Depsides: lichen metabolites active against hepatitis C virus

Abstract

A thorough phytochemical study of Stereocaulon evolutum was conducted, for the isolation of structurally related atranorin derivatives. Indeed, pilot experiments suggested that atranorin (1), the main metabolite of this lichen, would interfere with the lifecycle of hepatitis C virus (HCV). Eight compounds, including one reported for the first time (2), were isolated and characterized. Two analogs (5, 6) were also synthesized, to enlarge the panel of atranorin-related structures. Most of these compounds were active against HCV, with a half-maximal inhibitory concentration of about 10 to 70 µM, with depsides more potent than monoaromatic phenols. The most effective inhibitors (1, 5 and 6) were then added at different steps of the HCV lifecycle. Interestingly, atranorin (1), bearing an aldehyde function at C-3, inhibited only viral entry, whereas the synthetic compounds 5 and 6, bearing a hydroxymethyl and a methyl function, respectively, at C-3 interfered with viral replication.

Fig 1. Structures of compounds isolated from the lichen S. evolutum and of the semi-synthetic compounds 5–6.

Fig 2. HMBC key correlations for compound 2.

Fig 3. LC-MS analysis.

Compound 2 (extracted at Rt = 19.48 min) was detected through PDA chromatograms (total scan at λ = 220–600 nm), base-peak mass chromatograms and MS spectra of extemporaneously prepared acetone extract of S. evolutum (a), an ethyl acetate extract of S. evolutum (b) and pure atranorin macerated in acetone for one week (c).

Fig 4. Effects of compounds 1, 5 and 6 on cell viability and HCV propagation in vitro.

One day after seeding at 50,000 cells/cm², Huh-7.5.1 cells were infected with HCVcc at a MOI ~ 0.03 for 17 h (inoculation phase: I). The inoculum was removed and cell cultures were washed before the addition of new medium and incubation for an additional 30 h (replication phase: R). Compounds were present at various concentrations either during both phases (I/R: black circles and solid lines) or during only one phase (I: black squares and dotted lines; R: black triangles and dashed lines). (A) Cell viability and (B) viral replication were assessed at the end of the incubation period. Results are expressed as percentages (mean ± SEM; n = 3) of the mean values obtained for the respective control cultures, in which cells were incubated with vehicle (0.1% DMSO) alone. Erlotinib and telaprevir were used as positive antiviral drug controls inhibiting the entry and replication steps, respectively.