AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor
BIOCHEMICAL JOURNAL(2016)
摘要
The chemokine receptor CX(3)CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX(3)CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX(3)CR1, 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 (CX(3)CL1), 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 [S-35] GTP gamma S (guanosine 5'-[gamma-thio] triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak G(ai)-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX(3)CL1 response at sub-micromolar concentrations in a beta-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of I-125-CX(3)CL1 without affecting K-d. Kinetic experiments, determining the k(on) and k(off) 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 GTP. S increase the rate with which CX3CL1 dissociates from CX(3)CR1 in a similar manner, indicating a connection between AZD8797 and the CX(3)CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX(3)CL1, binding CX(3)CR1 and effecting G-protein signalling and beta-arrestin recruitment in a biased way.
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关键词
allosteric modulator,CX3CR1,fractalkine,G-protein,kinetic binding,radioligand binding
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