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 and G_βγ) that, in turn, will further initiate downstream signaling pathways^2,3. When the G_α-GTP becomes hydrolyzed, re-association of the G_α-GDP and G_βγ subunits will convert the G protein back into its resting state^3,4. Distinct types of G proteins exist (G_s, G_i/o, G_q, G_12/13), which are categorized based on sequence similarity with the G_α subunit⁵. All of these G proteins induce defined intracellular signaling pathways that underlie the biological response to receptor activation. Subsequent to receptor activation, GPCR kinases (GRKs) phosphorylate the intracellular tail of GPCRs, thereby promoting interaction with β-arrestins. This process leads to the termination of G protein signaling, receptor desensitization and internalization⁶. β-arrestins are also part of multi-molecular complexes that trigger signaling cascades independent of G protein signaling⁷.

GPCRs are amongst the most validated molecular targets for therapeutic intervention, as deregulated GPCR-mediated signaling, for instance due to gain-of-function mutations in the receptor gene or receptor overexpression, contributes to the etiology of many human diseases⁸. Therefore, GPCRs represent one of the most important classes of drug targets investigated by the pharmaceutical industry^8,9,10. A notable example of a clinically relevant GPCR is the CXC chemokine receptor 4 (CXCR4), which can be activated by a sole natural ligand, the CXC chemokine ligand 12 (CXCL12)¹¹. Due to its established role as a major co-receptor for human immunodeficiency virus 1 (HIV-1) entry and infection in cluster of differentiation 4 (CD4) positive T-lymphocytes¹², CXCR4 was first investigated as an antiviral drug target. CXCL12-CXCR4 interaction in the bone marrow further regulates the retention and homing of stem and progenitor cells¹³. Also, given its involvement in many aspects of cancer biology (e.g., tumor cell survival, metastasis, tumor-related angiogenesis)¹⁴ and several other human diseases (e.g., inflammatory diseases)¹⁵, CXCR4 raised significant interest as a promising target for drug discovery. AMD3100, a small molecule that specifically targets CXCR4, was initially discovered as an anti-HIV drug candidate¹⁶ and is still one of the most potent CXCR4 antagonists described to date¹⁷. Its development as an antiviral drug was, however, discontinued¹⁸. Currently this molecule is used as a stem cell mobilization agent during the treatment of multiple myeloma and lymphoma patients¹⁸. Several other chemically unrelated small molecules and biologics that inhibit CXCR4 function with varying potency have been described¹⁹.

Receptor binding methods are valuable tools in pharmacology that allow the identification of compounds (e.g., small molecules) that directly interact with the GPCR of interest. In order to perform binding studies, there is no need for prior knowledge concerning the intracellular signaling properties or functionality of a given GPCR. Although this can be considered to be an advantage, it implies that compounds for which receptor binding can be demonstrated need to be further characterized by evaluating their potential agonistic or antagonistic activity. This activity can be evaluated using pharmacological or biological assays related to the GPCR under study. Dependent on their activity profile, receptor binding molecules might then potentially evolve to become novel lead compounds for investigation in pre-clinical and clinical studies. Molecules that specifically bind to a receptor with high affinity can also serve as scaffolds to generate therapeutic or diagnostic tools, for instance by radiolabeling them for noninvasive in vivo imaging of tumor cells²⁰, or as potential vehicles for targeted delivery of therapeutics²¹. In case of CXCR4, in vivo imaging of tumor cells has already been demonstrated using mouse models wherein labeled CXCR4-targeting molecules allowed the visualization of human cancer xenografts^20,22,23.

In this report, we describe a detailed protocol for a competition binding assay that enables the identification of small molecules and biologics that directly interfere with agonist (CXCL12) binding to CXCR4. The basic principle of the assay is the competition between a fixed amount of fluorescently labeled ligand (CXCL12^AF647, see Table of Materials and Reagents) and unlabeled compounds for binding to the receptor protein^17,24. The specific fluorescent signal from labeled ligand bound to single cells expressing CXCR4 is then analyzed by flow cytometry. This fluorescent signal will be reduced when unlabeled small molecules disrupt the interaction between CXCL12^AF647 and CXCR4. The assay uses non-manipulated living cells that endogenously express CXCR4 (i.e., Jurkat cells). Hence, no cell membrane preparation is required, which makes the assay convenient, fast and compatible with increased throughput. Since a fluorescently labeled ligand is used, radioactivity is avoided.

Because CXCL12 is the natural agonist for CXCR4, small molecule compounds that interfere with CXCL12^AF647 binding in the assay are likely to interact with the orthosteric receptor binding site (i.e., the binding site occupied by the natural agonist). Molecules that would interact with receptor binding sites topographically distinct from the orthosteric binding site remain undetected, if they do not influence binding of CXCL12. For instance, positive and negative allosteric modulators, an important and emerging category of GPCR targeting molecules acting on allosteric binding sites²⁵, will potentially not be picked up with this assay. In addition, whether the compounds identified with this binding assay function as receptor antagonists or as agonists cannot be derived. Investigation of the identified compounds in additional pharmacological or functional receptor-related assays will thus be required. These assays might include (a combination of) cellular fluorescence- or luminescence-based assays for the detection of second messengers (e.g., Ca²⁺, cyclic adenosine monophosphate (cAMP)), phenotypic or biological assays and β-arrestin recruitment assays, the choice of which depends on the specific signaling properties of the GPCR under study. Hence, the competitive binding assay described herein mainly serves as an initial screening assay that needs to be complemented with other cell-based assays to enable an in-depth characterization of compounds with receptor binding potency.

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, not within the scope of this protocol, but has been described previously¹⁷.
2. Let Jurkat cells grow in suspension until they reach 80 - 85% confluency. Before passaging the cells to a novel flask, allow all reagents to reach room temperature (RT).
3. Add 20 mL of fresh complete growth medium (RPMI-1640 medium, 10% fetal bovine serum (FBS), 2 mM glutamine) to a novel T75 culture flask.
4. Add 5 mL of Jurkat cell suspension from the original T75 flask (containing 25 mL of cell suspension) to the novel T75 culture flask. Incubate at 37 °C and 5% CO₂ in a humidified incubator.

2. Preparation of CXCL12, Assay Buffer, and Jurkat Cells for the Competition Binding Assay.

1. Prepare a stock solution of CXCL12^AF647 (20 µg/mL; see Table of Materials and Reagents) by dissolving the lyophilized reagent (stored at -80 °C, in the dark) in ultrapure water supplemented with 0.01% (volume/volume) of Polysorbate 20. Store single use aliquots from this stock solution at -80 °C, protected from light.
2. Prepare assay buffer by adding 40 mL HEPES (1 M, 20 mM final concentration) to 200 mL Hank's Balanced Salt Solution (HBSS, 10x, without phenol red and without sodium bicarbonate, 1x final concentration). Add ultrapure water to obtain a final volume of 2 L. Add 4 g (0.2% weight/volume) bovine serum albumin (BSA), and dissolve the BSA via magnetic stirring. Finally, adjust the pH to 7.4 (use NaOH for this) and filter the solution through 0.2 µm pores (see Table of Materials and Reagents) using a vacuum manifold.
   NOTE: This assay buffer will be used in all further steps of the protocol.
3. Count the number and the viability of the cells. For this, take a sample of the cell suspension and dilute it in phosphate buffered saline (PBS).
   NOTE: We routinely use an automated cell viability analyzer (see Table of Materials and Reagents) capable of counting cell suspensions at varying concentrations. For cell counting, dilute 0.5 mL of cell suspension in 1.5 mL PBS (other dilutions, e.g., 0.1 mL in 1.9 mL PBS, are also possible). The use of this method, which is based on the trypan blue dye exclusion method, has been described previously²⁶. Several other devices are commercially available for counting cell number and viability that should work equally well.
4. Collect the desired number of cells (i.e., ~24 x 10⁶ cells to run the assay with one complete 96-well plate) in a sterile 50 mL tube by centrifugation (see Table of Materials and Reagents for the type of centrifuge used) at 400 x g for 5 min at RT.
5. Gently pour off the supernatant without disturbing the cell pellet. Add fresh assay buffer (e.g., 20 mL) and resuspend the cells by gently pipetting up and down.
6. Centrifuge the cells again at 400 x g for 5 min at RT.
7. Pour off the supernatant again and resuspend the cell pellet in fresh assay buffer to obtain a density of 5 x 10⁶ cells/mL.

3. Competition Binding Assay

NOTE: The actual competition binding assay is performed at RT and can be performed under non-sterile conditions.

1. Dilute the compounds under investigation in assay buffer (see 2.2) to obtain the desired concentration. Either prepare a fixed concentration of compound for initial screening (e.g., 10 µM final concentration) or, alternatively, a serial dilution series of concentrations for more detailed characterization of the compounds (e.g., a 1/3, 1/4 or 1/5 dilution series starting at 1 µM, final concentration). Keep in mind that the compound solution will ultimately become 2x diluted in the assay; therefore, prepare a 2x concentrated solution.
2. Dispense 100 µL of compound solution (2x concentrated) into a clear 96-well round bottom plate (see Table of Materials and Reagents) according to a pre-defined experimental lay out (e.g., Figure 1C).
   NOTE: At this stage, negative and positive control samples are included in the assay. In the negative control sample, 100 µL of assay buffer is added instead of compound to the wells of the 96-well plate. For the positive control sample, assay buffer is also added during this step. See also Figure 1C for a typical experimental layout in which a dilution series of several compounds is tested.
3. Add 50 µL of cell suspension (see 2.7; 0.25 x 10⁶ cells) from a reagent reservoir into the 96-well plate using a multichannel pipette. Incubate the plate for 15 min at RT in the dark.
4. Add 50 µL of fluorescently labeled CXCL12 (i.e., 100 ng/mL of CXCL12^AF647 in assay buffer, 4x concentrated, 25 ng/mL final concentration) from a similar reagent reservoir to the wells of the 96-well plate. Incubate for 30 min at RT in the dark.
   NOTE: For the negative control samples, add assay buffer instead. Hence, the fluorescent signal detected in the negative control samples will correspond to the autofluorescent background signal (Figure 1A). For the positive control samples, add 50 µL/well of CXCL12^AF647. The positive control samples will yield the maximal fluorescence signal detected, since no potential inhibition by pre-incubation with compounds was included (Figure 1A).
5. Centrifuge the 96-well plate at 400 x g for 5 min at RT. Remove the supernatant from the pelleted cells by flipping over the plate. Dry the plate on a tissue.
6. Add 200 µL of fresh assay buffer from a reagent reservoir to the wells using a multichannel pipette. Immediately proceed.
7. Centrifuge the plate again for 5 min at 400 x g at RT. Remove the supernatant by flipping over the plate and again dry it on tissue.
8. Gently resuspend the cell pellet in 200 µL of 1% paraformaldehyde dissolved in PBS. This step will fix the cells.
9. Continue the protocol immediately with the quantification of the fluorescence by flow cytometry.

4. Analysis of the samples by flow cytometry

CXCL12^AF647 stained and fixated cells are now ready to be analyzed using flow cytometry. Several types of flow cytometers can be used, but they need to be equipped with the correct laser (i.e., a red laser, excitation range ~630 nm) for excitation and suitable filters for fluorophore detection (emission filters ~660 nm). They need to be capable of handling samples in a 96-well plate format. Examples of suitable flow cytometry devices are given in the Table of Materials and Reagents.

1. Start up the device and open the corresponding software (see Table of Materials and Reagents).
2. Select the following cellular parameters to be visualized in a dot blot format: forward scatter (FSC), side scatter (SSC) and the fluorophore (CXCL12^AF647) detection channel.
   NOTE: With the FSC parameter, cells are discriminated based on their size, since the detected light absorption is proportional to the cell's diameter. The SSC parameter, measuring light scattering at a 90° angle, provides information about the granularity of the cells.
3. Choose one sample (e.g., a negative control sample) to perform gating of a defined homogenous cell population based on the FSC and SSC parameters.
   1. Select automatic injection of ~100 µL of fixated cells from this negative control sample into the flow cytometer.Select the option "mixing" before injection and use a sample flow rate of 1.5 µL/s.
   2. Run this sample by selecting "Acquire Data." The FSC and SSC parameters for this sample will now appear on the screen.
   3. Select the software's gating tool. Based on the FSC and SSC dot blot visualization, pre-define a homogenous and viable cell population by gating. To do so, create a polygon (using the software's gating tool) that includes the homogenously distributed single cells ("events") based on these two dimensions.
      NOTE: The gating procedure aims to define a homogenous and viable cell population that will be used for further analysis. Gating relies on the assumption that the majority of viable cells will form a homogenous cell population based on the FSC and SSC parameters. By performing this step, cellular debris, dead cells, and cell aggregates can largely be excluded from further analysis. An illustration of the gating process is given in Figure 1B.
4. Select to analyze 20,000 "events" (i.e., single cells) per sample.
   NOTE: This means that for each sample, 20,000 cells that fall within the pre-defined gate will eventually be analyzed. Data acquisition for each sample will continue until this number of events is analyzed.
5. Start the run (select "Record Data"). The flow cytometry device will now analyze all samples one-by-one by recording the mean fluorescence intensity (MFI) for each sample. This MFI corresponds to the mean fluorescent signal corresponding to the 20,000 cells that fall within the pre-defined gate.

5. Data analysis

Use the MFI obtained for each sample to perform all further calculations. Analysis of the flow cytometry data can be performed by several commercially available software packages (see Table of Materials and Reagents).

1. To determine the percentage inhibition of the fluorescent binding signal, as a result of compound pre-incubation, apply the following formula:
   
   Where:
   MFI_Exp = the MFI of the experimental (= compound-treated) sample
   MFI_PC = the MFI of the positive control
   MFI_NC = the MFI of the negative control
2. To determine the IC₅₀ value of a compound (i.e., the concentration of compound that can reduce the fluorescent binding signal by 50%), apply the following formula:
   
   Where:
   Log_conc.2 = the log of a concentration of compound that results in less than 50% inhibition of the difference between the MFI value of the PC and NC
   Log_conc.1 = the log of a concentration of compound that results in more than 50% inhibition of the difference between the MFI value of the PC and NC
   NOTE: Alternatively, in case of highly active compounds, a dose-response curve covering several logs of magnitude can be generated based on the MFI corresponding to each tested concentration of compound. By applying non-linear regression curve fitting using appropriate software (see Table of Materials and Reagents), IC₅₀ values can then be deduced from the generated curves. An example of this analysis approach is shown in Figure 3.

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

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