Discovery and characterization of vicriviroc (SCH 417690), a CCR5 antagonist with potent activity against human immunodeficiency virus type 1

Abstract

Inhibiting human immunodeficiency virus type 1 (HIV-1) infection by blocking the host cell coreceptors CCR5 and CXCR4 is an emerging strategy for antiretroviral therapy. Currently, several novel coreceptor inhibitors are being developed in the clinic, and early results have proven promising. In this report, we describe a novel CCR5 antagonist, vicriviroc (formerly SCH-D or SCH 417690), with improved antiviral activity and pharmacokinetic properties compared to those of SCH-C, a previously described CCR5 antagonist. Like SCH-C, vicriviroc binds specifically to the CCR5 receptor and prevents infection of target cells by CCR5-tropic HIV-1 isolates. In antiviral assays, vicriviroc showed potent, broad-spectrum activity against genetically diverse and drug-resistant HIV-1 isolates and was consistently more active than SCH-C in inhibiting viral replication. This compound demonstrated synergistic anti-HIV activity in combination with drugs from all other classes of approved antiretrovirals. Competition binding assays revealed that vicriviroc binds with higher affinity to CCR5 than SCH-C. Functional assays, including inhibition of calcium flux, guanosine 5'-[35S]triphosphate exchange, and chemotaxis, confirmed that vicriviroc acts as a receptor antagonist by inhibiting signaling of CCR5 by chemokines. Finally, vicriviroc demonstrated diminished affinity for the human ether a-go-go related gene transcript ion channel compared to SCH-C, suggesting a reduced potential for cardiac effects. Vicriviroc represents a promising new candidate for the treatment of HIV-1 infection.

Figures

FIG. 1.

Structure of SCH-C (SCH 351125) and vicriviroc (SCH-D or SCH 417690).

FIG. 2.

Inhibition of CCR5 function by vicriviroc. (A) Chemotaxis assay. Ba/F3-CCR5 cells incubated in the presence or absence of antagonists were induced to migrate through a 5-μm ChemoTx filter by MIP-1α (0.3 nM). Cell migration was measured by quantifying the number of cells migrating through the membrane using the Cell Titer Glow luminescence kit. The data from a representative experiment are expressed in relative luminescence units, and each point represents an average of 3 wells (±standard deviations [SD]). Both SCH-C and vicriviroc inhibited migration of the cells with similar efficacy. (B) Ca flux assay. U-87-CCR5 cells in 96-well plates were loaded with the calcium-sensitive dye Fluo4. Compounds or buffer alone was added to the cells at the indicated concentrations, and calcium signal was immediately read on the FLIPR. The cells were incubated with compound for an additional 5 min prior to the addition of 10 nM RANTES, and the plates were again read on the FLIPR. Data are expressed as the mean (±SD) percentage of calcium signal obtained following addition of RANTES in compound-treated wells (n = 4) compared with no compound control wells (RANTES only). Both compounds inhibited calcium signaling with similar potency in a dose-dependent fashion. Calcium signal measured in wells treated with SCH-C or vicriviroc alone were similar to background (no RANTES) controls. Data shown are representative of four experiments. C. GTPγS binding assay. Membranes (4 μg/well) from HTS-hCCR5 cells were incubated in binding buffer in the presence of the indicated concentrations of SCH-C or vicriviroc for 24 h at 4°C. The samples were warmed to room temperature and further incubated with 3 μM GDP and 1 nM RANTES for 1 h. Following the addition of 0.1 nM [35S]GTPγS, the incubation continued for 1 h. [35S]GTPγS binding to the membranes was measured by WGA-SPA scintillation. Data represent the mean specific binding ± standard errors of the means of triplicate determinations from a representative experiment (n = 3).

FIG. 3.

Saturation and competition binding analysis of hCCR5 with [3H]SCH-C. (A) Membranes (1 μg/well) from HTS-hCCR5 cells were incubated for the indicated times in binding buffer with increasing concentrations of [3H]SCH-C in the absence (total binding) or presence of 1 μM SCH-C (nonspecific binding). Radioligand binding to the membranes was measured by WGA-SPA scintillation. Data represent the mean specific binding ± standard errors of the means of triplicate determinations from a representative experiment (n = 2). (B) Membranes (2 μg/well) from HTS-hCCR5 cells were incubated in binding buffer containing 4 nM [3H]SCH-C and the indicated concentrations of SCH-C and vicriviroc. Radioligand binding to the membranes was measured by WGA-SPA scintillation. The Cheng-Prusoff equation (1) was used to calculate compound affinities from binding IC50 at the indicated times. Data from representative experiments (n = 2) represent the compound Ki relative to incubation time.

FIG. 4.

Comparative antiviral activity of SCH-C and vicriviroc. The antiviral potencies of SCH-C (hatched bars) and vicriviroc (solid bars) were evaluated under identical conditions in the PBMC infection assay against a panel of HIV-1 isolates. The bars represent the geometric mean EC50s determined for the indicated number experiments. Error bars represent the upper and lower boundaries of the 95% confidence interval (CI). Mean EC50s for vicriviroc were 2- to 40-fold lower than SCH-C against all viruses tested.