Flavonoids from Pterogyne nitens Inhibit Hepatitis C Virus Entry

Abstract

Hepatitis C virus (HCV) is one of the leading causes of liver diseases and transplantation worldwide. The current available therapy for HCV infection is based on interferon-α, ribavirin and the new direct-acting antivirals (DAAs), such as NS3 protease and NS5B polymerase inhibitors. However, the high costs of drug design, severe side effects and HCV resistance presented by the existing treatments demonstrate the need for developing more efficient anti-HCV agents. This study aimed to evaluate the antiviral effects of sorbifolin (1) and pedalitin (2), two flavonoids from Pterogyne nitens on the HCV replication cycle. These compounds were investigated for their anti-HCV activities using genotype 2a JFH-1 subgenomic replicons and infectious virus systems. Flavonoids 1 and 2 inhibited virus entry up to 45.0% and 78.7% respectively at non-cytotoxic concentrations. The mechanism of the flavonoid 2 block to virus entry was demonstrated to be by both the direct action on virus particles and the interference on the host cells. Alternatively, the flavonoid 1 activity was restricted to its virucidal effect. Additionally, no inhibitory effects on HCV replication and release were observed by treating cells with these flavonoids. These data are the first description of 1 and 2 possessing in vitro anti-HCV activity.

Figure 1

Flavonoids from Pterogynes nitens. The structure of sorbifolin (1) and pedalitin (2) (a). Cell viability and virus infectivity of flavonoids in näive Huh-7.5 cell line, cells were treated with compounds (1) and (2) at two-fold dilution 500 to3.9 µM for 72 h (b). DMSO 0.1% was used as non-treated control. Mean values of experiments are shown.

Figure 2

Inhibitory effect of compounds 1 and 2 against HCV genotype 3. Huh-7.5 cells were infected with S52/JFH-1 HCVcc and compounds at 50 μM concentration were immediately added to cells. The intracellular virus was titrated 72 h.p.i. by analyzing focus-forming units per milliliters (FFU/mL). DMSO 0.1% was used as non-treated control. P < 0.01 vs DMSO was considered significant.

Figure 3

Effects of (1) and (2) on HCV infectivity. Infectious supernatant and compounds were added at different times to the cells, and the intracellular virus was titrated 72 h post-infection by analyzing focus-forming units per milliliters (FFU/mL). The percentage of infection was calculated using as reference the DMSO non-treated control. For entry assay, Huh-7.5 cells were infected with JFH-1 HCVcc and compounds 1 and 2 were immediately added. After 4 h, the supernatant was removed and replaced with fresh medium after repeated washes with PBS to remove the inoculum (a). For virucidal assay, JFH-1 HCVcc were incubated with (1) or (2) for 1 h prior to the infection. After that, the inoculum was used to infect naïve Huh-7.5 cells for 4 h. Cells were exhaustively washed and medium replaced (b). In the pre-treatment assay, cells were previously treated with compounds (1) and (2) for 1 h prior to the infection. Cells were washed to remove compounds and infected with JFH-1 virus for 4 h. Supernatants were removed, cells were washed to complete virus removal and were incubated with fresh media for up to 72 h post-infection (c). DMSO 0.1% was used as non-treated control and EGCG was used as control for entry blockade. Mean values of three independent experiments each measured in triplicate including the standard deviation are shown. P < 0.001 vs DMSO was considered significant.

Figure 4

Effect on HCV replication. Huh-7.5 cells stably harboring subgenomic replicon SGR-FEO-JFH-1 were treated with (1) (a) and (2) (b) at 50, 10, 2 and 0.4 µM for 72 h. Cell viability (▴) and replication (●) were evaluated by MTT and luciferase assays, respectively. DMSO 0.1% was used as non-treated control and cyclosporine A (CsA) at 1 µM was used as control of inhibition of replication. Mean values of experiments are shown and P < 0.001 vs DMSO was considered significant.

Figure 5

The capacity of compounds 1 and 2 to inhibit HCV release. Huh-7.5 cells previously infected with JFH-1 virus were plated 48 h prior treatment. Compounds (1) or (2) were added at 50 μM and incubated for 24 h. Supernatant was collected and cells were harvested, and intra and extracellular RNA were quantified by qPCR. DMSO 0.1% was used as non-treated control and naringenin (NR) at 400 μM was used as positive control of HCV secretion. Mean values of two independent experiments each measured in triplicate including the standard deviation are shown. ***P < 0.005 vs extracellular. ***P < 0.001 vs DMSO.

Figure 6

Effect of compounds 1 and 2 in monotherapy or combined with Sofosbuvir. Huh-7.5 cells were infected with JFH-1 HCVcc and compounds at specific concentration were immediately added to cells. After 72 h, cell viability (a) and virus infectivity (b) were measured. Compound 1, 2 and Sofosbuvir (SOF) were tested at 10 µM, 20 µM and 0.6 µM, respectively. DMSO was used as non-treated control. P < 0.01 vs DMSO was considered significant (***).