The Discovery of a Novel Chemical Scaffold that Binds Dengue Virus Non‐structural Protein 5
Dengue viruses (DENV) are mosquito‐borne flaviviruses that pose a continued and growing threat to global health. There are estimated to be 390 million DENV infections each year, and because there is no vaccine or approved therapeutic treatment, developing a small‐molecule treatment is imperative. Possible small‐molecule drug therapies for DENV could be immune system modulators, inhibitors of DENV‐required host factor, or inhibitors of a viral gene product. In this study, we chose to take the latter approach and focused our drug discovery efforts on the most highly conserved flaviviral protein, non‐structural protein 5 (NS5). NS5 contains two major domains, each with different enzymatic activities. The N‐terminus has methyltransferase activity, and the C terminus, an RNA‐dependent RNA polymerase (RdRp). The activities of both domains are purine‐dependent, and therefore both domains contribute to the purine‐binding properties of NS5. Inhibition of either of these domains in NS5 results in inadequate propagation of DENV, and the purine‐binding domains present ideal drug targets for disrupting these activities. These factors make NS5 protein an ideal candidate target for our small‐molecule library screen.
A high‐throughput fluorescence‐based screen was employed to identify anti‐DENV compounds based on their ability to competitively bind NS5. The screen was performed by binding green fluorescent protein NS5 fusion protein (GFP‐NS5) to immobilized ATP resin, and then performing parallel elutions using over 3,000 distinct compounds. One compound in particular, HS‐205020, was able to competitively elute GFP‐NS5 from the ATP resin and also exhibited antiviral activity in both the U937+DCSIGN human monocyte cell line and BHK‐21 cells. Additionally, HS‐205020 was able to inhibit DENV NS5 RNA polymerase activity in vitro. HS‐205020 is chemically distinct from the majority of previously reported NS5 inhibitors, which are nucleoside analogs that can cause severe toxicity in animal studies. In contrast, over the concentration range that produced anti‐DENV effects, HS‐205020 showed comparable viabilities to ribavirin, an FDA approved hepatitis C virus (HCV) therapeutic. These findings support HS‐205020 as a potential dengue antiviral candidate, and its chemical scaffold represents as an ideal starting compound for future structure‐activity relationship studies.
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