Inhibition of dengue virus by an ester prodrug of an adenosine analog

Abstract

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2'-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3'- and 5'-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3',5'-O-diisobutyryl-2'-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.

FIG. 1.

Antiviral activities of NITD449 and NITD203. (A) Compound structure. (B) Cytotoxicity analysis. A549 cells were incubated with NITD449 or NITD203 at various concentrations for 48 h. Cell viability was then measured using an MTT assay and presented as a percentage of colorimetric absorbance derived from the compound-treated cells, compared with that from the mock-treated cells. (C) Antiviral activities in cell culture. CFI assays were performed to determine the EC₅₀s for different DENV strains. Plaque assays were used to determine the EC₅₀ for infection of human PBMC cells. Averages of results from two independent experiments are presented. See Materials and Methods for details. The EC₅₀s for NITD008, previously reported in reference , are included for comparison.

FIG. 2.

Pharmacokinetic analysis of NITD449 and NITD203. Mice (A) and rats (B) were intravenously injected (i.v.) with 5 mg/kg of NITD449, orally dosed (p.o.) with 25 mg/kg of NITD449, or orally dosed with NITD203. Plasma concentrations of NITD449 (ng/ml) over time are presented. The dotted line in panel A indicates the lower limit of quantification (LOQ); the LOQ for NITD449 and NITD203 was 9.2 ng/ml; values below the LOQ are not included in the graphs. Plasma concentrations are indicated with standard deviations (n = 3).

FIG. 3.

Antiviral spectrum and mode-of-action analysis. (A) Vero cells were infected with the indicated viruses at an MOI of 0.1 and treated immediately with NITD203. For DENV-2 and YFV, culture medium was collected at 48 h postinfection and measured for viral titers using plaque assays. For Chikungunya virus, culture medium was collected at 22 h postinfection and measured for viral titer. For WNV, Vero cells were infected with virus-like particles (containing a luciferase replicon) at an MOI of about 0.1; the infected cells were assayed for luciferase activity at 24 h postinfection; relative luciferase activities were presented, with the level for the mock-treated replicon cells set as 100%. For HCV, Huh-7 cells carrying a luciferase replicon HCV (5) were incubated with NITD203 and assayed for luciferase activities at 48 h posttreatment. Average results and standard deviations (n = 3) are presented. (B) Transient DENV-2 replicon assay. A luciferase reporter replicon of DENV-2 was transfected into BHK-21 cells. The transfected cells were immediately incubated with the indicated concentrations of the compound and measured for luciferase activities at various time points posttransfection (p.t.). The effects of compound (1 μM and 3 μM) on viral translation and RNA synthesis were quantified by the luciferase signals at 2 to 4 h p.t. and at 24 to 40 h p.t., respectively. Average results and standard deviations (n = 3) are presented.

FIG. 4.

In vivo efficacy of NITD203. AG129 mice were intraperitoneally inoculated with 2 × 10⁶ PFU of DENV-2 (strain TSV01) on day 0. The infected mice (6 or 8 animals per group) were immediately treated with the indicated doses of NITD203 orally twice per day for three consecutive days. The peak viremia on day 4 postinfection was quantified by a plaque assay. For comparison, the infected mice were treated with 25 mg/kg of NITD008, and the reduction of peak viremia is presented.