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.

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

African sleeping sickness is a potentially deadly illness caused by the parasite Trypanosoma brucei. The disease is treatable, but many of the current treatments are old and are becoming increasingly ineffective. For instance, resistance is growing against pentamidine, a drug used in the early stages in the disease, as well as against melarsoprol, which is deployed when the infection has progressed to the brain. Usually, cases resistant to pentamidine are also resistant to melarsoprol, but it is still unclear why, as the drugs are chemically unrelated. Studies have shown that changes in a water channel called aquaglyceroporin 2 (TbAQP2) contribute to drug resistance in African sleeping sickness; this suggests that it plays a role in allowing drugs to kill the parasite. This molecular 'drain pipe' extends through the surface of T. brucei, and should allow only water and a molecule called glycerol in and out of the cell. In particular, the channel should be too narrow to allow pentamidine or melarsoprol to pass through. One possibility is that, in T. brucei, the TbAQP2 channel is abnormally wide compared to other members of its family. Alternatively, pentamidine and melarsoprol may only bind to TbAQP2, and then 'hitch a ride' when the protein is taken into the parasite as part of the natural cycle of surface protein replacement. Alghamdi et al. aimed to tease out these hypotheses. Computer models of the structure of the protein were paired with engineered changes in the key areas of the channel to show that, in T. brucei, TbAQP2 provides a much broader gateway into the cell than observed for similar proteins. In addition, genetic analysis showed that this version of TbAQP2 has been actively selected for during the evolution process of T. brucei. This suggests that the parasite somehow benefits from this wider aquaglyceroporin variant. This is a new resistance mechanism, and it is possible that aquaglyceroporins are also larger than expected in other infectious microbes. The work by Alghamdi et al. therefore provides insight into how other germs may become resistant to drugs.

Sustainable Elimination (Zero Cases) of Sleeping Sickness: How Far Are We from Achieving This Goal?

The recent massive reduction in the numbers of fresh Human African Trypanosomiasis (HAT) infection has presented an opportunity for the global elimination of this disease. To prevent a possible resurgence, as was the case after the reduced transmission of the 1960s, surveillance needs to be sustained and the necessary tools for detection and treatment of cases need to be made available at the points of care. In this review, we examine the available resources and make recommendations for improvement to ensure the sustenance of the already achieved gains to keep the trend moving towards elimination.

Urogenital schistosomiasis is associated with signatures of microbiome dysbiosis in Nigerian adolescents.

Urogenital schistosomiasis is a neglected tropical disease caused by the parasite Schistosoma haematobium, which resides in the vasculature surrounding the urogenital system. Previous work has suggested that helminthic infections can affect the intestinal microbiome, and we hypothesized that S. haematobium infection could result in an alteration of immune system-microbiota homeostasis and impact the composition of the gut microbiota. To address this question, we compared the fecal microbiomes of infected and uninfected schoolchildren from the Argungu Local Government Area of Kebbi State, Nigeria, detecting significant differences in community composition between the two groups. Most remarkably, we observed a decreased abundance of Firmicutes and increased abundance of Proteobacteria - a shift in community structure which has been previously associated with dysbiosis. More specifically, we detected a number of changes in lower taxa reminiscent of inflammation-associated dysbiosis, including decreases in Clostridiales and increases in Moraxellaceae, Veillonellaceae, Pasteurellaceae, and Desulfovibrionaceae. Functional potential analysis also revealed an enrichment in orthologs of urease, which has been linked to dysbiosis and inflammation. Overall, our analysis indicates that S. haematobium infection is associated with perturbations in the gut microbiota and may point to microbiome disruption as an additional consequence of schistosome infection.

The individual components of commercial isometamidium do not possess stronger trypanocidal activity than the mixture, nor bypass isometamidium resistance.

The four components present in the trypanocidal treatment Samorin, the commercially available formulation of isometamidium, were separated and purified by column chromatography. These compounds as well as the Samorin mixture and the other phenanthridine trypanocide, homidium, were tested on Trypanosoma congolense and wild type, diamidine- and isometamidium-resistant Trypanosoma brucei brucei strains using an Alamar blue drug sensitivity assay. EC50 values obtained suggest that M&B4180A (2) was the most active of the components, followed by M&B38897 (1) in all the strains tested, whereas M&B4596 (4) was inactive. Samorin was found to be significantly more active than any of the individual components alone, against T. congolense and all three T. b, brucei strains. Samorin and all its active constituents displayed reduced activity against the previously characterised isometamidium-resistant strain ISMR1.

Typhoid Fever Diagnosis in Endemic Countries: A Clog in the Wheel of Progress?

Typhoid fever causes significant morbidity and mortality in developing countries, with inaccurate estimates in some countries affected, especially those situated in Sub-Saharan Africa. Disease burden assessment is limited by lack of a high degree of sensitivity and specificity by many current rapid diagnostic tests. Some of the new technologies, such as PCR and proteomics, may also be useful but are difficult for low-resource settings to apply as point-of-care diagnostics. Weak laboratory surveillance systems may also contribute to the spread of multidrug resistant Salmonella serovar Typhi across endemic areas. In addition, most typhoid-endemic countries employ serological tests that have low sensitivity and specificity making diagnosis unreliable. Here we review currently available typhoid fever diagnostics, and advances in serodiagnosis of S. Typhi.

Tools for Detection of Schistosomiasis in Resource Limited Settings.

Schistosomiasis is a debilitating disease affecting over 200 million people, with the highest burden of morbidity and mortality in African countries. Despite its huge impact on the health and socio-economic burden of the society, it remains a neglected tropical disease, with limited attention from governments and stakeholders in healthcare. One of the critical areas that is hugely under-developed is the development of accurate diagnostics for both intestinal and urogenital schistosomiasis. Diagnosis of schistosomiasis is important for the detection and treatment of disease in endemic and non-endemic settings. A conclusive detection method is also an indispensable part of treatment, both in the clinic and during mass drug administration (MDA), for the monitoring efficacy of treatment. Here, we review the available diagnostic methods and discuss the challenges encountered in diagnosis in resource limited settings. We also present the available diagnostics and cost implications for deployment in resource limited settings. Lastly, we emphasize the need for more funding directed towards the development of affordable diagnostic tools that is affordable for endemic countries as we work towards the elimination of the disease.

Self-Medication with Antibiotics, Attitude and Knowledge of Antibiotic Resistance among Community Residents and Undergraduate Students in Northwest Nigeria.

This study set out to evaluate self-medicated antibiotics and knowledge of antibiotic resistance among undergraduate students and community members in northern Nigeria. Antibiotic consumption pattern, source of prescription, illnesses commonly treated, attitude towards antibiotics, and knowledge of antibiotic resistance were explored using a structured questionnaire. Responses were analyzed and summarized using descriptive statistics. Of the 1230 respondents from undergraduate students and community members, prescription of antibiotics by a physician was 33% and 57%, respectively, amongst undergraduate students and community members. We tested the respondents’ knowledge of antibiotic resistance (ABR) and found that undergraduate students displayed less knowledge that self-medication could lead to ABR (32.6% and 42.2% respectively). Self-medication with antibiotics is highly prevalent in Northwest Nigeria, with most medicines being purchased from un-licensed stores without prescription from a physician. We also observed a significant gap in respondents’ knowledge of ABR. There is an urgent need for public health authorities in Nigeria to enforce existing laws on antibiotics sales and enlighten the people on the dangers of ABR.

Prevalence of trypanosome infection in tsetse flies from Oji River and Emene axis of Enugu State, Nigeria: A preliminary report.

INTRODUCTION: Trypanosomes are protozoan parasites of vertebrates transmitted by blood-sucking tsetse fly. Trypanosomes remain a constant threat to the lives of humans and animals throughout large regions of Africa. AIMS AND OBJECTIVES: This study investigated the presence, prevalence, and species of trypanosome parasite in tsetse flies caught in two areas of no previous documented history of trypanosome infection. MATERIALS AND METHODS: For this purpose, 63 and 77 nonterenal tsetse flies were collected from Oji River and Emene areas of Enugu State Nigeria, respectively. Genomic DNA was isolated from the whole tsetse fly using genomic DNA extraction kit. Identification and characterization of trypanosome were done using two approaches: the amplification of internal transcribed spacer 1 of ribosomal DNA and the use of primers specific to Trypanozoon. RESULTS: In Oji River, of 63 tsetse flies collected, the identification of trypanosome parasite was done on 57 flies and 6 (10.71%) tsetse flies were infected with trypanosome parasite. Six flies were infected with Trypanosoma Congolense, 2 with Trypanosoma Vivax, and 1 with Trypanosoma brucei. Two mixed infections of T. vivax and T. congolense and 1 mixed infection of T. brucei and T. congolense was also identified. In Emene, of 77 tsetse flies collected, the identification of trypanosome parasite was done on 66 flies and 11 (16.6%) tsetse flies were infected with trypanosome parasite. Nine flies were infected with T. congolense, 2 with T. vivax, and 3 with T. brucei. Mixed infections identified include 2 mixed infections of T. brucei and T. congolense and 1 mixed infections of T. vivax and T. brucei. None of the subspecies of T. brucei were detected using species specific primers. DISCUSSION: This study shows the parasitological evidence on the occurrence of animal African trypanosomiasis and also demonstrated that there is likely no active transmission of human African trypanosomiasis in the study areas. CONCLUSION: This study shows that there is likely no active transmission of human African trypanosomiasis going on in these localities since no human infective form of the parasite was detected.

Monotherapy with amlodipine or hydrochlorothiazide in patients with mild to moderate hypertension: Comparison of their efficacy and effects on electrolytes.

BACKGROUND: Amlodipine and hydrochlorothiazide (HCTZ) are commonly prescribed in Nigeria either as a monotherapy or in combination with other drugs. The present study was designed to investigate the antihypertensive efficacy of monotherapy with amlodipine or HCTZ and their effects on electrolyte profile in patients with mild to moderate hypertension. METHODS: A single-blind randomized clinical study was used; fifty patients newly diagnosed with mild to moderate hypertension (aged 33 to 60 years) were recruited and divided into two groups: amlodipine or hydrochlorothiazide each comprising of 25 subjects. The subjects received 5mg of amlodipine or 25mg of hydrochlorothiazide in their respective group once daily for 4 weeks. Blood pressure, serum and urine electrolytes were measured at baseline and weekly throughout the experiment. RESULTS: At the end of follow up, amlodipine reduced systolic and diastolic blood pressure significantly more (p<0.001) than HCTZ. At the end of follow up, blood pressure was reduced to normal in 80% of the subjects in amlodipine group compared to 50% in HCTZ. Amlodipine had no significant effect on electrolyte profile of subjects unlike HCTZ which significantly changed both their serum and urine electrolytes. CONCLUSIONS: Monotherapy with amlodipine was more effective than HCTZ in black patients with mild to moderate hypertension and in addition maintained electrolyte balance.

Functional and genetic evidence that nucleoside transport is highly conserved in Leishmania species: Implications for pyrimidine-based chemotherapy.

Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy.

Bioassay-guided isolation of active principles from Nigerian medicinal plants identifies new trypanocides with low toxicity and no cross-resistance to diamidines and arsenicals.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles. AIM OF THE STUDY: To investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro. MATERIAL AND METHODS: Plants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin. RESULTS: Seven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38µg/mL, while a triterpenoid (3ß,13ß-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC50 of 1.58µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense. CONCLUSION: We have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species.

Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase.

BACKGROUND: Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. METHODOLOGY/PRINCIPAL FINDINGS: Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. CONCLUSIONS/SIGNIFICANCE: Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential.

Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei adenosine transporter 1 (TbAT1) and the proposal of a structural model for the protein.

The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1(-/-) T. brucei line. Out of a set of six reported SNPs from a reported 'resistance allele', none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [(3)H]-adenosine and [(3)H]-diminazene, identical to the tbat1(-/-) control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [(3)H]-diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.

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.

A new nonpolar N-hydroxy imidazoline lead compound with improved activity in a murine model of late-stage Trypanosoma brucei brucei infection is not cross-resistant with diamidines.

Treatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four new N-hydroxy and 12 new N-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluated in vitro against Trypanosoma brucei rhodesiense and in vivo in murine models of first- and second-stage sleeping sickness. Resistance profile, physicochemical parameters, in vitro BBB permeability, and microsomal stability also were determined. The N-hydroxy imidazoline analogues were the most effective in vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was found in vitro, suggesting that N-hydroxy imidazolines are metabolically stable and have intrinsic activity against T. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d in T. brucei was independent of known drug transporters (i.e., T. brucei AT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, the N-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.

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.

Structure-activity relationships of synthetic cordycepin analogues as experimental therapeutics for African trypanosomiasis.

Novel methods for treatment of African trypanosomiasis, caused by infection with Trypanosoma brucei are needed. Cordycepin (3'-deoxyadenosine, 1a) is a powerful trypanocidal compound in vitro but is ineffective in vivo because of rapid metabolic degradation by adenosine deaminase (ADA). We elucidated the structural moieties of cordycepin required for trypanocidal activity and designed analogues that retained trypanotoxicity while gaining resistance to ADA-mediated metabolism. 2-Fluorocordycepin (2-fluoro-3'-deoxyadenosine, 1b) was identified as a selective, potent, and ADA-resistant trypanocidal compound that cured T. brucei infection in mice. Compound 1b is transported through the high affinity TbAT1/P2 adenosine transporter and is a substrate of T. b. brucei adenosine kinase. 1b has good preclinical properties suitable for an oral drug, albeit a relatively short plasma half-life. We present a rapid and efficient synthesis of 2-halogenated cordycepins, also useful synthons for the development of additional novel C2-substituted 3'-deoxyadenosine analogues to be evaluated in development of experimental therapeutics.

Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake.

It has long been established that the Trypanosoma brucei TbAT1/P2 aminopurine transporter is involved in the uptake of diamidine and arsenical drugs including pentamidine, diminazene aceturate and melarsoprol. Accordingly, it was proposed that the closest Trypanosoma congolense paralogue, TcoAT1, might perform the same function in this parasite, and an apparent correlation between a Single Nucleotide Polymorphism (SNP) in that gene and diminazene tolerance was reported for the strains examined. Here, we report the functional cloning and expression of TcoAT1 and show that in fact it is the syntenic homologue of another T. brucei gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The T. congolense genome does not seem to contain a syntenic equivalent to TbAT1. Two TcoAT1 alleles, differentiated by three independent SNPs, were expressed in the T. brucei clone B48, a TbAT1-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); TbAT1 was also expressed as a control. The TbAT1 and TcoAT1 transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only TbAT1 increased sensitivity to diamidines and to cymelarsan. Uptake of [(3)H]-diminazene was detectable only in the B48 cells expressing TbAT1 but not TcoAT1, whereas uptake of [(3)H]-inosine was increased by both TcoAT1 alleles but not by TbAT1. Uptake of [(3)H]-adenosine was increased by all three ENT genes. We conclude that TcoAT1 is a P1-type purine nucleoside transporter and the syntenic equivalent to the previously characterised TbNT10; it does not mediate diminazene uptake and is therefore unlikely to play a role in diminazene resistance in T. congolense.

Synthesis and structure-activity analysis of new phosphonium salts with potent activity against African trypanosomes.

A series of 73 bisphosphonium salts and 10 monophosphonium salt derivatives were synthesized and tested in vitro against several wild type and resistant lines of Trypanosoma brucei (T. b. rhodesiense STIB900, T. b. brucei strain 427, TbAT1-KO, and TbB48). More than half of the compounds tested showed a submicromolar EC(50) against these parasites. The compounds did not display any cross-resistance to existing diamidine therapies, such as pentamidine. In most cases, the compounds displayed a good selectivity index versus human cell lines. None of the known T. b. brucei drug transporters were required for trypanocidal activity, although some of the bisphosphonium compounds inhibited the low affinity pentamidine transporter. It was found that phosphonium drugs act slowly to clear a trypanosome population but that only a short exposure time is needed for irreversible damage to the cells. A comparative molecular field analysis model (CoMFA) was generated to gain insights into the SAR of this class of compounds, identifying key features for trypanocidal activity.