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1.
Pharm Res ; 37(1): 13, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31873817

RESUMO

PURPOSE: Multiple time-point sampling is required in transcellular transport studies to accurately calculate the appropriate efflux ratio (ER). Our study sought to develop a simplified method to determine the ER in Caco-2 cells. METHODS: The equation for the ER was derived from a three-compartment model of apical to basal and basal to apical transport. Transcellular transport studies were conducted with 10 non-P-glycoprotein (P-gp) and 6 P-gp substrates in Caco-2 cells, and the ER was calculated using this equation. RESULTS: The equation for the ER used the concentration ratio in the receiver compartment at the same time-point; therefore, the ER can theoretically be calculated using only a single point. The ER of all non-P-gp substrates tested was close to 1 at all sampling times. The ERs of cyclosporine A calculated from the concentration ratio at 30, 60, 90, and 120 min incubation were 2.93, 6.43, 7.12, and 9.57, respectively, and the ER at 120 min was almost identical to the theoretical value (9.62) calculated using three-compartment model analysis. The other 5 P-gp substrates showed a similar tendency. Single-point sampling can be used to accurately calculate ER at 120 min. CONCLUSIONS: Single-point sampling is a promising approach for calculating appropriate ERs in the drug discovery stage.


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 , Modelos Biológicos , Transporte Biológico , Técnicas Biossensoriais/métodos , Células CACO-2 , Ciclosporina , Interações Medicamentosas/fisiologia , Humanos , Permeabilidade , Ligação Proteica , Fatores de Tempo , Transcitose
2.
Int J Pharm ; 570: 118653, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31472218

RESUMO

Co-amorphous mixtures have been demonstrated to represent a promising approach for enhancing the dissolution of poorly water-soluble drugs. However, little is known of their permeability properties, especially through biological membranes, or about the relationship between their dissolution and permeability. In the present study, co-amorphous glibenclamide (GBC) mixtures with two amino acids, arginine (ARG) and serine (SER), in molar ratios of 1:1 were prepared by cryomilling. Their dissolution and permeability properties were studied in side-by-side diffusion chambers using cell layers containing Madine Darby kidney cells overexpressing P-glycoprotein (Pgp) transporters (MDCKII-MDR1), as Pgp may influence the absorption of GBC. Furthermore, two other compounds, the flavonoid quercetin (QRT) which is a Pgp inhibitor and the surfactant, sodium lauryl sulfate (SLS), were used as excipients to investigate if they improved either passive or active diffusion of GBC. In addition, amorphous QRT and a co-amorphous mixture of GBC and QRT (1:1) were characterized with respect to their solid-state properties and physical stability. It was demonstrated that co-amorphous GBC mixtures exhibited superior dissolution properties over the corresponding physical mixtures and amorphous GBC. Furthermore, the co-amorphous GBC-ARG-SLS mixture exhibited a 9-fold increase in permeating through the MDCKII-MDR1 cell layer as compared to the corresponding physical mixture. There was a correlation between the dissolution and permeability area under curve (AUC) values, evidence that the main mechanism behind the improved permeability of co-amorphous mixtures was their improved dissolution. The simultaneous dissolution/permeation testing with side-by-side diffusion chambers and MDCKII-MDR1 cells proved to be a feasible method for evaluating the dissolution/permeation interplay of amorphous compounds.


Assuntos
Glibureto/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Animais , Arginina/química , Cães , Composição de Medicamentos/métodos , Flavonoides/química , Células Madin Darby de Rim Canino , Permeabilidade/efeitos dos fármacos , Difração de Pó/métodos , Quercetina/química , Serina/química , Solubilidade/efeitos dos fármacos
3.
Int J Mol Sci ; 20(18)2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527404

RESUMO

Heat Shock Protein 90 (Hsp90) chaperone interacts with a broad range of client proteins involved in cancerogenesis and cancer progression. However, Hsp90 inhibitors were unsuccessful as anticancer agents due to their high toxicity, lack of selectivity against cancer cells and extrusion by membrane transporters responsible for multidrug resistance (MDR) such as P-glycoprotein (P-gp). Recognizing the potential of new compounds to inhibit P-gp function and/or expression is essential in the search for effective anticancer drugs. Eleven Hsp90 inhibitors containing an isoxazolonaphtoquinone core were synthesized and evaluated in two MDR models comprised of sensitive and corresponding resistant cancer cells with P-gp overexpression (human non-small cell lung carcinoma and colorectal adenocarcinoma). We investigated the effect of Hsp90 inhibitors on cell growth inhibition, P-gp activity and P-gp expression. Structure-activity relationship analysis was performed in respect to cell growth and P-gp inhibition. Compounds 5, 7, and 9 directly interacted with P-gp and inhibited its ATPase activity. Their potential P-gp binding site was identified by molecular docking studies. In addition, these compounds downregulated P-gp expression in MDR colorectal carcinoma cells, showed good relative selectivity towards cancer cells, while compound 5 reversed resistance to doxorubicin and paclitaxel in concentration-dependent manner. Therefore, compounds 5, 7 and 9 could be promising candidates for treating cancers with P-gp overexpression.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Relação Estrutura-Atividade
4.
Molecules ; 24(10)2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31130601

RESUMO

P-glycoprotein (P-gp) is a transmembrane protein that actively transports a wide variety of chemically diverse compounds out of the cell. It is highly associated with the ADMET (absorption, distribution, metabolism, excretion and toxicity) properties of drugs/drug candidates and contributes to decreasing toxicity by eliminating compounds from cells, thereby preventing intracellular accumulation. Therefore, in the drug discovery and toxicological assessment process it is advisable to pay attention to whether a compound under development could be transported by P-gp or not. In this study, an in silico multiclass classification model capable of predicting the probability of a compound to interact with P-gp was developed using a counter-propagation artificial neural network (CP ANN) based on a set of 2D molecular descriptors, as well as an extensive dataset of 2512 compounds (1178 P-gp inhibitors, 477 P-gp substrates and 857 P-gp non-active compounds). The model provided a good classification performance, producing non error rate (NER) values of 0.93 for the training set and 0.85 for the test set, while the average precision (AvPr) was 0.93 for the training set and 0.87 for the test set. An external validation set of 385 compounds was used to challenge the model's performance. On the external validation set the NER and AvPr values were 0.70 for both indices. We believe that this in silico classifier could be effectively used as a reliable virtual screening tool for identifying potential P-gp ligands.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Animais , Descoberta de Drogas , Humanos , Camundongos , Modelos Moleculares , Modelos Teóricos
5.
Phytomedicine ; 62: 152945, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31132750

RESUMO

BACKGROUND: Cucurbitacin E (CuE) is an oxygenated tetracyclic triterpenoid isolated from the fruits of Citrullus colocynthis (L.) Schrad. PURPOSE: This study outlines CuE's cytotoxic activity against drug-resistant tumor cell lines. Three members of ABC transporters superfamily, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and ABCB5 were investigated, whose overexpression in tumors is tightly linked to multidrug resistance. Further factors of drug resistance studied were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). METHODS: Cytotoxicity assays (resazurin assays) were used to investigate the activity of Citrullus colocynthis and CuE towards multidrug resistant cancer cells. Molecular docking (In silico) has been carried out to explore the CuE's mode of binding to ABC transporters (P-gp, BCRP and ABCB5). The visualization of doxorubicin uptake was done by a Spinning Disc Confocal Microscope. The assessment of proteins expression was done by western blotting analysis. COMPARE and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to cucurbitacins (CuA, CuB, CuE, CuD, CuI, and CuK). RESULTS: Multidrug-resistant cells overexpressing P-gp or BCRP were cross-resistant to CuE. By contrast, TP53 knock-out cells were sensitive to CuE. Remarkably, resistant cells transfected with oncogenic ΔEGFR or ABCB5 were hypersensitive (collateral sensitive) to CuE. In silico analyses demonstrated that CuE is a substrate for P-gp and BCRP. Immunoblot analyses highlighted that CuE targeted EGFR and silenced its downstream signaling cascades. The most striking result that emerged from the doxorubicin uptake by ABCB5 overexpressing cells is that CuE is an effective inhibitor for ABCB5 transporter when compared with verapamil. The COMPARE analyses of transcriptome-wide expression profiles of tumor cell lines of the NCI identified common genes involved in cell cycle regulation, cellular adhesion and intracellular communication for different cucurbitacins. CONCLUSION: CuE represents a potential therapeutic candidate for the treatment of certain types of refractory tumors. To best of our knowledge, this is the first time to identify CuE and verapamil as inhibitors for ABCB5 transporter.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Citrullus colocynthis/química , Leucemia/tratamento farmacológico , Triterpenos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , 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/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/química , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Leucemia/metabolismo , Leucemia/patologia , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Triterpenos/química , Triterpenos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
Drug Dev Res ; 80(5): 655-665, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31050027

RESUMO

Beta-secreatse (BACE-1) and cholinesterases are clinically validated targets of Alzheimer's disease (AD), for which natural products have provided immense contribution. The multifaceted nature of AD signifies the need of multitargeted agents to tackle this disease. In the search of new natural products as dual BACE-1/cholinesterase inhibitors, a library of pure natural products was screened for inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE-1. The screening efforts have identified 1,4-benzoquinone "embelin," a natural product derived from Embelia ribes displaying inhibition of all three enzymes, with IC50 values of 2.5, 5.4, and 2.1 µM, respectively. This screen has also identified isoquinoline alkaloids papaverine and L-tetrahydropalmatine as AChE inhibitors. Kinetic study has shown that embelin inhibits EeAChE and EqBChE with ki values of 4.59 and 0.57 µM, in an uncompetitive and noncompetitive manner, respectively. The interactions of embelin with allosteric peripheral anionic site of cholinesterases, has further supported the results of kinetic study. Embelin has also enhanced the activity of P-gp in LS-180 cells, the efflux pump which is involved in the clearance of amyloid-ß from AD brain. Further, the cell viability study in neuronal cell line has indicated the excellent therapeutic window of embelin. These results are indicative of the fact that embelin is a multitargeted agent playing role in stopping the formation of amyloid-ß oligomers (via inhibition of BACE-1), improves cholinergic-transmission (via inhibition of AChE/BChE) and increases amyloid-ß clearance (via P-gp induction).


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Acetilcolinesterase/química , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/química , Benzoquinonas/farmacologia , Butirilcolinesterase/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Benzoquinonas/química , Butirilcolinesterase/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Embelia/química , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular
7.
Molecules ; 24(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035631

RESUMO

P-glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance (MDR) in cancer cells. In order to enhance the uptake of chemotherapy drugs, larger amounts of P-gp inhibitors are required. Besides several chemically synthesized P-gp inhibitors, flavonoids as P-gp inhibitors are being investigated, with their advantages including abundance in our daily diet and a low toxicity. The cytotoxicity of daunorubicin (as a substrate of P-gp) to KB/MDR1 cells and the parental KB cells was measured in the presence or absence of flavonoids. A two-dimensional quantitative structure-activity relationship (2D-QSAR) model was built with a high cross-validation coefficient (Q2) value of 0.829. Descriptors including vsurf_DW23, E_sol, Dipole and vsurf_G were determined to be related to the inhibitory activity of flavonoids. The lack of 2,3-double bond, 3'-OH, 4'-OH and the increased number of methoxylated substitutions were shown to be beneficial for the inhibition of P-gp. These results are important for the screening of flavonoids for inhibitory activity on P-gp.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Flavonoides/química , Flavonoides/farmacologia , Relação Quantitativa Estrutura-Atividade , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Células KB , Modelos Moleculares , Conformação Molecular , Ligação Proteica
8.
Eur J Pharmacol ; 852: 231-243, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959046

RESUMO

Multidrug resistance (MDR) remains an obstacle to chemotherapy related with the overexpression of several efflux membrane proteins, and p-glycoprotein (P-gp) is the most studied among them. Thus, continuous investigational efforts are necessary to find valuable MDR reversal agents, and the flavonoid compound glabridin (GBD) seems to be a promising candidate. This study aimed to investigate the potential of GBD against MDR and explore the possible mechanisms. First, we found that GBD could decrease the half maximal inhibitory concentration of paclitaxel and doxorubicin (DOX) in breast cancer cells like MDA-MB-231/MDR1 cells and MCF-7/ADR cells. It was further explained that GBD enhanced the apoptosis of MDA-MB-231/MDR1 cells induced by DOX, due to the increased accumulation of DOX. Then, tests were performed to explore the possible MDR reversal mechanisms. On one hand, GBD can suppress the expression of P-gp. On the other hand, GBD can downregulate the activity of P-gp ATPase when cotreated with DOX or verapamil, revealing that GBD was a substrate of P-gp. Moreover, the obtained kinetic inhibition parameters proved that GBD was a competitive inhibitor of P-gp, and in molecular docking simulation modeling, GBD exhibited stronger binding affinity with P-gp than DOX. In conclusion, GBD can increase the accumulation of DOX in MDA-MB-231/MDR1 cells by suppressing the expression of P-gp and competitively inhibiting the P-gp efflux pump and enhance the apoptosis of MDA-MB-231/MDR1 cells induced by DOX, and thus realize reversal effects on MDR. Therefore, the combination therapy of anticancer drugs and flavonoid-like GBD is a promising strategy to overcome P-gp-mediated MDR.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoflavonas/farmacologia , Fenóis/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Isoflavonas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Fenóis/metabolismo , Conformação Proteica
9.
J Agric Food Chem ; 67(17): 4967-4975, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30955332

RESUMO

Rooibos tea ( Aspalathus linearis) is a well-known South African herbal tea enjoyed worldwide. Limited reports indicate the potential of rooibos tea to alter the activity of certain cytochrome P450 (CYP450) isozymes. In this study, the phytochemical investigation of MeOH extract of A. linearis (leaves and stems) resulted in the isolation and characterization of 11 phenolic compounds. The MeOH extract exhibited significant inhibition of the major human CYP450 isozymes (CYP3A4, CYP1A2, CYP2D6, CYP2C9, and CYP2C19). The strongest inhibition was observed by the extract for CYP3A4 (IC50 1.7 ± 0.1 µg/mL) followed by CYP2C19 (IC50 4.0 ± 0.3 µg/mL). Among the tested phytochemicals, the most potent inhibitors were isovitexin on CYP3A4 (IC50 3.4 ± 0.2 µM), vitexin on CYP2C9 (IC50 8.0 ± 0.2 µM), and thermopsoside on CYP2C19 (IC50 9.5 ± 0.2 µM). The two major, structurally related compounds aspalathin and nothofagin exhibited a moderate pregnane-X receptor (PXR) activation, which was associated with increased mRNA expression of CYP3A4 and CYP1A2, respectively. These results indicate that a high intake of nutraceuticals containing rooibos extracts may pose a risk of herb-drug interactions when consumed concomitantly with clinical drugs that are substrates of CYP enzymes.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Aspalathus/química , Sistema Enzimático do Citocromo P-450/química , Preparações de Plantas/química , Receptor de Pregnano X/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aspalathus/metabolismo , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Inocuidade dos Alimentos , Humanos , Folhas de Planta/química , Preparações de Plantas/metabolismo , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Chás de Ervas/análise
10.
ACS Appl Mater Interfaces ; 11(18): 16296-16310, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-30997984

RESUMO

The enhanced endo-lysosomal sequestration still remains a big challenge in overcoming multidrug resistance (MDR). Herein, a dual-responsive polyplex with effective endo-lysosomal escape based on methoxypoly(ethylene glycol)-polylactide-polyhistidine-ss-oligoethylenimine (mPEG- b-PLA-PHis-ssOEI) was developed for codelivering MDR1 siRNA and doxorubicin (DOX). The polyplex showed good encapsulation of DOX and siRNA as well as triggered payload release in response to pH/redox stimuli because of the PHis protonation and the disulfide bond cleavage. The polyplex at an N/P ratio of 7 demonstrated a much higher payload delivery efficiency, MDR1 gene silence efficiency, cytotoxicity against MCF-7/ADR cell, and stronger MCF-7/ADR tumor growth inhibition than the polyplexes at higher N/P ratios. This was attributed to the stronger electrostatic attraction between siRNA and OEIs at higher N/P ratios that suppressed the release of MDR1 siRNA and OEIs, which played a dominant role in overcoming payload endo-lysosomal sequestration by the OEI-induced membrane permeabilization effect. Consequently, the polyplex with effective endo-lysosomal escape provides a rational approach for codelivery of siRNAs and chemotherapy agents for MDR reversal.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/farmacologia , Doxorrubicina/química , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endossomos/química , Humanos , Concentração de Íons de Hidrogênio , Lipídeos/química , Células MCF-7 , Nanopartículas/química , Neoplasias/patologia , Poliésteres/química , Polietilenoglicóis/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia
11.
Int J Mol Sci ; 20(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013627

RESUMO

Overcoming P-glycoprotein (P-gp) efflux is a strategy to improve the absorption and pharmacokinetics of its substrate drugs. Berberine inhibits P-gp and thereby increases the bioavailability of the P-gp substrate digoxin in rodents. However, the effects of berberine on P-gp in chickens are still unclear. Here, we studied the role of berberine in modulating broilers P-gp expression and function through both in situ and in vitro models. In addition, molecular docking was applied to analyze the interactions of berberine with P-gp as well as with chicken xenobiotic receptor (CXR). The results showed that the mRNA expression levels of chicken P-gp and CXR decreased in the ileum following exposure to berberine. The absorption rate constant of rhodamine 123 increased after berberine treatment, as detected using an in situ single-pass intestinal perfusion model. Efflux ratios of P-gp substrates (tilmicosin, ciprofloxacin, clindamycin, ampicillin, and enrofloxacin) decreased and the apparent permeability coefficients increased after co-incubation with berberine in MDCK-chAbcb1 cell models. Bidirectional assay results showed that berberine could be transported by chicken P-gp with a transport ratio of 4.20, and this was attenuated by verapamil (an inhibitor of P-gp), which resulted in a ratio of 1.13. Molecular docking revealed that berberine could form favorable interactions with the binding pockets of both CXR and P-gp, with docking scores of -7.8 and -9.5 kcal/mol, respectively. These results indicate that berberine is a substrate of chicken P-gp and down-regulates P-gp expression in chicken tissues, thereby increasing the absorption of P-gp substrates. Our findings suggest that berberine increases the bioavailability of other drugs and that drug-drug interactions should be considered when it is co-administered with other P-gp substrates with narrow therapeutic windows.


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 , Berberina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Berberina/química , Galinhas , Cães , Células Madin Darby de Rim Canino , Modelos Moleculares , Conformação Proteica , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade
12.
Curr Comput Aided Drug Des ; 15(3): 212-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30280673

RESUMO

BACKGROUND: Permeability glycoprotein (P-gp) is one of the cell membrane proteins that can push some drugs out of the cell causing drug tolerance and its inhibition can prevent drug resistance. OBJECTIVE: In this study, we used image-based Quantitative Structure-Activity Relationship (QSAR) models to predict the P-gp inhibitory activity of epigallocatechin and gallocatechin derivatives. METHODS: The 2D-chemical structures and their P-gp inhibitory activity were taken from literature. The pixels of images and their Principal Components (PCs) were calculated using MATLAB software. Principle Component Regression (PCR), Artificial Neural Network (ANN) and Support Vector Machine (SVM) approaches were used to develop QSAR models. Statistical parameters included the leave one out cross-validated correlation coefficient (q2) for internal validation of the models and R2 of test set, Root Mean Square Error (RMSE) and Concordance Correlation Coefficient (CCC) were applied for external validation. RESULTS: Six PCs from image analysis method were selected by stepwise regression for developing linear and non-linear models. Non-linear models i.e. ANN (with the R2 of 0.80 for test set) were chosen as the best for the established QSAR models. CONCLUSION: According to the result of the external validation, ANN model based on image analysis method can predict the P-gp inhibitory activity of epigallocatechin and gallocatechin derivatives better than the PCR and SVM models.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Catequina/análogos & derivados , Catequina/química , Catequina/farmacologia , Modelos Lineares , Dinâmica não Linear , Permeabilidade , Relação Quantitativa Estrutura-Atividade , Software , Máquina de Vetores de Suporte
13.
Curr Med Chem ; 26(10): 1746-1760, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29189117

RESUMO

The P-glycoprotein is an efflux transporter that expels substances out of the cells and has an important impact on the pharmacokinetic and pharmacodynamic properties of drugs. The study of the interactions between ligands and the P-glycoprotein has implications in the design of Central Nervous System drugs and their transport across the blood-brain barrier. Moreover, since the P-glycoprotein is overexpressed in some types of cancers, the protein is responsible for expelling the drug therapies from the cells, and hence, for drug resistance. In this review, we describe different P-glycoprotein binding sites reported for substrates, inhibitors and modulators, and focus on molecular docking studies that provide useful information about drugs and P-glycoprotein interactions. Docking in crystallized structures and homology models showed potential in the detection of the binding site and key residues responsible for ligand recognition. Moreover, virtual screening through molecular docking discriminates P-glycoprotein ligands from decoys. We also discuss challenges and limitations of molecular docking simulations applied to this particular protein. Computational structure-based approaches are very helpful in the study of novel ligands that interact with the P-glycoprotein and provide insights to understand the P-glycoprotein molecular mechanism of action.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Compostos Orgânicos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Animais , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Compostos Orgânicos/química , Ligação Proteica
14.
J Med Chem ; 62(2): 974-986, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30584838

RESUMO

P-Glycoprotein is a well-known membrane transporter responsible for the efflux of an ample spectrum of anticancer drugs. Its relevance in the management of cancer chemotherapy is increased in view of its high expression in cancer stem cells, a population of cancer cells with strong tumor-promoting ability. In the present study, a series of compounds were synthesized through structure modulation of [4'-(6,7-dimethoxy-3,4-dihydro-1 H-isoquinolin-2-ylmethyl)biphenyl-4-ol] (MC70), modifying the phenolic group of the lead compound. Among them, compound 5b emerged for its activity against the transporter (EC50 = 15 nM) and was capable of restoring doxorubicin antiproliferative activity at nontoxic concentration. Its behavior was rationalized through a molecular modeling study consisting of a well-tempered metadynamics simulation, which allowed one to identify the most favorable binding pose, and of a subsequent molecular dynamics run, which indicated a peculiar effect of the compound on the motion pattern of the transporter.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Tetra-Hidroisoquinolinas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cães , Doxorrubicina/farmacologia , Edição de Genes , Humanos , Ligantes , Células Madin Darby de Rim Canino , Simulação de Dinâmica Molecular , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Permeabilidade/efeitos dos fármacos , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/metabolismo , Tetra-Hidroisoquinolinas/farmacologia
15.
BMC Struct Biol ; 18(1): 17, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30545335

RESUMO

BACKGROUND: P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. Although several P-glycoprotein structures are available, these are either at low resolution, or represent mutated and/or quiescent states of the protein. RESULTS: In the post-hydrolytic state the structure of the wild-type protein has been resolved at about 8 Å resolution. The cytosolic nucleotide-binding domains (NBDs) are separated but ADP remains bound, especially at the first NBD. Gaps in the transmembrane domains (TMDs) that connect to an inner hydrophilic cavity are filled by density emerging from the annular detergent micelle. The NBD-TMD linker is partly resolved, being located between the NBDs and close to the Signature regions involved in cooperative NBD dimerization. This, and the gap-filling detergent suggest steric impediment to NBD dimerization in the post-hydrolytic state. Two central regions of density lie in two predicted drug-binding sites, implying that the protein may adventitiously bind hydrophobic substances even in the post-hydrolytic state. The previously unresolved N-terminal extension was observed, and the data suggests these 30 residues interact with the headgroup region of the lipid bilayer. CONCLUSION: The structural data imply that (i) a low basal ATPase activity is ensured by steric blockers of NBD dimerization and (ii) allocrite access to the central cavity may be structurally linked to NBD dimerization, giving insights into the mechanism of drug-stimulation of P-glycoprotein activity.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Microscopia Crioeletrônica , Humanos , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
16.
Life Sci ; 214: 118-123, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30449449

RESUMO

P-glycoprotein (P-gp) is a member of ATP-binding cassette (ABC) superfamily which extrudes chemotherapeutic agents out of the cell. Suppression of this efflux activity has been the subject of numerous attempts to develop P-gp inhibitors. The aim of this review is to present up-to-date information on the structural and functional aspects of P-gp and its known inhibitors. The data presented also provide some information on drug discovery approaches for candidate P-gp inhibitors. Nucleotide-binding domains (NBDs) and drug-binding domains (DBDs) have been extensively studied to gain more information about P-gp inhibition and it looks that the ATPase activity of this pump has been the most attractive target for designing inhibitors. Hydrophobic and π-π (aromatic) interactions between P-gp binding domains and inhibitors are dominant intermolecular forces that have been reported in many studies using different methods. Many synthetic and natural products have been found to possess inhibitory or modulatory effects on drug transporter proteins. Log P value is an important factor in studying these inhibitors and has a crucial role on absorption, distribution, metabolism, and excretion (ADME) properties of candidate P-gp inhibitors.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Acridinas/farmacologia , Antineoplásicos/química , Sítios de Ligação , Produtos Biológicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Bicamadas Lipídicas , Terapia de Alvo Molecular/métodos , Piperidinas/farmacologia , Conformação Proteica , Quinolinas/química , Quinolinas/farmacologia , Tetra-Hidroisoquinolinas/farmacologia
17.
BMC Cancer ; 18(1): 1168, 2018 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-30477461

RESUMO

BACKGROUND: Wnt signaling has been linked with P-glycoprotein (P-gp) overexpression and which was mainly mediated by ß-catenin nuclear translocation. Flavonoids have already been reported as modulators of the Wnt/ß-catenin pathway and hence they may serve as promising agents in the reversal of P-gp mediated cancer multi drug resistance (MDR). METHODS: In this study, we screened selected flavonoids against Wnt/ß-catenin signaling molecules. The binding interaction of flavonoids (theaflavin, quercetin, rutin, epicatechin 3 gallate and tamarixetin) with GSK 3ß was determined by molecular docking. Flavonoids on P-gp expression and the components of Wnt signaling in drug-resistant KBCHR8-5 cells were analyzed by western blotting and qRT-PCR. The MDR reversal potential of these selected flavonoids against P-gp mediated drug resistance was analyzed by cytotoxicity assay in KBCHR8-5 and MCF7/ADR cell lines. The chemosensitizing potential of flavonoids was further analyzed by observing cell cycle arrest in KBCHR8-5 cells. RESULTS: In this study, we observed that the components of Wnt/ß-catenin pathway such as Wnt and GSK 3ß were activated in multidrug resistant KBCHR8-5 cell lines. All the flavonoids selected in this study significantly decreased the expression of Wnt and GSK 3ß in KBCHR8-5 cells and subsequently modulates P-gp overexpression in this drug-resistant cell line. Further, we observed that these flavonoids considerably decreased the doxorubicin resistance in KBCHR8-5 and MCF7/ADR cell lines. The MDR reversal potential of flavonoids were found to be in the order of theaflavin > quercetin > rutin > epicatechin 3 gallate > tamarixetin. Moreover, we observed that flavonoids pretreatment significantly induced the doxorubicin-mediated arrest at the phase of G2/M. Further, the combinations of doxorubicin with flavonoids significantly modulate the expression of drug response genes in KBCHR8-5 cells. CONCLUSION: The present findings illustrate that the studied flavonoids significantly enhances doxorubicin-mediated cell death through modulating P-gp expression pattern by targeting Wnt/ß-catenin signaling in drug-resistant KBCHR8-5 cells.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Flavonoides/farmacologia , Expressão Gênica , Via de Sinalização Wnt/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Flavonoides/química , Glicogênio Sintase Quinase 3 beta/química , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Modelos Biológicos , Conformação Molecular , Ligação Proteica , Relação Estrutura-Atividade
18.
Sci Rep ; 8(1): 14643, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279588

RESUMO

P-glycoprotein (Pgp) is an ATP-binding cassette transporter that eliminates toxins from the cell but causes multidrug resistance in chemotherapies. The crystal structures of Pgp revealed drug-like compounds bound to an inward-facing conformation in which the energy-harnessing nucleotide binding domains (NBDs) were widely separated with no interfacial interaction. Following drug binding, inward-facing Pgp must transition to an NBD dimer conformation to achieve ATP binding and hydrolysis at canonical sites defined by both halves of the interface. However, given the high degree of flexibility shown for this transporter, it is difficult to envision how NBDs overcome entropic considerations for achieving proper alignment in order to form the canonical ATP binding site. We explored the hypothesis that substrate occupancy of the polyspecific drug-binding cavity plays a role in the proper alignment of NBDs using computational approaches. We conducted twelve atomistic molecular dynamics (MD) simulations (100-300 ns) on inward-facing Pgp in a lipid bilayer with and without small molecule substrates to ascertain effects of drug occupancy on NBD dimerization. Both apo- and drug-occupied simulations showed NBDs approaching each other compared to the crystal structures. Apo-Pgp reached a pseudo-dimerization in which NBD signature motifs for ATP binding exhibited a significant misalignment during closure. In contrast, occupancy of three established substrates positioned by molecular docking achieved NBD alignment that was much more compatible with a canonical NBD dimerization trajectory. Additionally, aromatic amino acids, known to confer the polyspecific drug-binding characteristic of the internal pocket, may also govern polyspecific drug access to the cavity. The enrichment of aromatics comprising the TM4-TM6 portal suggested a preferential pathway over the aromatic-poor TM10-TM12 for lateral drug entry from the lipid bilayer. Our study also suggested that drug polyspecificity is enhanced due to a synergism between multiple drug-domain interactions involving 36 residues identified in TM1, 5, 6, 7, 11 and 12.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Simulação de Dinâmica Molecular , Nucleotídeos/química , Animais , Sítios de Ligação , Bicamadas Lipídicas/química , Camundongos , Simulação de Acoplamento Molecular/métodos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica
19.
PLoS One ; 13(9): e0202749, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30183750

RESUMO

Drug resistant tuberculosis (TB) is a major worldwide health problem. In addition to the bacterial mechanisms, human drug transporters limiting the cellular accumulation and the pharmacological disposition of drugs also influence the efficacy of treatment. Mycobacterium tuberculosis topoisomerase-I (MtTopo-I) is a promising target for antimicrobial treatment. In our previous work we have identified several hit compounds targeting the MtTopo-I by in silico docking. Here we expand the scope of the compounds around three scaffolds associated with potent MtTopo-I inhibition. In addition to measuring the effect of newly generated compounds on MtTopo-I activity, we characterized the compounds' antimicrobial activity, toxicity in human cells, and interactions with human multidrug transporters. Some of the newly developed MtTopo-I inhibitors have strong antimicrobial activity and do not harm mammalian cells. Moreover, our studies revealed significant human ABC drug transporter interactions for several MtTopo-I compounds that may modify their ADME-Tox parameters and cellular effects. Promising new drug candidates may be selected based on these studies for further anti-TB drug development.


Assuntos
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/metabolismo , Mycobacterium tuberculosis/enzimologia , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase I/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Animais , Linhagem Celular , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Inibidores da Topoisomerase I/toxicidade
20.
Biochemistry (Mosc) ; 83(8): 907-929, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30208829

RESUMO

This review considers one of the most clinically relevant representatives of the ABC transporters - multidrug resistance protein 1 (P-glycoprotein 1 or Pgp). Data on the primary, secondary, and tertiary structure of the protein, its synthesis and degradation, and roles of its fragments in transporter activity are presented. Particular attention is given to the mechanism of functioning of Pgp. In view of the absence of a generally recognized mechanism of action of Pgp, several existing models of the protein transport cycle are discussed. Epigenetic regulation of the ABCB1 gene and modulation of Pgp expression by microRNAs are discussed.


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 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Epigênese Genética , Humanos , Domínios Proteicos , Transporte Proteico , Proteólise
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