Background:  Maraviroc (UK-427,857) is a slow offset, non-competitive antagonist of the human chemokine receptor CCR5 (hCCR5) in clinical development for the treatment of HIV-1 infection. The aim of this study was to evaluate the functional activity of maraviroc at hCCR5 using MIP-1b evoked responses in a CRE luciferase (CRE-luc) reporter gene assay and to relate this to the direct dissociation kinetics of maraviroc from the receptor.

Methods:  Functional activity at the hCCR5 was determined in HEKGa15 cells transiently expressing the CRE-luc construct and hCCR5, pre-incubated with maraviroc or an alternative small-molecule CCR5 antagonist, and then incubated with MIP-1b in the presence of forskolin. Radioligand dissociation assays were carried out using custom-synthesized tritiated compounds.

Results:  In the functional assay, maraviroc (1, 3, 10 nM) and an alternative CCR5 antagonist (3, 10, 30 nM) caused rightward shifts of the dose-response curve to MIP-1b. Maraviroc also caused a marked dose-dependent reduction of the maximum response to MIP-1b (9.9, 36.7, and 69.4% reduction at 1, 3, and 10 nM maraviroc, respectively), indicating insurmountable antagonism. In contrast, the second CCR5 antagonist induced only a 20% suppression of the maximum response at the highest concentration tested. The dissociation half-life of [³H]-maraviroc was estimated to be 15.95 hours (n = 3, 95%CI 9.36 to 27.16) whereas the dissociation of the second compound was significantly faster at 0.92 hours (n = 4, 0.68 to 1.26).

Conclusions:  The insurmountable profile displayed by maraviroc is likely to be a consequence of its slow rate of dissociation from hCCR5, giving rise to hemi-equilibrium conditions. This contrasts with the observations with the more rapid off-rate compound for which there was only a minor suppression of the maximum response. The dissociation constant (pKb) of maraviroc based on the assumption of hemi-equilibrium conditions was calculated to be 9.4 (n = 4, 95%CI 9.01 to 9.75) and is in good agreement with the reported binding affinity of maraviroc for hCCR5.