Inhibition of chemokine binding to CCR5

Binding of 125I-labeled MIP-1α, MIP-1β, and RANTES to CCR5 was measured using intact HEK-293 cells stably expressing the receptor or membrane preparations thereof. Briefly, cells were 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 were 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 mins), and resuspension in binding buffer to a protein concentration of 0.25 mg/mL (12.5 μg of membrane protein was used in each well of a 96-well plate). 125I-radiolabeled MIP-1α, MIP-1β, and RANTES were prepared and diluted in binding buffer to a final concentration of 400 pM in the assay. Maraviroc dilutions were added to each well to a final volume of 100 μL, the assay plates were incubated for 1 hr, and the contents were filtered through preblocked and washed Unifilter plates which were then counted and dried overnight.

Cell lines

PBMC or PM-1 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 ℃ for several months.

Reaction Conditions

0.01 ~ 10 nM; 5 days

Applications

In PBMC or PM-1 cells, Maraviroc was active against HIV-1 Ba-L even at low nanomolar concentrations, with the IC90 values ranging from 1.1 nM to 3.1 nM. Meanwhile, Maraviroc showed no effect on cell proliferation at concentrations up to 10 μM.

Animal models

Rats and dogs

Dosage form

74 and 21 ml/min/kg, respectively; i.v.

Applications

In rats and dogs, Maraviroc showed moderate to high clearance values, as well as moderate volumes of distribution (4.3 to 6.5 liters/kg). For rats and dogs, the half-life values of Maraviroc were 0.9 hr and 2.3 hrs, respectively.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Maraviroc is a potent and selective antagonist of Chemokine Receptor CCR5 [1].

CCR5 is a receptor for chemokines and exists on the surface of white blood cells that are involved in the immune system. Many forms of HIV enter and infect host cells through CCR5 [1].

Maraviroc is a potent and selective inhibitor of CCR5 with potent anti-HIV-1 activity. Maraviroc exhibited antiviral activity against all CCR5-tropic HIV-1 viruses tested with IC90 of 2.0 nM. In HeLa P4 cells, maraviroc inhibited binding of viral envelope gp120 to CCR5 with IC50 value of 11 nM and prevented the membrane fusion events, which were necessary for viral entry. While, maraviroc didn’t affect CCR5 levels or associated intracellular signaling, which suggested it is an antagonist of CCR5 [1].

In a patient infected with both R5-tropic and X4-tropic HIV-1 viruses, treatment with maraviroc suppressed all R5-tropic viruses. And the R5-tropic viruses were replaced by more X4-tropic viruses [2].

References:
[1].  Dorr P, Westby M, Dobbs S, et al. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother, 2005, 49(11): 4721-4732.
[2].  Symons J, van Lelyveld SF, Hoepelman AI, et al. Maraviroc is able to inhibit dual-R5 viruses in a dual/mixed HIV-1-infected patient. J Antimicrob Chemother, 2011, 66(4): 890-895.