The New World alphaviruses, Venezuelan (VEEV), Eastern and Western Equine Encephalitis viruses, are emerging pathogens that are endemic throughout the Americas. They cause encephalitis in humans and equines, thus presenting serious health and economic problems. Despite the urgent need, neither approved drugs nor vaccines are available for treatment of these diseases in humans. Broad, long-term objectives: The research objectives stated herein, and in future studies that build upon these findings, will deliver analogs of ML336, a promising lead compound, as small molecule therapeutic candidates for future clinical evaluation targeting diseases caused by VEEV and other alphaviruses. Specific aims/hypothesis: The specific aims address two hypotheses. In aims 1 and 2, we hypothesize that medicinal chemical optimization of ML336 can afford a safe and specific therapeutic preclinical candidate to treat New World alphaviral diseases. In aim 3, we test the hypothesis that ML336 blocks VEEV RNA synthesis through disruption of interactions required for replicase assembly and/or directly inhibits an enzymatic activity required for viral RNA synthesis. Research design and methods: Aim 1 will derive a robust pharmacological profile suitable for in vivo efficacy through structure-activity and structure-property relationshi (SAR-SPR) studies. Specifically, Dr. Golden will lead efforts to generate analogs of ML336 with an improved pharmacological profile that includes increased solubility and BBB permeability and reduced metabolic liability to improve exposure and clearance parameters. In Aim 2, Dr. Guo will assess the biodistribution and pharmacokinetics/dynamics of ML336 and 2-3 lead compounds each year showing suitable and robust pharmacological and antiviral (in vitro and in vivo) profiles. Dr. Jonsson will lead in vivo efficacy testing studies of two of the most promising lead candidates to define the optimal dosing regimen using the VEEV Trinidad Donkey strain. Finally, Dr. Chung will dissect the mechanism of action of ML336 using in vitro assays focused on replication (Aim 3) in consultation with Dr. Sawicki.