POTENT AND SELECTIVE INHIBITION OF THYMELAEA HIRSUTA EXTRACTS AGAINST HUMAN IMMUNODEFICIENCY VIRUS (HIV) -1

Introduction: Historically, natural products have been the most successful source of inspiration for development of new drugs. Members of the Thymelaeaceae family have been of interest owing to their excellent medicinal value. Different classes of natural products have been isolated from these species, including flavonoids, coumarins, and diterpenoids. Given the successful history of natural product-based drug discovery, extracts from the aerial parts of Thymelaea hirsuta, were evaluated for their anti-HIV activity. Materials and Methods: Ethylacetate extracts from leaves (71 B) and branches (72B) of Thymelaea hirsuta were tested in cell-based assays for their cytotoxicity and anti-HIV-1 wt, resistant strains and HIV-1 BaL properties. The mode of action of 72B extract, chosen as lead, was characterized evaluating potential virucidal activity, its ability to inhibit HIV-1 reverse transcriptase (RT), protease activity and syncytia formation. Furthermore, safety profile of the extract was determined on viability of Lactobacillus sp. and transepithelial electrical resistance (TEER). Results: Both leaves and branches extracts showed potent and selective activity against HIV-1 wt (EC50 = 0.8 µg/ml) at not cytotoxic concentrations (CC50 = >100 µg/ml). They proved to be active against HIV-1 variants carrying clinically relevant NNRTI and NRTI mutations at low concentration (0.3-4 µg/ml range) and against the M-tropic strain HIV-1 BaL. 72B extract prevented syncytia formation at low concentration (0.4 µg/ml). 72B did not affect the Caco-2 epithelial cells monolayer integrity. The extract tested up to 100 µg/ml did not reduce the viability of Lactobacilli. Discussion and Conclusions: In our studies, ethyl acetate extracts from T. hirsuta turned out interestingly active against HIV-1 with either one of the two main T- and M-tropic viruses that use as co-receptor for cell entry CXCR4 or CCR5, respectively. Furthermore, extract turned to be potently active against resistant strains and it was not able to inhibit the RT and PI enzymatic functions. The potent inhibitory effect on the syncytia formation in co-cultures showed that 72B inhibits an early event in the replication cycle of HIV. Pre-clinical safety profile of 72B showed no adverse effect on the growth of Lactobacilli, and non-toxic concentration of the extract did not influence the TEER. These observations are encouraging and further safety and efficacy in vitro and in vivo studies will be performed in order to identify the chemical constituents of this extract and to better define the mode of action.