Chemical and Genetic Validation of an Essential Calcium Entry Channel of Trypanosoma brucei as a Therapeutic Target

The Trypanosoma brucei group of parasites causes Nagana in cattle and human African trypanosomiasis, or sleeping sickness, in humans. Current drugs against these parasites have severe toxicity, vaccines are not available, and development of drug resistance makes finding new chemotherapeutic targets imperative. Ion channels, which are involved in several biological processes, are targets of many therapeutically useful agents, and they remain significantly underexplored as therapeutic targets in parasites. Here, we report the presence of a voltage gated Ca²⁺ channel (VGCC, TbCa_v), which is localized in the flagellar plasma membrane (PM) of T. brucei and is essential for proliferation of both bloodstream (BSF) and procyclic forms (PCF) of the parasite. TbCa_V is a single subunit channel capable of transporting Ca²⁺ when expressed in mutant yeast lacking PM Ca²⁺ channels or in HEK293T cells. Through the virtual screening of a commercial chemical library using dynamic ensembles of various conformations of TbCa_v and associated docking analyses, several inhibitors of TbCa_v were discovered. As pharmacological validation of the essential roles of TbCa_v, these compounds were shown to inhibit T. brucei growth with the most potent agent, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) acetamide (NBD-A), exhibiting an EC₅₀ of 25 ± 3 nM and no cytotoxicity in Vero cells possessing related channels. Thus, such studies constitute pharmacological validation of TbCa_v as a viable therapeutic target of T. brucei.