FIG 2.
Pyrimethamine inhibits dengue virus and NS2B/3 proteinase cleavage like ARDP0006. (A) Chemical structures and (B) ROCS overlay of ARDP0006 and pyrimethamine. (C) Dose-response curves for ARDP0006 and pyrimethamine treatment of DENV2-infected Huh7 cells revealed IC50 values of 0.59 μM and 1.2 μM, respectively. Compounds were added 1 h prior to infection, removed, and then readministered after removal of viral inoculum. Infectious virus in cell supernatant was quantified by plaque assay at 24 hpi (left y axis, black or red). Cell viability was measured at the same pyrimethamine doses 24 h posttreatment by WST-1 assay (right y axis, gray). Viral titer and cell viability were normalized to DMSO-treated samples. (D) Splenic titers of DENV2-infected AGB6 mice at 4 days postinfection. Mice were infected with 1 × 106 PFU DENV2 and treated twice daily with 20 mg/kg pyrimethamine (n = 9) or a vehicle control (n = 10). Data were analyzed by two-tailed unpaired t test. (E) Cleavage kinetics of NS2B/3/4A in the presence of 100 μM pyrimethamine. NS2B/3/4A protein was expressed in vitro and radiolabeled with [35S]methionine, followed by treatment with 100 μM pyrimethamine or DMSO. Reaction samples were obtained over a 45-min time course, separated by SDS-PAGE, and the fraction of uncleaved proteinase precursor remaining was determined as described previously (23). Data from three replicates were fit using single-phase exponential decay functions, and P value was determined by comparing the rate constant, K, of the models using the extra sum-of-squares F test.