Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis
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 the 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 adapt 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 the 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.
Resolving metabolic connections and mapping dynamics of glucose utilization in pleomorphic Trypanosoma brucei using multiplexed biosensors
The diseases caused by members of the class Kinetoplastea, which includes the African trypanosome Trypanosoma brucei and Leishmania spp., affected ~1.4 million people in 2017 according to the World Health Organization. The differentiation from the long slender (LS) to short stumpy (SS) to procyclic form (PF) is a highly regulated developmental cascade necessary to complete the parasite lifecycle because premature or inappropriate differentiation to the PF while in the bloodstream is lethal to the parasite. However, the signals and regulatory pathway that governs these differentiation steps are poorly understood. Recently, we showed that low glucose induces SS to PF differentiation and it has been known that adenosine monophosphate (AMP) activated protein kinase (AMPK) is involved in the regulation of SS to PF differentiation. Hence, we believe that AMPK is both a central regulator of metabolism and intimately involved in the signaling cascades that lead to SS to PF differentiation. Resolving the metabolic homeostasis signals of parasites during SS to PF differentiation is, therefore, a necessary step in the drive towards new therapies for trypanosomiasis and other kinetoplastid diseases. Our focus is to use multiplexed fluorescent protein biosensors in living trypanosomes to make near-simultaneous observations of the dynamic changes in critically important metabolites that activate SS to PF differentiation (glucose, ATP, ADP, AMP, and fructose-1,6-bisphosphate (FBP)). We will initially monitor these molecules in near-live time in response to developmental cues. This highly innovative new approach will allow us to better understand the mechanisms by which parasites perceive glucose. We will then establish the signaling pathway using genetic tools combined with the biosensors that will identify critical signaling molecules that may become targets for a new generation of therapeutic targets. We will also assess other potential glucose perception sensing pathways and identify additional genes involved in the process for future drug discovery campaigns. This work is expected to provide a focused picture of the relationship between metabolism, regulatory systems, and differentiation to adapt to the multiple environments that trypanosomes inhabit and may be directly applicable to other kinetoplastid parasites including Leishmania and T. cruzi.