability of the membrane protein CXCR2 in a 384-well format.
• The assay successfully detected and characterized interactions between CXCR2 and 14 different low molecular weight (LMW) ligands across a 4 log unit potency range.
• Results from the BODIPY-based DSF assay were consistent with label-free differential static light scattering (DSLS) thermal denaturation data.

Conclusions:

• The developed BODIPY-based DSF assay is a sensitive and reliable tool for studying membrane protein stability.
• This method facilitates the high-throughput screening and characterization of low molecular weight (LMW) ligands targeting integral membrane proteins (IMPs).
• The assay format shows significant potential as a generalizable method for investigating membrane protein-ligand interactions in drug discovery.