Fluorescence Polarization Screening for Allosteric Small Molecule Ligands of the Cholecystokinin Receptor

Abstract

The success in screening for drug candidates is highly dependent on the power of the strategy implemented. In this work, we report and characterize a novel fluorescent benzodiazepine antagonist of the type 1 cholecystokinin receptor (3-(3-]-diazepin-3-yl)ureido)benzoic acid) that can be used as a receptor ligand in a fluorescence polarization assay, which is ideally suited for the identification of small molecule allosteric modulators of this physiologically important receptor. By binding directly to the small molecule-docking region within the helical bundle of this receptor, this indicator can be displaced by many small molecule candidate drugs, even those that might not affect the binding of an orthosteric cholecystokinin-like peptide ligand. The biological, pharmacological, and fluorescence properties of this reagent are described, and proof-of-concept is provided in a fluorescence polarization assay utilizing this fluorescent benzodiazepine ligand.

Fluorescence‐ and bioluminescence‐based approaches to study GPCR ligand binding

“…This difference in polarization can be used to differentiate bound from free ligand. The early examples used a cuvette‐based spectrofluorometer and were therefore very low throughput (Tota et al ., ; Tota et al ., ; Turcatti et al ., ), but advances in plate reader technology has led to FA and FP experiments being performed in 96‐ (Cornelius et al ., ; Kecskes et al ., ; Veiksina et al ., ; Harikumar et al ., ), 384‐ (Allen et al ., ) and even 1536‐ (Harris et al ., ) well plate formats. A range of different GPCRs, both peptide and non‐peptide, has been successfully studied using FP experiments.…”

“…A range of different GPCRs, both peptide and non‐peptide, has been successfully studied using FP experiments. The GPCRs with low MW endogenous ligands include the 5‐HT 2C receptor (Cornelius et al ., ), adenosine A 2A receptor (Kecskes et al ., ) and muscarinic M 1 receptor (Harris et al ., ), and the peptide receptors include the cholecystokinin CCK 1 receptor (Harikumar et al ., ), vasopressin V 1A and δ opioid receptors (Allen et al ., ). As FA and FP can distinguish between bound and unbound ligand, there is no requirement for a wash or filtration step.…”

Cholecystokinin-induced satiety, a key gut servomechanism that is affected by the membrane microenvironment of this receptor

“…This was achieved with photoaffinity labeling using 14C-labeled benzodiazepine agonist and antagonist ligands. 40 It was also confirmed using fluorescence quenching of ligand binding 41 and the impact of receptor mutagenesis on ligand binding. 42,43 The allosteric nature of this small-molecule-binding site was further confirmed using pharmacologic analyses, including the kinetics of ligand dissociation and the impact of orthosteric and possible allosteric ligands on the functions of each other.…”

Biophysical screening methods for extracellular domain peptide receptors, application to natriuretic peptide receptor C ligands

“…The fluorescence polarization technique is limited, however, to interrogate molecules that compete with the reporter molecule for the orthosteric binding site, and so this particular assay set‐up would not be able to identify non‐competitive, allosteric modulators. In order to remedy this, an FP probe would need to be designed to bind at the identified allosteric site of interest, and while this would be challenging for this particular project, it has been achieved with other examples . The thermal shift technique is equally amenable to high throughput, as plate reading takes only just over an hour; however, this technique is rather protein‐intensive and thus more expensive.…”

“…In order to remedy this, an FP probe would need to be designed to bind at the identified allosteric site of interest, and while this would be challenging for this particular project, it has been achieved with other examples. [35,36] The thermal shift technique is equally amenable to high throughput, as plate reading takes only just over an hour; however, this technique is rather protein-intensive and thus more expensive. The full contrast and comparison of the three biophysical methods compared with traditional radiolabelled ligand binding studies are outlined in Table 3.…”