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Abstract
Human African trypanosomiasis (HAT) is caused by the protozoan parasiteTrypanosoma brucei. This disease affects millions of people throughout Africa. The drugs used against this disease are toxic, have shown signs of resistance, and are difficult to administer, prompting the need for safer more effective drugs. There is evidence of both tyrosine kinase activity and tyrosine phosphorylated proteins in T. brucei. Bioinformatic analysis suggests that tyrosine kinase-like domains are also present in T. brucei. Protein tyrosine kinases have become a target to fight some cancers; tyrosine kinases inhibitors (TKI) are currently being used in some treatments. Lastly, tyrphostin A47, a general tyrosine kinase inhibitor has been shown to inhibit growth of T. brucei.To elucidate how TKIs kill T. brucei, we used 2 TKIs lapatinib and SU14813, with different targets/pathways in mammalian cells, and measured their function in inhibition of growth, cell morphology, protein phosphorylation, and endocytosis. Lapatinib and SU14813 were used in cell growth inhibition experiments and shown to both have IG50 in the 1-2 micromolar range. To establish whether or not a cell shape change was evident, we useddifferential interference contrast (DIC) along with light scatter plots from flow cytometry and found that cells treated with lapatinib, but not SU14813 demonstrated a rounded up phenotype compared to control cells. Effect in tyrosine phosphorylation was revealed through western blot analysis using PTyr100, an antibody that recognizes phosphorylated tyrosines. Both TKIsexhibited decrease in tyrosine phosphorylated proteins, with lapatinib having a stronger effect. Receptor-mediated endocytosis was tested on cells treated with the TKIs and only lapatinib seemed to exhibit a block in uptake of transferrin. These data confirm PTKs as drug discovery targets in T. brucei and gives some clues as to the mechanisms by which they cause cell death.