Figure 4. Confirmation of anti-HCV activity for top 17 hits. 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 Renilla Luciferase or HCV Core Cellomics ArrayScan readouts. doi:10.1371/journal.pone.0042609.g004

96 h pi was attributed to subsequent rounds of replication. As expected, control entry (EI) and genome replication (BMS-339) inhibitors exhibited similar potency at 48 and 96 h pi, while the late-stage inhibitor (LY411575) was significantly more potent (.200 fold) at 96 h (Fig. 5A). Of the 17 hits selected for further analysis, 13 showed similar potencies (,3-fold difference) at both 48 and 96 h pi (Fig. 5B) while 4 hits (Inhs 14, 15, 16 & 17) showed a marked increase in potency (32?45 fold) at 96 h (Fig. 5C). These results suggested those 13 inhibitors with similar potencies at 48 and 96 h act coincident with early or genome replication inhibition, while the 4 with increased potency at 96 h with latestage inhibition. To discriminate between early and genome replication inhibitors, the entry process was bypassed by direct transfection of viral RNA (vRNA). Renilla luciferase signals were measured at 48 h pi of HCVcc or post-transfection of the corresponding vRNA. As expected for an entry inhibitor, EI blocked the luciferase signal following HCVcc infection but not transfection of vRNA, while the control genome replication inhibitor (BMS-339) showed similar potency in both assays (Fig. 5D). Of the 13 hits that showed similar potency in the single- and multi-cycle assays, 5 (Inhs 1?) were less active following transfection of vRNA (Fig. 5E) while 8 (Inhs 6?3) exhibited similar potency in both formats (Fig. 5F). Altogether, this triage strategy segregated the 17 hits into three temporally & mechanistically-distinct virus replication stage Table 2. Primary Screen Results.

categories including early (5), genome replication (8) and late (4) inhibitors. To identify entry-specific compounds, the 5 early-stage hits were tested against HCV pseudo-particles (HCVpp) harboring the HCV genotype 1a envelope glycoproteins matching those of the screening virus. Three of the 5 hits exhibited a similar profile as EI with similar potency against both HCVcc and HCVpp viruses (Fig. 6A). HCV selectivity was confirmed by counter-screening for activity against VSVpp and cytotoxicity (Fig. 6A). Together, these findings confirmed that Inh-1, 2 and 3 were HCV entry inhibitors. On the contrary, Inhs 4 and 5 showed no activity against HCVpp (Fig. 6B) suggesting that these hits could target an HCVcc-specific entry event or an early event not recapitulated by the pseudoparticle system (e.g., nucleocapsid uncoating). Next, genotype coverage of the inhibitors was determined using HCVcc chimeras harboring genotype 1a, 1b (432? isolate) or 2a (J6 isolate) structural proteins. Of the 3 entry inhibitors, Inh-1 was selective for genotype 1a, while Inhs 2 and 3 exhibited activity against genotype 1a and 1b but not 2a (Fig. 6C). A similar profile was observed using HCVpp harboring genotype 1a,1b or 2a envelopes (data not shown). For the HCVcc-specific inhibitors, both Inh-4 and Inh-5 exhibited similar potency against all 3 genotypes (Fig. 6D).

Screening Tier Primary Screen & Re-Tests Potency Filter Dose-Response Curves Therapeutic Index $10c Renilla luciferase counterscreen Chemical analysis Hit Confirmation
Figure 5. Life cycle stage triage. A. Performance (EC50) of control entry (EI), genome replication (BMS-339) and late-stage (LY411575) inhibitors in single- vs. multi-cycle replication assay formats. B. Screen hits (Inhs 1?3) that demonstrated similar potency in single- and multi-cycle replication formats (single- & multi-cycle inhibitors). C. Screen hits (Inhs 14?7) that demonstrated greater potency in multi-cycle replication assay format (multicycle specific inhibitors). D. Performance of control entry (EI) and genome replication (BMS-339) inhibitors following infection with gt 1a/2a-Rluc virus or direct transfection of the corresponding virus genomic RNA. E. Screen hits (Inhs 1?) that demonstrated reduced potency following direct transfection of virus genomic RNA (early inhibitors). F. Screen hits (Inhs 6?3) that demonstrated similar potency in infection and transfection assays (genome replication inhibitors)
Viral Genome Replication Inhibitors
HCV and Bovine viral diarrhea virus (BVDV) replicons were used to determine if Inhs 7?3 were HCV-selective genome replication inhibitors. All of the hits exhibited similar potency against HCVcc and a genotype 2a replicon (Fig. 7A), confirming that these compounds block replication of the viral genome. Of the 8 inhibitors, 6 (Inhs 6?1) exhibited reduced potency against both genotype 1a HCV and BVDV replicons which was indistinguishable from cytotoxicity (Fig. 7B) showing that these agents were selective for genotype 2a HCV. On the contrary, Inhs 12 and 13

exhibited similar activity against genotype 2a and 1a HCV as well as BVDV replicons (Fig. 7C) and these activities were separable from cytotoxicity. Taken together, these results demonstrated that these inhibitors were not selective for HCV but rather blocked the replication of both viruses.

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