Background: Combination antiretroviral therapy (cART) durably suppresses HIV replication, but virus persists in cellular reservoirs. Latency reversal agents (LRAs) capable of reactivating HIV-infected cells may promote their elimination through host or viral cytopathic effects; an approach termed “shock-and-kill”. However, current LRAs have shown limited clinical success, and no single LRA reactivates all latent cells. Conversely, “deep-latency” agents (DLAs) that promote long-term suppression of viral transcription that is refractory to subsequent reactivation are few in number and not yet assessed in vivo. Thus additional LRAs and DLAs are needed.
Methods: We examined 433 pure compounds from marine natural products and medicinal plants using J-Lat 8.4 GFP-reporter T cells containing an NL4.3Δenv/nef genome. Compounds that induced GFP in >5% of cells while retaining >30% cell viability at 5µg/mL were assessed for synergism with established LRAs. Compounds that blocked >50% GFP expression induced by 50ng/mL TNFα were assessed for antagonism of LRAs and ability to block a doxycycline-induced, Tat/TAR-deficient provirus. Select compounds were also tested in infected, primary resting CD4 T-cells using the Tat/Rev-Induced Limiting Dilution Assay (TILDA).
Results: We identified 8 new LRAs and 2 DLAs. Two novel phorbol esters induced GFP in 6.0 ± 0.6 and 6.9 ± 0.4% of cells at 0.03µg/mL, respectively, having ~10-fold less activity than PMA but ~100-fold greater than prostratin. Interestingly, these compounds originate from a plant used by African traditional healers to treat AIDS symptoms. Anthrone “p61” reactivated 9.9 ± 0.9% of cells at 5µg/mL and synergized with panobinostat (HDAC inhibitor), prostratin (PKC activator), and TNFα, suggesting a distinct mechanism of action. p61-containing plants are traditionally used to treat malaria, and p61 does not synergize with the anti-malarial artemisinin, suggesting related modes of targeting latent HIV. Two novel flavonoids at 5µg/mL suppressed >85% of GFP induction by TNFα, control LRAs, and production of Tat/TAR-deficient provirus, suggesting mechanism(s) distinct from previously-reported DLAs. Finally, both phorbol esters induced multiply-spliced vRNA expression in resting CD4 T-cells, while one flavonoid suppressed >80% of PMA/ionomycin-induced vRNA.
Conclusions: We identified potential new LRAs and DLAs of natural origin, including some supported by traditional medicine, that display synergy with established LRAs and/or distinct mechanisms of action.