4-deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat.

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposed to eliminate these viral reservoirs is the pharmacological reactivation of latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4beta-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4beta-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to diterpene esters such SJ23B. Our data suggest 4beta-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4beta-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4beta-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCtheta/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4beta-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4beta-dPE A. Further, NF-kappab transcription factor is involved in 4beta-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4beta-dPE A in combination with other LRAs. When 4beta-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistic diminishing the EC50 value but also increasing the efficacy showed by the drugs alone. Further, combination of 4beta-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not induce undesirable effects on antiretroviral therapy (ART). Finally, 4beta-dPE A induced latent HIV reactivation in CD4+ T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.