Production of ammonia by Tritrichomonas foetus and Trichomonas vaginalis

Production of ammonia is difficult to find among the various studies of amino acid metabolism in protozoa. Several studies suggest that catabolism of arginine to ammonium is important for the growth of trichomonads. Trichomonads are amitochondriate zooflagellates that thrive under microaerophilic and anaerobic conditions. We were able to detect accumulation of ammonium ions and ammonia in cultures of Tritrichomonas foetus and Trichomonas vaginalis including those resistant to metronidazole. Ammonium ions and ammonia were detected using the indophenol colorimetric method. Aerobic overnight cultures had 0.9 mM of soluble ammonium (NH4+ and NH3) or a 20% greater concentration of ammonium relative to sterile tryptose, yeast extract maltose medium with heat inactivated horse serum that was incubated similarly. Production of ammonia itself was confirmed by analysis of a wick that was moistened with sulfuric acid (40 mM) and placed above the liquid in sealed cultures of a strain of T. vaginalis. The wicks from these cultures captured the equivalent of 0.048mM of volatile ammonia (NH3) from the liquid as compared to 0.021mM volatile ammonia from sterile medium after overnight incubation. Intact cells of trichomonads (0.1mg protein) incubated in Doran’s buffer and with or without (1 mM) L-arginine produced significant amounts of soluble ammonium (0.07 mM, 0.035 mM respectively) during 60 minutes. These amounts are similar to those reported for the metabolism of carbohydrates by trichomonads. The results indicate that ammonium ions and the more irritating ammonia are significant metabolites of trichomonads

Eicosapentaenoic Acid Modulates Trichomonas 1 vaginalis Activity

Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in 50 males, the parasite is associated with disease in both sexes. Metronidazole is an effective 51 treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become 52 necessary to investigate other possible therapies. In this study, we examined the effects of native 53 and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic and arachidonic 54 acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 μM and 380 55 μM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In 56 contrast, oxidized eicosapentaenoic acid was the least toxic, requiring \u3e3 mM to inhibit activity, 57 while low levels (10μM) were associated with increased parasite density. Mass spectrometric 58 analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six 59 additional oxygen atoms and various degrees of bond saturation. These results indicate that 60 eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is 61 present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis 62 may facilitate the design of synthetic fatty acids that are effective for the treatment of 63 metronidazole-resistant T. vaginalis

Arginine metabolism in Trichomonas vaginalis infected with Mycoplasma hominis

Both Mycoplasma hominis and Trichomonas vaginalis utilize arginine as an energy source via the arginine dihydrolase (ADH) pathway. It has been previously demonstrated that M. hominis forms a stable intracellular relationship with T. vaginalis; hence, in this study we examined the interaction of two localized ADH pathways by comparing T. vaginalis strain SS22 with the laboratory-generated T. vaginalis strain SS22-MOZ2 infected with M. hominis MOZ2. The presence of M. hominis resulted in an approximately 16-fold increase in intracellular ornithine and a threefold increase in putrescine, compared with control T. vaginalis cultures. No change in the activity of enzymes of the ADH pathway could be demonstrated in SS22-MOZ2 compared with the parent SS22, and the increased production of ornithine could be attributed to the presence of M. hominis. Using metabolic flow analysis it was determined that the elasticity of enzymes of the ADH pathway in SS22-MOZ2 was unchanged compared with the parent SS22; however, the elasticity of ornithine decarboxylase (ODC) in SS22 was small, and it was doubled in SS22-MOZ2 cells. The potential benefit of this relationship to both T. vaginalis and M. hominis is discussed

Pharmacokinetics and pharmacodynamics utilizing unbound target tissue exposure as part of a disposition-based rationale for lead optimization of benzoxaboroles in the treatment of Stage 2 Human African Trypanosomiasis

This review presents a progression strategy for the discovery of new anti-parasitic drugs that uses in vitro susceptibility, time-kill and reversibility measures to define the therapeutically relevant exposure required in target tissues of animal infection models. The strategy is exemplified by the discovery of SCYX-7158 as a potential oral treatment for stage 2 (CNS) Human African Trypanosomiasis (HAT). A critique of current treatments for stage 2 HAT is included to provide context for the challenges of achieving target tissue disposition and the need for establishing pharmacokinetic-pharmacodynamic (PK-PD) measures early in the discovery paradigm. The strategy comprises 3 stages. Initially, compounds demonstrating promising in vitro activity and selectivity for the target organism over mammalian cells are advanced to in vitro metabolic stability, barrier permeability and tissue binding assays to establish that they will likely achieve and maintain therapeutic concentrations during in-life efficacy studies. Secondly, in vitro time-kill and reversibility kinetics are employed to correlate exposure (based on unbound concentrations) with in vitro activity, and to identify pharmacodynamic measures that would best predict efficacy. Lastly, this information is used to design dosing regimens for pivotal pharmacokinetic-pharmacodyamic studies in animal infection model

2,4-Diaminopyrimidines as Potent Inhibitors of Trypanosoma brucei and Identification of Molecular Targets by a Chemical Proteomics Approach

The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT) or sleeping sickness, a fatal disease affecting nearly half a million people in sub-Saharan Africa. Current treatments for HAT have very poor safety profiles and are difficult to administer. There is an urgent need for new, safe and effective treatments for sleeping sickness. This work describes the discovery of 2,4-diaminopyrimidines, exemplified by 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid phenylamide or SCYX-5070, as potent inhibitors of T. brucei growth in vitro and also in animal models for HAT. To determine the parasite proteins responsible for interaction with SCYX-5070 and related compounds, affinity pull-downs were performed followed by sequence analysis and parasite genome database searching. The work revealed that mitogen-activated protein kinases (MAPKs) and cdc2-related kinases (CRKs) are the major proteins specifically bound to the immobilized compound, suggesting their potential participation in the pharmacological effects of 2,4-diaminopyrimidines against trypanosomatid protozoan parasites. These data strongly support the use of 2,4-diminipyrimidines as leads for the development of new drug candidates for the treatment of HAT

SCYX-7158, an Orally-Active Benzoxaborole for the Treatment of Stage 2 Human African Trypanosomiasis

Human African trypanosomiasis (HAT) is caused by infection with the parasite Trypanosoma brucei and is an important public health problem in sub-Saharan Africa. New, safe, and effective drugs are urgently needed to treat HAT, particularly stage 2 disease where the parasite infects the brain. Existing therapies for HAT have poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. Through an integrated drug discovery project, we have discovered and optimized a novel class of boron-containing small molecules, benzoxaboroles, to deliver SCYX-7158, an orally active preclinical drug candidate. SCYX-7158 cured mice infected with T. brucei, both in the blood and in the brain. Extensive pharmacokinetic characterization of SCYX-7158 in rodents and non-human primates supports the potential of this drug candidate for progression to IND-enabling studies in advance of clinical trials for stage 2 HAT