A GPI-phospholipase C and protein tyrosine kinases in Trypanosoma brucei
Hardin, Clyde Franklin
MetadataShow full item record
Trypanosoma brucei causes human African trypanosomiasis (HAT). The disease threatens over 60 million people in 36 African countries. Chemotherapy is the only method of treatment available against HAT. Generally, the drugs are toxic and difficult to administer. Today, the need for safer, orally administered drugs is urgent. T. brucei is covered with a glycosyl phosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG). VSG is essential for the viability of T. brucei in the host’s bloodstream. The parasite also expresses a GPI-specific phospholipase C that is able to cleave the GPI-anchor of VSG. Interestingly, little VSG is released in non-differentiating bloodstream cells. The intracellular localization of GPI-PLCp is not well characterized and the function(s) of the enzyme are just beginning to be elucidated. To gain better perspective of the functions of GPIPLCp, it is important to determine the intracellular location of the enzyme. Tyrosine kinase activity is present in T. brucei. Preliminary investigations suggest that drugs that inhibit protein tyrosine kinases (PTKs) kill the parasite. Given their role in cancer, PTKs have become the focus of an enormous effort to discover new drugs against the disease. Several drugs that inhibit PTKs have been developed and are being used to treat some cancers. The goal of this dissertation is two-fold. First, to further understand the function of GPIPLCp in T. brucei, we aimed to determine the intracellular location of the enzyme. Towards this, we used two approaches; (i) fluorescence microscopy, and (ii) iodixanol density-gradient centrifugation and found GPI-PLCp to be a glycosome protein. In a second project, we wanted to determine if PTK-specific drugs used to treat cancer could kill T. brucei. To this end, we treated T. brucei with the drugs Canertinib, PKI166 and AEE788 and found that all kill cultured trypanosomes. Further we found AEE788, a pyrrolopyrimidine, to be the most potent drug tested and within 90 minutes of exposure it: (i) reduces tyrosine phosphorylation, (ii) blocks endocytosis of transferrin, (iii) causes a morphological change, and (iv) triggers cell death in T. brucei. Finally, to understand which processes may be influenced by tyrosine phosphorylation in bloodstream T. brucei, we identified tyrosine-phosphorylated proteins by pTyr-affinity chromatography and LC-MS/MS. Using this technique, we identified 132 putative pTyr proteins and two proteins with confirmed pTyr residues. Collectively, these data validate PTKs as targets for drug discovery in T. brucei and introduce pyrrolopyrimidines as a lead scaffold for antitrypanosome drug discovery.