Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness.

African sleeping sickness is a fatal disease caused by two parasite subspecies: Trypanosoma brucei gambiense and T. b. rhodesiense. We previously reported that trypanosomes have extraordinary low CTP pools compared with mammalian cells. Trypanosomes also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. In this study, we have expressed and purified recombinant T. brucei CTP synthetase. The enzyme has a higher K(m) value for UTP than the mammalian CTP synthetase, which in combination with a lower UTP pool may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase is irreversibly inhibited by the glutamine analogue acivicin, a drug extensively tested as an antitumor agent. There is a rapid uptake of acivicin in mice both given intraperitoneally and orally by gavage. Daily injection of acivicin in trypanosome-infected mice suppressed the infection up to one month without any significant loss of weight. Experiments with cultured bloodstream T. brucei showed that acivicin is trypanocidal if present at 1 mum concentration for at least 4 days. Therefore, acivicin may qualify as a drug with "desirable" properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi.