Project Summary The Zika virus (ZIKV) is a member of the Flavivirus genus that includes other arthropod-borne human pathogens such as dengue virus and West Nile virus, among others. ZIKV’s link to microcephaly in newborn infants and the Guillan-Barré syndrome in adults has invigorated measures to develop a vaccine, as well as efforts to develop antivirals based on targeting enzymatic activities central to the life cycle and survival of ZIKV. One such enzymatic activity is encoded by the methyltransferase (MTase) domain, located at the N-terminus of the nonstructural protein NS5. Taking a structure informed approach, we have succeeded in identifying a novel “lead-like” compound (MS2045) for the inhibition of ZIKV’s NS5 MTase activity and for blocking its replication. MS2045 provides a basis for further chemistry and the development of even more potent inhibitors. In aim 1, we will a) design MS2045 analogs with the capacity to establish specific interactions with unique amino acids of ZIKV MTase as a means to provide additional selectivity against the human RNA 5’-cap MTases; b) chemically synthesize these analogs and produce them to a purity of 95% for in vitro and cell-based assays, and for structural studies. In aim 2, we will a) perform biophysical assays to assess the ability of these analogs to selectively bind the ZIKV NS5-MTase as compared to the human RNA 5’-cap MTases and test their ability to inhibit RNA methylation; b) test these analogs in viral cell-based assays to assess their efficacy in blocking viral replication; c) determine structures ZIKV NS5-Mtase with select analogs for additional, iterative rounds of structure activity relationships (SARs). Collectively, these studies will help to identify analogs of MS2045 that can be potentially developed into potent and selective inhibitors of NS5-MTase from ZIKV (and other pathogenic flaviviruses)