In vitro activity: Maraviroc inhibits MIP-1β-stimulated γ-S-GTP binding to HEK-293 cell membranes, indicating its ability to inhibit chemokine-dependent stimulation of GDP-GTP exchange at the CCR5/G protein complex. Maraviroc also inhibits the downstream event of chemokine-induced intracellular calcium redistribution, with IC50s ranging from 7 to 30 nM obtained against MIP-1β, MIP-1α and RANTES. In the same experiments, Maraviroc does not trigger release of intracellular calcium at concentrations up to 10 μM, indicating that it is devoid of CCR5 agonist activity. Consistent with this, Maraviroc fails to induce CCR5 internalization. Maraviroc is active at low nanomolar concentrations against HIV-1 Ba-L. Maraviroc inhibits all 200 pseudotyped viruses with a geometric mean IC90 of 13.7 nM.

Kinase Assay: Binding of 125I-labeled MIP-1α, MIP-1β, and RANTES to CCR5 is measured using intact HEK-293 cells stably expressing the receptor or membrane preparations thereof. Briefly, cells are resuspended in binding buffer (50 mM HEPES containing 1 mM CaCl2, 5 mM MgCl2, and 0.5% bovine serum albumin [BSA] and adjusted to pH 7.4) to a density of 2 × 106 cells/ml. For membrane preparations, phosphate-buffered saline (PBS)-washed cells are resuspended in lysis buffer (20 mM HEPES, 1 mM CaCl2, 1 tablet COMPLETE per 50 mL, pH 7.4) prior to homogenization in a Polytron hand-held homogenizer, ultracentrifugation (40,000× g for 30 min), and resuspension in binding buffer to a protein concentration of 0.25 mg/mL (12.5 μg of membrane protein is used in each well of a 96-well plate). 125I-radiolabeled MIP-1α, MIP-1β, and RANTES are prepared and diluted in binding buffer to a final concentration of 400 pM in the assay. Maraviroc dilutions are added to each well to a final volume of 100 μL, the assay plates incubate for 1 hour, and the contents filter through preblocked and washed Unifilter plates which are counted following overnight drying.

Cell Assay: Drug susceptibility assays are performed in 24-well tissue culture plates. Duplicate eight-point dilution series of Maraviroc are prepared in DMSO and medium to yield a final DMSO concentration of 0.1% (vol/vol) in the assay. PHA-stimulated PBMC or PM-1 cells are infected with virus for 1 hour at 37 °C. Cells are subsequently washed once, and 3.6 × 105 PBMC or 2.0 × 105 PM-1 cells are added to each well of assay plates containing diluted Maraviroc. Plates are incubated for 5 days (lab-adapted strains) or 7 days (primary isolates) at 37 °C in a humidified 5% CO2 (vol/vol) atmosphere.

Antimicrob Agents Chemother. 2005 Nov;49(11):4721-32; PLoS One. 2011;6(6):e20209.