title: CXCR4 Antagonists: A Screening Strategy for Identification of Functionally Selective Ligands.
abstract: 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.
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Castaldo
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy TES Pharma S.r.l., Corciano, Italy.
authors.forename: T
authors.initials: T
authors.lastname: Benicchi
authors.affiliation: Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Otrocka
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy Novartis Vaccines and Diagnostics S.r.l., Siena, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Mori
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Pilli
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Ferruzzi
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Valensin
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Diamanti
authors.affiliation: Department of Primary Pharmacology, UCB Pharma, Slough, UK.
authors.forename: W
authors.initials: W
authors.lastname: Fecke
authors.affiliation: Department of Medicinal Chemistry, Siena Biotech S.p.A., Siena, Italy GlaxoSmithKline R&D Neural Pathways DP Neurosciences TAU, Singapore.
authors.forename: M
authors.initials: M
authors.lastname: Varrone
authors.affiliation: Department of Pharmacology, Siena Biotech S.p.A., Siena, Italy vporcari@sienabiotech.it.
authors.forename: V
authors.initials: V
authors.lastname: Porcari
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002469:Cell Separation; D054377:Chemokine CXCL12 / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000737:chemistry; D018450:Disease Progression; D005434:Flow Cytometry; D006801:Humans; D008024:Ligands; D008403:Mass Screening; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D000071557:beta-Arrestins / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: D019390:Arrestins; C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D008024:Ligands; D010455:Peptides; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D000071557:beta-Arrestins; D000242:Cyclic AMP; D001616:beta-Galactosidase
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