FIG 2.

In vitro antiviral profile of the spiropyrazolopyridone compounds. (A) Structures of spiropyrazolopyridone compound 1a (R), compound 1b (S), and compound 14a (R). (B) Antiviral activities of two enantiomers (compounds 1a and 1b) of compound 1 against DENV-2. A549 cells were infected with DENV-2 (New Guinea C strain) at an MOI of 0.5 in the presence of 2-fold serial dilutions of compounds. After incubation at 37°C for 48 h, cell culture fluids were harvested for a plaque assay. The data are plotted as logarithm (log₁₀) values of average viral titers from triplicates versus the compound concentration. The error bars represent standard deviations (n = 3). (C) Antiviral spectra of compound 1a and its analog compound 14a. A549 cells were infected with DENV-1, -2, -3, or -4 (MOI of 0.5). Vero cells were infected with WNV, YFV, JEV, POW, WEEV, or VSV (MOI of 0.1). EC₅₀s were calculated by nonlinear regression analysis using Prism software (GraphPad). See Materials and Methods for assay details. (D) Transient-replicon assay. A549 cells were transfected with 10 μg of DENV-2 luciferase replicon RNA. The transfected cells were immediately treated with different concentrations of compound 1a or 0.9% DMSO (as a negative control). At the indicated time points p.i., cells were assayed for luciferase signals (quantified as relative light units [RLU]). The log₁₀ values of average luciferase signals and standard deviations are presented (n = 3). (E) Time-of-addition analysis. A549 cells were infected with DENV-2 (New Guinea C strain) at an MOI of 2 at 4°C for 1 h. After three washes with PBS to remove unbound viruses, cells were incubated at 37°C. At the indicated time points, compound 1a (1 μM) was added to the infected cells. As controls, the infected cells were treated with 0.5% DMSO. At 24 h p.i., the culture medium was collected, and viral titers were determined by a plaque assay. Average results and standard errors (n = 3) are presented. (F) Antiviral activity in mosquito C6/36 cells. C3/36 cells were infected with DENV-2 at an MOI of 1. Compound 1a was added at the indicated concentrations to cells immediately after infection. The culture supernatant was collected at 48 h p.i., and viral titers were quantified by a plaque assay.