The authors would like to thank Eric Fonteyn and Geert Schoofs for excellent technical assistance. This work has been supported by the KU Leuven (grant no. PF/10/018), the Fonds voor Wetenschappelijk Onderzoek (FWO, grant no. G.485.08), and the Fondation Dormeur Vaduz.

NOTE: All steps described under sections 1 and 2 are carried out under sterile conditions in a laminar flow cabinet.
1. Maintenance of U87.CD4.hCXCR4 Cells
<ol>
	<li>Grow the cells in T75 culture flasks at 37 &#176;C and 5% CO2 in a humidified incubator. 
	NOTE: The in vitro cell line used in this protocol is a U87 glioblastoma cell line stably expressing cluster of differentiation 4 (CD4) and human CXCR4 and has been previously described17. C...

<ol>
	<li>Pierce, K. L., Premont, R. T., Lefkowitz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven-transmembrane+receptors.">Seven-transmembrane receptors.</a> Nat Rev Mol Cell Biol. 3, 639-650 (2002).</li><li>Fredriksson, R., Lagerstrom, M. C., Lundin, L. G., Schioth, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+G-protein-coupled+receptors+in+the+human+genome+form+five+main+families.+Phylogenetic+analysis,+paralogon+groups,+and+fingerprints.">The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.</a> Mol Pharmacol. 63, 1256-1272 (2003).</li><li>Lappano, R., Maggiolini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-coupled+receptors:+novel+targets+for+drug+discovery+in+cancer.">G protein-coupled receptors: novel targets for drug discovery in cancer.</a> Nat Rev Drug Discov. 10, 47-60 (2011).</li><li>Zhang, R., Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+GPCR+drug+discovery.">Tools for GPCR drug discovery.</a> Acta Pharmacol Sin. 33, 372-384 (2012).</li><li>Milligan, G., Kostenis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotrimeric+G-proteins:+a+short+history.">Heterotrimeric G-proteins: a short history.</a> Br J Pharmacol. 147, S46-S55 (2006).</li><li>Tuteja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+G+protein+coupled+receptors.">Signaling through G protein coupled receptors.</a> Plant Signal Behav. 4, 942-947 (2009).</li><li>Neves, S. R., Ram, P. T., Iyengar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein+pathways.">G protein pathways.</a> Science. 296, 1636-1639 (2002).</li><li>Smith, J. S., Rajagopal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-Arrestins:+Multifunctional+Regulators+of+G+Protein-coupled+Receptors.">The beta-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.</a> J Biol Chem. 291, 8969-8977 (2016).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+non-peptide+small+molecular+CXCR4+antagonists+as+anti-HIV+agents:+Recent+advances+and+future+opportunities.">Discovery of non-peptide small molecular CXCR4 antagonists as anti-HIV agents: Recent advances and future opportunities.</a> Eur J Med Chem. 114, 65-78 (2016).</li><li>Chatterjee, S., Behnam Azad, B., Nimmagadda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intricate+role+of+CXCR4+in+cancer.">The intricate role of CXCR4 in cancer.</a> Adv Cancer Res. 124, 31-82 (2014).</li><li>Debnath, B., Xu, S., Grande, F., Garofalo, A., Neamati, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+inhibitors+of+CXCR4.">Small molecule inhibitors of CXCR4.</a> Theranostics. 3, 47-75 (2013).</li><li>Tsou, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+CXCR4+antagonists+in+stem+cell+mobilization,+HIV+infection,+ischemic+diseases,+and+oncology.">Harnessing CXCR4 antagonists in stem cell mobilization, HIV infection, ischemic diseases, and oncology.</a> Med Res Rev. , (2017).</li><li>Keating, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plerixafor:+a+review+of+its+use+in+stem-cell+mobilization+in+patients+with+lymphoma+or+multiple+myeloma.">Plerixafor: a review of its use in stem-cell mobilization in patients with lymphoma or multiple myeloma.</a> Drugs. 71, 1623-1647 (2011).</li><li>DiPersio, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+III+prospective+randomized+double-blind+placebo-controlled+trial+of+plerixafor+plus+granulocyte+colony-stimulating+factor+compared+with+placebo+plus+granulocyte+colony-stimulating+factor+for+autologous+stem-cell+mobilization+and+transplantation+for+patients+with+non-Hodgkin's+lymphoma.">Phase III prospective randomized double-blind placebo-controlled trial of plerixafor plus granulocyte colony-stimulating factor compared with placebo plus granulocyte colony-stimulating factor for autologous stem-cell mobilization and transplantation for patients with non-Hodgkin's lymphoma.</a> J Clin Oncol. 27, 4767-4773 (2009).</li><li>Balabanian, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+SDF-1/CXCL12+binds+to+and+signals+through+the+orphan+receptor+RDC1+in+T+lymphocytes.">The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes.</a> J Biol Chem. 280, 35760-35766 (2005).</li><li>Burns, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chemokine+receptor+for+SDF-1+and+I-TAC+involved+in+cell+survival,+cell+adhesion,+and+tumor+development.">A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.</a> J Exp Med. 203, 2201-2213 (2006).</li><li>Van Hout, A., D'Huys, T., Oeyen, M., Schols, D., Van Loy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell-based+assays+for+the+identification+and+evaluation+of+competitive+CXCR4+inhibitors.">Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors.</a> PLoS One. 12, e0176057 (2017).</li><li>Levoye, A., Balabanian, K., Baleux, F., Bachelerie, F., Lagane, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR7+heterodimerizes+with+CXCR4+and+regulates+CXCL12-mediated+G+protein+signaling.">CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling.</a> Blood. 113, 6085-6093 (2009).</li><li>Wootten, D., Christopoulos, A., Sexton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+paradigms+in+GPCR+allostery:+implications+for+drug+discovery.">Emerging paradigms in GPCR allostery: implications for drug discovery.</a> Nat Rev Drug Discov. 12, 630-644 (2013).</li><li>Burford, N. T., Watson, J., Bertekap, R., Alt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+identification+of+allosteric+modulators+of+G-protein-coupled+receptors.">Strategies for the identification of allosteric modulators of G-protein-coupled receptors.</a> Biochem Pharmacol. 81, 691-702 (2011).</li><li>Hendrix, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety,+pharmacokinetics,+and+antiviral+activity+of+AMD3100,+a+selective+CXCR4+receptor+inhibitor,+in+HIV-1+infection.">Safety, pharmacokinetics, and antiviral activity of AMD3100, a selective CXCR4 receptor inhibitor, in HIV-1 infection.</a> J Acquir Immune Defic Syndr. 37, 1253-1262 (2004).</li><li>Schols, D., Este, J. A., Henson, G., De Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicyclams,+a+class+of+potent+anti-HIV+agents,+are+targeted+at+the+HIV+coreceptor+fusin/CXCR-4.">Bicyclams, a class of potent anti-HIV agents, are targeted at the HIV coreceptor fusin/CXCR-4.</a> Antiviral Res. 35, 147-156 (1997).</li><li>Perry, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc:+a+review+of+its+use+in+the+management+of+CCR5-tropic+HIV-1+infection.">Maraviroc: a review of its use in the management of CCR5-tropic HIV-1 infection.</a> Drugs. 70, 1189-1213 (2010).</li><li>Mehlin, C., Crittenden, C., Andreyka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No-wash+dyes+for+calcium+flux+measurement.">No-wash dyes for calcium flux measurement.</a> Biotechniques. 34, 164-166 (2003).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR4+over-expression+and+survival+in+cancer:+a+system+review+and+meta-analysis.">CXCR4 over-expression and survival in cancer: a system review and meta-analysis.</a> Oncotarget. 6, 5022-5040 (2015).</li><li>De Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AMD3100+story:+the+path+to+the+discovery+of+a+stem+cell+mobilizer+(Mozobil).">The AMD3100 story: the path to the discovery of a stem cell mobilizer (Mozobil).</a> Biochem Pharmacol. 77, 1655-1664 (2009).</li><li>Milligan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+activity+and+inverse+agonists+of+G+protein-coupled+receptors:+a+current+perspective.">Constitutive activity and inverse agonists of G protein-coupled receptors: a current perspective.</a> Mol Pharmacol. 64, 1271-1276 (2003).</li><li>Kenakin, T., Miller, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+transmembrane+receptors+as+shapeshifting+proteins:+the+impact+of+allosteric+modulation+and+functional+selectivity+on+new+drug+discovery.">Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery.</a> Pharmacol Rev. 62, 265-304 (2010).</li><li>Conn, P. J., Christopoulos, A., Lindsley, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+modulators+of+GPCRs:+a+novel+approach+for+the+treatment+of+CNS+disorders.">Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders.</a> Nat Rev Drug Discov. 8, 41-54 (2009).</li><li>Burford, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+positive+allosteric+modulators+and+silent+allosteric+modulators+of+the+mu-opioid+receptor.">Discovery of positive allosteric modulators and silent allosteric modulators of the mu-opioid receptor.</a> Proc Natl Acad Sci U S A. 110, 10830-10835 (2013).</li><li>Bartfai, T., Wang, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+allosteric+modulators+to+peptide+GPCRs:+a+promising+class+of+drugs.">Positive allosteric modulators to peptide GPCRs: a promising class of drugs.</a> Acta Pharmacol Sin. 34, 880-885 (2013).</li><li>Hatse, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+CXCL12AF647+as+a+novel+probe+for+nonradioactive+CXCL12/CXCR4+cellular+interaction+studies.">Fluorescent CXCL12AF647 as a novel probe for nonradioactive CXCL12/CXCR4 cellular interaction studies.</a> Cytometry A. 61, 178-188 (2004).</li><li>Seifert, R., Wenzel-Seifert, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+activity+of+G-protein-coupled+receptors:+cause+of+disease+and+common+property+of+wild-type+receptors.">Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors.</a> Naunyn Schmiedebergs Arch Pharmacol. 366, 381-416 (2002).</li><li>Zhang, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+point+mutation+that+confers+constitutive+activity+to+CXCR4+reveals+that+T140+is+an+inverse+agonist+and+that+AMD3100+and+ALX40-4C+are+weak+partial+agonists.">A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists.</a> J Biol Chem. 277, 24515-24521 (2002).</li><li>Tamamura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-molecular-weight+inhibitor+against+the+chemokine+receptor+CXCR4:+a+strong+anti-HIV+peptide+T140.">A low-molecular-weight inhibitor against the chemokine receptor CXCR4: a strong anti-HIV peptide T140.</a> Biochem Biophys Res Commun. 253, 877-882 (1998).</li><li>Wang, Z. -. x., Neote, K., Letts, G. 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The authors have nothing to disclose.

The Ca2+-mobilization assay described herein has previously been shown to be a valuable tool to identify and characterize receptor antagonists targeting CXCR417. It is, however, anticipated that this method can be more generally applied to a large group of other GPCRs that trigger a cytosolic Ca2+ release upon their activation, as illustrated for the related chemokine receptor CCR5. Whereas in the case of CCR5 exactly the same experimental conditions could be applied, several...

GPCRs are an important superfamily of cell surface proteins that activate signal transduction cascades upon extracellular ligand binding. They can be activated by a large variety of stimuli including peptides, protein hormones, biogenic amines, and lipids, which results in the initiation of diverse intracellular signaling pathways and eventually biological responses1,2. Furthermore, GPCRs are involved in many, if not all, developmental and physiological processes and many human diseases are associated with dysfunctional GPCR signaling or receptor overexpression. GPCRs are therefore amongst the most validated pharmacological targets in medicine1,3.
Typically, a GPCR drug discovery workflow starts with cellular screening assays enabling the identification of compounds such as small molecules, monoclonal antibodies, and peptides that can modulate the activity of a particular GPCR. In GPCR drug discovery many different types of assays exist to search for such compounds, most of which are compatible with mid- to high-throughput screening campaigns. The most used assays include receptor binding experiments, fluorescence or luminescence based assays detecting fluctuations in the level of so-called secondary messengers (e.g., Ca2+, cyclic adenosine monophosphate (AMP)), phenotypic screening assays, and &#946;-arrestin recruitment assays4. The choice for a particular type of assay may depend on multiple factors, but is also determined by prior knowledge concerning the signaling properties of a given GPCR. Agonist binding to a GPCR induces a conformational change catalyzing the exchange of guanidine diphosphate (GDP) for guanidine triphosphate (GTP) on the &#945;-subunit of heterotrimeric G proteins. Subsequently the G&#945;-GTP subunit dissociates from the G&#946;&#947; subunit and both subunits will initiate further signaling pathways. Hydrolysis of the GTP-molecule and subsequent re-association of the G&#945;-GDP and G&#946;&#947; subunits will restore the G protein into its inactive conformation5,6. Based on sequence similarity of the G&#945; subunit different types of G proteins are defined (Gs, Gi, Gq, G12/13)7. Signaling via the G&#945; subunit gives rise to several typical responses such as the increase (via Gs) or decrease (via Gi) of cyclic AMP production and intracellular Ca2+ mobilization (via Gq)5,7. G&#946;&#947; subunits are also able to induce intracellular effector pathways. For instance, upon activation of Gi-coupled GPCRs, G&#946;&#947; can directly stimulate phospholipase C (PLC-&#946;) to produce inositol triphosphate (IP3) that triggers the release of Ca2+ from intracellular stores7. Following receptor activation, GPCRs are phosphorylated by GPCR kinases (GRKs) which promotes interaction with &#946;-arrestins. This process terminates G protein signaling and leads to receptor desensitization and eventually internalization. &#946;-arrestins are also able to form multi-molecular complexes that can trigger other signaling pathways independent of G protein signaling8.
Within the subfamily of chemokine receptors, the Gi-coupled CXCR4 is a GPCR that has raised much interest as a promising target for drug discovery. Given its established role as a major co-receptor for human immunodeficiency virus 1 (HIV-1) viral entry and infection, compounds targeting CXCR4 were initially developed as anti-HIV drug candidates9. More recently, a growing body of evidence has pointed to an important role for CXCR4 in tumorigenesis and cancer metastasis making it a well-validated therapeutic target in oncology as well10. CXCR4 is highly expressed in more than twenty types of human cancer and controls tumor cell survival, proliferation, and migration as well as tumor-related angiogenesis10. CXCR4 antagonists of different chemical classes have previously been described11,12, but only the small molecule AMD3100 is currently approved for use in the clinic as a stem cell mobilization agent used during treatment of lymphoma and myeloma patients13,14. Clinical trials are ongoing to evaluate the safety and efficacy of several other CXCR4 antagonists in different human diseases, but with a strong focus on oncology12. Given the many potential applications for CXCR4 antagonists, the search for novel compounds with improved pharmacokinetic properties, improved bioavailability, or potentially less side effects is warranted.
Herein, a kinetic fluorescence-based cellular assay primarily used to screen for compounds capable of inhibiting CXCR4 is described. The fluorescent measurement of this method is based on the transient increase of the intracellular Ca2+ concentration evoked upon CXCR4 activation by its endogenous agonist, the chemokine ligand CXCL12 (formerly known as stromal cell derived factor 1&#945; (SDF1-&#945;)), and the potential inhibition of this CXCL12-induced Ca2+ response by particular compounds. In this assay, U87 human glioblastoma cells stably expressing the human CXCR4 receptor are used. At the same time, these cells lack endogenous expression of CXCR7, a related chemokine receptor that also binds CXCL1215,16,17. CXCR7 has previously also been shown to be capable of forming heterodimers with CXCR4, thereby modulating the signaling properties of this latter receptor18. Fluctuations in the level of intracellular Ca2+ mediated by CXCR4 are monitored by loading the CXCR4+ cells with fluo-2 acetoxymethyl (AM) ester, a cell-permeable high affinity fluorescent Ca2+-binding dye. Fluo-2 AM is a single wavelength fluorescent molecule that can be excited at 490 nm while its emission fluorescence is measured at 520 nm. This emission fluorescence increases upon Ca2+ binding, with a large dynamic range between the Ca2+-bound and unbound state. The increase in fluorescent signal is transient, occurring within a time interval of a few minutes, and will decay afterwards. The peak height of the fluorescent emission further correlates with the level of receptor activation. The assay itself is performed using a fluorescence microplate reader equipped with an Intensified CCD (ICCD) camera that possesses an integrated pipetting system that allows standardization of the pipetting steps in the assay (see Table of Materials). In addition, the simultaneous measurement of the fluorescent signal in all wells of a microplate is another key advantage of the fluorescence reader that is used. During the first part of the assay the compounds under investigation (e.g., a panel of small molecules at a fixed concentration or in a dilution series) are added to the fluo-2 AM loaded CXCR4+ U87 cells followed by a ~ 10 min incubation period during which the potential agonistic effect of the compounds is continuously measured in real time. Then, the endogenous agonist (i.e., CXCL12) is added to the cells to evoke a CXCR4-mediated transient increase in the level of intracellular Ca2+. During this part of the assay the potential antagonistic activity of the tested compounds can be evaluated. A schematic overview of the assay&#39;s general workflow is presented in Figure 1.
Although this Ca2+ mobilization assay has primarily been used to identify and determine the inhibitory potency of competitive CXCR4 antagonists (i.e., compounds that prevent the endogenous agonist to bind and stimulate the receptor), it also can identify receptor agonists and, in addition, compounds that exert their function by binding at allosteric sites (i.e., sites that topographically differ from the orthosteric binding site occupied by the endogenous agonist). Examples of the latter category of compounds include allosteric agonists and PAMs and NAMs19,20. Whereas receptor-specific antagonists as well as NAMs would inhibit the CXCL12-induced Ca2+ response, PAMs would enhance this response (see also Discussion section). Although the assay described herein specifically targets CXCR4, it is anticipated that this method can be applied to other GPCRs with minimal optimization effort, at least if they signal via the release of intracellular Ca2+.

<table>
	<tbody>
		<tr>
			<td>Fluo-2 AM</td>
			<td>Abcam</td>
			<td>ab142775</td>
			<td>fluorescent Ca2+ sensitive dye</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250G</td>
			<td>pluronic acid</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>AMD3100</td>
			<td>Sigma</td>
			<td>A5602-5 mg</td>
			<td>specific CXCR4 antagonist</td>
		</tr>
		<tr>
			<td>Maraviroc</td>
			<td>kind gift of AnorMed</td>
			<td></td>
			<td>antiretroviral drug, CCR5 antagonist</td>
		</tr>
		<tr>
			<td>Chemokine ligand CXCL12</td>
			<td>PeproTech</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemokine ligand CCL5</td>
			<td>PeproTech</td>
			<td>300-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco (Life Technologies)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1933-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco (Life Technologies)</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10 x), calcium, magnesium, no phenol red</td>
			<td>Gibco (Life Technologies)</td>
			<td>14065-049</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco (Life Technologies)</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,25 %), phenol red</td>
			<td>Gibco (Life Technologies)</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>Gibco (Life Technologies)</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>227 261</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flask (T75)</td>
			<td>Corning</td>
			<td>353024</td>
			<td></td>
		</tr>
		<tr>
			<td>Black plate, 96-well, clear bottom, with lid</td>
			<td>Costar/Fisher Scientific</td>
			<td>10530753</td>
			<td>assay plate (96-well), for cell seeding</td>
		</tr>
		<tr>
			<td>Polypropylene (PP) plates</td>
			<td>Thermo Scientific (VWR)</td>
			<td>732-2661</td>
			<td>plates used to prepare the compound plates and chemokine plates, round bottom</td>
		</tr>
		<tr>
			<td>FLIPR Tetra high throughput cellular screening system</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Fluorescent plate reader with integrated pipettor head and ICCD camera</td>
		</tr>
		<tr>
			<td>FLIPR Tetra LED Module 470 - 495 nm</td>
			<td>Molecular Devices</td>
			<td>0200-6128</td>
			<td>Light emitting diodes for excitation of the fluorescent Ca2+ sensitive dye</td>
		</tr>
		<tr>
			<td>FLIPR Tetra Emission Filter 515 - 575 nm</td>
			<td>Molecular Devices</td>
			<td>0200-6203</td>
			<td>emission filter compatible with the fluorescent dye</td>
		</tr>
		<tr>
			<td>FLIPR Tetra 96 Head</td>
			<td>Molecular Devices</td>
			<td>0310-4536</td>
			<td>96-well pipettor head, integrated within the fluorescent plate reader</td>
		</tr>
		<tr>
			<td>ScreenWorks</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>software package used for data analysis and visualization on the FLIPR Tetra</td>
		</tr>
		<tr>
			<td>Vi-CELL</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>cell viability analyzer</td>
		</tr>
		<tr>
			<td>Corning CellBIND 96 Well Flat Clear Bottom Black Polystyrene Microplates, with Lid, Sterile</td>
			<td>Corning</td>
			<td>3340</td>
			<td>Pre-coated 96-well assay plates that may represent an alternative for manual coating of the assay plate.</td>
		</tr>
	</tbody>
</table>

a kinetic fluorescence-based ca2+ mobilization assay to identify g protein-coupled receptor agonists, antagonists, and allosteric modulators

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&#39;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 Ca2+ 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 Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;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&#39;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 Ca2+.

The effect of CXCL12 stimulation on the intracellular Ca2+ mobilization in U87.CD4.CXCR4+ and U87.CD4 cells was evaluated with the Ca2+ mobilization assay. Instead of 20 &#181;L of test compound that would normally be added during the first pipetting step of the protocol (Figure 1), assay buffer was added to the fluo-2 AM loaded U87.CD4.CXCR4+ cells in the measurement plate. During the second dispensing step, differ...

Watch this Scientific Journal Video about A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators at JoVE.com

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

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

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

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

The overall goal of this cell-based assay is to identify and characterize molecules capable of stimulating or inhibiting G protein-coupled receptor-mediated intracellular calcium mobilization. This method can be a valuable assay for G protein-coupled receptor drug discovery because it can be used to screen for molecules that modify receptor-mediated intracellular calcium bodies. The main advantage of this technique is that it allows molecules with agonistic or antagonistic properties to be identified within the same assay.This assay may help in the discovery of novel drug candidates that act on G protein-coupled receptors promising therapeutic targets that play a role in many human diseases. On the day of cell passaging, coat each well of a black walled polystyrene 96-well plate with clear bottoms with 100 microliters of 0.1%gelatin solution and incubate the plates for two hours at room temperature. While the gelatin is solidifying, use a trypsin EDTA solution to lift the cells of interest from the bottom of their culture flask, resuspending the cells in fresh growth medium in a 50 milliliter conical tube once they have detached.After counting, collect the cells by centrifugation and resuspend the pellet in fresh growth medium to a one times 10 to the fifth viable cells per milliliter concentration. When the plates are ready, discard any excess gelatin solution and dry the plates on a lab tissue. Then wash the wells with 200 microliters of PBS and replace the wash with 200 microliters of cells per well for an overnight incubation at 37 degrees Celsius and 5%carbon dioxide.The next morning, tap the plate to discard the growth medium from the wells and dry the plate upside on a paper towel. Then label the cells in each well with 100 microliters of loading dye solution per well and incubate the plate for 45 minutes in the dark at room temperature. While the cells are incubating, add 75 microliters of CXCL12 per well to the chemokine plate and 50 microliters of the compound of interest to each well of the compound plate.Next, turn on the cooler unit of the fluorescence plate reader followed by the plate reader itself. After allowing the system a few minutes to initiate, open the plate reader software. To set up the assay protocol, under the settings tab select read mode and the appropriate excitation and emission wavelengths for the loading dye.Select the mix with transfer fluid tab for automatic mixing of the compounds in the compound plate. In the transfer fluid tab, set 20 microliters of each well from the compound plate to be transferred into the measurement plate and position the pipette tips at 20 microliters below the liquid surface. Set the speed of aspiration to 50 microliters per second and the speed of dispensing to 25 microliters per second and click read with transfer fluid.During the first interval, select 60 reads with a read interval time of one second with 10 reads to be recorded before the compounds are dispensed into the measurement plate and 50 reads to be completed after. Set the second interval with a read interval time of 30 seconds and 18 reads. Select three tip washes for the protocol, setting each wash step to include the appropriate wash fluid, one wash cycle, a fast pump speed, and five strokes per wash.Use the pause pipettor function to include the pipettor pause of 300 seconds and use the mix with transfer fluid setting to allow automatic mixing of the CXCL12 solution within the chemokine plate. In the subsequent transfer fluid tab, set 25 microliters from each well of the chemokine plate to be transferred to the measurement plate and position the tips 20 microliters below the liquid surface. Set the aspiration speed to 50 microliters per second and the dispensing speed to 25 microliters per second.Use the read with transfer fluid button to set the first interval to include 145 reads with a read interval time of one second. Set the second interval to a read interval time of six seconds and 20 reads. Select three more wash tip cycles.Then click set stage temperature and set the device temperature to 37 degrees Celsius. At the end of the loading dye incubation, discard the buffer from the measurement plate and dry the plate on a lab tissue. Wash the cells with 150 microliters per well of assay buffer replacing the buffer with 80 microliters of assay buffer after two minutes.Place the measurement plate in the read position, the compound plate at the source two position, the chemokine plate at the source three position, and a box of black tips at the source one position. Close the device and click protocol signal test to determine the background relative light units and fluorescence variance over the plate. Select test signal in the popup window.If background values of 8, 000 to 10, 000 relative light units are obtained, select update and click save to save the main protocol. Then push run to start the assay. In this graph, a dose-dependent increase in fluorescence correlating with the dose-dependent increase in the release of calcium can be observed.Increasing levels of CXCL12-induced fluorescence allow the generation of a dose response curve based on the difference between the minimum and maximum response over baseline between the first measurement after addition addition of the chemokine and the final measurement in the protocol. No fluorescent calcium-related response was evoked by CXCL12 even at a high concentration when cells lacking the corresponding functional chemokine receptor CXCR4 were used demonstrating the receptor specificity of the CXCL12-evoked measurement. A key application for this cell-based assay is to identify small molecules that, following pre-incubation, dose dependently inhibit CXCL12-induced calcium mobilization in the cells as compared to control non-active compounds that do not inhibit the response.To determine the inhibitory potency of small molecules, a dose response curve can be generated for the inhibitory molecules using nonlinear regression. This assay can be used to study other G protein-coupled receptors with no or only minimal further optimization as well as in this experiment in which a dose-dependent increase in fluorescence was observed when another chemokine receptor, CCR5, was stimulated with its natural chemokine agonist CCL5. Once mastered, this technique can be used to study many other G-protein coupled receptors that signal via the mobilization of intracellular calcium ions upon receptor activation.After watching this video, you should have a good understanding of how this cellular assay can be used to identify agonists and antagonists of the CXCL4 chemokine receptor.

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8.7K visualizações.  KU Leuven. The overall goal of this cell-based assay is to identify and characterize molecules capable of stimulating or inhibiting G protein-coupled receptor-mediated intracellular calcium mobilization. This method can be a valuable assay for G protein-coupled receptor drug discovery because it can be used to screen for molecules that modify receptor-mediated intracellular calcium bodies. The main advantage of this technique is that it allows molecules with agonistic or antagonistic properties to be id...