RESUMEN
The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.
Asunto(s)
Neoplasias de la Mama , Placenta , Animales , Niño , Femenino , Humanos , Embarazo , Ratas , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Transporte de Nucleósidos/metabolismo , Proteínas de Transporte de Nucleósidos/farmacología , Nucleósidos/metabolismo , Nucleósidos/farmacología , Placenta/metabolismo , Ratas Wistar , UridinaRESUMEN
PURPOSE: We aimed to compare the effects of P-glycoprotein (ABCB1) on the intestinal uptake of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), and metabolites, tenofovir isoproxil monoester (TEM) and tenofovir (TFV), and to study the molecular mechanism of drug-drug interaction (DDI) between sofosbuvir (SOF) and TDF/TAF. METHODS: Bidirectional transport experiments in Caco-2 cells and accumulation studies in precision-cut intestinal slices prepared from the ileal segment of rodent (rPCIS) and human (hPCIS) intestines were performed. RESULTS: TDF and TAF were extensively metabolised but TAF exhibited greater stability. ABCB1 significantly reduced the intestinal transepithelial transfer and uptake of the TFV(TDF) and TFV(TAF)-equivalents. However, TDF and TAF were absorbed more efficiently than TFV and TEM. SOF did not inhibit intestinal efflux of TDF and TAF or affect intestinal accumulation of TFV(TDF) and TFV(TAF)-equivalents but did significantly increase the proportion of absorbed TDF. CONCLUSIONS: TDF and TAF likely produce comparable concentrations of TFV-equivalents in the portal vein and the extent of permeation is reduced by the activity of ABCB1. DDI on ABCB1 can thus potentially affect TDF and TAF absorption. SOF does not inhibit ABCB1-mediated transport of TDF and TAF but does stabilise TDF, albeit without affecting the quantity of TFV(TDF)-equivalents crossing the intestinal barrier. Our data thus suggest that reported increases in the TFV plasma concentrations in patients treated with SOF and TDF result either from a DDI between SOF and TDF that does not involve ABCB1 or from a DDI involving another drug used in combination therapy.
Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , Humanos , Tenofovir , Sofosbuvir/uso terapéutico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Células CACO-2 , Infecciones por VIH/tratamiento farmacológico , Adenina/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Fumaratos , AlaninaRESUMEN
Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.
Asunto(s)
Antivirales/farmacocinética , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Microextracción en Fase Sólida , Espectrometría de Masas en Tándem , Antivirales/química , Monitoreo de Drogas , Humanos , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is routinely used at concentrations of 0.10 µM and 0.10 mM to specifically inhibit transport of nucleosides mediated by equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2), respectively. We recently showed that NBMPR (0.10 mM) might also inhibit placental active efflux of [3H]zidovudine and [3H]tenofovir disoproxil fumarate. Here we test the hypothesis that NBMPR abolishes the activity of P-glycoprotein (ABCB1) and/or breast cancer resistance protein (ABCG2). METHODS: We performed accumulation assays with Hoechst 33342 (a model dual substrate of ABCB1 and ABCG2) and bi-directional transport studies with the ABCG2 substrate [3H]glyburide in transduced MDCKII cells, accumulation studies in choriocarcinoma-derived BeWo cells, and in situ dual perfusions of rat term placenta with glyburide. RESULTS: NBMPR inhibited Hoechst 33342 accumulation in MDCKII-ABCG2 cells (IC50 = 53 µM) but not in MDCKII-ABCB1 and MDCKII-parental cells. NBMPR (0.10 mM) also inhibited bi-directional [3H]glyburide transport across monolayers of MDCKII-ABCG2 cells and blocked ABCG2-mediated [3H]glyburide efflux by rat term placenta in situ. CONCLUSION: NBMPR at a concentration of 0.10 mM abolishes ABCG2 activity. Researchers using NBMPR to evaluate the effect of ENTs on pharmacokinetics must therefore interpret their results carefully if studying compounds that are substrates of both ENTs and ABCG2.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Tioinosina/análogos & derivados , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Antivirales/metabolismo , Antivirales/farmacocinética , Línea Celular , Perros , Femenino , Humanos , Células de Riñón Canino Madin Darby , Proteínas de Neoplasias/metabolismo , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Ratas , Ratas Wistar , Tioinosina/farmacologíaRESUMEN
P-glycoprotein (ABCB1), an ATP-binding-cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions (DDIs). ABCB1 has been suggested to interact with many antivirals used to treat HIV and/or chronic hepatitis C virus (HCV) infections. Using bidirectional transport experiments in Caco-2 cells and a recently established ex vivo model of accumulation in precision-cut intestinal slices (PCIS) prepared from rat ileum or human jejunum, we evaluated the potential of anti-HIV and anti-HCV antivirals to inhibit intestinal ABCB1. Lopinavir, ritonavir, saquinavir, atazanavir, maraviroc, ledipasvir, and daclatasvir inhibited the efflux of a model ABCB1 substrate, rhodamine 123 (RHD123), in Caco-2 cells and rat-derived PCIS. Lopinavir, ritonavir, saquinavir, and atazanavir also significantly inhibited RHD123 efflux in human-derived PCIS, while possible interindividual variability was observed in the inhibition of intestinal ABCB1 by maraviroc, ledipasvir, and daclatasvir. Abacavir, zidovudine, tenofovir disoproxil fumarate, etravirine, and rilpivirine did not inhibit intestinal ABCB1. In conclusion, using recently established ex vivo methods for measuring drug accumulation in rat- and human-derived PCIS, we have demonstrated that some antivirals have a high potential for DDIs on intestinal ABCB1. Our data help clarify the molecular mechanisms responsible for reported increases in the bioavailability of ABCB1 substrates, including antivirals and drugs prescribed to treat comorbidity. These results could help guide the selection of combination pharmacotherapies and/or suitable dosing schemes for patients infected with HIV and/or HCV.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Fármacos Anti-VIH/farmacología , Antivirales/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Anciano , Animales , Sulfato de Atazanavir/farmacología , Bencimidazoles/farmacología , Células CACO-2/efectos de los fármacos , Células CACO-2/metabolismo , Carbamatos , Interacciones Farmacológicas , Femenino , Fluorenos/farmacología , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Hepatitis C/complicaciones , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Imidazoles/farmacología , Intestinos/efectos de los fármacos , Lopinavir/farmacología , Masculino , Maraviroc/farmacología , Persona de Mediana Edad , Pirrolidinas , Ratas , Ratas Wistar , Ritonavir/farmacología , Saquinavir/farmacología , Valina/análogos & derivados , Zidovudina/farmacologíaRESUMEN
Abacavir is a preferred antiretroviral drug for preventing mother-to-child human immunodeficiency virus transmission; however, mechanisms of its placental transfer have not been satisfactorily described to date. Because abacavir is a nucleoside-derived drug, we hypothesized that the nucleoside transporters, equilibrative nucleoside transporters (ENTs, SLC29A) and/or Na+-dependent concentrative nucleoside transporters (CNTs, SLC28A), may play a role in its passage across the placenta. To test this hypothesis, we performed uptake experiments using the choriocarcinoma-derived BeWo cell line, human fresh villous fragments, and microvillous plasma membrane (MVM) vesicles. Using endogenous substrates of nucleoside transporters, [3H]-adenosine (ENTs, CNT2, and CNT3) and [3H]-thymidine (ENTs, CNT1, and CNT3), we showed significant activity of ENT1 and CNT2 in BeWo cells, whereas experiments in the villous fragments and MVM vesicles, representing a model of the apical membrane of a syncytiotrophoblast, revealed only ENT1 activity. When testing [3H]-abacavir uptakes, we showed that of the nucleoside transporters, ENT1 plays the dominant role in abacavir uptake into placental tissues, whereas contribution of Na+-dependent transport, most likely mediated by CNTs, was observed only in BeWo cells. Subsequent experiments with dually perfused rat term placentas showed that Ent1 contributes significantly to overall [3H]-abacavir placental transport. Finally, we quantified the expression of SLC29A in first- and third-trimester placentas, revealing that SLC29A1 is the dominant isoform. Neither SLC29A1 nor SLC29A2 expression changed over the course of placental development, but there was considerable interindividual variability in their expression. Therefore, drug-drug interactions and the effect of interindividual variability in placental ENT1 expression on abacavir disposition into fetal circulation should be further investigated to guarantee safe and effective abacavir-based combination therapies in pregnancy.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Didesoxinucleósidos/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Proteínas de Transporte de Nucleósidos/metabolismo , Placenta/metabolismo , Adenosina/metabolismo , Animales , Transporte Biológico/fisiología , Línea Celular Tumoral , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Femenino , Humanos , Proteínas de Transporte de Membrana/metabolismo , Nucleósidos/metabolismo , Embarazo , Ratas , Ratas WistarRESUMEN
Equilibrative ( SLC29A) and concentrative ( SLC28A) nucleoside transporters contribute to proper placental development and mediate uptake of nucleosides/nucleoside-derived drugs. We analyzed placental expression of SLC28A mRNA during gestation. Moreover, we studied in choriocarcinoma-derived BeWo cells whether SLC29A and SLC28A mRNA levels can be modulated by activity of adenylyl cyclase, retinoic acid receptor activation, CpG islands methylation, or histone acetylation, using forskolin, all- trans-retinoic acid, 5-azacytidine, and sodium butyrate/sodium valproate, respectively. We found that expression of SLC28A1, SLC28A2, and SLC28A3 increases during gestation and reveals considerable interindividual variability. SLC28A2 was shown to be a dominant subtype in the first-trimester and term human placenta, while SLC28A1 exhibited negligible expression in the term placenta only. In BeWo cells, we detected mRNA of SLC28A2 and SLC28A3. Levels of the latter were affected by 5-azacytidine and all- trans-retinoic acid, while the former was modulated by sodium valproate (but not sodium butyrate), all- trans-retinoic acid, 5-azacytidine, and forskolin that caused 25-fold increase in SLC28A2 mRNA; we documented by analysis of syncytin-1 that the observed changes in SLC28A expression do not correlate with the morphological differentiation state of BeWo cells. Upregulated SLC28A2 mRNA was reflected in elevated uptake of [3H]-adenosine, high-affinity substrate of concentrative nucleoside transporter 2. Using KT-5720 and inhibitors of phosphodiesterases, we subsequently confirmed importance of cAMP/protein kinase A pathway in SLC28A2 regulation. On the other hand, SLC29A genes exhibited constitutive expression and none of the tested compounds increased SLC28A1 expression to detectable levels. In conclusion, we provide the first evidence that methylation status and activation of retinoic acid receptor affect placental SLC28A2 and SLC28A3 transcription and substrates of concentrative nucleoside transporter 2 might be taken up in higher extent in placentas with overactivated cAMP/protein kinase A pathway and likely in the term placenta.
Asunto(s)
Diferenciación Celular/fisiología , Edad Gestacional , Proteínas de Transporte de Membrana/metabolismo , Placenta/metabolismo , Carbazoles/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Transporte de Nucleósido Equilibrativas/genética , Proteínas de Transporte de Nucleósido Equilibrativas/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Proteínas de Transporte de Membrana/genética , Placenta/efectos de los fármacos , Embarazo , Pirroles/farmacología , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Didesoxinucleósidos/metabolismo , Interacciones Farmacológicas/fisiología , Absorción Intestinal/fisiología , Proteínas de Transporte de Membrana/metabolismo , Rilpivirina/metabolismo , Animales , Transporte Biológico/fisiología , Células CACO-2 , Línea Celular , Línea Celular Tumoral , Didesoxinucleósidos/farmacología , Perros , Humanos , Lamivudine/metabolismo , Lamivudine/farmacología , Células de Riñón Canino Madin Darby , Masculino , Ratas , Ratas Wistar , Inhibidores de la Transcriptasa Inversa/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Rilpivirina/farmacologíaRESUMEN
1. Emtricitabine is a nucleoside reverse transcriptase inhibitor used in combination antiretroviral therapy of HIV (cART). Although active transport mechanisms are believed to mediate tubular secretion of the drug into urine, the responsible transporter and its potential to cause pharmacokinetic drug--drug interactions (DDI) has not been identified so far. The aim of this study was to investigate whether drug transporters P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), OCT1 (SLC22A1), OCT2 (SLC22A2) or MATE1 (SLC47A1) can mediate active transcellular transfer of emtricitabine. 2. We employed transport assays in polarized monolayers of MDCK cells stably expressing P-gp, BCRP, MRP2, OCT1, OCT2 and/or MATE1. Among the transporters studied only MATE1 accelerated basal-to-apical transport of emtricitabine over a wide range of concentrations (6 nM to 1 mM). The transport was enhanced by an oppositely directed pH gradient and significantly reduced (p < 0.001) at low temperature in MDCK-MATE1, MDCK-OCT1/MATE1 and MDCK-OCT2/MATE1 cells. Co-administration of cimetidine or ritonavir decreased MATE1-mediated transport of emtricitabine by up to 42 and 39%, respectively (p < 0.01) and augmented intracellular accumulation of emtricitabine (p < 0.05). 3. We demonstrate emtricitabine as a substrate of MATE1 and suggest that MATE1 might cause DDI between emtricitabine and other co-administrated drugs including antiretrovirals.
Asunto(s)
Emtricitabina/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Inhibidores de la Transcriptasa Inversa/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Perros , Células de Riñón Canino Madin DarbyRESUMEN
Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of drug efflux transporters P-glycoprotein (P-gp) (MDR1 [ABCB1]), BCRP (ABCG2), MRP2 (ABCC2), and MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of lamivudine. We employed in vitro accumulation and transport experiments on MDCK cells overexpressing drug efflux transporters, in situ-perfused rat term placenta, and vesicular uptake in microvillous plasma membrane (MVM) vesicles isolated from human term placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-MDR1, MDCK-MRP2, and MDCK-BCRP monolayers. MATE1-mediated efflux of lamivudine appeared to be a low-affinity process (apparent Km of 4.21 mM and Vmax of 5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP, or MRP2. However, lamivudine transfer across dually perfused rat placenta and the uptake of lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible involvement of MATE1 in the fetal-to-maternal efflux of the drug. To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2, or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction. We suggest that MATE1 might be, at least partly, responsible for this transport.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Lamivudine/metabolismo , Placenta/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico/fisiología , Línea Celular , Perros , Femenino , Humanos , Células de Riñón Canino Madin Darby , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Embarazo , Ratas , Ratas WistarRESUMEN
Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP-binding cassette (ABC) transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co-administered with AZT, did not affect ABC transporter-mediated AZT transfer.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Fármacos Anti-VIH/farmacocinética , Placenta/metabolismo , Zidovudina/farmacocinética , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Acridinas/farmacología , Animales , Perros , Interacciones Farmacológicas , Femenino , Técnicas In Vitro , Indometacina/farmacología , Lamivudine/farmacología , Células de Riñón Canino Madin Darby , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Embarazo , Ratas Wistar , Tetrahidroisoquinolinas/farmacologíaRESUMEN
Francisella tularensis is a highly infectious bacterium that causes the potentially lethal disease tularemia. This extremely virulent bacterium is able to replicate in the cytosolic compartments of infected macrophages. To invade macrophages and to cope with their intracellular environment, Francisella requires multiple virulence factors, which are still being identified. Proteins containing tetratricopeptide repeat (TPR)-like domains seem to be promising targets to investigate, since these proteins have been reported to be directly involved in virulence-associated functions of bacterial pathogens. Here, we studied the role of the FTS_0201, FTS_0778, and FTS_1680 genes, which encode putative TPR-like proteins in Francisella tularensis subsp. holarctica FSC200. Mutants defective in protein expression were prepared by TargeTron insertion mutagenesis. We found that the locus FTS_1680 and its ortholog FTT_0166c in the highly virulent Francisella tularensis type A strain SchuS4 are required for proper intracellular replication, full virulence in mice, and heat stress tolerance. Additionally, the FTS_1680-encoded protein was identified as a membrane-associated protein required for full cytopathogenicity in macrophages. Our study thus identifies FTS_1680/FTT_0166c as a new virulence factor in Francisella tularensis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Francisella tularensis/fisiología , Sitios Genéticos , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Citosol/microbiología , Modelos Animales de Enfermedad , Femenino , Francisella tularensis/genética , Francisella tularensis/crecimiento & desarrollo , Técnicas de Inactivación de Genes , Macrófagos/microbiología , Ratones Endogámicos BALB C , Mutagénesis Insercional , Tularemia/microbiología , Tularemia/patología , Virulencia , Factores de Virulencia/genéticaRESUMEN
FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.
Asunto(s)
Proteínas Fimbrias/metabolismo , Francisella tularensis/metabolismo , Antígenos O/metabolismo , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Francisella tularensis/genética , Francisella tularensis/patogenicidad , Glicosilación , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Antígenos O/química , Antígenos O/genética , Espectrometría de Masas en Tándem , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
The tetratricopeptide repeat (TPR) structural motif is known to occur in a wide variety of proteins present in prokaryotic and eukaryotic organisms. The TPR motif represents an elegant module for the assembly of various multiprotein complexes, and thus, TPR-containing proteins often play roles in vital cell processes. As the TPR profile is well defined, the complete TPR protein repertoire of a bacterium with a known genomic sequence can be predicted. This provides a tremendous opportunity for investigators to identify new TPR-containing proteins and study them in detail. In the past decade, TPR-containing proteins of bacterial pathogens have been reported to be directly related to virulence-associated functions. In this minireview, we summarize the current knowledge of the TPR-containing proteins involved in virulence mechanisms of bacterial pathogens while highlighting the importance of TPR motifs for the proper functioning of class II chaperones of a type III secretion system in the pathogenesis of Yersinia, Pseudomonas, and Shigella.
Asunto(s)
Secuencias de Aminoácidos/genética , Bacterias/patogenicidad , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , VirulenciaRESUMEN
In our previous study, we described synchronized activity of organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the passage of organic cations across the rat placenta and the role of these transporters in fetal defense; in this study, we hypothesized that changes in placental levels of OCT3 and MATE1 throughout gestation might affect the fetal protection and detoxification. Using quantitative RT-PCR, Western blot analysis, and immunohistochemistry, we were able to detect Oct3/OCT3 and Mate1/MATE1 expression in the rat placenta as early as on Gestation Day (gd) 12 with increasing tendency toward the end of pregnancy. Comparing first versus third trimester human placenta, we observed stable expression of OCT1 and decreasing expression of OCT2 and OCT3 isoforms. Contrary to the current literature, we were able to detect also MATE1/MATE2 isoforms in the human placenta, however, with considerable inter- and intraindividual variability. Using infusion of 1-methyl-4-phenylpyridinium (MPP(+)), a substrate of OCT and MATE transporters, into pregnant dams, we investigated the protective function of the placenta against organic cations at different gds. The highest amount of MPP(+) reached the fetus on gd 12 while from gd 15 onward, maternal-to-fetal transport of MPP(+) decreased significantly. We conclude that increased expression of placental OCT3 and MATE1 along with general maturation of the placental tissues results in significantly lower transport of MPP(+) from mother to fetus. In contrast, decreasing expression of OCT3 and MATE1 in human placenta indicates these transporters may play a role in fetal protection preferentially at earlier stages of gestation.
Asunto(s)
Antiportadores/metabolismo , Feto/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Placenta/metabolismo , Animales , Western Blotting , Femenino , Desarrollo Fetal , Humanos , Inmunohistoquímica , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
The inhibition of P-glycoprotein (ABCB1) could lead to increased drug plasma concentrations and hence increase drug toxicity. The evaluation of a drug's ability to inhibit ABCB1 is complicated by the presence of several transport-competent sites within the ABCB1 binding pocket, making it difficult to select appropriate substrates. Here, we investigate the capacity of antiretrovirals and direct-acting antivirals to inhibit the ABCB1-mediated intestinal efflux of [3H]-digoxin and compare it with our previous rhodamine123 study. At concentrations of up to 100 µM, asunaprevir, atazanavir, daclatasvir, darunavir, elbasvir, etravirine, grazoprevir, ledipasvir, lopinavir, rilpivirine, ritonavir, saquinavir, and velpatasvir inhibited [3H]-digoxin transport in Caco-2 cells and/or in precision-cut intestinal slices prepared from the human jejunum (hPCIS). However, abacavir, dolutegravir, maraviroc, sofosbuvir, tenofovir disoproxil fumarate, and zidovudine had no inhibitory effect. We thus found that most of the tested antivirals have a high potential to cause drug-drug interactions on intestinal ABCB1. Comparing the Caco-2 and hPCIS experimental models, we conclude that the Caco-2 transport assay is more sensitive, but the results obtained using hPCIS agree better with reported in vivo observations. More inhibitors were identified when using digoxin as the ABCB1 probe substrate than when using rhodamine123. However, both approaches had limitations, indicating that inhibitory potency should be tested with at least these two ABCB1 probes.
RESUMEN
The host-pathogen interaction represents a complex and dynamic biological system. The outcome of this interaction is dependent on the microbial pathogen properties to establish infection and the ability of the host to control infection. Although bacterial pathogens have evolved a variety of strategies to subvert host defense functions, several general mechanisms have been shown to be shared among these pathogens. As a result, host effectors that are critical for pathogen entry, survival and replication inside the host cells have become a new paradigm for antimicrobial targeting. This review focuses on the potential utility of a proteomics approach in defining the host-pathogen interaction from the host's perspective.
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Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Proteoma/inmunología , Proteoma/metabolismo , Proteómica/métodos , Humanos , Proteínas/análisisRESUMEN
An estimated 1.3 million pregnant women were living with HIV in 2018. HIV infection is associated with adverse pregnancy outcomes and all HIV-positive pregnant women, regardless of their clinical stage, should receive a combination of antiretroviral drugs to suppress maternal viral load and prevent vertical fetal infection. Although antiretroviral treatment in pregnant women has undoubtedly minimized mother-to-child transmission of HIV, several uncertainties remain. For example, while pregnancy is accompanied by changes in pharmacokinetic parameters, relevant data from clinical studies are lacking. Similarly, long-term adverse effects of exposure to antiretrovirals on fetuses have not been studied in detail. Here, we review current knowledge on HIV effects on the placenta and developing fetus, recommended antiretroviral regimens, and pharmacokinetic considerations with particular focus on placental transport. We also discuss recent advances in antiretroviral research and potential effects of antiretroviral treatment on placental/fetal development and programming.
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Antirretrovirales/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/transmisión , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Animales , Femenino , Humanos , Placenta/virología , Embarazo , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Complicaciones Infecciosas del Embarazo/virologíaRESUMEN
Depression is a prevalent condition affecting up to 20% of pregnant women. Hence, more than 10% are prescribed antidepressant drugs, mainly serotonin reuptake inhibitors (SSRIs) and selective serotonin and noradrenaline reuptake inhibitors (SNRIs). We hypothesize that antidepressants disturb serotonin homeostasis in the fetoplacental unit by inhibiting serotonin transporter (SERT) and organic cation transporter 3 (OCT3) in the maternal- and fetal-facing placental membranes, respectively. Paroxetine, citalopram, fluoxetine, fluvoxamine, sertraline, and venlafaxine were tested in situ (rat term placenta perfusion) and ex vivo (uptake studies in membrane vesicles isolated from healthy human term placenta). All tested antidepressants significantly inhibited SERT- and OCT3-mediated serotonin uptake in a dose-dependent manner. Calculated half-maximal inhibitory concentrations (IC50) were in the range of therapeutic plasma concentrations. Using in vitro and in situ models, we further showed that the placental efflux transporters did not compromise mother-to-fetus transport of antidepressants. Collectively, we suggest that antidepressants have the potential to affect serotonin levels in the placenta or fetus when administered at therapeutic doses. Interestingly, the effect of antidepressants on serotonin homeostasis in rat placenta was sex dependent. As accurate fetal programming requires optimal serotonin levels in the fetoplacental unit throughout gestation, inhibition of SERT-/OCT3-mediated serotonin uptake may help explain the poor outcomes of antidepressant use in pregnancy.
RESUMEN
P-glycoprotein (ABCB1), an ATP-binding cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions. Drug-mediated induction of intestinal ABCB1 is a clinically relevant phenomenon associated with significantly decreased drug bioavailability. Currently, there are no well-established human models for evaluating its induction, so drug regulatory authorities provide no recommendations for in vitro/ex vivo testing drugs' ABCB1-inducing activity. Human precision-cut intestinal slices (hPCISs) contain cells in their natural environment and express physiological levels of nuclear factors required for ABCB1 induction. We found that hPCISs incubated in William's Medium E for 48 h maintained intact morphology, ATP content, and ABCB1 efflux activity. Here, we asked whether rifampicin (a model ligand of pregnane X receptor, PXR), at 30 µM, induces functional expression of ABCB1 in hPCISs over 24- and 48-h incubation (the time to allow complete induction to occur). Rifampicin significantly increased gene expression, protein levels, and efflux activity of ABCB1. Moreover, we described dynamic changes in ABCB1 transcript levels in hPCISs over 48 h incubation. We also observed that peaks of induction are achieved among donors at different times, and the extent of ABCB1 gene induction is proportional to PXR mRNA levels in the intestine. In conclusion, we showed that hPCISs incubated in conditions comparable to those used for inhibition studies can be used to evaluate drugs' ABCB1-inducing potency in the human intestine. Thus, hPCISs may be valuable experimental tools that can be prospectively used in complex experimental evaluation of drug-drug interactions.