Target immobilization on glass microfiber membranes as a label-free strategy for hit identification

The discovery of novel chemical entities targeting G protein-coupled receptors (GPCRs) is usually guided by their receptor affinity. However, traditional affinity assay methods and hit identification procedures are usually laborious and expensive. In this work, the type-2 vasopressin receptor (V₂R) was chosen as a prototypical GPCR. Membrane fragments from cells highly expressing SNAP-V₂R were immobilized on the surface of a glass microfiber (GMF) coated with O⁶-benzylguanine (BG). This was achieved by transferring the benzyl group of BG to the active site of the SNAP-tag through a nucleophilic substitution reaction. As a result, a biofilm called SNAP-V₂R@GMF-BG was produced that showed good specificity and stability. The adsorption ratio for each V₂R ligand treated with SNAP-V₂R@GMF-BG was determined by HPLC and exhibited a good linear correlation with the K_i value determined by displacement assays. Furthermore, a K_i prediction assay was performed by comparing the data with that generated by a homogeneous time-resolved fluorescence (HTRF) assay. SNAP-V₂R@GMF-BG was also used to screen hit compounds from natural products. After SNAP-V₂R@GMF-BG was incubated with the total extract, the ligand that binds to V₂R could be separated and subjected to LC‒MS analysis for identification. Baicalein was screened from Clerodendranthus spicatus and verified as a potential V₂R antagonist. This V₂R-immobilized GMF platform can help determine the affinity of V₂R-binding hit compounds and screen the compounds efficiently and accurately.