ABSTRACT Zika Virus (ZIKV) has emerged as a global public health threat because of its recent outbreaks and its link to microcephaly in newborns and Guillain-Barré syndrome in adults. To date, there is no vaccine or treatment available, highlighting an urgent need to develop effective therapeutics. Inhibition of ZIKV NS5 methyltransferase (MTase) is a promising anti-ZIKV strategy. However, current ZIKV MTase inhibitors generally suffer from low activity, lack of selectivity, or unfavorable drug-like properties, hindering testing of MTase inhibitors in the animal models of ZIKV infection. We have designed and synthesized a structurally novel inhibitor of ZIKV NS5 MTase, which possessed anti-ZIKV activity without significant toxicity. In this application, we will perform lead optimization to improve the potency, selectivity and drug-like properties of our ZIKV MTase inhibitors, aiming to identify MTase inhibitors that can be used in the ZIKV animal disease models. To accomplish the goal, we propose the following specific aims: Specific Aim 1. Design and synthesize ZIKV NS5 MTase inhibitors and evaluate them in biochemical and antiviral assays; Specific Aim 2. Assess selected ZIKV NS5 MTase inhibitors for their physiochemical and in vitro absorption, distribution, metabolism, and excretion (ADME) properties; and Specific Aim 3. Determine in vivo PK parameters of advanced ZIKV NS5 MTase inhibitors. We expect that the proposed research will significantly contribute to efforts in developing ZIKV MTase inhibitors as anti-ZIKV therapeutics.