Quinic acid derivatives inhibit dengue virus replication in vitro

Abstract

Background: Dengue is the most prevalent arboviral disease in tropical and sub-tropical areas of the world. The incidence of infection is estimated to be 390 million cases and 25,000 deaths per year. Despite these numbers, neither a specific treatment nor a preventive vaccine is available to protect people living in areas of high risk.

Results: With the aim of seeking a treatment that can mitigate dengue infection, we demonstrated that the quinic acid derivatives known as compound 2 and compound 10 were effective against all four dengue virus serotypes and safe for use in a human hepatoma cell line (Huh7.5). Both compounds were non-virucidal to dengue virus particles and did not interfere with early steps of the dengue virus life cycle, including binding and internalization. Experiments using a replicon system demonstrated that compounds 2 and 10 impaired dengue virus replication in Huh7.5 cells. Additionally, the anti-dengue virus effects of the quinic acid derivatives were preserved in human peripheral blood mononuclear cells.

Conclusions: Taken together, these data suggest that quinic acid derivatives represent a novel chemical class of active compounds that could be used to combat dengue virus infection.

Fig. 1

Antiviral screening. Huh7.5 cells were infected and treated during and after infection. After 72 h, the results from the in situ ELISA assay indicated the most promising substances (*p < 0.05 compared to DENV control). Data represent the mean ± standard error (SE) from three independent experiments (*p < 0.05 compared to DENV control)

Fig. 2

Antiviral activity confirmation by secondary assays. Huh7.5 cells were infected with DENV-1 through −4 and treated during and after infection with each substance (NTC). After 72 h, the cells were submitted to FACS (a), and supernatants were used in focus-forming assays in C6/36 cells (b). Data represent the mean ± standard error (SE) from three independent experiments (*p < 0.05 compared to DENV control)

Fig. 3

Virucidal activity. DENV serotypes 1 through 4 were incubated with compounds 2 and 10 (NTC) in the presence or absence of RNase. RNA samples were extracted and submitted to RT-PCR and gel electrophoresis. Representative data from 4 independent assays. M: 1 kb DNA ladder; RNA: viral RNA control; bp: base pairs

Fig. 4

Adsorption and internalization assays. Binding inhibition assay (a) and inhibition of virus internalization (b) for each DENV serotype. After 72 h incubation period the in situ ELISA was performed. Data represents mean ± standard error of 3 independent assays (*p < 0.05 compared to DENV control)

Fig. 5

DENV subgenomic replicon system. Huh7.5 cells were transfected with either dengue virus serotype-1 replicon RepDV1 or dengue virus serotype-3 replicon RepDV3 RNA, and after one hour elapsed they were treated with compounds 2 and 10 at the NTC. After 72 h, the cells were submitted to FACS analysis (anti-NS3 staining using the monoclonal antibody 1722). Pseudocolor plots (a) and histograms showing the mean fluorescent intensity (MFI) (b) are representative of one experiment, and relative percentages as mean ± standard error (SE) from three independent experiments (c). In parallel, cell viability was evaluated by neutral red assay (d). *p < 0.05 compared to the untreated control

Fig. 6

Antiviral activity in PBMCs. PBMCs were infected with DENV-4 (MOI 10) and treated with each compound. After 5 days in culture, cells were assayed for apoptosis (a, d) and infection (b, c and e) by flow cytometry. The figure includes the pseudocolor plot data from one representative blood donor and the mean from 6 healthy donors. *p < 0.05 compared to the untreated control