PMID- 29578516
OWN - NLM
STAT- MEDLINE
DCOM- 20180713
LR  - 20200310
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 133
DP  - 2018 Mar 10
TI  - A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of 
      Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4.
LID - 10.3791/57271 [doi]
LID - 57271
AB  - 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.
FAU - Schoofs, Geert
AU  - Schoofs G
AD  - Laboratory of Virology and Chemotherapy, Department of Microbiology and 
      Immunology, Rega Institute for Medical Research, KU Leuven.
FAU - Van Hout, Anneleen
AU  - Van Hout A
AD  - Laboratory of Virology and Chemotherapy, Department of Microbiology and 
      Immunology, Rega Institute for Medical Research, KU Leuven.
FAU - D'huys, Thomas
AU  - D'huys T
AD  - Laboratory of Virology and Chemotherapy, Department of Microbiology and 
      Immunology, Rega Institute for Medical Research, KU Leuven.
FAU - Schols, Dominique
AU  - Schols D
AD  - Laboratory of Virology and Chemotherapy, Department of Microbiology and 
      Immunology, Rega Institute for Medical Research, KU Leuven.
FAU - Van Loy, Tom
AU  - Van Loy T
AD  - Laboratory of Virology and Chemotherapy, Department of Microbiology and 
      Immunology, Rega Institute for Medical Research, KU Leuven; 
      tom.vanloy@kuleuven.be.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20180310
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (CXCL12 protein, human)
RN  - 0 (CXCR4 protein, human)
RN  - 0 (Chemokine CXCL12)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Ligands)
RN  - 0 (Receptors, CXCR4)
RN  - 0 (Small Molecule Libraries)
SB  - IM
MH  - Binding, Competitive
MH  - Chemokine CXCL12/*antagonists & inhibitors/*metabolism
MH  - Drug Evaluation, Preclinical/methods
MH  - Flow Cytometry/*methods
MH  - Fluorescent Dyes
MH  - Humans
MH  - Jurkat Cells
MH  - Ligands
MH  - Protein Binding
MH  - Receptors, CXCR4/*antagonists & inhibitors/*metabolism
MH  - Small Molecule Libraries/*pharmacology
PMC - PMC5931669
EDAT- 2018/03/27 06:00
MHDA- 2018/07/14 06:00
PMCR- 2020/03/10
CRDT- 2018/03/27 06:00
PHST- 2018/03/27 06:00 [entrez]
PHST- 2018/03/27 06:00 [pubmed]
PHST- 2018/07/14 06:00 [medline]
PHST- 2020/03/10 00:00 [pmc-release]
AID - 57271 [pii]
AID - 10.3791/57271 [doi]
PST - epublish
SO  - J Vis Exp. 2018 Mar 10;(133):57271. doi: 10.3791/57271.