Outline of primary screen, re-test and hit triage strategy.

<p>Potent and selective hits identified from a primary screen were subjected to a myriad of life cycle stage assays, genotype coverage and resistance studies to group hits according to target.</p

Early/Entry Inhibitors.

<p>A. The potencies (EC₅₀) of early inhibitors (Inhs 1–5) against gt 1a/2a-Rluc virus (HCVcc) and HCV pseudo-particles (HCVpp) with the corresponding genotype 1 envelope glycoproteins were compared to identify virus entry inhibitors. Selectivity for HCVpp was confirmed using Vesicular stomatitis virus glycoprotein pseudo-particles (VSVpp) and cytotoxicity (CC₅₀) using Cell-Titer Glo. A. Screen hits (Inhs 1–3) that demonstrated similar potency against HCVcc and HCVpp and exhibited selectivity relative to VSVpp and cytotoxicity (entry inhibitors). A control entry inhibitor (EI) was included to confirm the predicted outcome for a bonafide entry inhibitor. B. Screen hits (Inhs 4–5) that demonstrated reduced potency against HCVpp (HCVcc-specific early inhibitors). Genotype selectivity was assessed by comparing potency against HCVcc chimeras with genotype 1a, 1b or 2a structural proteins. C. Potency of the entry inhibitors (Inhs 1–3) and (D) HCVcc-specific early inhibitors (Inhs 4 & 5) against genotype 1a, 1b and 2a HCVcc and corresponding cytotoxicity.</p

Optimization of a high-throughput 384 well virus replication assay.

<p>A. Comparison of virus replication over time (cells/viral foci) following infection using a standard protocol where virus was added to adherent Huh-7.5 cells (pre-plated 24 h prior to infection) or a homogenous protocol where virus and trypsinized cells were co-dispensed into a well. B. Co-titration of genotype 1a/2a-Rluc virus and Huh-7.5 cells to determine Z factor values in a 96 h assay. C. Effect of MOI on % inhibition obtained with entry (EI), genome replication (BMS-339) and virus assembly (LY411575) inhibitors in a 96 h assay. D. HTS assay strategy. E & F. HTS assay quality control. Plate-to-plate variability in signal/backgroundr (E) and Z factor (F) was determined for the <i>Renilla</i> luciferase and HCV Core Cellomics ArrayScan readouts from 50 and 12 plates validation runs, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042609#s2" target="_blank">Results</a> are expressed as mean and standard deviation.</p

Combination indices of EI-1 with HCV replication inhibitors.

a<p>NS5A, BMS-790052; NS3, BMS-605339.</p>b<p>Mean of 8 independent experiments.</p>c<p>Loewe combination index at the combined EC₅₀ level.</p

E1 and E2 amino acid changes that emerged during selection with EI-1.

a<p>Fold increase in EC₅₀ compared to wild-type virus. Values represent the mean of ≥3 experiments.</p

Activity of EI-1 in infectious virus assays.

a<p>Mean of ≥4 independent experiments.</p

Late Stage Inhibitors.

<p>Inhibitors that showed enhanced potency in the multi-cycle virus replication assay (Inhs 14–16) were tested for their ability to block the production of infectious virus. A. Control inhibitors that block virus entry (EI) and genome replication (BMS-339) inhibited <i>Renilla</i> luciferase expression in both producer (black bars) and target (gray bars) cells while a control late-stage inhibitor (LY411575) only affected <i>Renilla</i> luciferase expression in target cells. Similar to LY411575, Inhs 14–17 exhibited less than 20% inhibition of <i>Renilla</i> luciferase expression in producer cells but >75% inhibition in target cells suggesting a block in the production of infectious virus. B. Genotype coverage was assessed by comparing the potency (EC₅₀) of Inhs 14–17 and LY411575 against HCVcc with genotype 1a, 1b or 2a structural proteins.</p

Effect of EI-1 on HCV cell-to-cell spread.

<p>(A) Huh-7.5 cells were infected with 0.001 ffu/cell HCVcc-1a/2a at 37°C. At 12 hrs post infection, the inoculum was removed and replaced with medium +1% agarose overlay containing EI-1 (0.5 µM) or DMSO and the cultures were incubated at 37°C for 2, 3 or 4 days. Infected cells were labeled by indirect immunofluorescence using an anti-HCV core monoclonal antibody (green) and nuclei were stained with Hoechst 3325 (red). Images were captured using a Nikon Eclipse TE300 inverted epi-fluorescence microscope. (B) The mean number and standard deviation of infected cells/focus was determined from visual counting of infected cells in ≥100 foci for each time point. (C) The mean number and standard deviation of foci/well was determined at 2 and 4 days post infection.</p

Structure-activity-relationship of the EI-1 chemotype.

a<p>Mean of ≥3 independent experiments.</p>b<p>R, aniline ring substituent (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001086#ppat-1001086-g001" target="_blank">Fig 1A</a>).</p

Confirmation of anti-HCV activity for top 17 hits.

<p>A. Potency (EC₅₀) against the gt 1a/2a-Rluc virus (EC₅₀) in a 96 h multi-cycle assay and corresponding Huh-7.5 cell cytotoxicity (CC₅₀) for each of the top 17 screen hits. B. Confirmation of anti-HCV activity through comparison of potency using <i>Renilla</i> Luciferase or HCV Core Cellomics ArrayScan readouts.</p