RESUMEN
Subspecies of the protozoan parasite Trypanosoma brucei are the causative agents of Human African Trypanosomiasis (HAT), a debilitating neglected tropical disease prevalent across sub-Saharan Africa. HAT case numbers have steadily decreased since the start of the century, and sustainable elimination of one form of the disease is in sight. However, key to this is the development of novel drugs to combat the disease. Acoziborole is a recently developed benzoxaborole, currently in advanced clinical trials, for treatment of stage 1 and stage 2 HAT. Importantly, acoziborole is orally bioavailable, and curative with one dose. Recent studies have made significant progress in determining the molecular mode of action of acoziborole. However, less is known about the potential mechanisms leading to acoziborole resistance in trypanosomes. In this study, an in vitro-derived acoziborole-resistant cell line was generated and characterised. The AcoR line exhibited significant cross-resistance with the methyltransferase inhibitor sinefungin as well as hypersensitisation to known trypanocides. Interestingly, transcriptomics analysis of AcoR cells indicated the parasites had obtained a procyclic- or stumpy-like transcriptome profile, with upregulation of procyclin surface proteins as well as differential regulation of key metabolic genes known to be expressed in a life cycle-specific manner, even in the absence of major morphological changes. However, no changes were observed in transcripts encoding CPSF3, the recently identified protein target of acoziborole. The results suggest that generation of resistance to this novel compound in vitro can be accompanied by transcriptomic switches resembling a procyclic- or stumpy-type phenotype.
Asunto(s)
Resistencia a Medicamentos , Proteínas Protozoarias/genética , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/parasitología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.
Asunto(s)
Compuestos de Boro/metabolismo , Carboxipeptidasas/metabolismo , Tripanocidas/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma congolense/enzimología , Trypanosoma vivax/enzimología , Tripanosomiasis Africana/veterinaria , Valina/análogos & derivados , Animales , Ácidos Carboxílicos/metabolismo , Resistencia a Medicamentos , Femenino , Ganado , Ratones , Parasitemia/veterinaria , Profármacos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma congolense/efectos de los fármacos , Trypanosoma vivax/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología , Valina/metabolismoRESUMEN
The global prevalence of antibacterial resistance requires new antibacterial drugs with novel chemical scaffolds and modes of action. It is also vital to design compounds with optimal physicochemical properties to permeate the bacterial cell envelope. We described an approach of combining and integrating whole cell screening and metabolomics into early antibacterial drug discovery using a library of small polar compounds. Whole cell screening of a diverse library of small polar compounds against Staphylococcus aureus gave compound 2. Hit expansion was carried out to determine structure-activity relationships. A selection of compounds from this series, together with other screened active compounds, was subjected to an initial metabolomics study to provide a metabolic fingerprint of the mode of action. It was found that compound 2 and its analogues have a different mode of action from some of the known antibacterial compounds tested. This early study highlighted the potential of whole cell screening and metabolomics in early antibacterial drug discovery. Future works will require improving potency and performing orthogonal studies to confirm the modes of action.
RESUMEN
Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.
Asunto(s)
Benzoxazoles/farmacología , ARN Protozoario/metabolismo , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/metabolismo , Animales , Benzoxazoles/química , Bovinos , Resistencia a Medicamentos/genética , Cabras , Humanos , Ratones , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , Trans-Empalme/efectos de los fármacos , Tripanocidas/química , Trypanosoma brucei brucei/genética , Trypanosoma congolense/efectos de los fármacos , Trypanosoma congolense/genética , Trypanosoma congolense/metabolismo , Trypanosoma vivax/efectos de los fármacos , Trypanosoma vivax/genética , Trypanosoma vivax/metabolismo , Tripanosomiasis/tratamiento farmacológico , Tripanosomiasis/parasitologíaRESUMEN
The parasitic protozoan Trypanosoma brucei causes Human African Trypanosomiasis and Nagana in other mammals. These diseases present a major socio-economic burden to large areas of sub-Saharan Africa. Current therapies involve complex and toxic regimens, which can lead to fatal side-effects. In addition, there is emerging evidence for drug resistance. AN5568 (SCYX-7158) is a novel benzoxaborole class compound that has been selected as a lead compound for the treatment of HAT, and has demonstrated effective clearance of both early and late stage trypanosomiasis in vivo. The compound is currently awaiting phase III clinical trials and could lead to a novel oral therapeutic for the treatment of HAT. However, the mode of action of AN5568 in T. brucei is unknown. This study aimed to investigate the mode of action of AN5568 against T. brucei, using a combination of molecular and metabolomics-based approaches.Treatment of blood-stage trypanosomes with AN5568 led to significant perturbations in parasite metabolism. In particular, elevated levels of metabolites involved in the metabolism of S-adenosyl-L-methionine, an essential methyl group donor, were found. Further comparative metabolomic analyses using an S-adenosyl-L-methionine-dependent methyltransferase inhibitor, sinefungin, showed the presence of several striking metabolic phenotypes common to both treatments. Furthermore, several metabolic changes in AN5568 treated parasites resemble those invoked in cells treated with a strong reducing agent, dithiothreitol, suggesting redox imbalances could be involved in the killing mechanism.
Asunto(s)
Benzoxazinas/farmacología , S-Adenosilmetionina/metabolismo , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/metabolismo , Humanos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/parasitologíaRESUMEN
Priming and activating immune stimuli have profound effects on macrophages, however, studies generally evaluate stimuli in isolation rather than in combination. In this study we have investigated the effects of pro-inflammatory and anti-inflammatory stimuli either alone or in combination on macrophage metabolism. These stimuli include host factors such as IFNγ and ovalbumin-immunoglobulin immune complexes, or pathogen factors such as LPS. Untargeted LC-MS based metabolomics provided an in-depth profile of the macrophage metabolome, and revealed specific changes in metabolite abundance upon either individual stimuli or combined stimuli. Here, by factoring in an interaction term in the linear model, we define the metabolome interactome. This approach allowed us to determine whether stimuli interact in a synergistic or antagonistic manner. In conclusion this study demonstrates a robust approach to interrogate immune-metabolism, especially systems that model host-pathogen interactions.
Asunto(s)
Inflamación/metabolismo , Macrófagos/metabolismo , Metaboloma , Animales , Células Cultivadas , Inmunoglobulinas/metabolismo , Interferón gamma/metabolismo , Lipopolisacáridos/metabolismo , Metabolómica/métodos , Ratones Endogámicos C57BL , Ovalbúmina/metabolismoRESUMEN
Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Amina Oxidasa (conteniendo Cobre)/metabolismo , Compuestos de Boro/metabolismo , Modelos Biológicos , Profármacos/metabolismo , Tripanocidas/metabolismo , Trypanosoma brucei brucei/enzimología , Activación Metabólica , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa/química , Aldehído Deshidrogenasa/genética , Aldehído Oxidorreductasas/antagonistas & inhibidores , Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Amina Oxidasa (conteniendo Cobre)/química , Amina Oxidasa (conteniendo Cobre)/genética , Sustitución de Aminoácidos , Animales , Compuestos de Boro/química , Compuestos de Boro/farmacología , Resistencia a Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Mutación , Filogenia , Profármacos/química , Profármacos/farmacología , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/fisiologíaRESUMEN
Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme's active site, consistent with the fact that the resistant line continues to produce the enzyme's product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.
Asunto(s)
Anfotericina B/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/enzimología , Mutación Missense , Esterol 14-Desmetilasa/genética , Ergosterol/análisis , Prueba de Complementación Genética , Genoma de Protozoos , Leishmania mexicana/química , Metabolómica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Polimorfismo de Nucleótido Simple , Esterol 14-Desmetilasa/metabolismoRESUMEN
Leishmania infantum is an etiological agent of the life-threatening visceral form of leishmaniasis. Liposomal amphotericin B (AmB) followed by a short administration of miltefosine (MF) is a drug combination effective for treating visceral leishmaniasis in endemic regions of India. Resistance to MF can be due to point mutations in the miltefosine transporter (MT). Here we show that mutations in MT are also observed in Leishmania AmB-resistant mutants. The MF-induced MT mutations, but not the AmB induced mutations in MT, alter the translocation/uptake of MF. Moreover, mutations in the MT selected by AmB or MF have a major impact on lipid species that is linked to cross-resistance between both drugs. These alterations include changes of specific phospholipids, some of which are enriched with cyclopropanated fatty acids, as well as an increase in inositolphosphoceramide species. Collectively these results provide evidence of the risk of cross-resistance emergence derived from current AmB-MF sequential or co-treatments for visceral leishmaniasis.
Asunto(s)
Anfotericina B/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos , Leishmania infantum/efectos de los fármacos , Leishmania infantum/enzimología , Leishmaniasis/parasitología , Mutación , Fosforilcolina/análogos & derivados , ATPasas de Translocación de Protón/metabolismo , Proteínas Protozoarias/genética , Humanos , Leishmania infantum/genética , Leishmaniasis/inmunología , Fosforilcolina/farmacología , ATPasas de Translocación de Protón/genética , Proteínas Protozoarias/metabolismoRESUMEN
Treatment for human African trypanosomiasis is dependent on the species of trypanosome causing the disease and the stage of the disease (stage 1 defined by parasites being present in blood and lymphatics whilst for stage 2, parasites are found beyond the blood-brain barrier in the cerebrospinal fluid (CSF)). Currently, staging relies upon detecting the very low number of parasites or elevated white blood cell numbers in CSF. Improved staging is desirable, as is the elimination of the need for lumbar puncture. Here we use metabolomics to probe samples of CSF, plasma and urine from 40 Angolan patients infected with Trypanosoma brucei gambiense, at different disease stages. Urine samples provided no robust markers indicative of infection or stage of infection due to inherent variability in urine concentrations. Biomarkers in CSF were able to distinguish patients at stage 1 or advanced stage 2 with absolute specificity. Eleven metabolites clearly distinguished the stage in most patients and two of these (neopterin and 5-hydroxytryptophan) showed 100% specificity and sensitivity between our stage 1 and advanced stage 2 samples. Neopterin is an inflammatory biomarker previously shown in CSF of stage 2 but not stage 1 patients. 5-hydroxytryptophan is an important metabolite in the serotonin synthetic pathway, the key pathway in determining somnolence, thus offering a possible link to the eponymous symptoms of "sleeping sickness". Plasma also yielded several biomarkers clearly indicative of the presence (87% sensitivity and 95% specificity) and stage of disease (92% sensitivity and 81% specificity). A logistic regression model including these metabolites showed clear separation of patients being either at stage 1 or advanced stage 2 or indeed diseased (both stages) versus control.
Asunto(s)
Biomarcadores/análisis , Trypanosoma brucei gambiense/metabolismo , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/parasitología , 5-Hidroxitriptófano/sangre , 5-Hidroxitriptófano/líquido cefalorraquídeo , 5-Hidroxitriptófano/aislamiento & purificación , 5-Hidroxitriptófano/orina , Adolescente , Adulto , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/orina , Barrera Hematoencefálica , Femenino , Humanos , Masculino , Metabolómica/métodos , Neopterin/sangre , Neopterin/líquido cefalorraquídeo , Neopterin/aislamiento & purificación , Neopterin/orina , Análisis de Regresión , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5'-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanidem-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5'-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Metaboloma/efectos de los fármacos , Metabolómica , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Aciltransferasas/metabolismo , Adenosina/metabolismo , Adenosina/farmacología , Antibacterianos/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Ceftazidima/metabolismo , Ceftazidima/farmacología , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Cromatografía Liquida , ADN Bacteriano/antagonistas & inhibidores , ADN Bacteriano/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/metabolismo , Fosfomicina/farmacología , Expresión Génica , Células HEK293 , Humanos , Ácidos Hidroxámicos/metabolismo , Ácidos Hidroxámicos/farmacología , Espectrometría de Masas , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacología , Terpenos/antagonistas & inhibidores , Terpenos/metabolismo , Treonina/análogos & derivados , Treonina/metabolismo , Treonina/farmacología , Triclosán/metabolismo , Triclosán/farmacologíaRESUMEN
Antimony (SbIII) and miltefosine (MIL) are important drugs for the treatment of Leishmania parasite infections. The mitochondrion is likely to play a central role in SbIII and MIL induced cell death in this parasite. Enriched mitochondrial samples from Leishmania promastigotes selected step by step for in vitro resistance to SbIII and MIL were subjected to differential proteomic analysis. A shared decrease in both mutants in the levels of pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, and isocitrate dehydrogenase was observed, as well as a differential abundance in two calcium-binding proteins and the unique dynamin-1-like protein of the parasite. Both mutants presented a shared increase in the succinyl-CoA:3-ketoacid-coenzyme A transferase and the abundance of numerous hypothetical proteins was also altered in both mutants. In general, the proteomic changes observed in the MIL mutant were less pronounced than in the SbIII mutant, probably due to the early appearance of a mutation in the miltefosine transporter abrogating the need for a strong mitochondrial adaptation. This study is the first analysis of the Leishmania mitochondrial proteome and offers powerful insights into the adaptations to this organelle during SbIII and MIL drug resistance.
RESUMEN
Metabolomics-based studies are proving of great utility in the analysis of modes of action (MOAs) and resistance mechanisms of drugs in parasitic protozoa. They have helped to determine the MOA of eflornithine, half of the gold standard combination therapy in use against human African trypanosomiasis (HAT), as well as the mechanism of resistance to this drug. In Leishmania, metabolomics has also given insight into the MOA of miltefosine, an alkylphospholipid. Several studies on antimony resistance in Leishmania have been conducted, analyzing the metabolic content of resistant lines, offering clues as to the MOA of this class of drugs. A study of chloroquine resistance in Plasmodium falciparum combined metabolomics techniques with other genetic and proteomic techniques to offer new insight into the role of the PfCRT protein. The MOA and mechanism of resistance to a group of halogenated pyrimidines in Trypanosoma brucei have also recently been elucidated. Effective as metabolomics techniques are, care must be taken in the design and implementation of these experiments, to ensure the resulting data are meaningful. This review outlines the steps required to conduct a metabolomics experiment as well as provide an overview of metabolomics-based drug research in protozoa to date.
Asunto(s)
Antiparasitarios/farmacología , Antiparasitarios/uso terapéutico , Descubrimiento de Drogas/métodos , Metabolómica/métodos , Parásitos/efectos de los fármacos , Parásitos/metabolismo , Animales , Resistencia a Medicamentos , Humanos , InvestigaciónRESUMEN
The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.
Asunto(s)
Bases de Datos de Compuestos Químicos , Trypanosoma brucei brucei/metabolismo , Minería de Datos , Internet , Redes y Vías Metabólicas , Proteómica , Trypanosoma brucei brucei/genéticaRESUMEN
BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between distinct morphological and functional forms during its life cycle. Axenic multiplication and differentiation processes of this protozoan parasite can be reproduced in vitro, enabling the isolation and study of the different evolutionary forms. Although there are several publications attempting the cultivation of T. cruzi under chemically defined conditions, in our experience none of the published media are capable of maintaining T. cruzi in continuous growth. RESULTS: In this work we modified a known chemically defined medium for Trypanosoma brucei growth. The resulting LM14 and LM14B defined media enabled cultivation of five different strains of T. cruzi for more than forty passages until now. The parasite's biological characteristics such as morphology and differentiation to metacyclic trypomastigotes were maintained when defined media is used. CONCLUSIONS: The establishment of a defined medium for T. cruzi cultivation is an important tool for basic biological research allowing several different approaches, providing new perspectives for further studies related to cell biology of this parasite.
Asunto(s)
Medios de Cultivo/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
There are many theories as to the mode of action of miltefosine against Leishmania including alterations to the membrane lipid content, induction of apoptosis and modulation of macrophage responses. Here we perform untargeted metabolomics to elucidate the metabolic changes involved in miltefosine action. Over 800 metabolites were detected, 10% of which were significantly altered after 3.75 h. Many of the changes related to an increase in alkane fragment and sugar release. Fragment release is synchronised with reactive oxygen species production, but native membrane phospholipids remain intact. Signs of DNA damage were also detected as were changes to the levels of some thiols and polyamines. After 5 h of miltefosine treatment the cells showed depleted levels of most metabolites, indicating that the cells' outer membrane integrity had become compromised and internal metabolites were escaping upon cell death. In miltefosine resistant cells, the drug was not internalised and the changes to the internal metabolite levels were not seen. In contrast, cells resistant to antimony (SbIII) had similar corresponding alterations to the levels of internal metabolites as wild-type cells. A detailed knowledge of the mode of action of miltefosine will be important to inform the design of combination therapies to combat leishmaniasis, something that the research community should be prioritising in the coming years.
RESUMEN
The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonine's contribution being â¼ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end-products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC 1.1.1.103), is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC 4.1.1.49) shows a 1.7-fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8-fold reduction in threonine-derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.
Asunto(s)
Carbono/metabolismo , Metabolismo de los Lípidos , Redes y Vías Metabólicas/genética , Treonina/metabolismo , Trypanosoma brucei brucei/metabolismo , Acetatos/metabolismo , Biotransformación , Medios de Cultivo/química , Eliminación de Gen , Glucosa , Espectroscopía de Resonancia Magnética , Genética Inversa , Trypanosoma brucei brucei/genéticaRESUMEN
A non-targeted metabolomics-based approach is presented that enables the study of pathways in response to drug action with the aim of defining the mode of action of trypanocides. Eflornithine, a polyamine pathway inhibitor, and nifurtimox, whose mode of action involves its metabolic activation, are currently used in combination as first line treatment against stage 2, CNS-involved, human African trypanosomiasis (HAT). Drug action was assessed using an LC-MS based non-targeted metabolomics approach. Eflornithine revealed the expected changes to the polyamine pathway as well as several unexpected changes that point to pathways and metabolites not previously described in bloodstream form trypanosomes, including a lack of arginase activity and N-acetylated ornithine and putrescine. Nifurtimox was shown to be converted to a trinitrile metabolite indicative of metabolic activation, as well as inducing changes in levels of metabolites involved in carbohydrate and nucleotide metabolism. However, eflornithine and nifurtimox failed to synergise anti-trypanosomal activity in vitro, and the metabolomic changes associated with the combination are the sum of those found in each monotherapy with no indication of additional effects. The study reveals how untargeted metabolomics can yield rapid information on drug targets that could be adapted to any pharmacological situation.
Asunto(s)
Eflornitina/farmacología , Metaboloma , Nifurtimox/farmacología , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Animales , Biotransformación , Cromatografía Liquida/métodos , Interacciones Farmacológicas , Eflornitina/metabolismo , Humanos , Espectrometría de Masas/métodos , Metabolómica/métodos , Nifurtimox/metabolismo , Tripanocidas/metabolismo , Trypanosoma brucei brucei/químicaRESUMEN
Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.
Asunto(s)
Resistencia a Medicamentos , Tripanocidas/farmacología , Trypanosoma/fisiología , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Resistencia a Medicamentos/genética , Eflornitina/farmacocinética , Eflornitina/farmacología , Eflornitina/uso terapéutico , Humanos , Proteínas de Transporte de Membrana/metabolismo , Nifurtimox/farmacocinética , Nifurtimox/farmacología , Nifurtimox/uso terapéutico , Pentamidina/metabolismo , Pentamidina/farmacología , Pentamidina/uso terapéutico , Proteínas Protozoarias/metabolismo , Insuficiencia del Tratamiento , Tripanocidas/farmacocinética , Tripanocidas/uso terapéutico , Trypanosoma/efectos de los fármacos , Trypanosoma/metabolismoRESUMEN
Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.