An adenosine nucleoside inhibitor of dengue virus

Abstract

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

Fig. 1.

Antiviral activity of NITD008. (A) Structure of NITD008. (B) Cytotoxicity in Vero cells. Vero cells were incubated with NITD008 at the indicated concentrations for 48 h. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide 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 spectrum. Vero cells were infected with indicated viruses at a multiplicity of infection of 0.1 and treated immediately with NITD008. For DENV-2, WNV, YFV, PWV, and WEEV, culture medium was collected at 48 h postinfection and measured for viral titers using plaque assays. For VSV, culture medium was collected at 16 h postinfection and measured for viral titer. For HCV, Huh-7 cells carrying a luciferase replicon of HCV (19) were incubated with NITD008 and assayed for luciferase activity at 48 h posttreatment; relative luciferase activities were presented with the mock-treated replicon cells set as 100%. Average results and SDs (n = 3) are presented.

Fig. 2.

Inhibition of RdRp activity by NITD008 triphosphate. (A) RNA template for RdRp assay. The template RNA contains the authentic initial 12 nucleotides of the DENV-2 minus-sense RNA, followed by a U and a (C)₁₉-track. (B) RdRp primer extension assay. The RdRp assays contained 500 nM RNA template, 100 nM DENV-2 NS5, 5 μM cold GTP, and 1 μCi [α³³P]GTP. ATP (0.25 μM), 3′d-ATP (1 μM), 3′d-GTP (1 μM), or ppp-NITD008 (1 μM for lane 3 and 0.25 nM–4 μM for lanes 6–20 with a 2-fold increment of compound concentration) was added to the RdRp reaction, as indicated. The RdRp products were analyzed on a polyacrylamide denaturing gel. (C) Quantification of the RdRp results from B using a PhosphorImager.

Fig. 3.

In vivo efficacy of NITD008. (A) Viremia mouse model (12). AG129 mice (defective in IFN-α/β and IFN-γ receptors) were inoculated i.p. with 2 × 10⁶ pfu DENV-2 (strain TSV01) on day 0. (Top) Depiction of different treatment regimens. The mice (6 or 8 animal per group) were dosed p.o. with NITD008 twice a day. The peak viremia on day 3 postinfection (p.i.) was quantified by plaque assay; viral NS1 level in serum was measured by ELISA (12). (Middle) Indication of viremia and NS1 levels for the mice with immediate treatment after infection. (Bottom) Viremia and NS1 level from the mice with delayed start of treatment after infection (dosed at 25 mg/kg). (B) Lethal mouse model (13). AG129 mice were inoculated i.v. with 3 × 10⁷ pfu/mL DENV-2 strain D2S10 (13) and immediately treated with the indicated amount of NITD008 twice daily (6 mice per group). (Top) Survival curve. (Middle) Peak viremia. (Bottom) Summary of cytokine levels in serum.