RESUMEN
Ticks, notorious blood-feeders and disease-vectors, have lost a part of their genetic complement encoding haem biosynthetic enzymes and are, therefore, dependent on the acquisition and distribution of host haem. Solute carrier protein SLC48A1, aka haem-responsive gene 1 protein (HRG1), has been implicated in haem transport, regulating the availability of intracellular haem. HRG1 transporter has been identified in both free-living and parasitic organisms ranging from unicellular kinetoplastids, nematodes, up to vertebrates. However, an HRG1 homologue in the arthropod lineage has not yet been identified. We have identified a single HRG1 homologue in the midgut transcriptome of the tick Ixodes ricinus, denoted as IrHRG, and have elucidated its role as a haem transporter. Data from haem biosynthesis-deficient yeast growth assays, systemic RNA interference and the evaluation of gallium protoporphyrin IX-mediated toxicity through tick membrane feeding clearly show that IrHRG is the bona fide tetrapyrrole transporter. We argue that during evolution, ticks profited from retaining a functional hrg1 gene in the genome because its protein product facilitates host haem escort from intracellularly digested haemoglobin, rendering haem bioavailable for a haem-dependent network of enzymes.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Sistema Digestivo/parasitología , Hemo/metabolismo , Hemoproteínas/metabolismo , Hemoglobinas/metabolismo , Ixodes/metabolismo , Infestaciones por Garrapatas/parasitología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Sistema Digestivo/metabolismo , Hemoproteínas/genética , Filogenia , Homología de Secuencia , TranscriptomaRESUMEN
The neglected tropical diseases Human African Trypanosomiasis and leishmaniasis are caused by infection with trypanosomatid parasites Trypanosoma brucei and Leishmania spp, respectively. The genomes of these organisms contain multiple putative G-quadruplex (G4) forming sequences which have recently been proposed to mediate processes relevant for parasite survival. Therefore, G4 could be considered as potential targets for a novel approach towards the development of antiparasitic drugs. Recently, we have demonstrated that G4 ligands such as carbohydrate naphthalene diimide conjugates (carb-NDIs) possess notable antiparasitic activity. Herein, we have synthesized a new family of carb-NDIs, characterized by significant structural variability, and evaluated their anti-parasitic activity, with special focus on T. brucei. The interaction with relevant G4 sequences was evaluated in vitro through independent biophysical methods (FRET melting assays under competing conditions with double stranded DNA, circular dichroism and fluorescence titrations). Finally, flow cytometry and confocal microscopy experiments demonstrated that the conjugates exhibit excellent uptake into T. brucei parasites, localizing in the nuclei and kinetoplasts. Promising antiparasitic activity and selectivity against control mammalian cells, together with their peculiar mechanism of action, render the carb-NDI conjugates as suitable candidates for the development of an innovative treatment of trypanosomiasis.
Asunto(s)
Antiparasitarios/síntesis química , Carbohidratos/química , Imidas/química , Naftalenos/química , Animales , Antiparasitarios/farmacología , Línea Celular , G-Cuádruplex/efectos de los fármacos , Humanos , Imidas/farmacocinética , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/genética , Naftalenos/farmacocinética , Relación Estructura-Actividad , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis/tratamiento farmacológico , Tripanosomiasis/genéticaRESUMEN
Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Neoplasias de la Mama/fisiopatología , Proteínas de Neoplasias/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Anticuerpos Monoclonales , Neoplasias de la Mama/patología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Clonación Molecular , Cartilla de ADN , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Cinética , Mitoxantrona/farmacocinética , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Vanadatos/metabolismoRESUMEN
Progesterone and the antiprogestin RU38486 have been reported as non-transported modulators of P-glycoprotein-mediated drug efflux. However, their hormonal properties limit their potential for clinical trials. The present work shows that some derivatives from either progesterone/RU38486 or estradiol, displaying differential interaction with hormone receptors, bind to P-glycoprotein and chemosensitize the growth of MDR1-transfected cells to vinblastine more strongly than does RU38486. Structure comparison of the compounds indicates that the highly hydrophobic estradiol derivative RU49953, which does not interact with any hormone receptor, inhibits P-glycoprotein-mediated drug efflux very efficiently, as monitored by flow cytometry, and prevents drug site photoaffinity labeling by azidopine. It induces a much higher chemosensitization than the well-known P-glycoprotein modulator verapamil, which is itself more efficient than RU38486. RU49953 therefore constitutes a promising new lead for steroid-type modulators of multidrug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Inhibidores Enzimáticos/farmacología , Mifepristona/análogos & derivados , Mifepristona/farmacología , Células 3T3 , Animales , Azidas/metabolismo , Daunorrubicina/metabolismo , Dihidropiridinas/metabolismo , Ratones , Vinblastina/metabolismoRESUMEN
Abstract: Protozoan parasites are responsible for important diseases that threaten the lives of nearly one-quarter of the human population world-wide. Among them, leishmaniasis has become the second cause of death, mainly due to the emergence of parasite resistance to conventional drugs. P-glycoprotein (Pgp)-like transporters overexpression is a very efficient mechanism to reduce the intracellular accumulation of many drugs in cancer cells and parasitic protozoans including Plasmodium and Leishmania, thus conferring a multidrug resistance (MDR) phenotype. Therefore, there is a great clinical interest in developing inhibitors of these transporters to overcome such a resistance. Pgps are active pumps belonging to the ATP-binding cassette (ABC) superfamily of proteins, and consist of two homologous halves, each containing a transmembrane domain (TMD) involved in drug efflux, and a cytosolic nucleotide-binding domain (NBD) responsible for ATP binding and hydrolysis. Most conventional cancer MDR modulators interact with the drug-binding sites on the TMDs of Pgps, but they are also usually transported and the required concentrations for a permanent inhibition produce subsequent side-effects that hamper their clinical use. Besides, they only poorly modulate the resistance in protozoan parasites. We review here a rational strategy developed to overcome the MDR phenotype in Leishmania, consisting in: i) the selection of an MDR Leishmania tropica line that overexpresses a Pgp-like transporter; ii) the use of their cytosolic NBDs as new pharmacological targets; iii) the search of new natural compounds that revert the MDR phenotype in Leishmania by binding to the TMDs; iv) the combination of subdoses of the above selected modulators directed to both targets in the transporter, NBDs and TMDs, to accumulate their reversal effects while diminishing their toxicity. In this way, we have reverted the MDR phenotype in Leishmania, including the resistance to the most promising new antileishmania agents, the alkyl-lysophospholipids. This approach might be extrapolated to be used in other eukaryotic cells.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Leishmania/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/genética , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacología , Sitios de Unión/genética , Resistencia a Múltiples Medicamentos/genética , Flavonoides/química , Flavonoides/farmacología , Humanos , Leishmania/genética , Leishmania/metabolismo , Leishmaniasis/tratamiento farmacológico , Estructura Molecular , Fenotipo , Transporte de Proteínas , Sesquiterpenos/química , Sesquiterpenos/farmacologíaRESUMEN
Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos , Flavonoides/farmacología , Animales , Farmacorresistencia Fúngica Múltiple , Resistencia a Antineoplásicos , Flavonoides/química , Flavonoides/metabolismo , Humanos , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Relación Estructura-ActividadRESUMEN
Parasite resistance to drugs has emerged as a major problem in current medicine, and therefore, there is great clinical interest in developing compounds that overcome these resistances. In an intensive study of South American medicinal plants, herein we report the isolation, structure elucidation, and biological activity of dihydro-beta-agarofuran sesquiterpenes from the roots of Maytenus magellanica (1-14) and M. chubutensis (14-17). This type of natural products may be considered as privileged structures. The structures of 10 new compounds, 1, 3, 6-9, and12-15, were determined by means of (1)H and (13)C NMR spectroscopic studies, including homonuclear (COSY and ROESY) and heteronuclear correlation experiments (HMQC and HMBC). The absolute configurations of eight hetero- and homochromophoric compounds, 1, 3,6-9, 12, and 13, were determined by means of CD studies. Fourteen compounds, 1-3 and 6-16, have been tested on a multidrug-resistant Leishmania tropica line overexpressing a P-glycoprotein-like transporter to determine their ability to revert the resistance phenotype and to modulate intracellular drug accumulation. From this series, 1, 2, 3, 14, and 15 showed potent activity, 1 being the most active compound. The structure-activity relationships of the different compounds are discussed.
Asunto(s)
Leishmania tropica/efectos de los fármacos , Maytenus/química , Sesquiterpenos/farmacología , Triterpenos/farmacología , Tripanocidas/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Dicroismo Circular , Resistencia a Múltiples Medicamentos , Fluoresceínas/metabolismo , Espectroscopía de Resonancia Magnética , Extractos Vegetales/química , Raíces de Plantas/química , Sesquiterpenos/química , Sesquiterpenos/aislamiento & purificación , Estereoisomerismo , Relación Estructura-Actividad , Triterpenos/química , Triterpenos/aislamiento & purificación , Tripanocidas/química , Tripanocidas/aislamiento & purificaciónRESUMEN
Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C(5)-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Leishmania tropica/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Animales , Antibióticos Antineoplásicos/farmacología , Antiprotozoarios/farmacocinética , Antiprotozoarios/farmacología , Daunorrubicina/farmacología , Fluorescencia , Humanos , Leishmania tropica/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Éteres Fosfolípidos/química , Éteres Fosfolípidos/farmacocinética , Éteres Fosfolípidos/farmacología , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologíaRESUMEN
P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos , Inhibidores Enzimáticos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas de Saccharomyces cerevisiae , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antiprotozoarios/farmacología , Sitios de Unión , Citosol/metabolismo , Daunorrubicina/farmacología , Metabolismo Energético/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/metabolismo , Proteínas Fúngicas/metabolismo , Leishmania tropica/efectos de los fármacos , Leishmania tropica/crecimiento & desarrollo , Leishmania tropica/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Naftalenos/metabolismo , Naftalenos/farmacología , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Pirofosfatasas/antagonistas & inhibidores , Pirofosfatasas/metabolismo , Rodaminas/antagonistas & inhibidores , Rodaminas/metabolismo , Saccharomyces cerevisiae , Estaurosporina/metabolismo , Estaurosporina/farmacologíaRESUMEN
In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/farmacología , Daunorrubicina/farmacología , Leishmania tropica/metabolismo , Nucleótidos/metabolismo , Silimarina/análogos & derivados , Silimarina/farmacología , Animales , Resistencia a Múltiples Medicamentos , Leishmania tropica/efectos de los fármacos , Oxidación-Reducción , Biosíntesis de Proteínas , Prenilación de Proteína , Proteínas/genética , Silimarina/químicaRESUMEN
ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Leishmania/metabolismo , Transportador 1 de Casete de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Resistencia a Medicamentos , Leishmania/parasitología , Leishmania/fisiología , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiologíaRESUMEN
The effects produced by nine dihydro-beta-agarofuran sesquiterpenes isolated from Crossopetalum tonduzii (1-8) and Maytenus macrocarpa (9) (Celastraceae) on the reversion of the resistant phenotype on a multidrug-resistant Leishmania line and their binding to recombinant C-terminal nucleotide-binding domain of Leishmania P-glycoprotein-like transporter were studied. The structures of the new compounds (1-5) were elucidated by spectroscopic methods, including (1)H-(13)C heteronuclear correlation (HMQC), long-range correlation spectra with inversal detection (HMBC), ROESY experiments, and chemical correlations. The absolute configuration of one of them (1) was determined by CD studies. The structure-activity relationship is discussed.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Daunorrubicina/farmacología , Leishmania tropica/efectos de los fármacos , Rosales/química , Sesquiterpenos/síntesis química , Tripanocidas/síntesis química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Dicroismo Circular , Resistencia a Múltiples Medicamentos , Escherichia coli/metabolismo , Leishmania tropica/metabolismo , Unión Proteica , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacología , Relación Estructura-Actividad , Tripanocidas/química , Tripanocidas/metabolismo , Tripanocidas/farmacologíaRESUMEN
The C-terminal nucleotide-binding domain (NBD2) of a P-glycoprotein-like transporter, encoded by the ltrmdr1 gene in Leishmania tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound fluorescent derivatives of ATP, the hydrophobic steroid analogue RU 486, and different classes of flavonoids with the following efficiency: flavone > flavanone > isoflavone > glucorhamnosyl-flavone > chromone. The affinity for flavones was dependent on the presence of hydroxyl groups at positions 5 and 3 and was further increased by a hydrophobic 1,1-dimethylallyl substituent at position 8. When flow cytometry was used to measure daunomycin accumulation in a MDR L. tropica line, a reversing effect was observed with flavones such as dimethylallyl-kaempferide at low concentration or apigenin at higher concentration, but neither with the glucorhamnosyl derivative rutin nor with the isoflavone genistein. The in vivo reversing effect of dimethylallyl-kaempferide was correlated to a high inhibition of MDR cell growth in the presence of daunomycin. The results suggest that flavone inhibition of both daunomycin efflux and parasite growth in the presence of the drug correlates to direct binding of the compound to cytosolic domain of the P-glycoprotein-like transporter.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Citosol/metabolismo , Daunorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Flavonoides/metabolismo , Leishmania tropica/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Animales , Línea Celular , Citosol/efectos de los fármacos , Daunorrubicina/metabolismo , Flavonoides/farmacología , Leishmania tropica/efectos de los fármacos , Leishmania tropica/crecimiento & desarrollo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania tropica/efectos de los fármacos , Meglumina/farmacología , Compuestos Organometálicos/farmacología , Compuestos de Sulfhidrilo/metabolismo , Animales , Butionina Sulfoximina/farmacología , Clorambucilo/farmacología , Resistencia a Medicamentos , Antimoniato de MegluminaRESUMEN
We selected a Leishmania tropica cell line resistant to daunomycin (DNM) that presents a multidrug-resistant (MDR) phenotype characterized by overexpression of a P-glycoprotein of 150 kDa. The resistant line overexpressed an MDR-like gene, called ltrmdr1, located in an extrachromosomal circular DNA. DNM uptake experiments using laser flow cytometry showed a significant reduction in drug accumulation in the resistant parasites. The initial stages of the interaction of DNM with membranes from wild-type and DNM-resistant parasites were defined by a rapid kinetic stopped-flow procedure which can be described by two kinetic components. On the basis of a previous similar kinetic study with tumor cells, we ascribed the fast component to rapid interaction of DNM with membrane surface components and the slow component to passive diffusion of the drug across the membranes. The results reported here indicate that entrance of DNM into wild-type parasites was facilitated in respect to the resistant ones. We propose that resistance to DNM in L. tropica is a multifactorial event involving at least two complementary mechanisms. an altered drug membrane permeability and the overexpression of a protein related to P-glycoprotein that regulates drug efflux.