Trypanosoma brucei CTP synthetase: A target for the treatment of African sleeping sickness

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

Myristate exchange on the Trypanosoma brucei variant surface glycoprotein.

The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.