Protein kinases are potentially attractive therapeutic targets for neglected parasitic diseases, including African trypanosomiasis caused by the protozoan, Trypanosoma brucei. How to prioritize T. brucei kinases and quantify their intracellular engagement by small-molecule inhibitors remain unsolved problems. Here, we combine chemoproteomics and RNA interference to interrogate trypanosome kinases bearing a Cys-Asp-Xaa-Gly motif (CDXG kinases). We discovered that hypothemycin, a fungal polyketide previously shown to covalently inactivate a subset of human CDXG kinases, kills T. brucei in culture and in infected mice. Quantitative chemoproteomic analysis with a hypothemycin-based probe revealed the relative sensitivity of endogenous CDXG kinases, including TbGSK3short and a previously uncharacterized kinase, TbCLK1. RNAi-mediated knockdown demonstrated that both kinases are essential, but only TbCLK1 is fully engaged by cytotoxic concentrations of hypothemycin in intact cells. Our study identifies TbCLK1 as a therapeutic target for African trypanosomiasis and establishes a new chemoproteomic tool for interrogating CDXG kinases in their native context.
Human African trypanosomiasis—commonly known as sleeping sickness—is a debilitating and potentially fatal tropical disease that is widespread in sub-Saharan Africa. It is caused by the single-celled parasite Trypanosoma brucei, which is transmitted to humans by the bite of the tsetse fly. The infection takes its name from the disruption of the circadian clock that occurs early on in the disorder and leads to sleep disturbances. If left untreated, T. brucei infection leads to coma, organ failure and death.
Most of the existing pharmaceutical treatments for sleeping sickness were developed more than 50 years ago. However, they are only weakly absorbed into the bloodstream—meaning that high doses must be used—and they lead to unpleasant side effects. Moreover, the T. brucei parasite is developing resistance to existing drugs, so further research is needed to identify new therapeutic targets.
One promising option could be the parasite’s protein kinases. These enzymes, which add phosphate-based chemical groups to proteins, have a key role in regulating protein function and many of them are already being investigated as therapeutic targets for cancers and autoimmune diseases. T. brucei has 182 different kinases, suggesting a wealth of potential new targets. However, many of these are similar to human enzymes, and inhibiting the latter could lead to harmful side effects.
Now, Nishino et al. have produced a synthetic version of a microbially derived kinase inhibitor, called hypothemycin, and have shown that it kills T. brucei cells grown in culture. Hypothemycin also killed T. brucei in infected mice, completely curing the infection in one third of animals, although high doses of the drug led to side effects. Using a chemical biology approach and quantitative mass spectrometry, Nishino et al. found that the main target of hypothemycin was a previously unknown kinase that is essential for T. brucei survival. Although hypothemycin itself is probably unsuitable as a treatment due to its lack of specificity, the work of Nishino et al. suggests that its kinase targets deserve further investigation.