RESUMO
Current research on antiretroviral therapy is mainly focused in the development of new formulations or combinations of drugs belonging to already known targets. However, HIV-1 infection is not cured by current therapy and thus, new approaches are needed. Bevirimat was developed by chemical modification of betulinic acid, a lupane-type pentacyclic triterpenoid (LPT), as a first-in-class HIV-1 maturation inhibitor. However, in clinical trials, bevirimat showed less activity than expected because of the presence of a natural mutation in Gag protein that conferred resistance to a high proportion of HIV-1 strains. In this work, three HIV-1 inhibitors selected from a set of previously screened LPTs were investigated for their targets in the HIV-1 replication cycle, including their maturation inhibitor effect. LPTs were found to inhibit HIV-1 infection acting as promiscuous compounds with several targets in the HIV-1 replication cycle. LPT12 inhibited HIV-1 infection mainly through reverse transcription, integration, viral transcription, viral proteins (Gag) production and maturation inhibition. LPT38 did it through integration, viral transcription or Gag production inhibition and finally, LPT42 inhibited reverse transcription, viral transcription or Gag production. The three LPTs inhibited HIV-1 infection of human primary lymphocytes and infections with protease inhibitors and bevirimat resistant HIV-1 variants with similar values of IC50. Therefore, we show that the LPTs tested inhibited HIV-1 infection through acting on different targets depending on their chemical structure and the activities of the different LPTs vary with slight structural alterations. For example, of the three LPTs under study, we found that only LPT12 inhibited infectivity of newly-formed viral particles, suggesting a direct action on the maturation process. Thus, the multi-target behavior gives a potential advantage to these compounds since HIV-1 resistance can be overcome by modulating more than one target.
RESUMO
OBJECTIVE: To investigate the toxicity and activity against HIV of 5-hydroxytyrosol as a potential microbicide. DESIGN: The anti-HIV-1 activity of 5-hydroxytyrosol, a polyphenolic compound, was tested against wild-type HIV-1 and viral clones resistant to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and integrase inhibitors. In addition to its activity against founder viruses, different viral subtypes and potential synergy with tenofovir disoproxil fumarate, lamivudine and emtricitabine was also tested. 5-Hydroxytyrosol toxicity was evaluated in vivo in rabbit vaginal mucosa. METHODS: We have cloned pol gene from drug-resistant HIV-1 isolated from infected patients and env gene from Fiebeg III/IV patients or A, C, D, E, F and G subtypes in the NL4.3-Ren backbone. 5-Hydroxytyrosol anti-HIV-1 activity was evaluated in infections of MT-2, U87-CCR5 or peripheral blood mononuclear cells preactivated with phytohemagglutininâ+âinterleukin-2 with viruses obtained through 293T transfections. Inhibitory concentration 50% and cytotoxic concentration 50% were calculated. Synergy was analysed according to Chou and Talalay method. In-vivo toxicity was evaluated for 14 days in rabbit vaginal mucosa. RESULTS: 5-Hydroxytyrosol inhibited HIV-1 infections of recombinant or wild-type viruses in all the target cells tested. Moreover, 5-hydroxytyrosol showed similar inhibitory concentration 50% values for infections with NRTIs, NNRTIs, protease inhibitors and INIs resistant viruses; founder viruses and all the subtypes tested. Combination of 5-hydroxytyrosol with tenofovir was found to be synergistic, whereas it was additive with lamivudine and emtricitabine. In-vivo toxicity of 5-hydroxytyrosol was very low even at the highest tested doses. CONCLUSION: 5-Hydroxytyrosol displayed a broad anti-HIV-1 activity in different cells systems in the absent of in-vivo toxicity, therefore supporting its candidacy as a potential new class of microbicides.
