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

Abstract

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 ␣-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. bruceiinfected 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. U p to 500,000 people are estimated to suffer from African sleeping sickness (1, 2), a fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. These pathogens are transmitted by tsetse flies to their mammalian hosts, where they first establish an infection in the blood and lymph. In the second stage of the disease, the parasites invade the central nervous system. Most drugs used to treat sleeping sickness do not cross the bloodbrain barrier; as soon as the trypanosomes have entered the central nervous system, only eflornithine and arsenicals such as melarsoprol are effective. However, the arsenicals are highly toxic, whereas eflornithine is ineffective against T. b. rhodesiense (3) and is very expensive.Purine metabolism has been extensively studied in trypanosomes (4), and it is well established that they lack the ability to form purines de novo. They therefore absolutely depend on preformed purine bases or nucleosides for survival. Any purine source is fine, because they have all of the enzymes needed to interconvert IMP, AMP, and GMP. Much less attention has been paid to the synthesis of pyrimidines in trypanosomes (4); so far, no study of the pathways for CTP synthesis has been published.We have previously reported that cultured bloodstream T. brucei have a very small CTP pool in comparison with other NTP pools and the CTP pools of other cell types (5). In this paper, we demonstrate that the low CTP pools are a consequence of slow synthesis by the parasite CTP synthetase. We suggest that this enzyme is an excellent target for chemotherapy, because we found that the trypanosomes, unlike mammalian cells, cannot compensate for the inhibition of CTP synthetase by the salvage of cytidine. The rest of our work is focused on the use of two irreversible glutamine analog CTP synthetase inhibitors, 6-diazo-5-oxo-L-norleucine (DON) and ␣-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin). These drugs have been used extensively in clinical trials against various cancers, and at least acivicin is known to penetrate the blood-brain barrier (6). We found that both drug...

Intracellular Positioning of Isoforms Explains an Unusually Large Adenylate Kinase Gene Family in the Parasite Trypanosoma brucei

“…Strikingly, the activity of ADKG with CMP is very high (2.5 times the activity seen with AMP) and reaches its V max at low CMP concentration. We believe this high activity is linked to the very low CTP/CDP pools detected in T. brucei (52). The intracellular CTP pool size in T. brucei is only 26 M, approximately 10 times lower than the corresponding pool in mouse fibroblasts, and the low K m value of ADKG toward CMP may be critical in maintaining CDP/CTP homeostasis.…”

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

“…CTP occupies a central role in the biosynthesis of nucleic acids, phospholipids and sialic acid, and CTP synthase serves as an attractive target for the development of anti-tumour, anti-viral and anti-protozoal agents [4,5,[27][28][29]. This surprising discovery thus presents many exciting opportunities for revealing hitherto unforeseen aspects of CTP synthase biology, from the cell biology of metabolic pathways to the mechanism of CTP synthase regulation.…”

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex,

“…Moreover, using these inhibitors to deplete the intracellular CTP concentration increases the efficacy of the anticancer/antiviral drugs cytosine arabinoside (26,27), 2′,3′-dideoxy-3′-thia-cytidine (28), and 2′,2′-difluoro-2′-deoxycytidine (29). Trypanosoma brucei CTPS is a validated African sleeping sickness drug target (30), and malaria (31), giardiosis (32), chlamydia (33), and hemorrhagic fevers (34) are also potentially treatable using anti-CTPS therapies. However, spontaneous resistance to these drugs arises frequently through clustered CTPS gene mutations that release CTP feedback inhibition and increase intracellular CTP levels (Figures 2 and 6) (18,25,35,36).…”