Optimization of a malachite green-pyrophosphatase assay for the Zika virus RNA-dependent RNA polymerase and preliminary identification of natural product modulators.

The Zika virus (ZIKV) is an Orthoflavivirus linked to several neurological and developmental diseases, and it remains a significant global health concern for which no treatments or vaccines are currently available. In this study, we optimized the malachite green colorimetric assay, previously used to measure the RNA-dependent RNA polymerase (RdRp) activity of the hepatitis C and foot-and-mouth disease viruses, and applied this assay for the preliminary identification of the ZIKV RdRp modulators. RdRp is an enzyme essential for viral replication. Modulator screening was conducted using 204 compounds from the Natural Product Set IV of the National Cancer Institute Developmental Therapeutics Program. We identified two compounds, purpurogallin and digallic acid, as preliminary modulators of the ZIKV RdRp. Future studies will validate these preliminary hits using orthogonal assays, perform dose-response analyses, and elucidate their mechanisms of action.