A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Summary

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Abstract

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor's function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

Introduction

G protein-coupled receptors (GPCRs) are cell surface proteins that can be activated by extracellular ligands (e.g., peptides, protein hormones, amines), thereby regulating many physiological and developmental processes¹. When an agonist occupies its GPCR binding pocket, the induced conformational change in the receptor protein promotes the binding of intracellular receptor-associated heterotrimeric G proteins, consisting of G_α-GDP and G_βγ subunits. The subsequent exchange of GTP for GDP on the G_α subunit results in the dissociation of the G protein subunits (G_α-GTP an....

Protocol

1. Maintenance of Cell Culture

NOTE: All steps described under 1 and 2 are carried out under sterile conditions in a laminar flow cabinet.

1. Grow cells in T75 culture flasks at 37 °C and 5% CO₂ in a humidified incubator.
   NOTE: In this assay, Jurkat cells (i.e., human leukemic T lymphocyte cells that endogenously express CXCR4¹⁷) are used. Expression of CXCR4 at the cell surface should be evaluated throughout cell culturing by means of flow cytometry. A description of the flow cytometry procedure and reagents to determine receptor expression levels at the cell surface is, however, no....

Results

The general workflow of the binding assay is presented in Figure 1A. An illustration of the type of flow cytometry data obtained for different sample types in a standard experiment (i.e., negative control, positive control, and experimental sample) is depicted in Figure 1B, and a possible plate layout to perform the assay in a 96-well plate format is given in Figure 1C. Incubation of Jurkat .......

Discussion

Compared to other types of binding assays (i.e., saturation binding and kinetic binding experiments), competition binding assays are best suited for screening purposes. Indeed, they allow evaluation of large batches of unlabeled compounds, for instance small molecules, by scoring their capability to interfere with the binding of a fixed amount of a labeled receptor ligand. Compounds that bind to other receptor sites than the labeled ligand might remain undetected in the assay. Although the competition bindi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD FACSCanto II	Becton Dickinson	Not applicable	Flow cytometry device
BD FACSDIVA Software
BD FACSArray	Becton Dickinson	Not applicable	Flow cytometry device
BD FACSArray System Software
Graphpad Prism	Graphpad		software package used for nonlinear regression analysis in Figure 2 and Figure 3
FlowJo	FlowJo is now a wholly owned subsidiary of BD.
Vi-CELL	Beckman Coulter	Not applicable	cell viability analyzer
Sigma 3-18 KS	Sigma	Not applicable	centrifuge
AMD3100	Sigma	A5602-5mg	specific CXCR4 antagonist
Maraviroc	Pfizer		antiretroviral drug, CCR5 antagonist, available for research at Selleckchem (cat#S2003), Sigma (cat#PZ0002)
h-SDF1a (AF647)	ALMAC	CAF-11-B-01	fluorescently labeled CXCL12, CXCL12AF647
Fetal Bovine Serum (FBS)	Gibco (Life Technologies)	10270-106
Bovine Serum Albumin (BSA)	Sigma	A1933-25G
HBSS (10x), calcium, magnesium, no phenol red	Gibco (Life Technologies)	14065-049
HEPES (1M)	Gibco (Life Technologies)	15630-056
Dulbecco's Phosphate Buffered Saline (DPBS)	Gibco (Life Technologies)	14190-094
Jurkat cells	ATCC
Reagent reservoir PP	Sigma	BR703411
Rapid flow filter: 0.2 µm aPES	Thermo Scientific	566-0020
Sterilin microtiter plate, 96-well, U bottom, clear	Thermo Scientific	611U96
Falcon tubes, 50ml	Greiner Bio-One	227 261
Tissue culture flask (T75)	Corning	353024