Natural Plant Alkaloid (Emetine) Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity

Abstract

Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine's potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT) Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT) to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V). Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.

Keywords: HIV-1; PBMC; Reverse Transcriptase; emetine; resistance.

Figure 1

Emetine blocks RT activity. (A) RT activity in vitro was measured by a colorimetric Reverse Transcriptase Assay. Inhibitory effects of emetine were presented as relative percentage of HIV-1 RT sample that does not contain any inhibitor. We presented the means and errors bars calculated from three independent experiments; (B) Emetine blocks intravirion RT activity. HIV viral particles were purified and normalized by ELISA p24. Virus particles treated or not treated with antiretroviral drugs were exposed to dNTPs to stimulate NERT activity. The ssDNA synthesis was evaluated by qPCR 3 h later. The absolute quantification was obtained by comparison with a DNA standard curve (HIV-1 NL4-3 plasmid). All the results are expressed as a percentage related to virus exposed to dNTPs (dNTPs +). At least three replicates of each sample were assayed, and data sets in which the linear correlation coefficient of the standard curve was less than 0.98 were not included for further analysis. dNTPs −: virus not exposed to dNTPs.

Figure 2

Emetine docking on RT-HIV in comparison with a pyridone analogue. (A) Representation of emetine and the pyridone non-nucleosidic analogue binding to RT. The main amino acid residues on compounds binding to RT are presented. Green lines represent hydrophobic pockets, while ref residues indicate interactions observed for both compounds; (B) Crystallographic complex between emetine pyridone analogue and RT, and (C) RT-emetine complex derived from docking calculations. Common amino acid residues for both complexes are indicated as yellow sticks.

Figure 2

Emetine docking on RT-HIV in comparison with a pyridone analogue. (A) Representation of emetine and the pyridone non-nucleosidic analogue binding to RT. The main amino acid residues on compounds binding to RT are presented. Green lines represent hydrophobic pockets, while ref residues indicate interactions observed for both compounds; (B) Crystallographic complex between emetine pyridone analogue and RT, and (C) RT-emetine complex derived from docking calculations. Common amino acid residues for both complexes are indicated as yellow sticks.

Figure 3

Emetine inhibits HIV wild type and HIV M184V replication in GHOST Cells. (A) Resistance profile of HIV WT and HIV M184V in GHOST cells in the presence of 3TC; (B) GHOST cells were infected with HIV WT and HIV M184V in the presence of emetine. The HIV infectivity was evaluated by luciferase activity 48 h post-infection from the cell lysates; (C) Cytotoxicity effects of emetine were evaluated using fluorescent live/dead staining. All the results are shown in percentage relative to positive controls corresponding to cells not exposed to emetine. Here we present the means and standard deviations of three independent experiments with internal triplicates. The EC₅₀ representative value is shown in the graphic.

Figure 4

Emetine inhibits HIV wild type and HIV M184V infectivity in PBMCs. Peripheral blood mononuclear cells were isolated from HIV-negative blood donors and infected with HIV WT or HIV M184V and seeded in the presence of an antiretroviral. Viruses' infectivity were evaluated through luciferase activity from PBMCs lysates 96 h post-infection. (A) Resistance profile of HIV WT and HIV M184V in PBMCs in the presence of 3TC; (B) Phenotypic assays of HIV viruses (WT and M181V) with emetine; (C) Cytotoxicity effects of emetine in PBMCs. Peripheral blood mononuclear cells were incubated with increasing concentrations of emetine, followed by propidium iodide staining and flow cytometry analysis. All the results represent the means calculated from three independent experiments with triplicates and all the results are expressed as percentage of untreated cells. The EC₅₀ representative value is shown in the graphics.