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Among HIV-1 reverse transcriptase (RT)-associated functions, DNA polymerase and Ribonuclease H (RNase H) are both essential for HIV replication and excellent targets for drug development. While all RT inhibitors approved for therapy target the DNA polymerase activity, there is the pressing need for new RT inhibitors possibly targeting the RNase H function. In the last 20 years, many natural substances have shown antiviral activity against HIV-1, but only a few against the RNase H function. In this study, we have tested the ethanolic extracts obtained by the Hypericum hircinum L. (Hypericaceae) growing in Sardinia (Italy) on the HIV-1 RT-associated RNase H function and found that they have inhibitory effects. Active extracts were fractionated up to obtain the main components that have been isolated, tested, and identified to be betulinic acid, shikimic acid, chlorogenic acid, quercetin, 5,7,3',5'-tetrahydroxyflavanone, and 5,7,3',5'-tetrahydroxyflavanone 7-O-glucoside. Betulinic acid and 5,7,3',5'-tetrahydroxyflavanone 7-O-glucoside were active on both RT-associated activities, and betulinic acid was also active on HIV-1 mutant RTs resistant to efavirenz. Overall, our results suggest that some of these compounds inhibit the HIV-1 RT binding to an allosteric site previously described for other natural compounds and are potential leads for further drug development of a single molecules having dual inhibitory activity.

The essential oils of Artemisia arborescens growing in Sardinia (Italy), collected during three plant growth stages, i.e., from the vegetative stage to post-blooming time, were characterized. Moreover, the in vitro antiproliferative and antioxidant activities of the oil isolated from aerial parts collected in February were evaluated. The essential oils belonged to the β-thujone/chamazulene chemotype, notably with the highest amount of chamazulene (ca. 52%) ever detected up to now in the genus Artemisia and, in general, in essential oils. Quantitative variations in the oil composition were observed as the plant passes from the vegetative to the blooming stage. The oil was tested for its potential tumor cell growth-inhibitory effect on T98G, MDA-MB 435S, A375, and HCT116 human cell lines, using the MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The highest activity was observed on A375 and HCT116 cell lines, with IC50 values of 14 μg/ml. Moreover, the in vitro antioxidant and free radical-scavenging assays revealed the oil to be an effective scavenger of the ABTS radical cation, with an activity comparable to that of Trolox(®) . These results support the use of A. arborescens oil for the treatment of inflamed skin conditions. Finally, the composition of the polar fraction of the A. arborescens aerial parts was also examined, and the main component detected was 5-O-caffeoylquinic acid, which was identified for the first time in this plant.

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Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome whose recent epidemic enlightened the urgent need of novel therapeutic agents, since no drug is currently approved. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which the viral protein VP35 plays a crucial role, blocking the interferon type I production, firstly by masking the viral dsRNA and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of VP35 interaction with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification, and a novel and robust fluorescence-based rVP35-RNA interaction assay (Z'-factor of 0.69). Taking advantage of such newlyestablished methods, we screened a small library of Sardinian natural extracts finding Limonium morisianum as the most potent inhibitor extract. A bio-guided fractionation led to the identification of myricetin as the component able to inhibit rVP35-dsRNA interaction with an IC 50 value of 2.7 µM.Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent VP35-dsRNA inhibitors.