Lassa fever virus (LASV) and Machupo virus (MACV) are hemorrhagic fever arenaviruses which are classified as Category A Agents. Currently, there are no licensed LASV or MACV vaccines and LASV/MACV therapy is limited to use of the nucleoside analog ribavirin, which is only partially effective and associated with significant side effects. The impact of arenaviruses on public health and biodefense readiness, coupled with limited treatment options to combat arenavirus infections, underscore the importance of developing novel and effective strategies to combat arenaviruses, especially LASV and MACV. New small molecule inhibitors would be of paramount importance for use during arenaviral outbreaks or bioterrorist attacks. This application defines a plan to develop potent, small molecule inhibitors, which block entry of LASV and MACV. Entry of LASV and MACV into the hosts is mediated by a single viral glycoprotein (GP), which is considered one of the major therapeutic targets. GP consists of three subunits, GP1, GP2, and a stable signal peptide (SSP). GP1 is responsible for receptor binding and host tropism, while GP2 mediates viral/cell membrane fusion and viral entry. We have used an HTS protocol targeting GP-mediated viral entry to screen a small molecule library, and we have identified compounds that inhibit entry of LASV and MACV (IC50 values 10 ?M). These hit compounds exhibit selectivity for arenavirus entry. The overall objective of this Phase I application is to develop these inhibitors as potentia antiviral therapeutics. This application will focus on the following three specific aims: (1) Synthesize structurally diverse analogs of the anti-arenavirus MBX406 hit series based on structure-activity relationships (SARs) to improve potency and selectivity. (2) Select LASV/MACV inhibitors with in vitro ADME properties suitable for i.v. and oral dosing. (3) Validate the lead inhibitor candidates in the infectious assays and investigate the mechanism of action (MOA) of the LASV/MACV inhibitors.