Searching for plant-derived antivirals against dengue virus and Zika virus

Abstract

Background: The worldwide epidemics of diseases as dengue and Zika have triggered an intense effort to repurpose drugs and search for novel antivirals to treat patients as no approved drugs for these diseases are currently available. Our aim was to screen plant-derived extracts to identify and isolate compounds with antiviral properties against dengue virus (DENV) and Zika virus (ZIKV).

Methods: Seven thousand plant extracts were screened in vitro for their antiviral properties against DENV-2 and ZIKV by their viral cytopathic effect reduction followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, previously validated for this purpose. Selected extracts were submitted to bioactivity-guided fractionation using high- and ultrahigh-pressure liquid chromatography. In parallel, high-resolution mass spectrometric data (MSn) were collected from each fraction, allowing compounds into the active fractions to be tracked in subsequent fractionation procedures. The virucidal activity of extracts and compounds was assessed by using the plaque reduction assay. EC₅₀ and CC₅₀ were determined by dose response experiments, and the ratio (EC₅₀/CC₅₀) was used as a selectivity index (SI) to measure the antiviral vs. cytotoxic activity. Purified compounds were used in nuclear magnetic resonance spectroscopy to identify their chemical structures. Two compounds were associated in different proportions and submitted to bioassays against both viruses to investigate possible synergy. In silico prediction of the pharmacokinetic and toxicity (ADMET) properties of the antiviral compounds were calculated using the pkCSM platform.

Results: We detected antiviral activity against DENV-2 and ZIKV in 21 extracts obtained from 15 plant species. Hippeastrum (Amaryllidaceae) was the most represented genus, affording seven active extracts. Bioactivity-guided fractionation of several extracts led to the purification of lycorine, pretazettine, narciclasine, and narciclasine-4-O-β-D-xylopyranoside (NXP). Another 16 compounds were identified in active fractions. Association of lycorine and pretazettine did not improve their antiviral activity against DENV-2 and neither to ZIKV. ADMET prediction suggested that these four compounds may have a good metabolism and no mutagenic toxicity. Predicted oral absorption, distribution, and excretion parameters of lycorine and pretazettine indicate them as candidates to be tested in animal models.

Conclusions: Our results showed that plant extracts, especially those from the Hippeastrum genus, can be a valuable source of antiviral compounds against ZIKV and DENV-2. The majority of compounds identified have never been previously described for their activity against ZIKV and other viruses.

Keywords: Alkaloids; Antiviral; Bioactivity-guided fractionation; Compounds; Dengue virus; Hippeastrum; Natural products; Plant extract; Virucidal; Zika virus.

Fig. 1

Isobolograms representing in vitro interactions of lycorine and pretazettine. The compounds were combined in fixed ration generating solutions which were tested for their cytotoxicity in BHK-21 (a) and Vero (c) cells and in parallel for their antiviral activity against DENV-2 (b) and ZIKV (d). The pair of doses were evaluated at the level of EC₅₀ and CC₅₀, which were determined by regression curves. Subsequently, their FIC was determined and plotted on the graphs above. All points on isobole represent dose pairs, FIC of pretazettine (x-axis) and lycorine (y-axis), that are expected to give the EC₅₀ (in the case of viruses) or CC₅₀ (in the case of cells). Plots are observed very close to the theoretical diagonal line, which means, in short, the indifferent effect of both compounds used together. The illustration strengthens the results obtained of statistical analyses, from which the fractional inhibitory concentration index (FICI) was obtained (shown at the upper right corner) and used to define interactions as indifferent for being between 0.5 and 4