3B. These data demonstrate that NM-107 efficiently inhibits both gt1b replication (reduction of GFP expression) as well as gt2 infection (reduction of translocated RFP) without affecting cell growth even at high concentrations (EC100) (nuclear parameters measured
in blue channel were unchanged). From these various outputs of total cell number (SumCellNumber), percent of GFP expressing cells (AvgPercentCellGFP), and RFP translocation cells (Ratio), DRCs can be derived to assess cytotoxicity, gt1b RNA replication and gt2 HCVcc infection, Forskolin clinical trial respectively as illustrated in Fig. 3C for NM-107 and A-837093. Both gt1 RNA replication and gt2 HCVcc infection were inhibited by NM-107 treatment in dose dependent manner as shown in green and red, respectively. This antiviral effect was not related to cytotoxicity that started to be detectable only at the learn more highest compound concentrations (grey area in Fig. 3C). The EC50 of NM-107 was calculated from each DRC by non-linear regression analysis using Prism (GraphPad Software, Inc.) at 4.06 μM against gt1 RNA replication and 6.1 μM against gt2 HCVcc versus more than 300 μM for CC50 (cytotoxic concentration giving 50% cell death) (Fig. 3C). These values were comparable to published data (Bassit et al., 2008) and non-multiplexed assays using the gt1 replicon (4.46 ± 1.5 μM) or gt2 HCVcc (8.8 ± 2.2 μM). Likewise,
Urease a DRC analysis with A-837093 (Fig. 3C) resulted in dose dependent antiviral activity against gt1 replicons but not against gt2 HCVcc as shown
by decreased GFP expression and unchanged RFP localization respectively (Fig. 3C lower chart). We tested several HCV inhibitors which have different mode of action to demonstrate that this assay is suitable to identify inhibitors targeting various steps in the viral life cycle (Fig. 3C table). Telaprevir, a NS3-4A protease inhibitor (Selleck Chemicals, USA) (Lin et al., 2006), GS-7977, a NS5B inhibitor (Medchem Express, China) (Murakami et al., 2010 and Sofia et al., 2010), LY-411575, a late step inhibitor (BOC Science, USA) (Wichroski et al., 2012), and an antibody serving as an entry inhibitor by targeting CD81 (BD Bioscience, USA) were tested by 10-points DRC analysis as described above. EC50 values of each inhibitor are comparable with previously reported data. In addition, we observed couple phenotype which is the result of primarily infection and cell division during the 72 h assay period in late step inhibitor treatment (Fig. 3D). The multiplex system presented here facilitates the simultaneous evaluation of not only antiviral activity and cytotoxicity but also provides basic mechanistic information. This strategy is time and cost effective, as more information can be acquired in comparison with classical assays using a single readout (e.g. luciferase values). Importantly, our multiplex assay is compatible with HTS.