RESUMEN
Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/análogos & derivados , Celastraceae/química , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Azidas/metabolismo , Bencimidazoles/metabolismo , Unión Competitiva , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Daunorrubicina/farmacocinética , Daunorrubicina/farmacología , Dihidropiridinas/metabolismo , Resistencia a Antineoplásicos , Fluoresceínas/farmacocinética , Humanos , Cinética , Liposomas/metabolismo , Moduladores del Transporte de Membrana , Proteínas de Transporte de Membrana/antagonistas & inhibidores , Proteínas de Transporte de Membrana/metabolismo , Ratones , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Células 3T3 NIH , Etiquetas de Fotoafinidad/metabolismo , Unión Proteica , Especificidad por Sustrato , Vinblastina/farmacocinética , Vinblastina/farmacologíaRESUMEN
A series of dihydro-beta-agarofuran sesquiterpenes isolated from the leaves of Maytenus cuzcoina (1-10) or semisynthetic derivatives (11-30) 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. Almost all natural compounds showed potent reversal activity with different degrees of selectivity. Compounds 2, 7, and 8 are the most effective reversal agents tested against the multidrug resistance phenotype of Leishmania. Three-dimensional quantitative structure-activity relationships using the comparative molecular similarity indices analysis (CoMSIA) were employed to characterize the steric (contribution of 5.4%), electrostatic (58.9%), lipophilic (10.0%), and hydrogen-bond-donor (13.3%) and -acceptor (7.5%) requirements of these sesquiterpenes as modulators at the P-glycoprotein-like transporter. The most salient features of these requirements are the H-bond interaction between the substituents at the C-2 and C-6 positions with the receptor.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Leishmania tropica/efectos de los fármacos , Sesquiterpenos/química , Tripanocidas/química , Animales , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Leishmania tropica/metabolismo , Modelos Moleculares , Fenotipo , Relación Estructura-Actividad Cuantitativa , Sesquiterpenos/síntesis química , Sesquiterpenos/farmacología , Tripanocidas/síntesis química , Tripanocidas/farmacologíaRESUMEN
The kavapyrone (+)-(7 R,8 S)-epoxy-5,6-didehydrokavain (1) and the chalcone flavokavain B (2) were isolated from Piper rusbyi as the bioactive components by bioassay-guided fractionation, using an in vitro assay against promastigote forms of three Leishmania strains. In addition, the new kavapyrone, (7 R,8 R/7 S,8 S)-dihydroxy-5,6-didehydrokavain (3), which is very likely an artifact, and four known compounds (4-7) were isolated. Their structures were elucidated on the basis of spectral analysis, and the absolute configurations of compounds 1 and 3 were established by CD studies and the modified Mosher ester procedure, respectively. All compounds were evaluated for in vitro leishmanicidal activity. The most active compounds 1 (IC50=81.9 microM) and 2 (IC50=11.2 microM) were also evaluated in vivo against a New World strain of cutaneous leishmaniasis, and the results showed the efficacy of 2 at a dose of 5 mg/kg/day. Compounds 1 and 3 were also assayed as reversal agents against a multidrug-resistant Leishmania tropica line, but were found to be inactive.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania/efectos de los fármacos , Fitoterapia , Piper , Extractos Vegetales/farmacología , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/uso terapéutico , Leishmania/clasificación , Leishmaniasis/tratamiento farmacológico , Pruebas de Sensibilidad Parasitaria , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Hojas de la PlantaRESUMEN
Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDR1. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide-binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-beta-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Acridinas/farmacología , Dibenzocicloheptenos/farmacología , Leishmania/efectos de los fármacos , Leishmania/metabolismo , Fosforilcolina/análogos & derivados , Quinolinas/farmacología , Tetrahidroisoquinolinas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Resistencia a Medicamentos , Humanos , Ratones , Células 3T3 NIH , Fosforilcolina/farmacocinética , Fosforilcolina/farmacología , Estructura Terciaria de Proteína , Células VeroRESUMEN
P-glycoprotein (Pgp) transporters play an important role in multidrug resistance in eukaryotic cells and in protozoan parasites such as Leishmania. To search for new reversal agents of the Leishmania tropica Pgp, we developed a screening assay using the Baculovirus-insect cell expression system. We demonstrated a MgATP-dependent, vanadate-sensitive transport of Hoechst 33342 in membrane preparations of Sf9 insect cells expressing Pgp. We have found that dihydro-beta-agarofuran sesquiterpenes from Maytenus cuzcoina inhibited Hoechst 33342 transport that correlates with their reversal effect in a multidrug-resistant L. tropica line overexpressing Pgp. The results suggest that Sf9 cell membrane Hoechst 33342 transport system represents an efficient tool for examining the interactions of Leishmania Pgp with pharmacological agents.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Bencimidazoles/farmacocinética , Membrana Celular/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Leishmania/metabolismo , Ingeniería de Proteínas/métodos , Spodoptera/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Leishmania/efectos de los fármacos , Sesquiterpenos/farmacología , Spodoptera/efectos de los fármacos , Spodoptera/genética , Transfección/métodosRESUMEN
Five new dihydro-beta-agarofuran sesquiterpenes (1-5) were isolated from the leaves of Maytenus chiapensis. The structures of 1-5 were determined by means of 1D and 2D NMR techniques. A semiselective HMBC technique was applied in order to obtain complete (13)C NMR assignments. Absolute configurations were determined by CD studies and chemical correlations and on biogenetic grounds. Compound 4 showed weak activity against a multidrug-resistant Leishmania tropica line.