Chemical genetics-based discovery of indole derivatives as HCV NS5B polymerase inhibitors

• Indole derivatives were discovered as HCV replication inhibitors.
• Two rounds of SAR studies resulted in the most potent and the least cytotoxic compound 12e.
• Chemical genetic study suggested an NS5B RNA polymerase as its potential target.
• Compound 12e directly inhibited an NS5B RNA polymerase activity in vitro.

In order to identify the inhibitors of hepatitis C virus (HCV) replication with a novel scaffold via a mechanistically unbiased approach, we screened our in-house library composed of ∼6000 compounds with various chemical structures by using the renilla luciferase-linked genotype 2a reporter virus, and we identified a series of compounds containing an indole moiety that were active against HCV replication. Based on this result, we further synthesized three groups of indole derivatives and evaluated their inhibitory effects on HCV replication. In the present structure–activity relationship study of these indole derivatives, we discovered that compound 12e was the most potent inhibitor of HCV replication with minimal cytotoxicity (EC50 = 1.1 μM, EC90 = 2.1 μM, and CC50 = 61.8 μM). We also confirmed that compound 12e caused a dose- and time-dependent reduction of viral RNA as well as viral protein levels in both genotype 2a J6/JFH1 RNA-transfected cells and genotype 1b Bart79I subgenomic replicon cells. Finally, a genetic mapping study of mutant viruses resistant to compound 12e revealed that NS5B RNA polymerase was the potential target. This finding was further validated by demonstration of inhibition of NS5B RNA polymerase in vitro by compound 12e (IC50 = 292 nM). Compound 12e may serve as a valuable candidate for the development of a new class of HCV NS5B RNA polymerase inhibitors in the future.

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