A hypothesis-generating chemical proteomics approach reveals phosphotyrosine pathways in the African trypanosome
Behera, Ranjan Kumar
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New drugs are needed for human African trypanosomiasis (HAT) caused by subspecies of Trypanosoma brucei as drug resistance and toxic side effects plague current therapies. The phosphotyrosine signaling in trypanosome could be a drug target because it is unique and divergent from the mammalian system. The 4-anilinoquinazoline class of tyrosine kinase inhibitors have been successfully developed as anticancer drugs. From a focused screen of 4-anilinoquinazolines, we discovered GW572016 that kills T. brucei at low micromolar concentrations. We developed a chemical biology approach with GW572016 to gain insight into the phosphotyrosine signaling pathways in the African trypanosome. We also performed a structure-activity relationship study to optimize 4-aninlinoquinazoline scaffold for antitrypanosmal lead drug discovery. Finally, efficacy of select tyrosine kinase inhibitors was tested in a mouse model of acute HAT. GW572016 as a tool inhibits tyrosine phosphorylation of trypanosome proteins. Subsequently, thirty-nine proteins were identified as a part of the GW572016-susceptible phosphotyrosine-signaling pathway. Predicted functions of these proteins include (i) endocytosis (e.g. actin A), (ii) cytoskeleton and cell morphogenesis (e.g. beta-tubulin), and (iii) flagellum biogenesis (e.g. PFR-1/PFR-C). We studied the effects of GW572016 on these three pathways. GW572016 inhibited transferrin endocytosis. Further, the drug causes loss of trypanosome polarity evident from (i) changes in the intracellular localization of beta-tubulin, (ii) redistribution of tyrosylated alpha-tubulin, and (iii) retraction of the flagellum into the cell body. From the 4-anilinoquinazoline scaffold optimization study, we discovered many compounds better (GI50) than GW572016. The in vivo study with tyrosine kinase inhibitors revealed that these compounds could control parasitemia, and they improved mean survival of the treated group. Also, GW572016 could cure the infection in 25% of mice. We propose that 4-anilinoquinazoline is a lead chemical scaffold for antitrypanosome drug discovery.