The African natural product knipholone anthrone and its analogue anthralin (dithranol) enhance HIV-1 latency reversal.

A sterilizing or functional cure for HIV is currently precluded by resting CD4+ T cells that harbor latent but replication-competent provirus. The "shock-and-kill" pharmacological ap-proach aims to reactivate provirus expression in the presence of antiretroviral therapy and target virus-expressing cells for elimination. However, no latency reversal agent (LRA) to date effectively clears viral reservoirs in humans, suggesting a need for new LRAs and LRA combinations. Here, we screened 216 compounds from the pan-African Natural Product Library and identified knipholone anthrone (KA) and its basic building block anthralin (dithranol) as novel LRAs that reverse viral latency at low micromolar concentrations in multiple cell lines. Neither agent's activity depends on protein kinase C; nor do they inhibit class I/II histone deacetylases. However, they are differentially modulated by oxidative stress and metal ions and induce distinct patterns of global gene expression from established LRAs. When applied in combination, both KA and anthralin synergize with LRAs representing multiple functional classes. Finally, KA induces both HIV RNA and protein in primary cells from HIV-infected donors. Taken together, we describe two novel LRAs that enhance the activities of multiple "shock-and-kill" agents, which in turn may inform ongoing LRA combination therapy efforts.

Inhibition of NF-κB-dependent HIV-1 replication by the marine natural product bengamide A.

HIV-1 inhibitors that act by mechanisms distinct from existing antiretrovirals can provide novel insights into viral replication and potentially inform development of new therapeutics. Using a multi-cycle HIV-1 replication assay, we screened 252 pure compounds derived from marine invertebrates and microorganisms and identified 6 (actinomycin Z2, bastadin 6, bengamide A, haliclonacyclamine A + B, keramamine C, neopetrosiamide B) that inhibited HIV-1 with 50% effective concentrations (EC50s) of 3.8 µM or less. The most potent inhibitor, bengamide A, blocked HIV-1 in a T cell line with an EC50 of 0.015 µM and in peripheral blood mononuclear cells with an EC50 of 0.032 µM. Bengamide A was previously described to inhibit NF-κB signaling. Consistent with this mechanism, bengamide A suppressed reporter expression from an NF-κB-driven minimal promoter and an HIV-1 long terminal repeat (LTR) with conserved NF-κB response elements, but lacked activity against an LTR construct with mutation of these elements. In single-cycle HIV-1 infection assays, bengamide A also suppressed viral protein expression when viruses encoded an intact LTR but exhibited minimal activity against those with mutated NF-κB elements. Finally, bengamide A did not inhibit viral DNA accumulation, indicating that it likely acts downstream of this step in HIV-1 replication. Our study identifies multiple new antiviral compounds including an unusually potent inhibitor of HIV-1 gene expression.

The Croton megalobotrys Müll Arg. traditional medicine in HIV/AIDS management: Documentation of patient use, in vitro activation of latent HIV-1 provirus, and isolation of active phorbol esters.

ETHNOPHARMACOLOGICAL RELEVANCE: Current HIV therapies do not act on latent cellular HIV reservoirs; hence they are not curative. While experimental latency reversal agents (LRAs) can promote HIV expression in these cells, thereby exposing them to immune recognition, existing LRAs exhibit limited clinical efficacy and high toxicity. We previously described a traditional 3-step medicinal plant regimen used for HIV/AIDS management in Northern Botswana that inhibits HIV replication in vitro. Here we describe use of one component of the regimen that additionally contains novel phorbol esters possessing HIV latency-reversal properties. AIM OF THE STUDY: We sought to document experiences of traditional medicine users, assess the ability of traditional medicine components to reverse HIV latency in vitro, and identify pure compounds that conferred these activities. MATERIALS AND METHODS: Experiences of two HIV-positive traditional medicine users (patients) were documented using qualitative interview techniques. Latency reversal activity was assessed using a cell-based model (J-Lat, clone 9.2). Crude plant extracts were fractionated by open column chromatography and reverse-phase HPLC. Compound structures were elucidated using NMR spectroscopy and mass spectrometry. RESULTS: Patients using the 3-step regimen reported improved health over several years despite no reported use of standard HIV therapies. Crude extracts from Croton megalobotrys Müll Arg. ("Mukungulu"), the third component of the 3-step regimen, induced HIV expression in J-lat cells to levels comparable to the known LRA prostratin. Co-incubation with known LRAs and pharmacological inhibitors indicated that the active agent(s) in C. megalobotrys were likely to be protein kinase C (PKC) activator(s). Consistent with these results, two novel phorbol esters (Namushen 1 and 2) were isolated as abundant components of C. megalobotrys and were sufficient to confer HIV latency reversal in vitro. CONCLUSION: We have identified novel LRAs of the phorbol ester class from a medicinal plant used in HIV/AIDS management. These data, combined with self-reported health effects and previously-described in vitro anti-HIV activities of this traditional 3-step regimen, support the utility of longitudinal observational studies of patients undergoing this regimen to quantify its effects on plasma viral loads and HIV reservoir size in vivo.

Croton megalobotrys Müll Arg. and Vitex doniana (Sweet): Traditional medicinal plants in a three-step treatment regimen that inhibit in vitro replication of HIV-1.

ETHNOPHARMACOLOGICAL RELEVANCE: Human Immunodeficiency Virus (HIV) strains resistant to licensed anti-retroviral drugs (ARVs) continue to emerge. On the African continent, uneven access to ARVs combined with occurrence of side-effects after prolonged ARV therapy have led to searches for traditional medicines as alternative or complementary remedies to conventional HIV/AIDS management. AIM OF THE STUDY: Here we characterize a specific three-step traditional HIV/AIDS treatment regimen consisting of Cassia sieberiana root, Vitex doniana root, and Croton megalobotrys bark by combining qualitative interviews of traditional medical knowledge users in Botswana with in vitro HIV replication studies. MATERIALS AND METHODS: Crude extracts from a total of seven medicinal plants were tested for in vitro cytotoxicity and inhibition of wild-type (NL4.3) and ARV-resistant HIV-1 replication in an immortalized GFP-reporter CD4+ T-cell line. RESULTS: C. sieberiana root, V. doniana root, and C. megalobotrys bark extracts inhibited HIV-1NL4.3 replication with dose-dependence and without concomitant cytotoxicity. C. sieberiana and V. doniana extracts inhibited HIV-1 replication by 50% at 84.8µg/mL and at 25µg/mL, respectively, while C. megalobotrys extracts inhibited HIV-1 replication by a maximum of 45% at concentrations as low as 0.05µg/mL. Extracts did not interfere with antiviral activities of licensed ARVs when applied in combination and exhibited comparable efficacies against viruses harboring major resistance mutations to licensed protease, reverse-transcriptase, or integrase inhibitors. CONCLUSIONS: We report for the first time a three-step traditional HIV/AIDS regimen, used alone or in combination with standard ARV regimens, where each step exhibited more potent ability to inhibit HIV replication in vitro. Our observations support the "reverse pharmacology" model where documented clinical experiences are used to identify natural products of therapeutic value.

Screening of the Pan-African natural product library identifies ixoratannin A-2 and boldine as novel HIV-1 inhibitors.

The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.

Modest decreases in NNRTI susceptibility do not influence virological outcome in patients receiving initial NNRTI-containing triple therapy.

OBJECTIVE: To assess the prevalence of modest (< 10-fold) decreases in baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) susceptibility and their impact on virological response to NNRTI-containing triple therapy in drug-naive individuals. METHODS: Baseline HIV resistance phenotype, genotype and response to therapy were examined retrospectively for all antiretroviral-naive individuals initiating therapy with two nucleoside analogues and an NNRTI in British Columbia, Canada, between 05/1997 and 08/1999 (n = 279), followed until July 31 2001. Time to viral suppression (first of at least two consecutive plasma viral loads < 400 copies HIV RNA copies/ml) and viral rebound (to > or = 400 copies/ml after first pVL < 400 copies HIV RNA copies/ml), were estimated by Kaplan-Meier methods. Multivariate analyses were performed using Cox proportional hazards regression. RESULTS: Nevirapine was the most commonly prescribed NNRTI (96%). Four- to 10-fold decreased susceptibility to NNRTIs was observed in > 30% of untreated individuals at baseline, an observation strongly driven by decreased susceptibility to delavirdine (22.4%). A > 10-fold decrease in susceptibility to any NNRTI was observed only rarely (< 2%). There was no association between four- and 10-fold decreased baseline susceptibility to NNRTIs and virological outcome (P > 0.05). In multivariate analyses, the strongest predictors of poor virological response to NNRTI-based therapy were baseline plasma viral load and the proportion of time on therapy in the first year of follow-up. There was no relationship between the presence of previously reported mutations associated with decreased NNRTI susceptibility (at codons 135 and 283 in HIV reverse transcriptase) and virological response. CONCLUSIONS: These data suggest that the clinically significant level of resistance to NNRTIs, particularly nevirapine, in drug-naive individuals is likely greater than four- to 10-fold.