Project Summary/Abstract Parasite infection with members of the class Kinetoplastea, including Trypanosoma brucei and Leishmania spp., places a tremendous burden on human health, with an estimated ~1.4 million cases of disease recorded in 2015 (World Health Organization). Despite the widespread impact of these organisms, there remain major gaps in our understanding of fundamental parasite biology. For example, it is appreciated that glucose is a critical metabolite that can also serve to regulate important developmental pathways in the parasites. However, our understanding of these areas, particularly in the context of living parasites, is extremely limited. We propose to elucidate key mechanistic aspects of kinetoplastid glucose metabolism in kinetoplastids using a validated, yet unprecedented, screening assay platform based on live parasites expressing protein sensors that measure glucose uptake, distribution, and metabolism. We will adopt these cells to a novel and effective multiplexed high-throughput screening assay to identify small molecule probes that disrupt glucose uptake, distribution and metabolism in live parasites. This unique approach will simultaneously identify inhibitors of glycolysis that possess chemical properties required for delivery to the cellular target. Hits from the screening of a structurally diverse 100,000 compound collection will be validated by resynthesis, reconfirmation and counter- screening. Target identification, which will be facilitated by mapping the impact of the inhibitor on the pathway using the read-out from the assay, will be confirmed using reverse genetic and proteomic approaches. Last, the identified small molecule glycolytic probe inhibitors will be improved by SAR and their activity against parasites scored. Expected outcomes include development of a screening strategy that will be applicable to many types of cells, along with the identification of validated probes that dissect an essential parasite metabolic pathway.