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

PRO0371155 inhibits HCV infection by both cell-free and cell-cell modes of transmission.

<p>The anti-viral activity of PRO0371155 was evaluated in an assay that measures both cell-free and cell-cell transmission of virus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#pone.0035351-Brimacombe1" target="_blank">[37]</a>. To evaluate transmission of GT 1a/2a HCV in culture, infected cells (90% HCV+) were stained with CMFDA green, according to the manufacturer's instructions (Invitrogen), and mixed at a ratio of 5∶1 with non-stained naïve cells. Mixed infected and naïve cells (7.5×10⁵ total) were seeded in T-75 flasks and subjected to treatment with 1 µM of PRO0371155 or DMSO vehicle for 72 h at 37°C. As a positive control for cell-cell transmission, PA-25, a mouse monoclonal antibody raised against sE2 was also evaluated in the viral spread assay at a single concentration of 10 µg/mL. This concentration is approximately 20-fold above its EC₅₀ concentration and is sufficient to completely inhibit HCV entry in a single-cycle infection assay. Cells were detached, fixed and permeabilized with BD Cytofix/cytoperm (BD Biosciences). Infected cells were stained with anti-NS3 antibody 1847 (Virostat) or mIgG1 (BD Biosciences) and counter-stained with 1 µg/mL AlexFluor 647 (Invitrogen). Single- and dual-labeled cells were quantified by flow cytometry.</p

Fusogenic properties of variant envelopes and sensitivity to representative triazines in the HCVpp assay.

<p>Individual variants, representing both drug-selected and natural polymorphisms, were introduced by site directed mutagenesis of plasmid vectors that express either the genotype 1a or 1b E1/E2 glycoproteins. Unique HCVpp, representing each variant were produced in 293T cells and normalized by p24 ELISA assay. Viral entry was measured by quantifying luciferase activity 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>. Luciferase activity was expressed as R.L.U. per nanogram of p24. Cell lysates were prepared from 293T cells that were previously co-transfected with envelope expression constructs and the HIV-1 vector. After normalizing for total protein content, proteins were then separated by electrophoresis through an SDS-polyacrylamide gel and subjected to western blot analysis with PA-25, a mouse monoclonal antibody raised against sE2 derived from a genotype 1a strain.</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

Structure and anti-viral activity of PRO037115.

<p>(A, left panel) Generic structure representing the N2-R1-phenyl-N4-R2-6-R3-2,4-diamine-1,3,5-triazine lead series of HCV entry inhibitors. (A, right panel) The structure of PRO0371155. (B, left panel) HCVpp pseudotyped with the GT1a envelope were added to Hep3B cells in the presence of various concentrations of PRO0371155. Luciferase activity was measured 72 hrs post-infection using BrightGlo reagent (Promega) 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 mean EC₅₀ across three independent assays was 2 nM. (B, right panel) PRO0371155 was tested for its inhibitory activity against GT 1a/2a HCV 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>. PRO0371155 demonstrated potent anti-viral activity against HCV genotype 1 in this assay: EC₅₀ = 9 nM across four independent assays; Positive Control JS-81: EC₅₀ = 0.18 µg/mL.</p

ImmunoPET Imaging of Endogenous and Transfected Prolactin Receptor Tumor Xenografts

Antibodies labeled with positron-emitting isotopes have been used for tumor detection, predicting which patients may respond to tumor antigen-directed therapy, and assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent. REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or labeled with I-131/I-124. In vitro determination of the half-maximal inhibitory concentration (IC50) of parental REGN2878, DFO-REGN2878, and iodinated REGN2878 was performed by examining the effect of the increasing amounts of these on uptake of trace-labeled I-131 REGN2878. REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution studies were performed in mice bearing tumor xenografts with various expression levels of PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89 REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in tumor xenografts with and without prior injection of 2 mg of nonradioactive REGN2878. The competition binding assay of DFO-REGN2878 at ratios of 3.53–5.77 DFO per antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ± 33.8%IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed a low uptake (7.0 ± 2.3%IA/mL) of control Zr-89 REGN1932 and a very low uptake and rapid clearance of I-124 REGN2878 (1.4 ± 0.6%IA/mL). Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a very rapid in vivo turnover half-life of ∼14 min from the cell membrane. Despite relatively modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final retention of the Zr-89 REGN2878 antibody, with an uptake that appeared to be related to PRLR expression. This reagent has the potential to be used in clinical trials targeting PRLR