Subtype selective fluorescent ligands based on ICI 118,551 to study the human β2-adrenoceptor in CRISPR/Cas9 genome-edited HEK293T cells at low expression levels

Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β₂ -adrenoceptor (β₂ AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β₂ AR (pK_D >7.0) with good selectivity over the β₁ AR (pK_D <6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; β₂ AR pK_D 7.48), 9c (ICI 118,551-βAla-βAla-BODIPY-FL-X; β₂ AR pK_D 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; β₂ AR pK_D 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; β₂ AR pK_D 7.42). 9a (ICI 118,551-βAla-βAla-BODIPY-X-630/650) had the highest affinity at recombinant β₂ ARs (pK_D 7.57), but also exhibited significant binding affinity to the β₁ AR (pK_D 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular β₂ ARs in HEK293 T cell expressing exogenous β₂ ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular β₂ ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T β₂ ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.