Identifying the Target of a Potent and Selective Inhibitor of Babesia Blood Stages.

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Project Summary Tick-borne diseases, including human babesiosis, are on the rise. Caused by protozoa in the genus Babesia, this infection can be life-threatening in asplenic or immunocompromised individuals. Based on the most recent CDC data, the number of annual reported cases in the US nearly doubled between 2011 and 2018, with some states experiencing a greater than 10-fold increase. Treatment options for babesiosis in immunocompromised individuals are very limited, have substantial side effects, and often fail to fully clear the parasites. This makes the development of new treatments imperative. We recently developed the small molecule inhibitor C10 that has low toxicity and excellent activity against multiple species of Babesia parasites that infect humans. The molecular target of this inhibitor is clearly druggable, essential, and likely novel but is currently unknown. Using a combination of genetic and biochemical approaches, the proposed experiments will identify and validate the parasite target of this inhibitor. In AIM1 we will identify what parasite proteins that bind tightly to C10 using µMap, a newly developed photocatalytic chemo-proteomics platform with greatly enhanced sensitivity and specificity over current methods. In a parallel effort, we have selected multiple independent parasite lines with partial resistance to C10. Since mutation in genes encoding a drug target is the most common resistance mechanism, AIM2 will use whole-genome resequencing to identify genes with newly acquired mutations in resistant lines but absent from the susceptible parent to identify an additional set of target candidates. Finally, we will validate target candidates from AIM1 and AIM2 by confirming that engineered mutations of the target, such as knockdown or overexpression, alters susceptibility to C10. If feasible, we will also test if C10 binds to and inhibits the activity of the recombinantly expressed target. Throughout these experiments will use C13, a nearly identical but inactive isomer of C10, to ensure target specificity.