Blockade of X4-tropic HIV-1 cellular entry by GSK812397, a potent noncompetitive CXCR4 receptor antagonist

Abstract

GSK812397 is a potent entry inhibitor of X4-tropic strains of HIV-1, as demonstrated in multiple in vitro cellular assays (e.g., in peripheral blood mononuclear cells [PBMCs] and a viral human osteosarcoma [HOS] assay, mean 50% inhibitory concentrations [IC50s]+/-standard errors of the means were 4.60+/-1.23 nM and 1.50+/-0.21 nM, respectively). The primary in vitro potency of GSK812397 was not significantly altered by the addition of serum proteins (2.55 [+/-0.12]-fold shift in the presence of human serum albumin and alpha-acid glycoprotein in the PBMC assay). Pharmacological characterization of GSK812397 in cell-based functional assays revealed it to be a noncompetitive antagonist of the CXCR4 receptor, with GSK812397 producing a concentration-dependent decrease in both an SDF-1-mediated chemotaxis and intracellular calcium release (IC50s were 0.34+/-0.01 nM and 2.41+/-0.50 nM, respectively). With respect to the antiviral activity of GSK812397, it was effective against a broad range of X4- and X4R5-utilizing clinical isolates. The potency and efficacy of GSK812397 were dependent on the individual isolate, with complete inhibition of infection observed with 24 of 30 isolates. GSK812397 did not show any detectable in vitro cytotoxicity and was highly selective for CXCR4, as determined using a wide range of receptors, enzymes, and transporters. Moreover, GSK812397 demonstrated acceptable pharmacokinetic properties and bioavailability across species. The data demonstrate that GSK812397 has antiviral activity against a broad range of X4-utilizing strains of HIV-1 via a noncompetitive antagonism of the CXCR4 receptor.

FIG. 1.

Chemical structure of GSK812397.

FIG. 2.

Effect of GSK812397 on the infection of CXCR4-expressing host cells by various clinical isolates of HIV-1. This assay was performed by Monogram Biosciences (San Francisco, CA) in accordance with the methodology described in reference . (A) The potency of GSK812397 in inhibiting infection was compared across X4 clinical isolates (filled bars) and X4R5 dual-tropic isolates (open bars). (B) The maximal inhibition of infection for each isolate was also examined and was compared to that of the infection by each isolate in the absence of compound. Data represent the mean calculated from a single curve fitted to a concentration-response of GSK812397 in which each point was performed in triplicate.

FIG. 3.

Pharmacological profiling of GSK812397. Inhibition of SDF-1-mediated calcium release (A) and chemotaxis of U937 cells by GSK812397 (B). Calcium release assays were performed in HEK-293 cells stably transfected to express the human CXCR4 receptor and the chimeric G protein Gqi5. Cells were preincubated with increasing concentrations of GSK812397 (▪, vehicle; ⋄, 0.94 nM; •, 1.88 nM; □, 3.75 nM) 15 min prior to the addition of a concentration-response curve of SDF-1. Intracellular calcium release was measured using FLIPR. Chemotaxis assays were performed using the U2OS cell line that endogenously expresses CXCR4. Cells were preincubated with increasing concentrations of GSK812397 (▪, vehicle; ⋄, 0.01 nM; •, 0.06 nM; □, 0.32 nM; ▴, 1.6 nM; ▾, 8.0 nM) 15 min prior to the addition of a concentration-response curve of SDF-1. Cells were allowed to chemotax for 2 h. Data represent the mean value of the results from at least three experiments. Error bars represent the standard error of the mean.

FIG. 4.

Measurement of the association and dissociation of GSK812397 from the CXCR4 receptor. (A) Association of GSK812397 from the receptor was examined by measuring the potency (IC₅₀) of the compound following increasing incubation periods (▪, 5 min; ⋄, 15 min; •, 30 min; □, 45 min) using the calcium release assay. Calcium release was stimulated by addition of 30 nM SDF-1. (B) Dissociation of GSK812397 from the receptor was also examined in the calcium release assay by examining the potency (IC₅₀) of the compound following removal of the compound by washing and examining the change in potency over time (▪, no wash; ⋄, 15 min; •, 30 min; □, 45 min). Data represent the mean value of the results from at least three experiments. Error bars represent the standard error of the mean.