Novel Small-Molecule Inhibitors of Hepatitis C Virus Entry Block Viral Spread and Promote Viral Clearance in Cell Culture

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection

A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120

The human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein (Env) complex, a homotrimer containing gp120 surface glycoprotein and gp41 transmembrane glycoprotein subunits, mediates the binding and fusion of the virus with susceptible target cells. The Env complex is the target for neutralizing antibodies (NAbs) and is the basis for vaccines intended to induce NAbs. Early generation vaccines based on monomeric gp120 subunits did not confer protection from infection; one alternative approach is therefore to make and evaluate soluble forms of the trimeric Env complex. We have directly compared the immunogenicity in rabbits of two forms of soluble trimeric Env and monomeric gp120 based on the sequence of HIV-1 JR-FL. Both protein-only and DNA-prime, protein-boost immunization formats were evaluated, DNA-priming having little or no influence on the outcome. One form of trimeric Env was made by disrupting the gp120–gp41 cleavage site by mutagenesis (gp140 UNC), the other contains an intramolecular disulfide bond to stabilize the cleaved gp120 and gp41 moieties (SOSIP.R6 gp140). Among the three immunogens, SOSIP.R6 gp140 most frequently elicited neutralizing antibodies against the homologous, neutralization-resistant strain, HIV-1 JR-FL. All three proteins induced NAbs against more sensitive strains, but the breadth of activity against heterologous primary isolates was limited. When antibodies able to neutralize HIV-1 JR-FL were detected, antigen depletion studies showed they were not directed at the V3 region but were targeted at other, undefined gp120 and also non-gp120 epitopes

Selection of viral variants with reduced susceptibility to triazines.

<p>Cell cultures that were ∼30% infected with cell culture-derived GT 1a/2a HCV were established and treated with 1 µM PRO0371155 or DMSO in the passage control. Cultures were split twice weekly to maintain sub-confluent levels. The percentage of HCV+ cells as a function of time was determined at 3–4 day intervals. Cells were subjected to staining with anti-NS3 antibodies and analysis by flow cytometry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>.</p

Effect of drug-induced and natural amino acid variants at position 719 on the growth kinetics of HCV genotype 1/2a in cell culture.

<p>The drug resistant variant, V719G and three natural amino acid polymorphisms, I719, L719, A719, were introduced into genotype 1a and 1b genetic backgrounds using the QuikChange II XL site-directed mutagenesis kit (Stratagene). Full-length HCV RNAs bearing various polymorphisms at position 719 were prepared and electroporated into naïve target cells. Infected cell cultures were maintained for up to 2.5 months. Every 3–4 days, viral supernatants were harvested and subsequently used to infect naïve target cells. After 72 hrs, <i>Renilla</i> luciferase activity was measured and viral titer/infectivity was expressed in R.L.U. (relative light units). Changes in viral titers were plotted on a logarithmic scale as a function of time. (A) A comparison of the growth kinetics of GT 1a/2a HCV (diamonds) with the drug-induced variant, GT 1a/2a-V719G (squares) (B) A comparison of the growth kinetics of GT 1a/2a HCV (diamonds) with a potential polymorphism, GT 1a/2a-V719A (triangles). (C) A comparison of the growth kinetics of GT 1b/2a HCV (diamonds) with the drug-induced polymorphism, GT 1b/2a-V719G (squares). (D) A comparison of the growth kinetics of GT 1b/2a HCV (diamonds) with a natural polymorphism, GT 1b/2a-V719I (circles) and two potential polymorphisms GT 1b/2a-V719L (squares) and GT 1b/2a-V719A (triangles).</p

Spectrum of activity of representative triazines against envelopes isolated from HCV⁺ patient sera.

<p>HCV RNA was isolated and purified from sera obtained from individuals infected with different strains of HCV, representing genotypes 1a, 1b, 2a, 2b. Envelopes representing genotypes 3, 4, and 6 were described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a> and published elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#pone.0035351-Lavillette1" target="_blank">[33]</a>. Genotype specific primers were used to amplify sequences encoding the E1/E2 glycoprotein from each of the strains by RT-PCR. Envelope sequences were ligated into expression constructs as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>. Unique HCVpp, representing different genotypes, were produced in 293T cells and validated with the anti-CD81 mAb, JS-81. Individual HCVpp were normalized based on total infectivity and added to Hep3B cells in the presence of various concentrations of PRO0371155 (Panel A) or PRO0502797 (Panel B) in 384-well microplates. Luciferase activity was measured 72 hrs post-infection using Bright Glo reagent (Promega). The potency of PRO0371155 and PRO0502797 against each fusogenic envelope was expressed as a mean EC₅₀. Each data point represents the average of multiple dose response experiments (n>3). The genotype specific panel consisted of the following strains: genotype 1a (n = 22), genotype 1b (n = 19), genotype 2a (n = 2), genotype 2b (n = 1), genotype 3, (n = 1), genotype 4, n = 1), genotype 6 (n = 1). Actual number of strains tested for each compound is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#pone-0035351-t001" target="_blank">Table 1</a>. The box extends from the 25^th to the 75^th percentile, with a line at the median EC₅₀. The whiskers mark the full range from the lowest to the highest EC₅₀.</p

Long term treatment of HCV infected cell cultures with a triazine compound promotes viral clearance.

<p>PRO0371155 was evaluated for its inhibitory activity against GT 1a/2a HCV over multiple weeks in culture. (A) Cell cultures were ∼90% infected with cell culture-derived GT 1a/2a HCV. Cultures were treated with 1 µM PRO0371155 or DMSO in the passage control. At 3–4 day intervals, cells were subjected to staining with anti-NS3 antibodies and analysis by flow cytometry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>. The percentage of HCV+ cells as a function of time was plotted for each experiment. (B) Results obtained in the protein expression analysis were confirmed and extended by qRT-PCR analysis of HCV RNA isolated from HCV infected cultures treated with PRO0371155 or DMSO control at various time points. HCV RNA was normalized to GAPDH mRNA and expressed in copies/cell. The limit of quantification of the qRT-PCR assay was found to be ∼0.5 copies/cell. (C) Cell cultures were ∼20% infected with cell culture-derived GT 1a/2a HCV. Cultures were treated with 1 µM PRO0371155, 10 IU/mL IFN-alpha (Sigma) or DMSO in the passage control. At 3–4 day intervals, cells were subjected to staining with anti-NS3 antibodies and analysis by flow cytometry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>. The percentage of HCV+ cells as a function of time was plotted for each experiment. (D) Data shown in panel C were re-plotted on a different scale to accentuate the differences between IFN-alpha and PRO0371155 treated HCV+ cultures.</p

Various triazine compounds exhibit broad activity against HCV genotype 1a and 1b strains.

<p>Various triazine compounds exhibit broad activity against HCV genotype 1a and 1b strains.</p

Natural polymorphisms at position 719 confer resistance to representative triazines in the HCVpp assay.

<p>Natural polymorphisms at position 719 confer resistance to representative triazines in the HCVpp assay.</p