In vitro activity: AZD8797 is a selective small-molecule and allosteric non-competitive modulator of the human CX3CR1 receptor; it antagonizes CX3CR1 and CXCR2 with Ki of 3.9 and 2800 nM, respectively. 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.

Kinase Assay: CHO-hCX3CR1 membranes together with different concentrations of AZD8797 are incubated in 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 10 μM GDP and 0.01% gelatin in a MicroWell 96-well plate. 0.56 μCi/mL [35S]GTPγS and EC80 of CX3CL1 are then added. The plate is incubated at 30°C for 1 h and subsequently unbound [35S]GTPγS is separated from bound by vacuum filtration to a Printed Filtermat B. The different AZD8797 concentrations are achieved by stepwise dilution in DMSO to achieve a final DMSO concentration of 1% in all wells after addition of assay buffer, regardless of AZD8797 concentration.

Cell Assay: In a flow adhesion assay, AZD8797 antagonizes 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 prevents G-protein activation in a [35S]GTPγS accumulation assay. AZD8797 positively modulates the CX3CL1 response at sub-micromolar concentrations in a β-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduces the maximal binding of 125I-CX3CL1 without affecting Kd. AZD8797 binds selectively with high affinity to human and rat CX3CR1 (Ki of hCX3CR1, 4 nM; Ki of rCX3CR1, 7 nM, respectively). The equilibrium dissociation constant, KB, demonstrates that AZD8797 is a very potent inhibitor for human CX3CR1 (10 nM). The potency is threefold lower for rat CX3CR1 (29 nM) and decreases even further at mouse CX3CR1 (54 nM).