Dual Role of Novel Ingenol Derivatives from <i>Euphorbia tirucalli</i> in HIV Replication: Inhibition of <i>De Novo</i> Infection and Activation of Viral LTR

<div><p>HIV infection is not cleared by antiretroviral drugs due to the presence of latently infected cells that are not eliminated with current therapies and persist in the blood and organs of infected patients. New compounds to activate these latent reservoirs have been evaluated so that, along with HAART, they can be used to activate latent virus and eliminate the latently infected cells resulting in eradication of viral infection. Here we describe three novel diterpenes isolated from the sap of <i>Euphorbia tirucalli,</i> a tropical shrub. These molecules, identified as ingenols, were modified at carbon 3 and termed ingenol synthetic derivatives (ISD). They activated the HIV-LTR in reporter cell lines and human PBMCs with latent virus in concentrations as low as 10 nM. ISDs were also able to inhibit the replication of HIV-1 subtype B and C in MT-4 cells and human PBMCs at concentrations of EC₅₀ 0.02 and 0.09 µM respectively, which are comparable to the EC₅₀ of some antiretroviral currently used in AIDS treatment. Control of viral replication may be caused by downregulation of surface CD4, CCR5 and CXCR4 observed after ISD treatment <i>in vitro</i>. These compounds appear to be less cytotoxic than other diterpenes such as PMA and prostratin, with effective dose versus toxic dose TI>400. Although the mechanisms of action of the three ISDs are primarily attributed to the PKC pathway, downregulation of surface receptors and stimulation of the viral LTR might be differentially modulated by different PKC isoforms.</p></div

Summarized data of chimeric virus utilized in <i>in vitro</i> selections.

<p>Summarized data of chimeric virus utilized in <i>in vitro</i> selections.</p

Graphics showing the selection process using 3TC (A) and ZDV (B) in MT4 cells infected with recombinant HIV-1 clones carrying the RT gene from subtype B and C.

<p>The virus load measured through real-time PCR from the culture supernatant is depicted on the y-axis. The time and concentration of the drugs utilized in each virus passage during the selection process are shown on the x-axis. Drug resistant mutations found in the clones during the passages are shown in the boxes below the curves. The white boxes contain mutations found in subtype B, and the black boxes contain the mutations found in subtype C.</p

ING-B blocks HIV <i>de novo</i> replication through downregulation of surface receptors.

<p>CD4+ T cells from three healthy donors were stimulated with PHA/IL-2 for 5 days and then treated with different concentrations of ING-B for 24 h. A portion of the cells was used for cytometry evaluation (A), and the graph depicts the mean and standard deviation of three independent experiments. The remaining cells were infected with HIV NL4-3-Luc for 24 h and cell lysates were analyzed by luciferase activity (B). Results are shown as relative light units (RLU). ZVD 1 µM was used as positive control and DMSO 1% as vehicle control. Results are representative of mean and standard deviation of triplicates for each blood donor.</p

ISDs reduce HIV replication in a dose-dependent manner.

<p>Lymphocytic cell line MT-4 (A and B) and human PBMCs (C and D) were infected with HIV subtype B NL-4.3 (A and C) or HIV subtype C ZM247Fv-1 (B and D) and treated with different concentrations of ISDs. After six days, cell viability was evaluated using the Cell Titer blue kit. Curves were derived by non-linear regression (dose-response curve by Hill for 3 parameters), and dotted lines represent EC₅₀. Experiments were done in triplicate for PBMCs and sextuplicate for MT-4. ZVD was used as positive control.</p

Drug resistance mutations found during the <i>in vitro</i> selection of RTB_′ and C_′ with escalating concentration of 3TC.

<p>NS - not sequenced,</p>*<p>- mutation not associated with 3TC resistance, () - replicate number.</p

Baseline characteristics of study subjects.

<p>(*) Time on HAART with documented continuous suppression of plasma viremia (< 50 copies/mL); (**) Drug abbreviations: ZDV, zidovudine; 3TC, lamivudine; EFV, efavirenz; TDF, tenofovir disoproxil fumarate.</p

ISDs upregulate the production of HIV particles in diverse cell models.

<p>A) Four J-Lat clones were treated with 1 µM of different diterpenes and p24 levels in the supernatant were quantitated by ELISA. Similar experiment was done in J-Lat clone 15.4 (B) and other latent cell models (C). Graphs show mean and standard deviation of 3 separate tests. In all experiments, TNF-α 10 ng/mL was used as positive control.</p

ING-B activates the HIV-LTR in cells isolated from virally suppressed HIV+ patients.

<p>PBMCs isolated from five HIV+ patients were treated with 1 µM of ING-B, prostratin or PMA for 24 h. Total RNA was isolated and HIV <i>pol</i> RNA was quantitate by RT-qPCR. All patients were HAART-treated and presented undetectable levels of plasma viral load.</p

ISDs upregulated HIV-LTR-driven GFP expression through PKC pathways.

<p>J-Lat clones 9.2 (A and B) and 10.6 (C and D) were incubated with different PKC inhibitors prior to stimulation with ISDs and other compounds. Graphs A and C depict levels of relative GFP expression for J-Lat clone 9.2 (A) and 10.6 (C) after treatment with different concentrations of GÖ6983 and subsequent compounds. Graphs B and D show the relative GFP expression for J-Lat clone 9.2 (B) and 10.6 (D), after treatment with two PKC inhibitors (GÖ6976 and RO-31-8220) at 0.1 µM followed by treatment with ING-B, prostratin, PMA or TNF-α. Results are representative of mean and standard deviation of three independent experiments.</p