Summary Chronic, long-term infection with herpes simplex virus (HSV) remains a significant unmet medical need. It is estimated that ~800,000 individuals in the US will be newly infected with HSV1 or HSV2 each year, and that the total infection rate approaches 80% for adults over 50 yrs of age. Long-term latent infection and chronic reactivation of HSV1 and HSV2 remain a significant source of morbidity, including a major source of blindness and encephalitis. Presently, there are no vaccines or small molecule drugs that selectively treat and eliminate the latent infection. Here, we propose to develop new chemical entity small molecule drugs to target and treat latent forms of HSV infection in human neurons. HSV express only one viral transcript, termed LAT, during neuronal latency. LAT is known to be a stable intron that is highly conserved among all viral strains across both HSV1 and HSV2 species. LAT is important for maintaining viral latency and confers resistance to apoptosis for latently infected neurons. The most highly conserved substructure of LAT is the stable 3’ stem loop containing a non- canonical branch point sequence that prevents debranching by the cellular enzyme DBR1. Genetic studies reveal that disruption of the stem-loop and branch point structure destabilizes LAT and eliminates its functional activity. Here, we propose to carry out high-throughput screenings with the recently developed FRET-based biochemical assay and will develop and implement cell-based assays to identify selective interacting, RNA-structure disrupting molecules that have favorable drug-like properties using industry standard principles of medicinal chemistry and fragment-based drug design. The product that ultimately results from this proposal is a small molecule inhibitor that selectively binds and disrupts the function of LAT RNA in HSV neuronal latency, and can be further developed for the treatment of HSV latency and associated diseases.