RESUMEN
The human multidrug transporter P-glycoprotein (Pgp or ABCB1) sets up pharmacological barriers to many clinically important drugs, a therapeutic remedy for which has yet to be formulated. For the rational design of mechanism-based inhibitors (or modulators), it is necessary to map the potential sites for modulator interaction and understand their modes of communication with the other functional domains of Pgp. In this study, combining directed mutagenesis with homology modeling, we provide evidence of two modulator-specific sites at the lipid protein interface of Pgp. Targeting 21 variant positions in the COOH-terminal transmembrane (TM) regions, we find residues M948 (in TM11) and F983, M986, V988, and Q990 (all four in TM12) critically involved in substrate-site modulation by a thioxanthene-based allosteric modulator cis-(Z)-flupentixol. Interestingly, for ATP-site modulation by the same modulator, only two (M948 and Q990) of those four residues appear indispensable, together with two additional residues, T837 and I864 in TM9 and TM10, respectively, suggesting independent modes of communication linking the allosteric site with the substrate binding and ATPase domains. None of the seven residues identified prove to be critical for modulation of the substrate or ATP sites by Pgp modulators that are transported by the pump, such as cyclosporin A or verapamil, indicating their specificity for cis-(Z)-flupentixol. On the other hand, ATP-site modulation by verapamil proves to be highly sensitive to replacement at positions F716 (in TM7) and I765 (in TM8), and to a more moderate extent at I764 and L772 (both in TM8). Homology modeling based on the known crystal structures of the bacterial multidrug transporter SAV1866 and the mouse Pgp homologue maps the identified residues primarily at the lipid-protein interface of Pgp, in two spatially distinct modulator-specific clusters. The two modulatory sites demonstrate negative synergism in influencing ATP hydrolysis, consolidating their spatial distinctness. Because Pgp is known to recruit drug molecules directly from the lipid bilayer, identification of modulatory sites at the lipid-protein interface and at the same time outside the conventional central drug binding cavity is mechanistically revealing.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Lípidos/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Animales , Secuencia de Bases , Cartilla de ADN , Flupentixol/farmacología , Humanos , Ratones , Mutagénesis Sitio-DirigidaRESUMEN
We had earlier demonstrated that chronic exposure (30 days) to micro-molar concentration (0.50 microM) of arsenic induced head kidney macrophage (HKM) death in Clarias batrachus. The purpose of the present study is to characterize the nature of HKM death induced by arsenic and elucidate the signal transduction pathways involved in the process. Arsenic-induced HKM death was apoptotic in nature as evident from DNA gel, Annexin V-propidium iodide, Hoechst 33342 staining and TdT-mediated dUTP nick end labeling (TUNEL) assays. Inhibitor studies and immunoblot analyses further demonstrated that arsenic-induced HKM apoptosis involved activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, a well-characterized caspase-3 substrate. Preincubation with antioxidants N-acetyl-cysteine or dimethyl sulfoxide significantly lowered reactive oxygen species (ROS) levels in arsenic-treated HKM and prevented caspase activation, malondialdehyde formation and HKM apoptosis. Arsenic induced membrane translocation of the NADPH oxidase subunit p47(phox). Preincubation with apocynin and diphenyleneiodonium chloride, both selective inhibitors of NADPH oxidases, prevented p47(phox) translocation, ROS production and HKM death. Exposure of HKM to arsenic induced the activation of mitogen-activated protein kinase family (MAPK) proteins including c-Jun NH(2)-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38). Preincubation of HKM with p38 inhibitor SB203580 and JNK inhibitor SP600125 protected the HKM against arsenic-induced apoptosis. We conclude that exposure to micro-molar concentration of arsenic induces ROS generation through the activation of NADPH oxidases, which in turn causes caspase-3 mediated HKM apoptosis. In addition, the study also indicates a role of p38-JNK pathway in arsenic-induced HKM apoptosis in C. batrachus.
Asunto(s)
Apoptosis/efectos de los fármacos , Arsénico/toxicidad , Caspasa 3/fisiología , Peces/fisiología , Riñón/patología , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anexina A5/metabolismo , Antioxidantes/metabolismo , Bencimidazoles , Fragmentación del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Etiquetado Corte-Fin in Situ , Riñón/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.
Asunto(s)
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/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/aislamiento & purificación , Regulación Alostérica , Animales , Azidas/metabolismo , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Flupentixol/metabolismo , Flupentixol/farmacología , Humanos , Fenotiazinas/metabolismo , Prazosina/análogos & derivados , Prazosina/metabolismo , Xantinas/metabolismoRESUMEN
Through an extensive herbal drug screening program, we found that gomisin A, a dibenzocyclooctadiene compound isolated from Schisandra chinensis, reversed multidrug resistance (MDR) in Pgp-overexpressing HepG2-DR cells. Gomisin A was relatively non-toxic but without altering Pgp expression, it restored the cytotoxic actions of anticancer drugs such as vinblastine and doxorubicin that are Pgp substrates but may act by different mechanisms. Several lines of evidence suggest that gomisin A alters Pgp-substrate interaction but itself is neither a Pgp substrate nor competitive inhibitor. (1) First unlike Pgp substrates gomisin A inhibited the basal Pgp-associated ATPase (Pgp-ATPase) activity. (2) The cytotoxicity of gomisin A was not affected by Pgp competitive inhibitors such as verapamil. (3) Gomisin A acted as an uncompetitive inhibitor for Pgp-ATPase activity stimulated by the transport substrates verapamil and progesterone. (4) On the inhibition of rhodamine-123 efflux the effects of gomisin A and the competitive inhibitor verapamil were additive, so were the effects of gomisin A and the ATPase inhibitor vanadate. (5) Binding of transport substrates with Pgp would result in a Pgp conformational change favoring UIC-2 antibody reactivity but gomisin A impeded UIC-2 binding. (6) Photocrosslinking of Pgp with its transport substrate [125I]iodoarylazidoprazosin was inhibited by gomisin A in a concentration-dependent manner. Taken together our results suggest that gomisin A may bind to Pgp simultaneously with substrates and alters Pgp-substrate interaction.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Ciclooctanos/farmacología , Dioxoles/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Lignanos/farmacología , 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/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Anticarcinógenos/química , Anticarcinógenos/farmacología , Azidas/farmacología , Western Blotting/métodos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclooctanos/química , Dioxoles/química , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Hidrólisis/efectos de los fármacos , Lignanos/química , Prazosina/análogos & derivados , Prazosina/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Rodamina 123/metabolismo , Vanadatos/farmacología , Verapamilo/farmacología , Vinblastina/farmacologíaRESUMEN
Vertex is developing biricodar as a chemosensitizing agent designed to restore the effectiveness of chemotherapeutic agents in tumor multidrug resistance. By November 1998, phase II trials had commenced for biricodar, in combination with chemotherapy, for five common cancer indications: breast, ovarian, soft-tissue sarcomas, small cell lung cancer and prostate cancer. Phase II trials were ongoing in January 2002. By March 2000, Vertex was the sole developer of biricodar, as an agreement made in 1996 with BioChem Pharma (now Shire Pharmaceuticals), for the development and marketing of biricodar in Canada was terminated. Biricodar is the free base compound, which also has a citrate salt analog known as VX-710-3. Vertex has published three patents, WO-09615101, WO-09636630 and WO-09736869, disclosing derivatives of biricodar that are claimed for the treatment of multidrug resistant protein and P-glycoprotein-mediated multidrug resistant tumors. In January 2002, a Banc of America analyst report forecast that biricodar had a 30% chance of reaching the market with a launch date in the second half of 2005, with peak sales estimated at $250 million.
Asunto(s)
Antineoplásicos/uso terapéutico , Piperidinas/uso terapéutico , Piridinas/uso terapéutico , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Ensayos Clínicos como Asunto , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Neoplasias/tratamiento farmacológico , Piperidinas/efectos adversos , Piperidinas/farmacología , Piridinas/efectos adversos , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
Recent studies have shown that dibenzocyclooctadiene lignans may reverse P-glycoprotein-mediated multidrug resistance (Pgp-MDR) in cancer cells; however, the mechanism of action remains unknown. Through screening of herbs, we found that schisandrol A (SCH) isolated from Fructus Schisandrae (the dried fruit of Schisandra chinensis (Turcz.) Baill.) sensitized Pgp-MDR HepG2-DR cells by interfering with the function of Pgp-substrate complexes. In Pgp-MDR cells, SCH enhanced the cytotoxicity of cancer drugs that are Pgp substrates and restored vinblastine-induced G2/M arrest without lowering Pgp expression. SCH increased cellular retention of Pgp substrates such as rhodamine 123. In Pgp-overexpressing membrane preparations, SCH stimulated basal Pgp-ATPase thus showing some substrate-like function. However, SCH was not a competitive inhibitor for verapamil or progesterone and decreased their Km. In the presence of substrates, SCH decreased the reactivity between Pgp and the monoclonal antibody UIC-2 which is normally increased with active substrate-Pgp complexes. The labeling of active Pgp transport sites by [125I]-iodoarylazidoprazosin was partially blocked by SCH. SCH did not affect the activity of the mutant Pgp F983A suggesting that SCH acted differently than the thioxanthene type of Pgp allosteric inhibitors. Our results suggest that SCH acts by affecting the normal formation and functioning of the Pgp-substrate complexes.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/farmacología , Ciclooctanos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Lignanos/farmacología , Fitoterapia , Extractos Vegetales/farmacología , Schisandra , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/uso terapéutico , Western Blotting , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclooctanos/administración & dosificación , Ciclooctanos/uso terapéutico , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Citometría de Flujo , Frutas , Humanos , Concentración 50 Inhibidora , Lignanos/administración & dosificación , Lignanos/uso terapéutico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/química , Sitio Alostérico , Animales , Azidas/química , Sitios de Unión , Dominio Catalítico , Línea Celular , Membrana Celular/metabolismo , Flupentixol/química , Humanos , Hidrólisis , Insectos , Ligandos , Conformación Molecular , Prazosina/análogos & derivados , Prazosina/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Tioxantenos/química , Factores de Tiempo , Tripsina/química , Vanadatos/químicaRESUMEN
The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Flupentixol/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Regulación Alostérica/genética , Sitio Alostérico/genética , Sustitución de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Humanos , Ratones , Células 3T3 NIH , Mutación Puntual , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Vinblastina/químicaRESUMEN
The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Sitio Alostérico/efectos de los fármacos , Animales , Azidas/química , Azidas/metabolismo , Unión Competitiva/efectos de los fármacos , Dominio Catalítico , Línea Celular , Relación Dosis-Respuesta a Droga , Flupentixol/análogos & derivados , Flupentixol/metabolismo , Flupentixol/farmacología , Humanos , Hidrólisis/efectos de los fármacos , Insectos/metabolismo , Modelos Biológicos , Etiquetas de Fotoafinidad/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z)-flupentixol, a thioxanthene derivative, occurs through an allosteric mechanism. Unlike competitive inhibitors, such as cyclosporin A and verapamil, cis-(Z)-flupentixol does not interfere with substrate ([(125)I]iodoarylazidoprazosin) recognition by Pgp, instead it prevents substrate translocation and dissociation, resulting in a stable but reversible Pgp-substrate complex. cis-(Z)-Flupentixol-induced complex formation requires involvement of the Pgp substrate site, because agents that either physically compete (cyclosporin A) for or indirectly occlude (vanadate) the substrate-binding site prevent formation of the complex. Allosteric modulation by cis-(Z)-flupentixol involves a conformational change in Pgp detectable by monoclonal antibody UIC2 binding to a conformation-sensitive external epitope of Pgp. The conformational change observed is distinct from that induced by Pgp substrates or competitive inhibitors. A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Taken together, our results describe the molecular mechanism by which the Pgp modulator cis-(Z)-flupentixol allosterically inhibits drug transport.