A novel class of meso-tetrakis-porphyrin derivatives exhibits potent activities against hepatitis C virus genotype 1b replicons in vitro

Abstract

Recent years have seen the rapid advancement of new therapeutic agents against hepatitis C virus (HCV) in response to the need for treatment that is unmet by interferon (IFN)-based therapies. Most antiviral drugs discovered to date are small molecules that modulate viral enzyme activities. In the search for highly selective protein-binding molecules capable of disrupting the viral life cycle, we have identified a class of anionic tetraphenylporphyrins as potent and specific inhibitors of the HCV replicons. Based on the structure-activity relationship studies reported herein, meso-tetrakis-(3,5-dicarboxy-4,4'-biphenyl) porphyrin was found to be the most potent inhibitor of HCV genotype 1b (Con1) replicon systems but was less effective against the genotype 2a (JFH-1) replicon. This compound induced a reduction of viral RNA and protein levels when acting in the low nanomolar range. Moreover, the compound could suppress replicon rebound in drug-treated cells and exhibited additive to synergistic effects when combined with protease inhibitor BILN 2061 or with IFN-alpha-2a. Our results demonstrate the potential use of tetracarboxyphenylporphyrins as potent anti-HCV agents.

FIG. 1.

Time- and dose-dependent reduction of viral parameters in genotype 1b (Con1) replicon cells induced by compound 6. Huh-luc/neo-ET cells were incubated with serially diluted compound 6 for 24, 48, or 72 h. Results are expressed as percentages of the level for mock-treated controls. (A) Reduction of reporter luciferase activity, which indirectly reflects the replication level of HCV replicons. (B) Reduction of HCV RNA level normalized against the mRNA level of human β-actin. (C) Reduction of the NS5A protein level as in one experiment. (D) NS5A protein level quantitated and normalized against the protein level of α-tubulin (means ± standard deviations [SD] of results from three independent experiments).

FIG. 2.

Effect of serum binding on the EC₅₀s of compounds 1, 4, and 6. The fold changes in EC₅₀ were plotted against percentages of serum by using the EC₅₀ at 5% FBS as the 1-fold level (means ± SD of results from three independent experiments). The antiviral activities of compounds 1 and 6 decreased linearly with increasing serum and also differed significantly from each other at the 95% confidence intervals. Change in the EC₅₀s of compound 4, which was the least affected by serum binding, was nonlinear. The P value was determined by a two-way ANOVA test, using GraphPad Prism 4.0.

FIG. 3.

Compound 6 could prevent the rebound of genotype 1b (Con1) replicons. Replicon cells were incubated with increasing concentrations of compound 6 for 12 days, free from G418. At the end of the incubation, compound 6 was removed and 250 μg/ml G418 was reintroduced. (A) The level of HCV RNA in cells was quantitated by qRT-PCR. RNA copy number per μg of total RNA was expressed as a ratio relative to the level for the mock-treated controls. During the rebound period, replicon cells incubated with 300 nM and 1 μM compound 6 were not confluent enough for sampling. (B) Cell viability was shown on the log₁₀ scale as a percentage of the level for mock-treated controls. Replicon cells that were treated with 300 nM and 1 μM compound 6 were no longer viable by day 21.

FIG. 4.

Compared with genotype 1b (Con1) replicons, genotype 2a (JFH-1) replicons were more resistant to both compound 6 and IFN-α-2a. Genotype 1b (Con1) replicon cells (Huh-luc/neo-ET) and genotype 2a (JFH-1) replicon cells (YSGR-JFH) were incubated with increasing concentrations of compound 6 (A) or IFN-α-2a (B). Cells were harvested 72 h after incubation. The HCV RNA level was quantitated by qRT-PCR and expressed as a percentage of the level for mock-treated controls (mean ± SD of results from three independent experiments).

FIG. 5.

Antiviral isobologram and CI₉₀ plot of compound 6 in combination with BILN 2061 or IFN-α-2a in vitro. Huh-luc/neo-ET cells were coincubated for 72 h with various concentrations of drug 1 or 2 alone or the two in combination at different potency ratios. (A, C) Ratios of the apparent EC₅₀ of each drug in combination over its EC₅₀ when applied alone were plotted against each other in isobolograms. The hypotenuse represents the linear additive response to the action of two therapeutic agents. Isoboles that bow below the hypotenuse indicate synergism, and isoboles that bow above the hypotenuse indicate antagonism. Experimental data points on the isobole represent a combination that inhibits the HCV replication by 50% and is hence isoeffective with the line of additivity. The EC₉₀ isobolograms of compound 6 in combination with BILN 2061 or IFN-α-2a are shown in panels B and D, respectively (means ± SD of results from at least four independent experiments). Different degrees of synergism/antagonism are expected at different effect levels. The combination indices (CI) at the 90% effect level for the combination of compound 6 with BILN 2061 and IFN-α-2a are plotted in panels E and F, respectively. Mean CI₉₀ values for each dose ratio are indicated above the bars. A CI value of 1 ± 0.1 suggests additivity, as indicated by the dashed line. CI values below and above the boundary indicate synergism and antagonism, respectively.