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, ß-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 ß-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.
Subject(s)
Receptors, CXCR4/antagonists & inhibitors , 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 , Recombinant Proteins/chemistry , Signal Transduction , Small Molecule Libraries/chemistry , Spectrometry, Fluorescence , beta-Arrestins/chemistry , beta-Galactosidase/chemistryABSTRACT
The objectives of the study were to characterize the production, function and survival of T lymphocytes of children with prolonged fetal exposure to dexamethasone for anti-Ro/SS-A antibodies associated congenital complete heart block. The analysis of thymic function, studied by measuring the level of T-cell receptor excision circles, was performed by real time PCR, the composition of T-cell subpopulation was evaluated by flow cytometry and the T-cell diversity was assayed by heteroduplex analysis. T-cell competence was gauged at two functional levels by determining the proliferation and the number of T-cell divisions and by measuring gamma-interferon production after mitogenic stimulation. We observed that the thymic output, distribution of T-cell subsets, thymidine incorporation, number of T-cell divisions, and y-interferon production were comparable to those of age-matched control. On the contrary, heteroduplex analysis demonstrated the presence of both polyclonal and oligoclonal peripheral T-cell repertoires. In conclusion, the analysis of the T-cell compartment in children with prolonged intrauterine exposure to high dose dexamethasone did not disclose any relevant abnormality, except a restriction of T-cell receptor diversity in some patients.
