In both replicon lines, BV showed significant antiviral activity at concentrations as low as 20 μM. In contrast, concentrations of BR-IX-α or BR mixed isomers required to suppress HCV replication were considerably higher (200 μM) (Fig. 2). For comparison, 20 μM of BV or BR corresponds to a circulating BR level of approximately 1.4 mg/dL. Western blots (Fig. 3A, B) confirmed decreased NS5A in both replicon lines after treatment with BV or BR. Levels of core protein were also reduced by BV or BR in full-length replicons,

consistent with reduced replication of HCV. Treatment with BV dose-dependently decreased NS5A when assayed by WB (Fig. 3C) or immunoprecipitation using specific NS5A antibody (Fig. 3D). In accord with prior reports,12, 18 FeCl2 (100 μM) also decreased NS5A and core protein (Fig. 3A, B) as well as diminishing HCV RNA (not shown). Cellular proliferation and toxicity profoundly affect replicon Caspase inhibitor expression of HCV RNA and protein.19 Consequently, we evaluated whether BV influenced cell growth or was toxic under the current assay

conditions. Presentation and description of these experiments are in the Supporting Data, available online. We observed no effect of BV or BR on cellular proliferation H 89 supplier or toxicity when cells were incubated with tetrapyrrole in medium containing 5% or 10% fetal bovine serum, the conditions used for incubation of cells throughout the manuscript. We next tested the effects of BV (20-200 μM) on HCV infection of Huh7.5 cells with J6/JFH infectious HCV construct.16 BV markedly decreased Huh7.5 cell infection

with J6/JFH, based on immunoreactivity of HCV polyvalent sera (Fig. 4A-C) and measurement of HCV RNA (Fig. 4D). Deconjugated bile pigments are known to inhibit serine-activated pancreatic proteases such as chymotrypsin and trypsin.20 This led us to evaluate the effects of BV and other tetrapyrroles on the HCV NS3/4A protease (Fig. 5A-C). These assays were conducted with wide wavelength excitation/emission (591 nm/622 nm, respectively) transfer peptides. Preliminary experiments established that shorter fluorescence wavelength transfer peptides (340 nm/490 nm or 490 nm/520 nm, excitation/emission, 上海皓元医药股份有限公司 respectively) could not be employed because BV, BR, and other tetrapyrroles showed unacceptable autofluorescence or quenching at the shorter wavelengths. In an assay using a recombinant protease, BV was a markedly more potent inhibitor than BR (either highly purified BR-IXα or BR mixed isomers) (Fig. 5A). BV also displayed the highest median inhibitory concentration (IC50) (9.3 μM) of any tetrapyrrole tested (Table 2), which was similar to that of the commercial NS3/4A inhibitor, AnaSpec #25346. Notably, the IC50 value for the commercial inhibitor in our hands (4.9 μM) was indistinguishable from the value reported by the manufacturer (5 μM), supporting the accuracy of our assay. Assays conducted in the presence of both BV and #25346 showed an additive effect (Fig.