Abstract
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.
Highlights
There are only two drugs known to be effective against the late stage of the disease, DL-␣-difluoromethylornithine (DFMO,2 eflornithine) and Melarsoprol
From the Gas-phase Electrophoretic Mobility Macromolecule/ Nanoparticle Analysis (GEMMA) experiments, we could observe that at an enzyme concentration of 20 g/ml, which is similar to the concentration in the activity assay, the dominating forms of the trypanosome CTP synthetase (CTPS) in the absence of nucleotides were monomers and tetramers
A similar outcome was obtained when a mouse was infected with the lower dose of 2 ϫ 104 trypanosomes and treated with daily intraperitoneal injections of 5 mg/kg acivicin advantage of the new GEMMA technique, which allows direct for 6 days
Summary
Plasmid Construction—The T. brucei CTPS gene was amplified from genomic DNA of the TC221 strain using primers with the following sequence: 5Ј-AGTTAAAACGGTAGACGAGGAG-3Ј and 5Ј-ACACATATATACACTAGACTCGTT-3Ј. Using the diluted (100ϫ) PCR product as a template, a second round of PCR was performed This time one of the primers contained an NdeI recognition sequence followed by a His coding region and a tobacco etch virus protease recognition sequence preceding the start codon: 5Ј-CATATGCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCATGGAAGGCAGTGCAACTGCG-3Ј. The Talon resin was washed twice with 50 mM Hepes buffer, pH 7.3, 0.2 M NaAc supplemented with 5 mM imidazole. Following the second imidazole wash, the resin was packed into a glass Econo-Column (Bio-Rad), and the CTPS was eluted from the column with 50 mM Hepes buffer pH 7.3, 0.2 M NaAc supplemented with 80 mM imidazole. The protein solution was loaded again on a Talon resin column but this time the flow-through, containing CTPS without His tag, was collected. The enzyme was purified as described under “Experimental Procedures.” The data are based on 90 g of wet cells corresponding to 6 liters of bacterial culture
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