A Kinetic Fluorescence-based Ca²⁺ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

Abstract

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor's function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca concentration in the absence of CXCL12) or compounds modulating the receptor's function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay's readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca.

Stimulation of the atypical chemokine receptor 3 (ACKR3) by a small-molecule agonist attenuates fibrosis in a preclinical liver but not lung injury model

“…The calcium (Ca 2+ ) mobilization assay was described in detail before [ 24 ]. Briefly, U87.CD4.CXCR4 or U87.CD4.CCR5 cells (2 × 10 4 cells per well in DMEM/10% FBS/0.01 M HEPES) were seeded in gelatin-coated (Sigma-Aldrich; 0.1% gelatin in DPBS) black-walled 96-well plates and incubated overnight at 37 °C and 5% CO 2 .…”

Synthesis and Anti-HIV Activity of a Novel Series of Isoquinoline-Based CXCR4 Antagonists

“…The calcium mobilization assay has been described in detail previously [ 31 ]. U87.CD4.CXCR4 cells (2 × 10 4 cells per well in DMEM/10% FBS/0.01 M HEPES) were seeded in gelatin-coated (Sigma-Aldrich; 0.1% gelatin in DPBS) black-walled 96-well plates and incubated overnight at 37 °C and 5% CO 2 .…”

A Set of Experimentally Validated Decoys for the Human CC Chemokine Receptor 7 (CCR7) Obtained by Virtual Screening

“…U87.CD4 cells that stably express either human CCR7, CXCR2, CCR5 or CXCR4 were seeded (20,000 cells/well) in gelatin-coated black-walled polystyrene 96-well plates with clear bottom and incubated overnight at 37 °C and 5% CO 2 . The next day, a fluorescent Ca 2+ -sensitive dye solution (Fluo-2 AM) was prepared as described before (Claes et al, 2018). Culture medium was removed, and cells were incubated for 45 min at room temperature in the dark.…”

“…Culture medium was removed, and cells were incubated for 45 min at room temperature in the dark. Meanwhile, 96-well polypropylene plates containing 5-fold concentrated compound dilutions and 5-fold concentrated solution of chemokine ligands (CCL19, CXCL8, LD78-β, CXCL12, respectively; all purchased from PeproTech) were prepared for use with the FLIPR Tetra device (Molecular Devices) as described before (Claes et al, 2018). The antagonistic properties of the compounds were calculated based on their capacity to inhibit the Ca 2+ release induced by a fixed concentration of chemokine (i.e.…”

“…The antagonistic properties of the compounds were calculated based on their capacity to inhibit the Ca 2+ release induced by a fixed concentration of chemokine (i.e. 50 ng/mL final concentration for CCL19, CXCL8 and CXCL12 and 100 ng/ml for LD78-β), as described (Claes et al, 2018). Exactly the same protocol was used to record calcium responses in Chinese hamster ovary (CHO)-K1 cells, upon stimulation with adenosine triphosphate (ATP, purchased from Sigma).…”