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1.
Adv Exp Med Biol ; 1141: 549-580, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571174

RESUMO

ATP-binding cassette (ABC) transporters are involved in active pumping of many diverse substrates through the cellular membrane. The transport mediated by these proteins modulates the pharmacokinetics of many drugs and xenobiotics. These transporters are involved in the pathogenesis of several human diseases. The overexpression of certain transporters by cancer cells has been identified as a key factor in the development of resistance to chemotherapeutic agents. In this chapter, the localization of ABC transporters in the human body, their physiological roles, and their roles in the development of multidrug resistance (MDR) are reviewed. Specifically, P-glycoprotein (P-GP), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP/ABCG2) are described in more detail. The potential of ABC transporters as therapeutic targets to overcome MDR and strategies for this purpose are discussed as well as various explanations for the lack of efficacy of ABC drug transporter inhibitors to increase the efficiency of chemotherapy.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Neoplasias , Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Neoplasias/fisiopatologia , Distribuição Tecidual
2.
BMC Biol ; 15(1): 108, 2017 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-29121928

RESUMO

BACKGROUND: Protein-protein interactions (PPIs) are fundamental to the growth and survival of cells and serve as excellent targets to develop inhibitors of biological processes such as host-pathogen interactions and cancer cell proliferation. However, isolation of PPI inhibitors is extremely challenging. While several in vitro assays to screen for PPI inhibitors are available, they are often expensive, cumbersome, and require large amounts of purified protein. In contrast, limited in vivo assays are available to screen for small-molecule inhibitors of PPI. METHODS: We have engineered a yeast strain that is suitable for screening of small-molecule inhibitors of protein-protein interaction using the Yeast 2-hybrid Assay. We have optimised and validated the assay using inhibitors of the p53-Mdm2 interaction and identified a hitherto unreported putative Mdm2-binding domain in p53. RESULTS: We report a significantly improved and thoroughly validated yeast two-hybrid (Y2H) assay that can be used in a high throughput manner to screen for small-molecule PPI inhibitors. Using the p53-Mdm2 interaction to optimize the assay, we show that the p53-Mdm2 inhibitor nutlin-3 is a substrate for the yeast ATP-binding cassette (ABC) transporter Pdr5. By deleting nine ABC transporter-related genes, we generated a ABC9Δ yeast strain that is highly permeable to small molecules. In the ABC9Δ strain, p53-Mdm2 interaction inhibitors, like AMG232 and MI-773, completely inhibited the p53-Mdm2 interaction at nanomolar concentrations in the Y2H assay. In addition, we identified a conserved segment in the core DNA-binding domain of p53 that facilitates stable interaction with Mdm2 in yeast cells and in vitro. CONCLUSION: The Y2H assay can be utilized for high-throughput screening of small-molecule inhibitors of PPIs and to identify domains that stabilize PPIs.


Assuntos
Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Técnicas do Sistema de Duplo-Híbrido
3.
Adv Cancer Res ; 125: 71-96, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25640267

RESUMO

ABCB1 (P-glycoprotein/P-gp) is an ATP-binding cassette transporter well known for its association with multidrug resistance in cancer cells. Powered by the hydrolysis of ATP, it effluxes structurally diverse compounds. In this chapter, we discuss current views on the molecular basis of the substrate polyspecificity of P-gp. One of the features that accounts for this property is the structural flexibility observed in P-gp. Several X-ray crystal structures of mouse P-gp have been published recently in the absence of nucleotide, with and without bound inhibitors. All the structures are in an inward-facing conformation exhibiting different degrees of domain separation, thus revealing a highly flexible protein. Biochemical and biophysical studies also demonstrate this flexibility in mouse as well as human P-gp. Site-directed mutagenesis has revealed the existence of multiple transport-active binding sites in P-gp for a single substrate. Thus, drugs can bind at either primary or secondary sites. Biochemical, molecular modeling, and structure-activity relationship studies suggest a large, common drug-binding pocket with overlapping sites for different substrates. We propose that in addition to the structural flexibility, the molecular or chemical flexibility also contributes to the binding of substrates to multiple sites forming the basis of polyspecificity.


Assuntos
Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/ultraestrutura , Animais , Sítios de Ligação/fisiologia , Transporte Biológico/fisiologia , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Conformação Proteica
4.
J Cell Biochem ; 115(8): 1381-91, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24939447

RESUMO

ARRY-334543 is a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases. We conducted this study to determine whether ARRY-334543 can enhance the efficacy of conventional anticancer drugs through interaction with ABC transporters. Lung cancer cell line NCI-H460 and its ABCG2-overexpressing NCI-H460/MX20, as well as the ABCG2-, ABCB1-, and ABCC10-overexpressing transfected cell lines were used for the reversal study. Our results demonstrated that ARRY-334543 (1.0 µM) significantly reversed ABCG2-mediated multidrug resistance (MDR) by directly inhibiting the drug efflux function of ABCG2, resulting in the elevated intracellular accumulation of chemotherapeutic drugs in the ABCG2-overexpressing cell lines. In addition, in isolated membranes, ARRY-334543 stimulated ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner indicating that this drug directly interacts at the drug-binding pocket of this transporter. ARRY-334543 (1.0 µM) only slightly reversed ABCB1- and partially reversed ABCC10-mediated MDR suggesting that it exhibits high affinity toward ABCG2. Moreover, homology modeling predicted the binding conformation of ARRY-334543 at Arg482 centroid-based grid of ABCG2. However, ARRY-334543 at reversal concentrations did not affect the expression level of ABCG2, AKT and ERK1/2 and regulate the re-localization of ABCG2. We conclude that ARRY-334543 significantly reverses drug resistance mediated by ABCG2.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinas/administração & dosagem , Tiazóis/administração & dosagem , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/química , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Proteínas Oncogênicas v-erbB/genética , Paclitaxel/administração & dosagem , Ligação Proteica , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética
5.
PLoS One ; 8(12): e82463, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24349290

RESUMO

P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Expressão Gênica , Células HeLa , Humanos , Hidrólise , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução Genética
6.
Biochemistry ; 52(41): 7327-38, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24053441

RESUMO

P-Glycoprotein (P-gp) is an ATP-binding cassette efflux transporter involved in the development of multidrug resistance in cancer cells. Although the mechanism of P-gp efflux has been extensively studied, aspects of its catalytic and transport cycle are still unclear. In this study, we used conserved C431 and C1074 in the Walker A motif of nucleotide-binding domains (NBDs) as reporter sites to interrogate the interaction between the two NBDs during the catalytic cycle. Disulfide cross-linking of the C431 and C1074 residues in a Cys-less background can be observed in the presence of M14M and M17M cross-linkers, which have spacer arm lengths of 20 and 25 Å, respectively. However, cross-linking with both cross-linkers was prevented in the ADP-vanadate trapped (closed) conformation. Both C431 and C1074 alone or together (double mutant) in the apo and closed conformations were found to be accessible to fluorescein 5-maleimide (FM) and methanethiosulfonate derivatives of rhodamine and verapamil. In addition, C1074 showed 1.4- and 2-fold higher degrees of FM labeling than C431 in the apo and closed conformations, respectively, demonstrating that C1074 is more accessible than C431 in both conformations. In the presence of P-gp substrates, cross-linking with M17M is still observed, suggesting that binding of substrate in the transmembrane domains does not change the accessibility of the cysteines in the NBDs. In summary, the cysteines in the Walker A motifs of NBDs of human P-gp are differentially accessible to thiol-specific agents in the apo and closed conformations.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Difosfato de Adenosina/metabolismo , Cisteína/metabolismo , Compostos de Sulfidrila/metabolismo , Vanadatos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cisteína/química , Humanos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Compostos de Sulfidrila/química , Vanadatos/química
7.
Biochem Pharmacol ; 86(7): 904-13, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23962445

RESUMO

The overexpression of the serine/threonine specific Polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. BI 2536 is the first selective inhibitor of Plk1 that inhibits cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to BI 2536 is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that overexpressing of either ABCB1 or ABCG2 is a novel mechanism of acquired resistance to BI 2536 in human cancer cells. Moreover, BI 2536 stimulates the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner, and inhibits the drug substrate transport mediated by these transporters. More significantly, the reduced chemosensitivity and BI 2536-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor or other chemotherapeutic agents that also interact with ABCB1 and ABCG2, such as tyrosine kinase inhibitors nilotinib and lapatinib. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to BI 2536, a combined regimen of BI 2536 and inhibitors or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Lapatinib , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Quinase 1 Polo-Like
8.
Int J Cancer ; 132(1): 224-35, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22623106

RESUMO

Saracatinib, a highly selective, dual Src/Abl kinase inhibitor, is currently in a Phase II clinical trial for the treatment of ovarian cancer. In our study, we investigated the effect of saracatinib on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters in vitro and in vivo. Our results showed that saracatinib significantly enhanced the cytotoxicity of ABCB1 substrate drugs in ABCB1 overexpressing HeLa/v200, MCF-7/adr and HEK293/ABCB1 cells, an effect that was stronger than that of gefitinib, whereas it had no effect on the cytotoxicity of the substrates in ABCC1 overexpressing HL-60/adr cells and its parental sensitive cells. Additionally, saracatinib significantly increased the doxorubicin (Dox) and Rho 123 accumulation in HeLa/v200 and MCF-7/adr cells, whereas it had no effect on HeLa and MCF-7 cells. Furthermore, saracatinib stimulated the ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. In addition, the homology modeling predicted the binding conformation of saracatinib within the large hydrophobic drug-binding cavity of human ABCB1. However, neither the expression level of ABCB1 nor the phosphorylation level of Akt was altered at the reversal concentrations of saracatinib. Importantly, saracatinib significantly enhanced the effect of paclitaxel against ABCB1-overexpressing HeLa/v200 cancer cell xenografts in nude mice. In conclusion, saracatinib reverses ABCB1-mediated MDR in vitro and in vivo by directly inhibiting ABCB1 transport function, without altering ABCB1 expression or AKT phosphorylation. These findings may be helpful to attenuate the effect of MDR by combining saracatinib with other chemotherapeutic drugs in the clinic.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Benzodioxóis/farmacologia , Quinazolinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Gefitinibe , Células HEK293 , Células HL-60 , Células HeLa , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases Associadas a rho/metabolismo
9.
Biochem Pharmacol ; 85(3): 325-34, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23153455

RESUMO

Melanoma is the most serious type of skin cancer with a high potential for metastasis and very low survival rates. The discovery of constitutive activation of the BRAF kinase caused by activating BRAF(V600E) kinase mutation in most melanoma patients led to the discovery of the first potent BRAF(V600E) signaling inhibitor, vemurafenib. Vemurafenib was effective in treating advanced melanoma patients and was proposed for the treatment of other BRAF(V600E) mutant cancers as well. Unfortunately, the success of vemurafenib was hampered by the rapid development of acquired resistance in different types of BRAF(V600E) mutant cancer cells. It becomes important to identify and evaluate all of the potential mechanisms of cellular resistance to vemurafenib. In this study, we characterized the interactions of vemurafenib with three major ATP-binding cassette (ABC) transporters, ABCB1, ABCC1 and ABCG2. We found that vemurafenib stimulated the ATPase activity and potently inhibited drug efflux mediated by ABCB1 and ABCG2. Vemurafenib also restored drug sensitivity in ABCG2-overexpressing cells. Moreover, we revealed that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF(V600E) mutant A375 cells. Taken together, our findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting involvement of this transporter in acquired resistance to vemurafenib. Thus, combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Indóis/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sulfonamidas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/genética , Vemurafenib
10.
Mol Pharmacol ; 82(1): 47-58, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22491935

RESUMO

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC(50) = 0.24 µM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Quinolinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Células HEK293 , Células HL-60 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Rodaminas/farmacologia , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
11.
Biochem Pharmacol ; 82(11): 1562-71, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21855533

RESUMO

Over-expression of ABCG2 is linked to multidrug resistance in cancer chemotherapy. We have previously shown that functionalized aurones effectively reduced the efflux of pheophorbide A (an ABCG2 substrate) from ABCG2 over-expressing MDA-MB-231/R ("R") cells. In the present report, we investigated the functional relevance of this observation and the mechanisms by which it occurs. Aurones and related analogs were investigated for re-sensitization of R cells to mitoxantrone (MX, a chemotherapeutic substrate of ABCG2) in cell-based assays, accumulation of intracellular MX by cell cytometry, interaction with ABCG2 by biochemical assays and in vivo efficacy in MX resistant nude mice xenografts. We found that methoxylated aurones interacted directly with ABCG2 to inhibit efflux activity, possibly by competing for occupancy of one of the substrate binding sites on ABCG2. The present evidence suggests that they are not transported by ABCG2 although they stimulate ABCG2-ATPase activity. Alteration of ABCG2 protein expression was also discounted. One member was found to re-sensitize R cells to MX in both in vitro and in vivo settings. Our study identified methoxylated aurones as promising compounds associated with low toxicities and potent modulatory effects on the ABCG2 efflux protein. Thus, they warrant further scrutiny as lead templates for development as reversal agents of multidrug resistance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzofuranos/farmacologia , Chalconas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Flavonas/farmacologia , Indanos/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Antineoplásicos/farmacologia , Benzofuranos/química , Sobrevivência Celular/efeitos dos fármacos , Chalconas/química , Clorofila/análogos & derivados , Clorofila/metabolismo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Feminino , Flavonas/química , Indanos/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitoxantrona/farmacologia , Transplante de Neoplasias , Transplante Heterólogo
12.
Bioorg Med Chem Lett ; 21(18): 5428-31, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21788136

RESUMO

The emergence and spread of multidrug-resistant strains of Staphylococcus aureus and Mycobacterium tuberculosis are generating a threat to public health worldwide. In the current study, a series of N(1)-benzyl and N(1)-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamine derivatives were synthesized and investigated for their antimicrobial activity against S. aureus, and Mycobacterium smegmatis which is taxonomically related to M. tuberculosis. Most of the compounds exhibited good activity against M. smegmatis as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 7o showed potent antimycobacterial activity against M. smegmatis without mammalian DHFR inhibition liability. The results from this study indicate that 1-benzyl derivatives of 1,6-dihydro-1,3,5-triazine-2,4-diamines may be used as lead compounds for the discovery of antimycobacterial agents.


Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Compostos de Espiro/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Triazinas/farmacologia , Antibacterianos/química , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Compostos de Espiro/síntese química , Compostos de Espiro/química , Estereoisomerismo , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/química
13.
ChemMedChem ; 6(4): 713-24, 2011 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-21302361

RESUMO

The ability of aurones to modulate the efflux activities of ABCG2 and ABCB1 was investigated by quantifying their effects on the accumulation of pheophorbide A (PhA) in ABCG2-overexpressing MDA-MB-231/R cells and calcein AM in ABCB1-overexpressing MDCKII/MDR1 cells. Key structural features for interactions at both ABCG2 and ABCB1 are a methoxylated ring A, an intact exocyclic double bond, and the location of the carbonyl bond on ring C. Modifications on rings B and C were less critical and served primarily to moderate activity and selectivity for one or both transporters. These SAR trends were quantified by Free-Wilson analyses and are reflected in a pharmacophore model for PhA accumulation. Several compounds were found to be equipotent with fumitremorgin C (FTC) in promoting PhA accumulation, and they also demonstrated strong affinities for ABCB1. These compounds were disubstituted on ring B with methoxy or a combination of methoxy and hydroxy groups. Taken together, our findings highlight the versatility of the aurone template as a lead scaffold for the design of dual-targeting ABCG2 and ABCB1 modulators.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Proteínas de Neoplasias/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos/síntese química , Antineoplásicos/química , Benzofuranos/síntese química , Benzofuranos/química , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Relação Estrutura-Atividade
14.
Eur J Pharm Sci ; 35(4): 293-306, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18725288

RESUMO

A series of 4,6-dimethoxyaurones were synthesized by reacting 4,6-dimethoxybenzofuran-3(2H)-one with various benzaldehydes in a base-catalyzed aldol reaction. A Z configuration was assigned to the aurones based on spectroscopic and crystallographic data. The aurones were tested for their ability to modulate ABCG2 (breast cancer resistance protein)-mediated multidrug resistance in vitro. Several members (0.5 microM) increased the accumulation of mitoxantrone (MX) in human breast cancer cells (MDA-MB-231) transfected with ABCG2 and re-sensitized these cells to the cytotoxic effects of MX. In the re-sensitization assay, aurones at 0.5 microM reduced the resistance of the transfected cells to MX to just twice that of the parental cells, exceeding fumitremorgin C (FTC) tested at the same concentration. The aurones (10 microM) also increased calcein-AM accumulation in MDCKII/MDR1 cells that were transfected with ABCB1 (P-glycoprotein), at levels comparable to verapamil tested at the same concentration. Structure-activity analysis showed that substitution of the benzylidene ring B of the aurone template was less important for ABCG2 inhibition, with little variation in activity noted for compounds with an unsubstituted ring B or one that was substituted. In contrast, substitution of ring B gave rise to better inhibitors of ABCB1. A preference for the 3' position of ring B was noted. There was also some indication from the data that aurones with good ABCG2 inhibitory activity were poor ABCB1 inhibitors and vice versa, but further confirmation would be required. Limited antiproliferative activity (>70% cell survival) was observed for many aurones on four different cell lines. Thus, functionalized 4,6-dimethoxyaurones are promising ABCG2 inhibitors that combine good activity at submicromolar concentrations with limited antiproliferative activity.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzofuranos/síntese química , Benzofuranos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Antineoplásicos/metabolismo , Benzofuranos/metabolismo , Western Blotting , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , Cristalografia por Raios X , DNA Complementar/biossíntese , DNA Complementar/genética , Cães , Feminino , Citometria de Fluxo , Fluoresceínas/metabolismo , Humanos , Indicadores e Reagentes , Mitoxantrona/metabolismo , Modelos Moleculares , Relação Estrutura-Atividade , Sais de Tetrazólio , Tiazóis , Células Tumorais Cultivadas
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