title: AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor.
abstract: The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [(35)S]GTPγS (guanosine 5'-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a β-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of (125)I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and β-arrestin recruitment in a biased way.
authors.affiliation: Reagents and Assay Development, Discovery Sciences, AstraZeneca R&D Gothenburg, Mölndal 431 83, Sweden.
authors.forename: Linda
authors.initials: L
authors.lastname: Cederblad
authors.affiliation: CVMD Bioscience, Cardiovascular & Metabolic Diseases iMed, AstraZeneca R&D Gothenburg, Mölndal 431 83, Sweden.
authors.forename: Birgitta
authors.initials: B
authors.lastname: Rosengren
authors.affiliation: CVMD Bioscience, Cardiovascular & Metabolic Diseases iMed, AstraZeneca R&D Gothenburg, Mölndal 431 83, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Ryberg
authors.affiliation: Reagents and Assay Development, Discovery Sciences, AstraZeneca R&D Gothenburg, Mölndal 431 83, Sweden Nils-Olov.Hermansson@astrazeneca.com.
authors.forename: Nils-Olov
authors.initials: NO
authors.lastname: Hermansson
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D001773:Blood Cells / Q000187:drug effects* / Q000378:metabolism; D016466:CHO Cells; D000073883:CX3C Chemokine Receptor 1; D054428:Chemokine CX3CL1 / Q000494:pharmacology; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D011743:Pyrimidines / Q000494:pharmacology*; D011869:Radioligand Assay; D019707:Receptors, Chemokine / Q000378:metabolism*; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology*; D000071557:beta-Arrestins
chemical_list: C000609791:AZD8797; D019390:Arrestins; D000073883:CX3C Chemokine Receptor 1; C095235:CX3CR1 protein, human; D054428:Chemokine CX3CL1; D011743:Pyrimidines; D019707:Receptors, Chemokine; D013844:Thiazoles; D000071557:beta-Arrestins; D019204:GTP-Binding Proteins
keywords: CX3CR1; G-protein; allosteric modulator; fractalkine; kinetic binding; radioligand binding