Enantioselective Synthesis, Stereochemical Correction, and Biological Investigation of the Rodgersinine Family of 1,4-Benzodioxane Neolignans

The enantioselective synthesis and chiroptic analysis of all members of the rodgersinine family of 1,4-benzodioxane neolignans has been achieved. ECD spectra and optical rotation analysis determined that the previously published stereochemistry of <i>trans</i>-rodgersinines A and B was incorrect. The <i>cis</i>-rodgersinines A and B did not follow the model ECD study commonly used to assign the absolute stereochemistry of 1,4-benzodioxane natural products. This finding has implications on the absolute stereochemistry of other natural products of this type. Additionally, the rodgersinines were found to have anti-HCV activities

Silibinin but not SIL inhibits innate inflammatory and antiviral signaling.

<p>A, B, effect of silibinin and SIL on NF-κB dependent transcription. Huh7 cells were transfected with an NF-κB responsive reporter plasmid (pRDII-luc) and twenty-four hours later, cells were pretreated with the indicated doses of silibinin (A) or SIL (B). Cells were then treated with 10 ng/ml TNF-α and luciferase activity measured by Britelite assay 3.5 hours later. C, D, effect of silibinin and SIL on IRF-3 driven transcription from the IFN-B promoter. Huh7.5.1 cells were co-transfected with a luciferase reporter plasmid under control of the IFN-B promoter and IRF-35D, a constitutively active mutant of IRF-3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016464#pone.0016464-Lin1" target="_blank">[28]</a>. Twenty-four hours later, cells were pretreated with the indicated doses of silibinin (C) or SIL (D). Luciferase activity measured by Britelite assay 24 hours later. Fluorescence is reported as relative light units (RLU). Error bars represent standard deviation from triplicate cultures.</p

Antiviral Activity of SIL and silibinin.

<p>A, effect of SIL on HCV replication in genotype 1b subgenomic replicon cells (BB7) and JFH-1 infection of Huh7.5.1 cells. The top panels show HCV NS5A protein expression detected by western blot, while the lower graph depicts HCV RNA levels determined by real time RT-PCR. Cells were treated with 0, 6.9, 27.6, 138, and 414 µM of SIL for 72 hours before protein and RNA isolations. B, effect of silibinin on HCV replication in genotype 1b subgenomic replicon cells (BB7) and JFH-1 infection of Huh7.5.1 cells. Cells were treated with DMSO, 15.5, 31.1, and 62.1 µM of silibinin for 72 hours before protein and RNA isolations. C, effects of SIL and silibinin on HCV replication in subgenomic JFH-1 replicon cells. Cells were treated with 0, 6.9, 27.6, 138, and 414 µM of SIL or DMSO, 20.7, 41.4, and 82.8 µM of silibinin for 72 hours before proteins were extracted and NS5A detected by western blot. D, effect of SIL and silibinin on progeny virus production. Huh7.5.1 cells were treated with 20 µg/ml silibinin, 300 µg/ml SIL or DMSO and PBS controls immediately after 5 hours of adsorption with JFH-1 at an m.o.i. of 0.05. Culture supernatants were harvested 72 hours later and carry over silibinin or SIL was removed by concentration through 10,000 molecular filters. Supernatants were diluted 1∶100 in Huh7 media and used to infect naïve Huh7.5.1 cells in triplicate and immunofluorescent detection of HCV core protein was performed. Foci were counted manually and used to calculate infectious virus yields expressed as focus forming units per milliliter (FFU/ml). Error bars represent standard deviations of triplicate cultures.</p

Chemical structures of SIL and silibinin.

<p>See text for details.</p

Silibinin and SIL inhibit HCVpp-mediated fusion.

<p>Membrane fusion between HCVpp and R₁₈-labeled liposomes was followed by fluorescence spectroscopy with excitation and emission at 560 and 590 nm, respectively. Fluorescent liposomes (12.5 µM final lipid concentration) were added to 20 µl of HCVpp in PBS pH 7.4 at 37°C, in the absence or presence of 5, 10 or 40 µg/ml of indicated compound, which corresponds to 6.9, 13.8 or 55 µM of SIL and 10.4, 20.7, and 82.8 µM of silibinin. After a 2 min-equilibration, lipid mixing was initiated by decreasing the pH to 5.0 with diluted HCl, and R₁₈ dequenching was recorded. Maximal fluorescence was obtained after addition of 0.1% final Triton X-100 to the cuvette. A, values of the last min of fusion (final extent of fusion) were used to calculate the percentage of fusion in the presence of the drug, relative to 100% fusion in the absence of drug. Results are expressed from the mean of 2 separate experiments. Compounds were added at 5 (black), 10 (dark grey) or 40 µg/ml (light grey). B, fusion kinetics of HCVpp with liposomes, in the absence (black) or presence of three concentrations of SIL: blue, 5 µg/ml; red, 10 µg/ml and green, 40 µg/ml.</p

SIL and silibinin dose-dependently inhibit T cell proliferation.

<p>For all assays, a single dose of silibinin or SIL was added at culture initiation and compared to parallel cultures treated with a single dose of silibinin vehicle (MetOH) or media (control for SIL). Freshly isolated PBMC were tested for proliferative responses to plate-bound anti-CD3 (10 µg/mL) stimulation measured using ³H-thymidine incorporation. <b>A</b>, PBMC cultures treated with SIL at various doses (black bars) and the media control (white bar). Cells were also separately treated with silibinin control (gray bar), and the corresponding methanol control (hatched bar). <b>B</b>, PBMC cultures were treated with silibinin at various doses (gray bars), or with methanol solvent controls (hatched bars). Cells were also treated with silymarin as a positive control for inhibition of proliferation (bar with vertical lines). Open bars represent proliferation of PBMC stimulated with anti-CD3 alone in media, which serves as the control for SIL treatment. Results are shown as mean cpm incorporated. Error bars indicate 1 standard deviation among 3–4 replicates for each condition.</p

Cytotoxicity profile of SIL on BB7 subgenomic replicon cells (A), Huh7.5.1 cells (B), and PBMC (C).

<p>Cells were plated in quadruplicate in 96 well plates and the indicated concentrations of SIL in PBS were added. Cells were incubated for 72 hours (for BB7 and Huh7.5.1 cells) and 24 hours for PBMCs before ATP levels were measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016464#s2" target="_blank">Materials and Methods</a>. Fluorescence is reported as relative light units (RLU).</p

Silibinin Inhibits HIV-1 Infection by Reducing Cellular Activation and Proliferation

<div><p>Purified silymarin-derived natural products from the milk thistle plant (<em>Silybum marianum</em>) block hepatitis C virus (HCV) infection and inhibit T cell proliferation in vitro. An intravenous formulation of silibinin (SIL), a major component of silymarin, displays anti-HCV effects in humans and also inhibits T-cell proliferation in vitro. We show that SIL inhibited replication of HIV-1 in TZM-bl cells, PBMCs, and CEM cells in vitro. SIL suppression of HIV-1 coincided with dose-dependent reductions in actively proliferating CD19+, CD4+, and CD8+ cells, resulting in fewer CD4+ T cells expressing the HIV-1 co-receptors CXCR4 and CCR5. SIL inhibition of T-cell growth was not due to cytotoxicity measured by cell cycle arrest, apoptosis, or necrosis. SIL also blocked induction of the activation markers CD38, HLA-DR, Ki67, and CCR5 on CD4+ T cells. The data suggest that SIL attenuated cellular functions involved in T-cell activation, proliferation, and HIV-1 infection. Silymarin-derived compounds provide cytoprotection by suppressing virus infection, immune activation, and inflammation, and as such may be relevant for both HIV mono-infected and HIV/HCV co-infected subjects.</p> </div

SIL suppresses HIV-1 Infection of TZM-bl cells.

<p><b>A</b>, Cytotoxicity profile of SIL in TZM-bl cells. Cells were infected with LAI, a CXCR4-using virus, or BAL, a CCR5-using virus, at an MOI of 0.05 in the presence of the indicated concentrations of SIL and ATP was measured using the ATPlite kit 48 hours later. The data are representative of 2 (BAL) and 3 (LAI) independent technical repeats. <b>B</b>, Antiviral profile of SIL in TZM-bl cells. Serial dilutions of SIL were tested for inhibition of infection in TZM cells. Following addition of compounds and virus, cells were incubated for 48 hours before luciferase activity was measured. Percent inhibition refers to percent reduction in luciferase activity of SIL versus untreated cultures. Error bars represent standard deviation of 3 independent technical repeats. <b>C</b>, SIL inhibits pseudovirus replication in TZM-bl cells. TZM-bl cells were infected with the indicated viruses in the presence of the indicated concentrations of SIL and luciferase activity was measured 48 hours post-infection. The D013M12 psuedovirus contains a subtype D envelope sequence, while the D769 psuedovirus contains a subtype A envelope sequence. Error bars represent standard deviations of triplicate wells per condition.</p

SIL inhibits activation marker expression on CD4+ T cells.

<p>PBMCs were activated with either SEB (0.5 µg/ml) or PHA (2 µg/ml) for 24 hours, and treated with the indicated concentrations of SIL for 12 hours. Representative flow cytometry dot plots showing expression of HLA-DR (A), CD38 (B), Ki67 (C), and CCR5 (D). Data are representative of 3 HIV-seronegative individuals tested for each marker.</p