RESUMEN
Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 µM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 µM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 µM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.
Asunto(s)
Bacillus subtilis/fisiología , Biopelículas/crecimiento & desarrollo , Espermidina/análogos & derivados , Secuencia de Aminoácidos , Bacillus subtilis/crecimiento & desarrollo , Datos de Secuencia Molecular , Plancton/crecimiento & desarrollo , Alineación de Secuencia , Espermidina/biosíntesis , Espermidina/metabolismo , Espermidina/fisiología , Vibrio cholerae/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.
Asunto(s)
Resistencia a Medicamentos , Leishmania donovani , Leishmaniasis Visceral , Esterol 14-Desmetilasa , Leishmania donovani/enzimología , Esterol 14-Desmetilasa/metabolismo , Esterol 14-Desmetilasa/genética , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/tratamiento farmacológico , Anfotericina B/farmacología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Antiprotozoarios/farmacología , Humanos , Ergosterol/metabolismoRESUMEN
Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Leishmania donovani/efectos de los fármacos , Leishmania donovani/enzimología , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Terapia Molecular Dirigida , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Quinasa 9 Dependiente de la Ciclina/química , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Simulación del Acoplamiento Molecular , Proteoma/efectos de los fármacos , Proteómica , Pirazoles/química , Pirazoles/uso terapéutico , Pirimidinas/química , Pirimidinas/uso terapéutico , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
Asunto(s)
Antiprotozoarios/administración & dosificación , Leishmania donovani/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Leishmaniasis Visceral/diagnóstico por imagen , Inhibidores de Proteasoma/administración & dosificación , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antiprotozoarios/química , Sitios de Unión , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Leishmania donovani/química , Leishmania donovani/enzimología , Leishmania infantum/química , Leishmania infantum/enzimología , Leishmaniasis Visceral/parasitología , Masculino , Ratones , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Trypanosomatid parasites are the infectious agents causing Chagas disease, visceral and cutaneous leishmaniasis and human African trypanosomiasis. Recent work of others has implicated an aldo-keto reductase (AKR) in the susceptibility and resistance of Trypanosoma cruzi to benznidazole, a drug used to treat Chagas disease. Here, we show that TcAKR and homologues in the related parasites Trypanosoma brucei and Leishmania donovani do not reductively activate monocyclic (benznidazole, nifurtimox and fexinidazole) or bicyclic nitro-drugs such as PA-824. Rather, these enzymes metabolise a variety of toxic ketoaldehydes, such as glyoxal and methylglyoxal, suggesting a role in cellular defence against chemical stress. UPLC-QToF/MS analysis of benznidazole bioactivation by T. cruzi cell lysates confirms previous reports identifying numerous drug metabolites, including a dihydro-dihydroxy intermediate that can dissociate to form N-benzyl-2-guanidinoacetamide and glyoxal, a toxic DNA-glycating and cross-linking agent. Thus, we propose that TcAKR contributes to benznidazole resistance by the removal of toxic glyoxal. In addition, three of the four enzymes studied here display activity as prostaglandin F2α synthases, despite the fact that there are no credible cyclooxygenases in these parasites to account for formation of the precursor PGH2 from arachidonic acid. Our studies suggest that arachidonic acid is first converted non-enzymatically in parasite lysates to (PGH2-like) regioisomers by free radical-mediated peroxidation and that AKRs convert these lipid peroxides into isoprostanes, including prostaglandin F2α and 8-iso-prostaglandin F2α.
Asunto(s)
Aldo-Ceto Reductasas/metabolismo , Dinoprost/análogos & derivados , Dinoprost/metabolismo , Isoprostanos/metabolismo , Leishmania donovani/metabolismo , Proteínas Protozoarias/metabolismo , Piruvaldehído/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/metabolismo , Aldo-Ceto Reductasas/genética , Dinoprost/genética , Isoprostanos/genética , Leishmania donovani/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genéticaRESUMEN
The lack of information regarding the mechanisms of action (MoA) or specific molecular targets of phenotypically active compounds can prove a barrier to their development as chemotherapeutic agents. Here, we report the results of our orthogonal genetic, molecular, and biochemical studies to determine the MoA of a novel 7-substituted 8-hydroxy-1,6-naphthyridine (8-HNT) series that displays promising activity against Trypanosoma brucei and Leishmania donovani High-throughput loss-of-function genetic screens in T. brucei highlighted two probable zinc transporters associated with resistance to these compounds. These transporters localized to the parasite Golgi apparatus. Directed by these findings, the role of zinc and other divalent cations in the MoA of these compounds was investigated. 8-HNT compounds were found to directly deplete intracellular levels of Zn2+, while the addition of exogenous Zn2+ and Fe2+ reduced the potency of compounds from this series. Detailed biochemical analyses confirmed that 8-HNT compounds bind directly to a number of divalent cations, predominantly Zn2+, Fe2+, and Cu2+, forming 2:1 complexes with one of these cations. Collectively, our studies demonstrate transition metal depletion, due to chelation, as the MoA of the 8-HNT series of compounds. Strategies to improve the selectivity of 8-HNT compounds are discussed.
Asunto(s)
Antiprotozoarios/farmacología , Proteínas de Transporte de Catión/genética , Quelantes/farmacología , Naftiridinas/farmacología , Proteínas Protozoarias/genética , Zinc/metabolismo , Antiprotozoarios/síntesis química , Proteínas de Transporte de Catión/metabolismo , Cationes Bivalentes , Quelantes/síntesis química , Cobre/metabolismo , Expresión Génica , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Hierro/metabolismo , Leishmania donovani/efectos de los fármacos , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/metabolismo , Mutación , Naftiridinas/síntesis química , Pruebas de Sensibilidad Parasitaria , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Drug discovery pipelines for the "neglected diseases" are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic nitro-compounds (R)-PA-824, DNDI-VL-2098 and delamanid have been identified as potential candidates for the treatment of visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative NAD(P)H oxidase as the activating nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic nitro-compounds, but only marginally to the monocyclic nitro-drugs, nifurtimox and fexinidazole sulfone, known to be activated by a mitochondrial oxygen-insensitive nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic nitro-compounds and only marginally resistant to nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series.
Asunto(s)
Leishmaniasis Visceral/parasitología , Nitrorreductasas/metabolismo , Proteínas Protozoarias/metabolismo , Tripanocidas/farmacología , Animales , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Espectrometría de Masas , Enfermedades Desatendidas/parasitología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pruebas de Sensibilidad Parasitaria , Reacción en Cadena de la PolimerasaRESUMEN
The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1-3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1-3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1-3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1-3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1-3 loss-of-function is a mechanism of antifolate drug resistance.
Asunto(s)
Transportadores de Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Metotrexato/farmacocinética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Transportadores de Ácido Fólico/genética , Estudio de Asociación del Genoma Completo , Metotrexato/farmacología , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genéticaRESUMEN
BACKGROUND: Protein kinases have been shown to be key drug targets, especially in the area of oncology. It is of interest to explore the possibilities of protein kinases as a potential target class in Plasmodium spp., the causative agents of malaria. However, protein kinase biology in malaria is still being investigated. Therefore, rather than assaying against individual protein kinases, a library of 4731 compounds with protein kinase inhibitor-like scaffolds was screened against the causative parasite, Plasmodium falciparum. This approach is more holistic and considers the whole kinome, making it possible to identify compounds that inhibit more than one P. falciparum protein kinase, or indeed other malaria targets. RESULTS: As a result of this screen, 9 active compound series were identified; further validation was carried out on 4 of these series, with 3 being progressed into hits to lead chemistry. The detailed evaluation of one of these series is described. DISCUSSION: This screening approach proved to be an effective way to identify series for further optimisation against malaria. Compound optimisation was carried out in the absence of knowledge of the molecular target. Some of the series had to be halted for various reasons. Mode of action studies to find the molecular target may be useful when problems prevent further chemical optimisation. CONCLUSIONS: Progressible series were identified through phenotypic screening of a relatively small focused kinase scaffold chemical library.
Asunto(s)
Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Evaluación Preclínica de MedicamentosRESUMEN
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
De novo synthesis of threonine from aspartate occurs via the ß-aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L-homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the ß-aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3-oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.
Asunto(s)
Acil-Butirolactonas/metabolismo , Liasas de Carbono-Oxígeno/metabolismo , Homoserina/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Trypanosoma brucei brucei/fisiología , Moscas Tse-Tse/microbiología , Animales , Liasas de Carbono-Oxígeno/genética , Mutación , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Percepción de Quorum , Simbiosis , Treonina/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Moscas Tse-Tse/parasitologíaRESUMEN
OBJECTIVES: The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes. METHODS: Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined. RESULTS: Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are â¼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen. CONCLUSIONS: NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype.
Asunto(s)
Antiprotozoarios/farmacología , Resistencia a Medicamentos , Nifurtimox/farmacología , Nitroimidazoles/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Genoma de Protozoos , Nitrorreductasas/genética , Nitrorreductasas/metabolismo , Pruebas de Sensibilidad Parasitaria , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
OBJECTIVES: In response to reports of Trypanosoma brucei resistance to the nitroaromatic drug nifurtimox, we evaluated the potential of antituberculosis nitrofuran isoxazolines as inhibitors of trypanosome growth. METHODS: The susceptibility of T. brucei brucei was assessed in vitro. The lowest effective concentration to inhibit growth (EC90) against drug-susceptible and -resistant parasites, time-kill kinetics, reversibility of inhibition and propensity for P-glycoprotein-mediated exclusion from the blood-brain barrier were determined. RESULTS: Nitrofuran isoxazolines were potent inhibitors of T. brucei brucei proliferation at nanomolar concentrations, with pentacyclic nitrofurans being 100-fold more potent than nifurtimox. Activity was sustained against nifurtimox-resistant parasites, suggesting the possibility of a unique mechanism of activation and potential for use in the treatment of drug-resistant infections. Exposure of parasites to the maximum concentrations of Compound 15 achieved in vivo with oral dosing yielded >2 logs of irreversible killing in <4 h, indicating rapid trypanocidal activity. CONCLUSIONS: Pentacyclic nitrofuran isoxazolines warrant further development for the treatment of drug-susceptible and nifurtimox-resistant trypanosome infections.
Asunto(s)
Nifurtimox/farmacología , Nitrofuranos/farmacología , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Resistencia a Medicamentos , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Nitrofuranos/síntesis química , Nitrofuranos/toxicidad , Tripanocidas/síntesis química , Trypanosoma brucei brucei/ultraestructuraRESUMEN
African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antiparasitarios/farmacología , Antiparasitarios/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología , Aciltransferasas/metabolismo , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Animales , Antiparasitarios/química , Antiparasitarios/metabolismo , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Estructura Molecular , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología , Pirazoles/uso terapéutico , Ratas , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Factores de Tiempo , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
The Indian subcontinent is the only region where arsenic contamination of drinking water coexists with widespread resistance to antimonial drugs that are used to treat the parasitic disease visceral leishmaniasis. We have previously proposed that selection for parasite resistance within visceral leishmaniasis patients who have been exposed to trivalent arsenic results in cross-resistance to the related metalloid antimony, present in the pentavalent state as a complex in drugs such as sodium stibogluconate (Pentostam) and meglumine antimonate (Glucantime). To test this hypothesis, Leishmania donovani was serially passaged in mice exposed to arsenic in drinking water at environmentally relevant levels (10 or 100 ppm). Arsenic accumulation in organs and other tissues was proportional to the level of exposure and similar to that previously reported in human liver biopsies. After five monthly passages in mice exposed to arsenic, isolated parasites were found to be completely refractory to 500 µg · mL(-1) Pentostam compared with the control passage group (38.5 µg · mL(-1)) cultured in vitro in mouse peritoneal macrophages. Reassessment of resistant parasites following further passage for 4 mo in mice without arsenic exposure showed that resistance was stable. Treatment of infected mice with Pentostam confirmed that resistance observed in vitro also occurred in vivo. We conclude that arsenic contamination may have played a significant role in the development of Leishmania antimonial resistance in Bihar because inadequate treatment with antimonial drugs is not exclusive to India, whereas widespread antimonial resistance is.
Asunto(s)
Antiprotozoarios/metabolismo , Arsénico/toxicidad , Agua Potable/análisis , Resistencia a Medicamentos/efectos de los fármacos , Exposición a Riesgos Ambientales/efectos adversos , Leishmania/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Contaminantes Químicos del Agua/toxicidad , Animales , Gluconato de Sodio Antimonio , Línea Celular , India , Macrófagos/efectos de los fármacos , Tamizaje Masivo , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB CRESUMEN
Dual submicromolar trypanocidal-antiplasmodial compounds have been identified by screening and chemical synthesis of 4-aminoquinoline-based heterodimeric compounds of three different structural classes. In Trypanosoma brucei, inhibition of the enzyme trypanothione reductase seems to be involved in the potent trypanocidal activity of these heterodimers, although it is probably not the main biological target. Regarding antiplasmodial activity, the heterodimers seem to share the mode of action of the antimalarial drug chloroquine, which involves inhibition of the haem detoxification process. Interestingly, all of these heterodimers display good brain permeabilities, thereby being potentially useful for late stage human African trypanosomiasis. Future optimization of these compounds should focus mainly on decreasing cytotoxicity and acetylcholinesterase inhibitory activity.
Asunto(s)
Aminoquinolinas/química , Aminoquinolinas/farmacología , Antimaláricos/química , Antimaláricos/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Aminoquinolinas/síntesis química , Aminoquinolinas/farmacocinética , Animales , Antimaláricos/síntesis química , Antimaláricos/farmacocinética , Encéfalo/metabolismo , Línea Celular , Dimerización , Hemoproteínas/metabolismo , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Ratas , Tripanocidas/síntesis química , Tripanocidas/farmacocinética , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitologíaRESUMEN
Co- and post-translational N-myristoylation is known to play a role in the correct subcellular localization of specific proteins in eukaryotes. The enzyme that catalyses this reaction, NMT (N-myristoyltransferase), has been pharmacologically validated as a drug target in the African trypanosome, Trypanosoma brucei. In the present study, we evaluate NMT as a potential drug target in Trypanosoma cruzi, the causative agent of Chagas' disease, using chemical and genetic approaches. Replacement of both allelic copies of TcNMT (T. cruzi NMT) was only possible in the presence of a constitutively expressed ectopic copy of the gene, indicating that this gene is essential for survival of T. cruzi epimastigotes. The pyrazole sulphonamide NMT inhibitor DDD85646 is 13-23-fold less potent against recombinant TcNMT than TbNMT (T. brucei NMT), with Ki values of 12.7 and 22.8 nM respectively, by scintillation proximity or coupled assay methods. DDD85646 also inhibits growth of T. cruzi epimastigotes (EC50=6.9 µM), but is ~1000-fold less potent than that reported for T. brucei. On-target activity is demonstrated by shifts in cell potency in lines that over- and under-express NMT and by inhibition of intracellular N-myristoylation of several proteins in a dose-dependent manner. Collectively, our findings suggest that N-myristoylation is an essential and druggable target in T. cruzi.
Asunto(s)
Aciltransferasas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Trypanosoma cruzi/enzimología , Aciltransferasas/genética , Aminopiridinas , Animales , Chlorocebus aethiops , Clonación Molecular , Eliminación de Gen , Cinética , Organismos Modificados Genéticamente , Proteínas Recombinantes , Sulfonamidas , Células VeroRESUMEN
Polyamine biosynthesis is a key drug target in African trypanosomes. The "resurrection drug" eflornithine (difluoromethylornithine), which is used clinically to treat human African trypanosomiasis, inhibits the first step in polyamine (spermidine) biosynthesis, a highly regulated pathway in most eukaryotic cells. Previously, we showed that activity of a key trypanosomatid spermidine biosynthetic enzyme, S-adenosylmethionine decarboxylase, is regulated by heterodimer formation with a catalytically dead paralog (a prozyme). Here, we describe an expansion of this prozyme paradigm to the enzyme deoxyhypusine synthase, which is required for spermidine-dependent hypusine modification of a lysine residue in the essential translation factor eIF5A. Trypanosoma brucei encodes two deoxyhypusine synthase paralogs, one that is catalytically functional but grossly impaired, and the other is inactive. Co-expression in Escherichia coli results in heterotetramer formation with a 3000-fold increase in enzyme activity. This functional complex is also present in T. brucei, and conditional knock-out studies indicate that both DHS genes are essential for in vitro growth and infectivity in mice. The recurrent evolution of paralogous, catalytically dead enzyme-based activating mechanisms may be a consequence of the unusual gene expression in the parasites, which lack transcriptional regulation. Our results suggest that this mechanism may be more widely used by trypanosomatids to control enzyme activity and ultimately influence pathogenesis than currently appreciated.
Asunto(s)
Lisina/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Proteínas Protozoarias/metabolismo , Espermidina/metabolismo , Trypanosoma brucei brucei/enzimología , Animales , Bovinos , Escherichia coli , Técnicas de Silenciamiento del Gen , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Ratones , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espermidina/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Bovina/enzimología , Tripanosomiasis Bovina/genéticaRESUMEN
African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.