Cloning and Characterization of Trypanosoma congolense and T. vivax Nucleoside Transporters Reveal the Potential of P1-Type Carriers for the Discovery of Broad-Spectrum Nucleoside-Based Therapeutics against Animal African Trypanosomiasis.

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei, T. vivax and T. congolense, is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line ('SUPKO') lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3'-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC50s were similar for T. b. brucei, T. congolense, T. evansi and T. equiperdum but correlated less well with T. vivax. However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

The Activity of Red Nigerian Propolis and Some of Its Components against Trypanosoma brucei and Trypanosoma congolense.

Propolis is a resin that is gathered by bees from exudates produced by various plants. Its exact chemical composition depends on the plants available near the hive. Bees use propolis to coat the surfaces of the hive, where it acts as an anti-infective. Regardless of the chemical composition of propolis, it is always anti-protozoal, probably because protozoan parasites, particularly Lotmarium passim, are widespread in bee populations. The protozoa Trypanosoma brucei and T. congolense cause disease in humans and/or animals. The existing drugs for treating these diseases are old and resistance is an increasingly severe problem. The many types of propolis present a rich source of anti-trypanosomal compounds-from a material gathered by bees in an environmentally friendly way. In the current work, red Nigerian propolis from Rivers State, Nigeria was tested against T. brucei and T. congolense and found to be highly active (EC50 1.66 and 4.00 µg/mL, respectively). Four isoflavonoids, vestitol, neovestitol, 7-methylvestitol and medicarpin, were isolated from the propolis. The isolated compounds were also tested against T. brucei and T. congolense, and vestitol displayed the highest activity at 3.86 and 4.36 µg/mL, respectively. Activities against drug-resistant forms of T. brucei and T. congolense were similar to those against wild type.

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter.

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.

Differences in Transporters Rather than Drug Targets Are the Principal Determinants of the Different Innate Sensitivities of Trypanosoma congolense and Trypanozoon Subgenus Trypanosomes to Diamidines and Melaminophenyl Arsenicals.

The animal trypanosomiases are infections in a wide range of (domesticated) animals with any species of African trypanosome, such as Trypanosoma brucei, T. evansi, T. congolense, T. equiperdum and T. vivax. Symptoms differ between host and infective species and stage of infection and are treated with a small set of decades-old trypanocides. A complication is that not all trypanosome species are equally sensitive to all drugs and the reasons are at best partially understood. Here, we investigate whether drug transporters, mostly identified in T. b. brucei, determine the different drug sensitivities. We report that homologues of the aminopurine transporter TbAT1 and the aquaporin TbAQP2 are absent in T. congolense, while their introduction greatly sensitises this species to diamidine (pentamidine, diminazene) and melaminophenyl (melarsomine) drugs. Accumulation of these drugs in the transgenic lines was much more rapid. T. congolense is also inherently less sensitive to suramin than T. brucei, despite accumulating it faster. Expression of a proposed suramin transporter, located in T. brucei lysosomes, in T. congolense, did not alter its suramin sensitivity. We conclude that for several of the most important classes of trypanocides the presence of specific transporters, rather than drug targets, is the determining factor of drug efficacy.

Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells' ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

The Antiprotozoal Activity of Papua New Guinea Propolis and Its Triterpenes.

Profiling a propolis sample from Papua New Guinea (PNG) using high-resolution mass spectrometry indicated that it contained several triterpenoids. Further fractionation by column chromatography and medium-pressure liquid chromatography (MPLC) followed by nuclear magnetic resonance spectroscopy (NMR) identified 12 triterpenoids. Five of these were obtained pure and the others as mixtures of two or three compounds. The compounds identified were: mangiferonic acid, ambonic acid, isomangiferolic acid, ambolic acid, 27-hydroxyisomangiferolic acid, cycloartenol, cycloeucalenol, 24-methylenecycloartenol, 20-hydroxybetulin, betulin, betulinic acid and madecassic acid. The fractions from the propolis and the purified compounds were tested in vitro against Crithidia fasciculata, Trypanosoma congolense, drug-resistant Trypanosoma congolense, Trypanosoma b. brucei and multidrug-resistant Trypanosoma b. brucei (B48). They were also assayed for their toxicity against U947 cells. The compounds and fractions displayed moderate to high activity against parasitic protozoa but only low cytotoxicity against the mammalian cells. The most active isolated compound, 20-hydroxybetulin, was found to be trypanostatic when different concentrations were tested against T. b. brucei growth.

Activity of Compounds from Temperate Propolis against Trypanosoma brucei and Leishmania mexicana.

Ethanolic extracts of samples of temperate zone propolis, four from the UK and one from Poland, were tested against three Trypanosoma brucei strains and displayed EC50 values < 20 µg/mL. The extracts were fractionated, from which 12 compounds and one two-component mixture were isolated, and characterized by NMR and high-resolution mass spectrometry, as 3-acetoxypinobanksin, tectochrysin, kaempferol, pinocembrin, 4'-methoxykaempferol, galangin, chrysin, apigenin, pinostrobin, cinnamic acid, coumaric acid, cinnamyl ester/coumaric acid benzyl ester (mixture), 4',7-dimethoxykaempferol, and naringenin 4',7-dimethyl ether. The isolated compounds were tested against drug-sensitive and drug-resistant strains of T. brucei and Leishmania mexicana, with the highest activities ≤ 15 µM. The most active compounds against T. brucei were naringenin 4',7 dimethyl ether and 4'methoxy kaempferol with activity of 15-20 µM against the three T. brucei strains. The most active compounds against L. mexicana were 4',7-dimethoxykaempferol and the coumaric acid ester mixture, with EC50 values of 12.9 ± 3.7 µM and 13.1 ± 1.0 µM. No loss of activity was found with the diamidine- and arsenical-resistant or phenanthridine-resistant T. brucei strains, or the miltefosine-resistant L. mexicana strain; no clear structure activity relationship was observed for the isolated compounds. Temperate propolis yields multiple compounds with anti-kinetoplastid activity.

Investigation of 5'-Norcarbocyclic Nucleoside Analogues as Antiprotozoal and Antibacterial Agents.

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein.

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.

Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis.

OBJECTIVES: To evaluate existing protocols, based on Alamar blue (resazurin), for the routine determination of drug susceptibility in trichomonads, develop new ones and validate these by screening small antiprotozoal libraries. METHODS: The resazurin-based assay was evaluated by determining fluorescence development in Trichomonas media with various cell densities after various intervals and in the presence of metronidazole. Similar investigations were performed with the alternative fluorophores propidium iodide (PI) and resorufin. The optimized protocols were used to screen for new antitrichomonal compounds. RESULTS: Anaerobic cultures of Trichomonas vaginalis rapidly reduced blue resazurin to red, fluorescent resorufin. However, the ascorbic acid in the culture medium produced similar effects, even in the absence of cells, causing high background fluorescence and variability. Moreover, T. vaginalis rapidly metabolized resorufin to the non-fluorescent and colourless metabolite dihydroresorufin, making the fluorescent signal transient. In contrast, resorufin proved to be an excellent viability probe for Trichomonas due to its chemical stability in media and rapid metabolism by the parasite. We also show that staining with PI after cell permeabilization similarly constitutes a reliable measurement of trophozoite numbers. Using the PI and resorufin assays we determined reproducible EC(50) values and identified potent antitrichomonal compounds from a limited screen of phosphodiesterase inhibitors and phosphonium salts. CONCLUSIONS: The resorufin- and PI-based assays are suitable for routine and high-throughput drug screening, whereas resazurin-based assays are not. These assays constitute a major advance in the current protocols as demonstrated by a successful screen for new antitrichomonal lead compounds.

Deazapurine Nucleoside Analogues for the Treatment of Trichomonas vaginalis.

Trichomoniasis is the most common nonviral sexually transmitted disease in humans, but treatment options are limited. Here, we report a resorufin-based drug sensitivity assay for high-throughput microplate-based screening under hypoxic conditions. A 5203-compound enamine kinase library and several specialized compound series were tested for the inhibition of Trichomonas growth at 10 µM with Z' values of >0.5. Hits were rescreened in serial dilution to establish an IC50 concentration. A series of 7-substituted 7-deazaadenosine analogues emerged as highly potent anti-T. vaginalis agents, with EC50 values in the low double digit nanomolar range. These analogues exhibited excellent selectivity indices. Follow-up medicinal chemistry efforts identified an optimal ribofuranose and C7 substituent. Several nucleosides rapidly cleared cultures of T. vaginalis at a concentrations of just 2 × EC50. Preliminary in vivo evaluation in a murine trichomoniasis model (Tritrichomonas foetus) revealed promising activity upon topical administration, validating purine nucleoside analogues as a new class of antitrichomonal agents.

Antiparasitic and Cytotoxic Activity of Bokkosin, A Novel Diterpene-Substituted Chromanyl Benzoquinone From Calliandra portoricensis.

Calliandra portoricensis is a medicinal plant growing freely in Nigeria. It is used traditionally to treat tuberculosis, as an anthelmintic and an abortifacient. Phytochemical fractionation and screening of its root extracts has yielded a novel (5-hydroxy-7-methoxy-4-oxo-1-chromanyl)-4-methoxy-p-benzoquinone (breverin)-substituted cassane diterpene, which was designated bokkosin. It was obtained from column chromatography of the ethyl acetate extract of the roots. The compound was characterized using IR, NMR (1D and 2D) and mass spectral data. Promising antiparasitic activity was observed against the kinetoplastid parasite Trypanosoma brucei brucei, as well as moderate activity against Trypanosoma congolense and Leishmania mexicana and low toxicity in mammalian cells, with the best in vitro EC50 values against T. b. brucei (0.69 µg/mL against a standard laboratory strain, and its multi-drug resistant clone (0.33 µg/mL). The effect on T. b. brucei in culture was rapid and dose-dependent, leading to apparently irreversible growth arrest and cell death after an exposure of just 2 h at 2 × or 4 × EC50. The identification of bokkosin constitutes the first isolation of this class of compound from any natural source and establishes the compound as a potential trypanocide that, considering its novelty, should now be tested for activity against other microorganisms as well.

Novel flavanones with anti-trypanosomal activity isolated from Zambian and Tanzanian propolis samples.

A bioassay-guided phytochemical investigation of propolis samples from Tanzania and Zambia that screened for activity against Trypanosoma brucei has led to the isolation of two novel flavanones with promising antitrypanosomal activity. The compounds were characterized based on their spectral and physical data and identified as 6-(1,1-dimethylallyl) pinocembrin and 5-hydroxy-4″,4″-dimethyl-5″-methyl-5″-H-dihydrofuranol [2″,3″,6,7] flavanone. The two compounds, together with the propolis extracts and fractions, were assayed against a standard drug-sensitive strain of T. b. brucei (s427 wild-type), multi-drug resistant-resistant T. b. brucei (B48), drug-sensitive T. congolense (1L300) and a derived diminazene-resistant T. congolense strain (6C3), and for toxicity against U947 human cells and RAW 246.7 murine cells. Activity against T. b. brucei was higher than against T. congolense. Interestingly, the Tanzanian propolis extract was found to be more active than its fractions and purified compounds in these assays, with an IC50 of 1.20 µg/mL against T. b. brucei. The results of a cytotoxicity assay showed that the propolis extracts were less toxic than the purified compounds with mean IC50 values > 165.0 µg/mL.

Protocols for the routine screening of drug sensitivity in the human parasite Trichomonas vaginalis.

Trichomonas vaginalis is a sexually transmitted protozoan parasite of humans. Treatment of trichomoniasis is almost completely dependent on the old drug metronidazole and is hampered by resistance. New drug development, like routine screening for drug resistance, has however been hampered by the lack of reliable screening protocols with sufficient throughput. Here we report on two separate in vitro protocols that use fluorescent dyes and allow for standardized drug sensitivity testing on the required scale.

Trypanosoma brucei: expression of multiple purine transporters prevents the development of allopurinol resistance.

Allopurinol is a hypoxanthine analogue used to treat Leishmania infections that also displays activity against the related parasite Trypanosoma brucei. We have investigated the ease by which resistance to this drug is established in Trypanosoma brucei brucei and correlated this to the mechanisms by which it is accumulated by the parasite. Long-term exposure of procyclic T. b. brucei to 3mM allopurinol did not induce resistance. This appears to be related to the fact that allopurinol was taken up through two distinct nucleobase transporters, H1 and H4, both with high affinity for the drug. The apparent Km for [3H]allopurinol transport by H4 (2.1+/-0.4 microM) was determined by expressing the encoding gene in Saccharomyces cerevisiae. Long-term allopurinol exposure did not change Km (hypoxanthine), Ki (allopurinol), or Vmax values of either H1 or H4 transporters and the cells retained their ability to proliferate with hypoxanthine as sole purine source. This study shows that transport-related resistance to purine antimetabolites is not easily induced in Trypanosoma spp. as long as uptake is mediated by multiple transporters.