CXCR4 Antagonists: A Screening Strategy for Identification of Functionally Selective Ligands

J Biomol Screen. 2014 Jul;19(6):859-69. doi: 10.1177/1087057114526283. Epub 2014 Mar 14.


The CXC chemokine receptor 4 (CXCR4) is a widely expressed G protein-coupled receptor implicated in several diseases. In cancer, an increased number of surface CXCR4 receptors, in parallel with aberrant signaling, have been reported to influence several aspects of malignancy progression. CXCR4 activation by the specific ligand C-X-C motif chemokine 12 (CXCL12) induces several intracellular signaling pathways that have been selectively related to malignancy depending on the tissue or cell type. We developed a panel of CXCR4 screening assays investigating Gα(i)-mediated cyclic adenosine monophosphate modulation, β-arrestin recruitment, and receptor internalization. All of the assays were set up in recombinant cells and were used to test four reported CXCR4 antagonists. Consequently, a set of hit compounds, deriving from a screening campaign of a 30,000-small-molecule internal library, was profiled with the different assays. We identified several compounds showing a pathway-selective activity: antagonists on a Gα(i)-dependent pathway; antagonists on both the β-arrestin and Gα(i)-dependent pathways, some of which induce receptor internalization; and compounds with an antagonist behavior in all of the readouts. The identified biased antagonists induce different functional states on CXCR4 and preferentially affect specific downstream responses from the activated receptor, thus providing an improved therapeutic profile for correction of CXCR4 abnormal signaling.

Keywords: AMD3100; CXC chemokine receptor 4 (CXCR4); G protein–coupled receptor (GPCR); biased antagonism; cyclic adenosine monophosphate (cAMP); glioblastoma multiforme (GBM); high-content screening (HCS); homogeneous time-resolved fluorescence (HTRF); receptor internalization; β-arrestin.

MeSH terms

  • Animals
  • Arrestins / chemistry
  • CHO Cells
  • Cell Line
  • Cell Line, Tumor
  • Cell Separation
  • Chemokine CXCL12 / chemistry
  • Cricetinae
  • Cricetulus
  • Cyclic AMP / chemistry
  • Disease Progression
  • Flow Cytometry
  • Humans
  • Ligands
  • Mass Screening
  • Peptides / chemistry
  • Phosphorylation
  • Receptors, CXCR4 / antagonists & inhibitors*
  • Recombinant Proteins / chemistry
  • Signal Transduction
  • Small Molecule Libraries / chemistry
  • Spectrometry, Fluorescence
  • beta-Arrestins / chemistry
  • beta-Galactosidase / chemistry


  • Arrestins
  • CXCL12 protein, human
  • CXCR4 protein, human
  • Chemokine CXCL12
  • Ligands
  • Peptides
  • Receptors, CXCR4
  • Recombinant Proteins
  • Small Molecule Libraries
  • beta-Arrestins
  • Cyclic AMP
  • beta-Galactosidase