In vitro and in vivo activities of the novel anticytomegalovirus compound AIC246

Abstract

Human cytomegalovirus (HCMV) remains a serious threat for immunocompromised individuals, including transplant recipients and newborns. To date, all drugs licensed for the treatment of HCMV infection and disease target the viral DNA polymerase. Although these drugs are effective, several drawbacks are associated with their use, including toxicity and emergence of drug resistance. Hence, new and improved antivirals with novel molecular targets are urgently needed. Here we report on the antiviral properties of AIC246, a representative of a novel class of low-molecular-weight compounds that is currently undergoing clinical phase II studies. The anti-HCMV activity of AIC246 was evaluated in vitro and in vivo using various cell culture assays and an engineered mouse xenograft model. In addition, antiviral properties of the drug were characterized in comparison to the current gold standard ganciclovir. We demonstrate that AIC246 exhibits excellent in vitro inhibitory activity against HCMV laboratory strains and clinical isolates, retains activity against ganciclovir-resistant viruses, is well tolerated in different cell types (median selectivity index, 18,000), and exerts a potent in vivo efficacy in a mouse xenograft model. Moreover, we show that the antiviral block induced by AIC246 is reversible and the efficacy of the drug is not significantly affected by cell culture variations such as cell type or multiplicity of infection. Finally, initial mode-of-action analyses reveal that AIC246 targets a process in the viral replication cycle that occurs later than DNA synthesis. Thus, AIC246 acts via a mode of action that differs from that of polymerase inhibitors like ganciclovir.

Figures

FIG. 1.

Chemical structure of AIC246.

FIG. 2.

Representative in vitro dose-response curves of AIC246 (A) and ganciclovir (B) determined by the GFP-RA. Error bars represent standard deviations of results for duplicate samples.

FIG. 3.

Effect of AIC246 on focal expansion of a cell-associated HCMV isolate in cell culture. Productively infected fibroblasts were cocultured with an excess of uninfected indicator fibroblasts for 5 days in the absence (A) or presence (B) of AIC246. Cells were fixed and stained for viral immediate-early antigen expression (green signals), and cell nuclei were counterstained with DAPI (blue signals). The number of infected cells per focus is indicated.

FIG. 4.

Kinetic block release assay. (A) Schematic representation of the experimental setup. HCMV AD169-infected NHDF cells were treated for 96 h with the indicated inhibitor (antiviral block). At 96 h postinfection (hpi), infected cells were released from the drug block, and the production of progeny virus was analyzed 24 h, 48 h, 72 h, and 96 h following drug release (hpr). (B) Production of progeny virus during antiviral virus block and following block release monitored via virus yield measurements.

FIG. 5.

Effect of the time of addition of AIC246, BAY 38-4766 or GCV on HCMV replication. (A) Schematic representation of the experimental setup. NHDF cells were infected with AD169-GFP and treated with fixed virus inhibitory concentrations (∼10 × EC50) of GCV, BAY 38-4766, and AIC246 at the indicated time points postinfection (hpi). After 7 days, cell supernatants were replaced by PBS and GFP units (GFPU) were determined. (B) GFPU in compound-treated cells were compared to those in untreated cells, and the percentage of activity is plotted. Results are averages for three experiments carried out in duplicate. Error bars indicate standard deviations.

FIG. 6.

Effect of VGCV or AIC246 therapy on HCMV replication in a mouse xenograft model. (A) Viral titers of HCMV-infected Gelfoam sponges harvested from transplanted mice receiving antiviral treatment with the indicated doses of placebo, VGCV, or AIC246. Drugs were given once daily per os for 9 days. Results are expressed as means ± standard errors of the means (SEM). Placebo, n = 24; VGCV, n = 20; AIC246 (1 mg/kg/day), n = 10, (3 mg/kg/day) n = 11, (10 mg/kg/day) n = 21, (30 mg/kg/day) n = 21, (100 mg/kg/day) n = 11. **, P value < 0.006 (unpaired t test); l.o.d., limit of detection; PFU, PFU. Data were derived from two additional experiments. (B) In vivo dose-response curves of VGCV and AIC246 therapy. AIC246 data were derived from animals described for panel A. VGCV data were derived from results shown in panel A and two separate experiments using mice receiving antiviral treatment with placebo and 1, 3, 10, 30, and 100 mg/kg VGCV given per os once daily. Results are expressed as means ± standard deviations (SD). For clarification, ED50 and ED90 values (50% or 10% of mean placebo values, respectively) are indicated by a dotted line. (C) ED50 and ED90 values calculated from the dose-response curves depicted in panel B. Since ED90 was not reached in the VGCV treatment group, the ED90 was set to >100 mg/kg/day.