RESUMEN
The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/metabolismo , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dicetopiperazinas , Resistencia a Antineoplásicos , Femenino , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos , Humanos , Mitoxantrona/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Alineación de SecuenciaAsunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Benzamidas/metabolismo , Sitios de Unión , Humanos , Mesilato de Imatinib , Piperazinas/metabolismoRESUMEN
ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are membrane proteins that couple the energy derived from ATP hydrolysis to efflux many chemically diverse compounds across the plasma membrane, thereby playing a critical and important physiological role in protecting cells from xenobiotics. These transporters are also implicated in the development of multidrug resistance (MDR) in cancer cells that have been treated with chemotherapeutics. One approach to blocking the efflux capability of an ABC transporter in a cell or tissue is inhibiting the activity of the transporters with a modulator. Since ABC transporter modulators can be used in combination with chemotherapeutics to increase the effective intracellular concentration of anticancer drugs, the possible impact of modulators of ABC drug transporters is of great clinical interest. Another possible clinical use of modulators that has recently attracted attention is their ability to increase oral bioavailability or increase tissue penetration of drugs transported by the transporters. Several preclinical and clinical studies have been performed to evaluate the feasibility and the safety of this approach. The primary focus of this review is to discuss progress made in recent years in the identification and applicability of compounds that may serve as ABC transporter modulators and the possible role of these compounds in altering the pharmacokinetics and pharmacodynamics of therapeutic drugs used in the clinic.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/uso terapéutico , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Administración Oral , Antineoplásicos/administración & dosificación , Antineoplásicos/metabolismo , Transporte Biológico , Humanos , Terapia Molecular Dirigida , Neoplasias/metabolismoRESUMEN
Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Epigénesis Genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Acetilación , Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Mitoxantrona/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaAsunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Piperazinas/metabolismo , Pirimidinas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Adenosina Trifosfato/metabolismo , Benzamidas , Sitios de Unión , Resistencia a Múltiples Medicamentos , Humanos , Mesilato de ImatinibRESUMEN
The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Resistencia a Antineoplásicos , Células Madre Hematopoyéticas , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/farmacocinética , Benzamidas , Sitios de Unión , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Proteínas de Neoplasias/genética , Inhibidores de Proteínas Quinasas , Recurrencia , Transducción GenéticaRESUMEN
P-glycoprotein (Pgp, ABCB1) is an ATP-dependent drug efflux pump linked to development of multidrug resistance (MDR) in cancer cells. Previously [Biochem Pharmacol 2002;64:573-82], we reported that a curcumin mixture could modulate both function and expression of Pgp. This study focuses on the effect of three major curcuminoids--curcumin I, II and III purified from a curcumin mixture--on modulation of Pgp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The similar IC(50) values for cytotoxicity of curcuminoids of KB-V1, and KB-3-1 (parental drug sensitive cell line) suggest that these curcuminoids may not be substrates for Pgp. Treating the cells with non-toxic doses of curcuminoids increased their sensitivity to vinblastine only in the Pgp expressing drug resistant cell line, KB-V1, and curcumin I retained the drug in KB-V1 cells more effectively than curcumin II and III, respectively. Effects of each curcuminoid on rhodamine123, calcein-AM, and bodipy-FL-vinblastine accumulation confirmed these findings. Curcumin I, II and III increased the accumulation of fluorescent substrates in a dose-dependent manner, and at 15 microM, curcumin I was the most effective. The inhibitory effect in a concentration-dependent manner of curcuminoids on verapamil-stimulated ATPase activity and photoaffinity labeling of Pgp with the [(125)I]-iodoarylazidoprazosin offered additional support; curcumin I was the most potent modulator. Taken together, these results indicate that curcumin I is the most effective MDR modulator among curcuminoids, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Curcumina/análogos & derivados , Curcumina/farmacología , Extractos Vegetales/química , Prazosina/análogos & derivados , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacología , Azidas/metabolismo , Supervivencia Celular/efectos de los fármacos , Curcuma , Diarilheptanoides , Combinación de Medicamentos , Interacciones Farmacológicas , Fluoresceínas/farmacocinética , Humanos , Radioisótopos de Yodo , Células KB , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Prazosina/metabolismo , Rodamina 123/farmacocinética , Vinblastina/farmacologíaRESUMEN
P-glycoprotein (Pgp), the ATP-binding cassette multidrug transporter, exhibits a drug (substrate)-stimulatable ATPase activity, and vanadate (Vi) inhibits this activity by stably trapping the nucleoside diphosphate in the Pgp.ADP.Vi conformation. We recently demonstrated that Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp in the absence of substrate occurs both in the presence of 8-azido-[alpha-(32)P]ATP (following 8-azido-ATP hydrolysis) or 8-azido-[alpha-(32)P]ADP (without hydrolysis) and, the transition state intermediates generated under either condition are functionally indistinguishable. In this study, we compare the effect of substrates on Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp under both non-hydrolysis and hydrolysis conditions. We demonstrate that whereas substrates stimulate the Vi-induced trapping of 8-azido-[alpha-(32)P]ADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of nucleoside diphosphate in the absence of Vi, indicating that the substrate-induced conformation exerts its effect at a step distinct from nucleoside diphosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition state, Pgp x ADP x P(i) (Vi), can be generated both via the hydrolysis of ATP or by directly providing ADP to the system, in the presence of substrate the reaction is driven in the forward direction, i.e. hydrolysis of ATP. These data suggest that substrate-stimulated ATP hydrolysis by Pgp is a vectorial process.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Baculoviridae/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Catálisis , Línea Celular , Membrana Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Hidrólisis/efectos de los fármacos , Insectos , Cinética , Modelos Químicos , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Vanadatos/farmacología , Verapamilo/farmacologíaRESUMEN
P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [alpha-(32)P]8-azido-ADP trapping into Pgp in the presence of [alpha-(32)P]8-azido-ATP (with ATP hydrolysis) or [alpha-(32)P]8-azido-ADP (without ATP hydrolysis). Vi mimics P(i) to trap the nucleotide tenaciously in the Pgp.[alpha-(32)P]8-azido-ADP.Vi conformation in either condition. Thus, by using [alpha-(32)P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [alpha-(32)P]8-azido-ATP or [alpha-(32)P]8-azido-ADP. The trapped [alpha-(32)P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [alpha-(32)P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([(125)I]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [alpha-(32)P]8-azido-ADP (in the absence of hydrolysis) displays an approximately 2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha-(32)P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the Pgp.[alpha-(32)P]8-azido-ADP (or ADP).Vi transition state complexes generated either in the absence of or accompanying [alpha-(32)P]8-azido-ATP hydrolysis are functionally indistinguishable.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacocinética , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacocinética , Azidas/farmacocinética , Vanadatos/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Marcadores de Afinidad/farmacocinética , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Células HeLa , Humanos , Hidrólisis , Insectos , Cinética , Radioisótopos de Fósforo , Proteínas Recombinantes/metabolismo , Termodinámica , TransfecciónRESUMEN
P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp.ADP.Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomes vis à vis the substrate, they show comparable t(12) for both incorporation and release of nucleotide, and the affinities for [alpha-(32)P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [alpha-(32)P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Prazosina/análogos & derivados , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Azidas/química , Azidas/metabolismo , Catálisis , Humanos , Hidrólisis , Radioisótopos de Yodo , Cinética , Prazosina/química , Conformación ProteicaRESUMEN
P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 micromol x min x mg(-1) with a K(m) for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K(m) for ATP or 8-azidoATP and K(i) for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [alpha-(32)P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. The K(m) for [alpha-(32)P]8-azidoATP (20 microm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [alpha-(32)P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, P(i) exhibits very low affinity for Pgp (K(i) approximately 30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [alpha-(32)P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Azidas/metabolismo , Vanadatos/farmacología , Catálisis , Humanos , Hidrólisis , CinéticaRESUMEN
ABC transporters are found in all known organisms, and approximately 1,100 different transporters belonging to this family have been described in the literature. The family is defined by homology within the ATP-binding cassette (ABC) region, which extends outside of the more typical Walker motifs found in all ATP-binding proteins. Most family members also contain transmembrane domains involved in recognition of substrates, which are transported across, into, and out of cell membranes, but some members utilize ABCs as engines to regulate ion channels. There are approximately 50 known ABC transporters in the human, and there are currently 13 genetic diseases associated with defects in 14 of these transporters. The most common genetic disease conditions include cystic fibrosis, Stargardt disease, age-related macular degeneration, adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome and progressive familial intrahepatic cholestasis. At least 8 members of this family are involved in the transport of a variety of amphipathic compounds, including anticancer drugs, and some appear to contribute to the resistance of cancer cells to chemotherapy.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Resistencia a Múltiples Medicamentos , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/fisiopatología , Humanos , Enfermedades del Sistema Inmune/genética , Enfermedades del Sistema Inmune/fisiopatología , Datos de Secuencia Molecular , Neoplasias/genética , Neoplasias/fisiopatologíaRESUMEN
P-glycoprotein (Pgp), the ATP-binding cassette (ABC) transporter, confers multidrug resistance to cancer cells by extruding cytotoxic natural product amphipathic drugs using the energy of ATP hydrolysis. Our studies are directed toward understanding the mechanism of action of Pgp and recent work deals with the assessment of interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified Pgp suggest that ADP release is the rate-limiting step in the catalytic cycle and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of Pgp, one in the transport of drug and the other in effecting conformational changes so as to reset the transporter for the next catalytic cycle. Detailed kinetic measurements determined that both nucleotide-binding domains behave symmetrically and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time, causing (in this site) a conformational change that drastically decreases (>30-fold) the affinity of the second site for ATP-binding. Thus, the blocking of ATP-binding to the second site while the first one is in catalytic conformation appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by Pgp, and this may be applicable as well to other ABC transporters linked with the development of multidrug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Adenosina Difosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Resistencia a Antineoplásicos/fisiología , Adenosina Difosfato/metabolismo , Animales , Azidas/metabolismo , Humanos , Hidrólisis , Modelos BiológicosRESUMEN
A benzophenone photoaffinity label 9 based on the polyene natural product (-)-stipiamide has been constructed using a diaminoethane spacer and the radioactive agent [3H]-BZDC (N-succinimidyl p-benzoyl-(2,3-3H)-dehydrocinnamate). Photoaffinity experiments show specific binding to human P-glycoprotein (Pgp) in the presence of cis-flupentixol but not with cyclosporin A.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Marcadores de Afinidad/síntesis química , Benzofenonas , Benzofenonas/síntesis química , Succinimidas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Marcadores de Afinidad/química , Marcadores de Afinidad/farmacocinética , Benzofenonas/química , Benzofenonas/farmacocinética , Diseño de Fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Polienos/química , Polienos/farmacocinética , TritioRESUMEN
A combinatorial library of polyenes, based on (-)-stipiamide, has been constructed and evaluated for the discovery of new multidrug resistance reversal agents. A palladium coupling was used to react each individual vinyl iodide with a mixture of the seven acetylenes at near 1:1 stoichiometry. The coupling was also used to react each individual acetylene with the mixture of six vinyl iodides to create 13 pools indexed in two dimensions for a total of 42 compounds. Individual compounds were detected at equimolar concentration. The vinyl iodides, made initially using a crotylborane addition to generate the anti1,2-hydroxylmethyl products, were now made using a more efficient norephedrine propionate boron enolate aldol reaction. The indexed approach, ideally suited for cellular assays that involve membrane-bound targets, allowed for the rapid identification of reversal agents using assays with drug-resistant human breast cancer MCF7-adrR cells. Intersections of potent pools identified new compounds with promising activity. Aryl dimension pools showed R = ph and naphthyl as the most potent. The acetylene dimension had R' = phenylalaninol and alaninol as the most potent. Isolated individual compounds, both active and nonpotent, were assayed to confirm the library results. The most potent new compound was 4ek (R = naphthyl, R' = phenylaninol) at 1.45 microM. Other nonnatural individual naphthyl-amide compounds showed potent MDR reversal including the morpholino-amide 4ej (1.69 microM). Synergistic activities attributed to the two ends of the molecule were also identified. Direct interaction with Pgp was established by ATPase and photoaffinity displacement assays. The results indicate that both ends of the polyene reversal agent are involved in Pgp interaction and can be further modified for increased potency.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Técnicas Químicas Combinatorias , Polienos/síntesis química , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Humanos , Espectroscopía de Resonancia Magnética , Modelos Químicos , Polienos/farmacología , Quinolinas/análisis , Soluciones , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the K(d) (412 nM) of the substrate analogue [(125)I]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5'-adenylylimididiphosphate and vanadate (K(d) = 403 nM). Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [(125)I]iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp x ADP x vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis. Additionally, there is an inverse correlation (R(2) = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Azidas/metabolismo , Membrana Celular/metabolismo , ADN Complementario/metabolismo , Genes MDR/genética , Humanos , Hidrólisis , Cinética , Modelos Biológicos , Etiquetas de Fotoafinidad/metabolismo , Unión Proteica , Conformación Proteica , Factores de Tiempo , Vanadatos/metabolismoRESUMEN
P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly(-)) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly(-) P-gp (91, 94, and 99N-->Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly(-) P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly(-) D (91, 94, 99N-->D) and Gly(-) Delta (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly(-) D and Gly(-) Delta Pgps were also expressed to the same level as the Gly(-) mutant protein. (35)S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of (35)S-methionine/cysteine in full length Gly(-) P-gp compared to wild-type protein, but the half-life ( approximately 3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of (35)S-methionine/cysteine in Gly(-) P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Adenosina Trifosfato/metabolismo , Azidas/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Citometría de Flujo , Glicosilación , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Complejos Multienzimáticos/metabolismo , Mutación , Etiquetas de Fotoafinidad/metabolismo , Prazosina/análogos & derivados , Prazosina/metabolismo , Complejo de la Endopetidasa Proteasomal , Transfección , Tripsina , Virus Vaccinia/genéticaRESUMEN
Considerable evidence has accumulated indicating that the multidrug transporter or P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. In recent years, various approaches such as mutational analyses and biochemical and pharmacological characterization have yielded significant information about the relationship of structure and function of P-glycoprotein. However, there is still considerable controversy about the mechanism of action of this efflux pump and its function in normal cells. This review summarizes current research on the structure-function analysis of P-glycoprotein, its mechanism of action, and facts and speculations about its normal physiological role.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Membrana Celular/metabolismo , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Humanos , Neoplasias/genética , Conformación Proteica , Relación Estructura-ActividadRESUMEN
P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer drugs. Biochemical and genetic studies with Pgp have identified the putative transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved among Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specificity, seven amino acid residues were identified within TM 12 that were not strictly conserved among the MDR1 and MDR2 family of proteins from different mammalian species. We replaced all seven of these amino acid residues with alanine, one at a time and in combinations, and used a vaccinia virus based transient expression system to analyze function. None of the single replacements caused any alteration in transport function. However, when residues L975, V981, and F983 were replaced collectively, drug transport, drug-stimulated ATP hydrolysis, and photoaffinity labeling with the drug analogue, [125I]iodoarylazidoprazosin (IAAP), were abrogated, with little effect on [alpha-32P]-8-azido-ATP labeling and basal ATPase activity. Pairwise alanine substitutuions showed variable effects on function. Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine substitutions of all four nonconserved residues M986, V988, Q990, and V991 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extrusion. These results suggest that nonconserved residues in the putative amino-proximal half of TM 12 of Pgp play a more direct role in determining specificity of drug transport function than those in the putative carboxy-terminal half of TM 12.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Secuencia Conservada , Fragmentos de Péptidos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Transporte Biológico/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Secuencia Conservada/genética , Cricetinae , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Genes MDR , Vectores Genéticos/metabolismo , Humanos , Hidrólisis , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Etiquetas de Fotoafinidad/metabolismo , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Especificidad por Sustrato/genética , Transfección , Células Tumorales Cultivadas , Vanadatos/metabolismoRESUMEN
P-Glycoprotein (Pgp), an energy-dependent drug efflux pump responsible for multidrug resistance of many cancer cells, is comprised of two homologous halves connected by a peptide segment approximately 75 amino acids (aa) in length. The effects of length and composition of this connecting region on Pgp cell surface expression and the ability of the two halves to interact were explored using both stable transfections of Pgp mutants in mammalian cell lines and a vaccinia virus transient expression system. A 17 aa insertion of predicted flexible structure between amino acids 681 and 682 resulted in a functional Pgp molecule that was capable of conferring drug resistance. In contrast, an 18 aa peptide insertion with a predicted alpha-helical structure was unstable when expressed transiently. A 34 aa deletion from the central core of the linker region (Delta653-686) resulted in a protein expressed at the cell surface in amounts comparable to that of wild-type Pgp but unable to confer drug resistance. No apparent differences in drug or [alpha-32P]-8-azido-ATP photoaffinity labeling were observed. However, both ATP hydrolysis and drug transport activities of the deletion mutant were completely abrogated, indicating that the linker deletion disconnected substrate binding from ATP hydrolysis and transport. This mutant also failed to exhibit an ATP hydrolysis-dependent enhancement of binding of a conformation-sensitive monoclonal antibody, UIC2. Upon replacement with a 17 aa linker peptide having a predicted flexible secondary structure, but bearing no homology to the deleted 34 aa segment, normal Pgp transport and basal and drug-stimulated ATPase activities were restored along with increased UIC2 binding in the presence of substrate, suggesting a dramatic conformational change between the nonfunctional and functional molecules. Taken together, these data suggest a flexible secondary structure of the connector region is sufficient for the coordinate functioning of the two halves of Pgp, likely specifically required for the proper interaction of the two ATP binding sites.