A Cytopathic Effect-Based High-Throughput Screening Assay Identified Two Novel Compounds that Inhibit Dengue Infection: Streptovitacin A and Nagilactone C

摘要

Dengue is an emerging infectious disease and is spreading world-wide at exponential levels. Two billion people in over 100 countries are at risk for infection from one of the four serotypes of the dengue virus. Those infected with dengue may develop diseases such as dengue fever and dengue hemorrhagic fever (DHF) and of the 500,000 cases that progress to DHF each year, more than 22,000 will result in fatality. Discovering new antivirals to treat DHF is essential to reducing this disease burden. Here, we have developed a cytopathic effect-based high-throughput screen (HTS) to discover possible inhibitors of Dengue viral infection of hepatocytes in vitro. Dengue virus infection of hepatocytes induces massive cell death, "cytopathic effect (CPE)", which we converted into a screening assay whereby inhibitors of Dengue infection prevent cells from dying. In this assay, the viral induced CPE is quantitated by monitoring cellular ATP levels, which positively correlates with cellular viability. ATP in the cell culture will drive the oxidation of luciferin resulting in the emission of light that is quantitated using a luminometer. The assay is simple and highly reproducible yielding a screening window coefficient, Z-factor, of 0.78±0.12 between plates. The Z-factor is a statistical parameter commonly accepted as an assay quality assessment and is reported as a value 0 to 1 and anything over 0.5 is considered excellent quality. This assay is advantageous to current methodology as it simultaneously screens possible inhibitory compounds while controlling for any unwanted toxicity triggered by these drugs. Our initial HTS of a 288 small compound library yielded a total of eleven hits that prevented the CPE of dengue infection. Further evaluation with an immunofluorescence assay showed that two of these compounds, Streptovitacin A and Nagilactone C, are highly potent inhibitors of dengue infection. At effective inhibitory doses, they did not appear to be cytotoxic, and therefore both of these compounds are possible antivirals and could be used to elucidate various cellular mechanisms utilized during the dengue life cycle. The discovery of these two inhibitors demonstrates the efficacy of our newly developed assay and the public health significance of this project.