Chagas disease, which is endemic to 18 Latin American countries, and is the major cause of heart failure in the region affecting 8 million people world-wide [1]. Chagas disease is caused by the parasite Trypanosoma cruzi which is transmitted to humans in 3 ways: 1) From fecal matter from blood-feeding insect vectors that belong to the Reduviidae family; 2) Through transfusion with infected blood; 3) Congenitally, from infected mother to fetus. The acute effects of the T. cruzi infection appear soon after infection and symptoms can include fever, swelling at the site of the insect bite and of the lymph glands, and enlargement of the liver and spleen. The chronic phase primarily affects cardiac and enteric cells and patients will suffer from irreversible damage to the heart and digestive tract which can occur even 20 years after infection [1-3]. This project aims to identify novel trypanocidal agents for the treatment of Chagas disease with little to no toxicity in mammalian host cells. An initial pilot high throughput screen of 22,378 compounds derived from the Broad Institute's Diveristy-Oriented Synthesis (DOS) collection (AID 651903) were tested against recombinant Tulahuen strain of T. cruzi stably expressing beta-galactosidase reporter co-cultured with host cell, mouse fibroblast NIH3T3 [4-7]. 1,092 ‘actives’ inhibited T. cruzi replication by 55% of control (at 5 μM). 800 compounds were retested at dose and tested for host cell toxicity. A SnAr 8-ortho (CID 44492448) scaffold compound exhibited greater than 1000-fold selectivity towards T. cruzi and was effective against amastigotes, the replicating form of the parasite inside the host cell. Activity against amastogotes is critical for the development of drugs targeting the chronic phase of Chagas disease. Preliminary SAR studies on the hit resulted in the probe ML341 which inhibited T. cruzi replication (IC50=0.9 nM). Potency of ML341 was confirmed against 2 strains of T. cruzi amastigotes (Tulahuen and CA-I/72). Moreover, ML341 is cidal at 750 nM after 20 days of compound treatment and cidal at the lowest dose tested (4 nM) after 29 days of compound treatment, and exhibited minimal toxicity to any mammalian cells tested (NIH3T3, L6, BESM, HepG2). Therefore, we have identified a potent and cidal inhibitor of T. cruzi that is non-toxic to mammalian host cell. Future studies to determine the efficacy in mouse models are currently being undertaken.