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Maraviroc is a pseudo-irreversible antagonist at the human CCR5 in a cre-luciferase reporter gene assay Maraviroc (UK-427,857) is an antagonist of the chemokine receptor CCR5, in clinical development for the treatment of HIV-1 infection. The compound is a non-competitive antagonist of chemokine binding to human CCR5 (hCCR5) with a slow rate of offset (Dorr et al., 2005; Watson et al., 2005). The aim of this study was to evaluate the functional activity of maraviroc at hCCR5 using MIP-1β evoked responses in a CRE-luciferase (CRE-luc) reporter gene assay. In addition, the direct dissociation kinetics of maraviroc from hCCR5 were determined using 3H-maraviroc. To perform the CRE-luc assay, HEK Gα15 cells were transiently transfected with the CRE-luc construct plus DNA encoding the hCCR5. Cells were pre-incubated with maraviroc for 30 min at 37°C in the presence of IBMX, and then incubated for a further 5 h at 37°C with MIP-1β (3 pM – 100 nM) plus forskolin (300 nM). Cells were then exposed to Steady-Glo Luciferase reagent and luminescence was read using an LJL Analyst Reader. Radioligand binding assays were performed and data analysed as described by Sale et al. (2005) except that the radioligand was 3H-maraviroc (Amersham, specific activity 16 Ci/mmol), and non-specific binding was defined by 10 μM cold maraviroc. 3H-maraviroc, at 5 nM, was incubated with hCCR5 cell homogenates (5 μg/well) at room temperature in 20 mM HEPES buffer (containing 5 mM MgCl2, 1 mM CaCl2 and 0.5% BSA, pH 7.4) until association reached a plateau. Dissociation of 3H-maraviroc was initiated by the addition of excess cold maraviroc (50 μM) and followed over time. Assays were terminated by filtration and bound radioactivity quantified by scintillation counting. In the CRE-luc assay, maraviroc at 1, 3, and 10 nM caused a dose dependent rightward shift of the dose response curve to MIP-1β . Maraviroc also caused a marked dose dependent reduction of the maximum response to MIP-1β (9.9, 36.7 and 69.4% reduction at 1, 3 and 10 nM maraviroc, respectively), indicating insurmountable antagonism. The dissociation half-life of 3H-maraviroc was estimated to be 15.95 h (n=3, 95% confidence interval 9.36-27.16). The insurmountable profile displayed by maraviroc is therefore likely to be a consequence of its slow rate of dissociation from the hCCR5, giving rise to pseudo-irreversible or hemi-equilibrium conditions (Paton and Rang, 1966; Kenakin, 1984). The dissociation constant (pKb) of maraviroc based on the assumption of hemi-equilibrium conditions was calculated to be 9.4 (9.01-9.75, n=4) and is in good agreement with the reported binding affinity of maraviroc for hCCR5 (Dorr et al., 2005).
Dorr P et al. (2005) Antimicrobial Agents & Chemother. In Press. |
