The Natural Stilbenoid (-)-Hopeaphenol Inhibits HIV Transcription by Targeting Both PKC and NF-κB Signaling and Cyclin-Dependent Kinase 9.

Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (-)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. In contrast, it does not substantially inhibit the early PKC-mediated T-cell activation marker CD69 production of IL-6 or NF-κB signaling induced by tumor necrosis factor alpha (TNF-α). We further show that hopeaphenol can inhibit cyclin-dependent kinase 9 (CDK9) enzymatic activity required for HIV transcription. Finally, it inhibits HIV replication in peripheral blood mononuclear cells (PBMCs) infected in vitro and dampens viral reactivation in CD4+ cells from PLWH. Our study identifies hopeaphenol as a novel inhibitor that targets a subset of PKC-mediated T-cell activation pathways in addition to CDK9 to block HIV expression. Hopeaphenol-based therapies could complement current antiretroviral therapy otherwise not targeting cell-associated HIV RNA and residual antigen production in PLWH.

Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus.

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.