RESUMEN
Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.
Asunto(s)
Antibacterianos , Proteínas de Escherichia coli , Animales , Ratones , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacología , Klebsiella pneumoniae/metabolismo , Escherichia coli , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacologíaRESUMEN
Multidrug resistance pathogens causing infections and illness remain largely untreated clinically. Efflux pumps are one of the primary processes through which bacteria develop resistance by transferring antibiotics from the interior of their cells to the outside environment. Inhibiting these pumps by developing efficient derivatives appears to be a promising strategy for restoring antibiotic potency. This investigation explores literature-reported inhibitors of E. coli efflux pump fusion proteins AcrB-AcrA and identify potential chemical derivatives of these inhibitors to overcome the limitations. Using computational and structure-guided approaches, a study was conducted with the selected inhibitors (AcrA:25-AcrB:59) obtained by data mining and their derivatives (AcrA:857-AcrB:3891) to identify their inhibitory effect on efflux pump using virtual screening, molecular docking and density functional theory (DFT) calculations. The finding indicates that Compound 2 (ZINC000072136376) has shown better binding and a significant inhibitory effect on AcrA, while Compound 3 (ZINC000072266819) has shown stronger binding and substantial inhibition effect on both non-mutant and mutated AcrB subunits. The identified derivatives could exhibit a better inhibitor and provide a potential approach for restoring the actions of resistant antibiotics.
Asunto(s)
Proteínas de Escherichia coli , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Antibacterianos/farmacología , Antibacterianos/químicaRESUMEN
BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].
Asunto(s)
AMP Cíclico , Miocitos Cardíacos , Ratones , Animales , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Adenosina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Péptidos/metabolismoRESUMEN
Background: Antibiotic-resistant pathogens became a real global threat to human and animal health. This needs to concentrate the efforts to minimize and control these organisms. Efflux pumps are considered one of the important strategies used by bacteria to exclude harmful materials from the cell. Inhibition of these pumps can be an active strategy against multidrug resistance pathogens. There are two sources of efflux pump inhibitors that can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin is characterized by a wide margin of safety for the host cell. Aim: In this study, the ability of some plant extracts like (propolis show rosemary, clove, capsaicin, and cumin) to potentiate the inhibitory activity of some antibiotics such as (ciprofloxacin, erythromycin, gentamycin, tetracycline, and ampicillin) against Staphylococcus aureus pathogen were tested. Methods: Efflux pump inhibitory activity of the selected plant extracts was tested using an ethidium bromide (EtBr) accumulation assay. Results: The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin, and gentamycin. While it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in a combination of the minimum inhibitory concentration for the selected plant extracts (rosemary, clove, capsaicin, and cumin) with any of the tested antibiotics. Interestingly, according to the results of the EtBr accumulation assay, Propolis has potent inhibitory activity against the S. aureus (MRS usa300) pump system. Conclusion: This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin, and gentamycin against S. aureus strains, in case of the efflux-mediated antimicrobial resistance mechanisms.
Asunto(s)
Própolis , Infecciones Estafilocócicas , Animales , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Staphylococcus aureus , Extractos Vegetales/farmacología , Capsaicina/farmacología , Capsaicina/uso terapéutico , Própolis/farmacología , Própolis/uso terapéutico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/uso terapéutico , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Tetraciclina/farmacología , Tetraciclina/uso terapéutico , Ciprofloxacina/farmacología , Ciprofloxacina/uso terapéutico , Eritromicina/farmacología , Eritromicina/uso terapéutico , Etidio/farmacología , Etidio/uso terapéutico , Ampicilina/farmacología , Ampicilina/uso terapéutico , Gentamicinas/farmacologíaRESUMEN
Of the many multidrug resistance (MDR) mechanisms, the ATP binding cassette (ABC) transporters expelling drug molecules out of cells is a major culprit in limiting the efficacy of present-day anticancer drugs. The present review offers an updated information on structure, function, and regulatory mechanisms of major MDR related ABC transporters such as P-glycoprotein, MRP1, BCRP and effect of modulators on their functions. An attempt has been made to provide a focused information on different modulators of ABC transporters so as utilize them in clinical practice for amelioration of emerging MDR crisis in cancer treatment. Finally, the importance ABC transporters as therapeutic targets has been discussed in the light of future strategic planning for translating the ABC transporter inhibitors in clinical practice.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/uso terapéutico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Resistencia a Antineoplásicos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacología , Proteínas de Neoplasias/uso terapéutico , Resistencia a Múltiples Medicamentos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Adenosina TrifosfatoRESUMEN
Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall devoted to efflux effectively cell toxic substances. Recent seminal work revealed the high-resolution structure of such a tripartic composition TolC-AcrA-AcrB suggesting to design inhibitors preventing efflux of antibiotics. To show that electrophysiology can provide supporting information here, we reconstitute single TolC homotrimer into a planar lipid membrane, apply a transmembrane voltage and follow the assembly of AcrA to TolC using the modulation of the ion current through TolC channel during binding. In particular, the presence of AcrA in solution increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we show that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex in presence of putative efflux pump inhibitors. Both characteristics, the average ion current across TolC and the current noise, taken into consideration together, point to a stiffening of the tip of TolC which might reduce the formation of the complex.
Asunto(s)
Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Electrofisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Lipoproteínas/metabolismoRESUMEN
Multidrug resistance is a primary factor in the poor response to chemotherapy and subsequent death in gastric cancer patients. However, the molecular mechanisms involved remain unclear. In this study, the high expression of special AT-rich sequence binding protein 1 (SATB1) in gastric cancer was found to be associated with reduced sensitivity to various chemotherapy drugs. Our results demonstrate that SATB1 can promote chemotherapy resistance in gastric cancer in vitro and in vivo. SATB1 exerts its effect by enhancing the activity of multiple ATP-binding cassette (ABC) transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) in gastric cancer cell lines. We also found that SATB1 affects ABC transporters by altering the subcellular localization of the ABC transporter rather than its expression. Subsequently, we confirmed that Ezrin binds to various ABC transporters and affects their subcellular localization. In addition, we found that SATB1 can also bind to the Ezrin promoter and regulate its expression. In the present study, we elucidate the mechanism of SATB1-mediated multidrug resistance in gastric cancer, providing a basis for SATB1 as a potential target for reversal of resistance.
Asunto(s)
Proteínas de Unión a la Región de Fijación a la Matriz , Neoplasias Gástricas , Humanos , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Resistencia a Antineoplásicos/genética , Proteínas de Neoplasias/metabolismo , Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacologíaRESUMEN
Recently, we have isolated and identified several bioactive flavonoids and stilbenoids with potential anticancer activity from Thai orchids. In this study, we further investigated the cytotoxic and chemosensitizing activities of these phytochemicals (namely, pinocembrin, cardamonin, isalpinin, galangin, pinosylvin monomethyl ether, 2,3'-dihydroxy-5'-methoxystilbene, (E)-2,5'-dihydroxy-2'-(4-hydroxybenzyl)-3'-methoxystilbene, 2,3-dihydroxy-3',5'-dimethoxystilbene, 2,3'-dihydroxy-5,5'-dimethoxystilbene, 3,4'-dihydroxy-5-methoxystilbene and batatasin III) against breast cancer MCF7 cells and its two multidrug resistant (MDR) sublines (MCF7/DOX and MCF7/MX). Cytotoxicity was determined with MTT assay for the estimation of the half maximal cytotoxic concentrations (IC50). Effects of the test compounds on activities of efflux transporters (BCRP, P-gp, MRP1, and MRP2) were evaluated with substrate accumulation assays using fluorometry and flow cytometry analysis. Out of these 11 test compounds, the stilbene pinosylvin monomethyl ether displayed its cytotoxicity specifically toward MCF7 cells (IC50 = 6.2 ± 1.2 µM, 72-h incubation) with 4.96 folds higher than normal fibroblast. Its potency decreased in MCF7/DOX and MCF7/MX cells by 3.94 and 7.38 folds, respectively. Our transporter assay indicated that this stilbene significantly reduced the activities of P-gp, MRP1, and MRP2, but not BCRP. After 48-h co-incubation, this stilbene (at 2 µM) synergistically increased doxorubicin- and mitoxantrone-mediated cytotoxicity in MCF7, MCF7/DOX, and MCF7/MX cells potentially by increasing the intracellular level of cytotoxic drug. Pinosylvin monomethyl ether could sensitize breast cancer cells to chemotherapy and overcome MDR, in part, via the inhibition of drug efflux transporters.
Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Estilbenos , Humanos , Femenino , Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Resistencia a Antineoplásicos , Antineoplásicos/farmacología , Doxorrubicina/farmacología , Estilbenos/farmacología , Éteres/farmacología , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
Efflux by resistance nodulation cell division transporters, such as AcrAB-TolC in Escherichia coli, substantially contributes to the development of Gram-negative multidrug resistance. Therefore, the finding of compounds that counteract efflux is an urgent goal in the fight against infectious diseases. Previously, an efflux inhibitory activity of the antimalarials mefloquine and artesunate was reported. In this study, we have investigated further antimalarials regarding efflux by AcrB, the pumping part of AcrAB-TolC, and their drug-enhancing potency in E. coli. We show that 10 of the 24 drugs tested are substrates of the multidrug efflux pump AcrB. Among them, tafenoquine and proguanil, when used at subinhibitory concentrations, caused an at least 4- and up to 24-fold enhancement in susceptibility to 6 and 14 antimicrobial agents, respectively. Both antimalarials are able to increase the intracellular accumulation of Hoechst 33342, with proguanil showing similar effectiveness as the efflux inhibitor 1-(1-naphthylmethyl)piperazine. In the case of proguanil, AcrB-dependent efflux inhibition could also be demonstrated in a real-time efflux assay. In addition to presenting new AcrB substrates, our study reveals two previously unknown efflux inhibitors among antimalarials. Particularly proguanil appears as a promising candidate and its chemical scaffold might be further optimized for repurposing as antimicrobial drug enhancer.
Asunto(s)
Antiinfecciosos , Antimaláricos , Farmacorresistencia Bacteriana Múltiple , Proteínas de Escherichia coli , Mefloquina , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Proguanil , Antiinfecciosos/farmacología , Antimaláricos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Proguanil/farmacología , Mefloquina/farmacologíaRESUMEN
Although there are a lot of chemical drugs to treat breast cancer, increasing drug resistance of cancer cells has strongly hindered the effectiveness of chemotherapy. ATP-binding cassette transporters represented by P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) play an important role in drug resistance. This study aims to investigate the effect of 7-O-geranylquercetin (GQ) combining microRNA-451(miR-451) on reversing drug resistance of breast cancer and reveal the mechanism related to P-gp. Real-time RT-PCR and western blot assays showed that miR-326, miR-328, miR-451 and miR-155 inhibitor down-regulated the expression of genes MRP1, BCRP, MDR1 and the corresponding proteins MRP1, BCRP, P-gp, respectively. Cell counting kit-8 (CCK-8) assay indicated that these miRNAs reversed the resistance of MCF-7/ADR cells to Adriamycin (ADR), and miR-451 showed the greatest reversal effect. Combination of GQ and miR-451 enhanced the inhibitory effects of ADR on the proliferation and migration of MCF-7/ADR cells, and attenuated the expression of MDR1 and P-gp in MCF-7/ADR cells. A xenograft tumor model was used to show that GQ and miR-451 amplified the antitumor effect of ADR in nude mice, while western blot and immunohistochemical assays revealed the decreased expression of P-gp in tumor tissues. These results suggest that GQ and miR-451 have synergistic effect on reversing drug resistance through reducing the expression of MDR1 and P-gp in breast cancer MCF-7/ADR cells.
Asunto(s)
Neoplasias de la Mama , MicroARNs , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , 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 , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Proteínas de Neoplasias/metabolismo , Quercetina/análogos & derivadosRESUMEN
Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Humanos , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Staphylococcus aureus/metabolismoRESUMEN
The epidemic of multidrug resistance (MDR) is a serious threat to public health, and new classes of antibiotics with novel mechanisms of action are in critical need. We rationally designed and efficiently synthesized three series of new chemical entities with potential antibacterial activity targeting filamenting temperature-sensitive mutant Z (FtsZ). Evaluation of these compounds against a panel of Gram-positive bacteria including MDR and vancomycin-resistant Enterococcus strains indicated that most compounds showed enhanced antibacterial efficacy, comparable or even superior to the reference drugs. The newly synthesized compounds proved to be substrates of the Escherichia coli efflux pump AcrB, thus affecting the activity. Their structure-activity relationships were summarized in detail. The most potent compound 10f quickly eliminated bacteria in a bactericidal mode, with low susceptibility to induce bacterial resistance. Further mechanistic studies with the BsFtsZ protein revealed that 10f functioned as an effective FtsZ inhibitor through altering the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which leads to inhibition of cell division and cell death. Besides, 10f not only displayed no obvious cytotoxicity to mammalian cells but also had a high efficacy in a murine model of bacteremia in vivo. Regarded as a whole, our findings highlight 10f as a promising new FtsZ-targeting bactericidal agent.
Asunto(s)
Proteínas del Citoesqueleto , Proteínas de Escherichia coli , Animales , Antibacterianos/química , Proteínas Bacterianas , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/farmacología , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacología , Mamíferos/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Relación Estructura-ActividadRESUMEN
It's known that APAP overdose often leads to hepatotoxicity and nephrotoxicity. In the present study, we investigated the preventative effect of Tan IIA on APAP-induced nephrotoxicity. Mice were orally administrated with Tan IIA (10 or 30 mg/kg/day) for 1 week and subsequently gavaged with 200 mg/kg of APAP. Tan IIA reduced APAP-induced nephrotoxicity as evidenced by histopathological evaluation and serum creatinine levels. Tan IIA pretreatment promoted the efflux of the toxic intermediate metabolite N-acetyl-p-benzoquinone imine (NAPQI), thus reduced its injury to mouse kidney. After Tan IIA pretreatment, a remarkable increase in mRNA and protein expression of Nrf2 and its target genes Mrp2 and Mrp4 was observed in Nrf2+/+ mice kidneys, however, no obvious change of Mrp2 and Mrp4 mRNA and protein expression was detected in Nrf2-/- mice kidneys. HK-2 cells were used for exploring the roles of Tan IIA in the Nrf2-MRPs pathway in vitro. Consistently, Tan IIA up-regulated the Nrf2-MRPs pathway and promoted the nuclear Nrf2 accumulation in HK-2 cells. Collectively, our findings suggested that Tan IIA facilitated the clearance of toxic intermediate metabolite NAPQI from the kidney through upregulation of the Nrf2-MRP2/4 pathway, thereby, performing preventive effects against APAP-induced nephrotoxicity.
Asunto(s)
Abietanos , Acetaminofén , Enfermedades Renales , Animales , Ratones , Abietanos/farmacología , Acetaminofén/farmacología , Acetaminofén/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/metabolismo , Enfermedades Renales/prevención & control , Ratones Endogámicos C57BL , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
One mechanism of ciprofloxacin resistance is attributed to chromosomal DNA-encoded efflux pumps such as the MepA and NorB proteins. The goal of this research is to find a way to bypass Staphylococcus aureus' efflux pumps. Because of its high membrane permeability and low association with NorB and MepA efflux proteins, a liposome-encapsulating antibiotic is one of the promising, cost-effective drug carriers and coating mechanisms for overcoming active transport of methicillin-resistant S. aureus (MRSA) multidrug-resistant efflux protein . The calculated "Log Perm RRCK" membrane permeability values of 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) ciprofloxacin liposome-encapsulated (CFL) showed a lower negative value of - 4,652 cm/s and greater membrane permeability than ciprofloxacin free (CPF). The results of RT-qPCR showed that cationic liposomes containing ciprofloxacin in liposome-encapsulated form (CFL) improved CPF antibacterial activity and affinity for negatively charged bacterial cell surface membrane in comparison to free drug and liposome, as it overcame several resistance mechanisms and reduced the expression of efflux pumps. Ciprofloxacin liposome-encapsulated (CFL) is therefore more effective than ciprofloxacin alone. Liposomes can be combined with a variety of drugs that interact with bacterial cell efflux pumps to maintain high sustained levels of antibiotics in bacterial cells.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Ciprofloxacina/metabolismo , Ciprofloxacina/farmacología , Liposomas/metabolismo , Liposomas/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacologíaRESUMEN
MRP-1 is implicated in multidrug resistance and was described as prognostic in high-risk patients with soft-tissue sarcoma (STS) in a previous study. The current research aimed to validate MRP-1 prognostic/predictive value in localized sarcomas treated with anthracyclines plus ifosfamide within the ISG-1001 phase III study. In addition, the inhibitory activity on MRP-1 was investigated in preclinical studies to identify new combinations able to increase the efficacy of standard chemotherapy in STS. MRP-1 expression was assessed by IHC in tissue microarrays from patients with STS and tested for correlation with disease-free survival (DFS) and overall survival (OS). In vitro studies tested the efficacy of MRP-1 inhibitors (nilotinib, ripretinib, selumetinib, and avapritinib) in sarcoma cell lines. The effect of combinations of the most active MRP-1 inhibitors and chemotherapy was measured on the basis of apoptosis. MRP-1 was evaluable in 231 of 264 cases who entered the study. MRP-1 expression (strong intensity) was independently associated with worse DFS [HR, 1.78; 95% confidence interval (CI), 1.11-2.83; P = 0.016], in the multivariate analysis, with a trend for a worse OS (HR, 1.78; 95% CI, 0.97-3.25; P = 0.062). In vitro studies showed that the addition of MRP-1 inhibitors (nilotinib or avapritinib) to doxorubicin plus palifosfamide, significantly increased cell death in SK-UT-1 and CP0024 cell lines. MRP-1 is an adverse predictive factor in localized high-risk patients with STS treated with neoadjuvant anthracyclines plus ifosfamide followed by surgery. In vitro findings support the clinical assessment of the combination of chemotherapy and MRP-1 inhibitors as a promising strategy to overcome the drug ceiling effect for chemotherapy.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/uso terapéutico , Sarcoma/tratamiento farmacológico , Investigación Biomédica Traslacional/métodos , Femenino , Humanos , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Valor Predictivo de las Pruebas , PronósticoRESUMEN
Cancer, a major public health problem, is one of the world's top leading causes of death. Common treatments for cancer include cytotoxic chemotherapy, surgery, targeted drugs, endocrine therapy, and immunotherapy. However, despite the outstanding achievements in cancer therapies during the last years, resistance to conventional chemotherapeutic agents and new targeted drugs is still the major challenge. In the present review, we explain the different mechanisms involved in cancer therapy and the detailed outlines of cancer drug resistance regarding multidrug resistance-associated proteins (MRPs) and their role in treatment failures by common chemotherapeutic agents. Further, different modulators of MRPs are presented. Finally, we outlined the models used to analyze MRP transporters and proposed a future impact that may set up a base or pave the way for many researchers to investigate the cancer MRP further.
Asunto(s)
Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Predicción , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/uso terapéutico , Neoplasias/tratamiento farmacológicoRESUMEN
Objectives. Characterization of a urine isolate of daptomycin non-susceptible Enterococcus faecium recovered from a patient with kidney transplantation and no history of daptomycin exposure. Methods. After isolation in a urine sample, identification of E. faecium was confirmed by amplification of the E. faecium-specific gene encoding D-alanyl-D-alanine ligase (ddl) and daptomycin susceptibility testing was performed by E-test on cation-adjusted Mueller-Hinton agar. In order to determine the genetic bases of daptomycin resistance, the open reading frames of five genes previously associated with daptomycin resistance in enterococci were sequenced. Results. Substitutions in the response regulator LiaR (S19F) and cardiolipin synthase (R218Q) were identified. Conclusions. To the best of our knowledge, this is the first characterization of emerging daptomycin resistance in E. faecium in a Spanish hospital in the absence of daptomycin exposure and in a renal transplant recipient (AU)
Objetivos. Presentamos la caracterización de un aislado de Enterococcus faecium no sensible a daptomicina, recuperado de una muestra de orina de un paciente con trasplante de riñón e infección urinaria y sin antecedentes de exposición previa a daptomicina. Métodos. Tras el aislamiento, la identificación de E. faecium fue confirmada por la amplificación del gen que codifica la región específica de la ligasa de la D-alanil-D-alanina (ddl) y la prueba de sensibilidad a daptomicina se realizó mediante E-test en agar Mueller-Hinton ajustado para cationes. Con el fin de determinar las bases genéticas de la resistencia a daptomicina, se secuenciaron las regiones de lectura abierta de cinco genes previamente asociados con la resistencia a daptomicina en enterococos. Resultados. Se identificaron cambios en el promotor de LiaR (S19F) y la sintetasa de la cardiolipina (R218Q). Conclusiones. Esta es la primera caracterización de un aislado clínico de E. faecium con resistencia a daptomicina en un hospital español, en ausencia de exposición previa y en un receptor de trasplante renal (AU)
Asunto(s)
Adulto , Femenino , Humanos , Daptomicina/administración & dosificación , Daptomicina/uso terapéutico , Bacterias Grampositivas , Bacterias Grampositivas/fisiología , Bacterias Grampositivas/patogenicidad , Diálisis Renal , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/análisis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacologíaRESUMEN
Oxidative stress induced in live HeLa cells by menadione (2-methyl-1,4-napthaquinone) was studied in real time by scanning electrochemical microscopy (SECM). The hydrophobic molecule menadione diffuses through a living cell membrane where it is toxic to the cell. However, in the cell it is conjugated with glutathione to form thiodione. Thiodione is then recognized and transported across the cell membrane via the ATP-driven MRP1 pump. In the extracellular environment, thiodione was detected by the SECM tip at levels of 140, 70, and 35 µM upon exposure of the cells to menadione concentrations of 500, 250, and 125 µM, respectively. With the aid of finite element modeling, the kinetics of thiodione transport was determined to be 1.6 10(-7) m/s, about 10 times faster than menadione uptake. Selective inhibition of these MRP1 pumps inside live HeLa cells by MK571 produced a lower thiodione concentration of 50 µM in presence of 500 µM menadione and 50 µM MK571. A similar reduced (50% drop) thiodione efflux was observed in the presence of monoclonal antibody QCRL-4, a selective blocking agent of the MRP1 pumps. The reduced thiodione flux confirmed that thiodione was transported by MRP1, and that glutathione is an essential substrate for MRP1-mediated transport. This finding demonstrates the usefulness of SECM in quantitative studies of MRP1 inhibitors and suggests that monoclonal antibodies can be a useful tool in inhibiting the transport of these MDR pumps, and thereby aiding in overcoming multidrug resistance.
Asunto(s)
Glutatión/análogos & derivados , Microscopía de Sonda de Barrido/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Estrés Oxidativo/fisiología , Vitamina K 3/análogos & derivados , Anticuerpos Monoclonales/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Técnicas Electroquímicas , Análisis de Elementos Finitos , Glutatión/metabolismo , Células HeLa , Humanos , Cinética , Simulación de Dinámica Molecular , Estructura Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Propionatos/farmacología , Quinolinas/farmacología , Vitamina K 3/metabolismoRESUMEN
Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (-)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2'-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (-)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (-)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.
Asunto(s)
Benzo(a)pireno/antagonistas & inhibidores , Benzo(a)pireno/toxicidad , Bronquios/citología , Carcinógenos/antagonistas & inhibidores , Carcinógenos/toxicidad , Aductos de ADN/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Alveolos Pulmonares/citología , Bronquios/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/citología , Línea Celular , ADN/genética , ADN/aislamiento & purificación , Dinoprostona/biosíntesis , Humanos , Inmunohistoquímica , Factor 2 Relacionado con NF-E2/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Probenecid/farmacología , Alveolos Pulmonares/efectos de los fármacos , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cross-Talk/efectos de los fármacos , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Fármacos Renales/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVES: In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND: Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS: Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS: Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/108 cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/108 cells vs. 1,670 ± 646 pg/108 cells TxB2-production). CONCLUSIONS: Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.