RESUMEN
Membrane transporters expressed in the choroid plexus (CP) are involved in the transport of substances between the blood and cerebrospinal fluid (CSF). Carnitine/organic cation transporter 1 (OCTN1, also known as SLC22A4) is expressed in rodent CP; however, its specific roles in blood-CSF transport remain unclear. Therefore, in this study, we aimed to evaluate the potential role of OCTN1 in the elimination of substances from CSF. Tritium-labeled ergothioneine ([3H]ERGO), a typical in vivo substrate of OCTN1, was injected into the lateral ventricles of wild-type and octn1 gene knockout (octn1-/-) mice. Clearance of [3H]ERGO from CSF was higher than that of the bulk flow marker, [14C]mannitol, in wild-type mice. However, [3H]ERGO clearance was significantly lower in octn1-/- mice than in wild-type mice. Furthermore, OCTN1 expression in CP was determined via immunohistochemical analysis. CP/CSF ratio of [3H]ERGO was significantly lower in octn1-/- mice than in wild-type mice. These results suggest that OCTN1 is functionally expressed in CP and involved in the elimination of ERGO from CSF in mice.
Asunto(s)
Plexo Coroideo , Ergotioneína , Ratones Noqueados , Proteínas de Transporte de Catión Orgánico , Animales , Plexo Coroideo/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Ergotioneína/metabolismo , Ergotioneína/líquido cefalorraquídeo , Ratones , Masculino , Ratones Endogámicos C57BL , SimportadoresRESUMEN
Previously, we reported a novel natural scaffold compound, isobavachin (4',7-dihydroxy-8-prenylflavanone), as a highly potent hURAT1 inhibitor with anti-hyperuricemia effect. However, the structure-activity relationship remains unknown and the poor pharmacokinetic (PK) parameters may limit further clinical use. Herein, a series of isobavachin derivatives were rationally designed and synthesized to explore the structure-activity relationship of isobavachin target hURAT1, and to improve their PK properties. Among them, compounds 15d, 15f, 15g, 27b and 27d showed promising hURAT1 inhibitory activities, which could comparable to that of isobavachin (IC50 = 0.24 µM). In addition, 27b also inhibited another urate reabsorption transporter GLUT9 with an IC50 of 4.47 µM. Compound 27b displayed greater urate-lowering activity in a hyperuricemia mouse model at a dose of 10 mg/kg compared to isobavachin and lesinurad. Overall, our results suggest that compound 27b represents a novel, safe hURAT1 and GLUT9 dual-target inhibitor with excellent drug availability and is worthy of further investigation as an anti-hyperuricemia agent.
Asunto(s)
Diseño de Fármacos , Hiperuricemia , Animales , Humanos , Masculino , Ratones , Relación Dosis-Respuesta a Droga , Hiperuricemia/tratamiento farmacológico , Estructura Molecular , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/metabolismo , Relación Estructura-Actividad , Ácido Úrico/sangreRESUMEN
OCTN1 and OCTN2 are membrane transport proteins encoded by the SLC22A4 and SLC22A5 genes, respectively. Even though several transcripts have been predicted by bioinformatics for both genes, only one functional protein isoform has been described for each of them. Both proteins are ubiquitous, and depending on the physiopathological state of the cell, their expression is regulated by well-known transcription factors, although some aspects have been neglected. A plethora of missense variants with uncertain clinical significance are reported both in the dbSNP and the Catalogue of Somatic Mutations in Cancer (COSMIC) databases for both genes. Due to their involvement in human pathologies, such as inflammatory-based diseases (OCTN1/2), systemic primary carnitine deficiency (OCTN2), and drug disposition, it would be interesting to predict the impact of variants on human health from the perspective of precision medicine. Although the lack of a 3D structure for these two transport proteins hampers any speculation on the consequences of the polymorphisms, the already available 3D structures for other members of the SLC22 family may provide powerful tools to perform structure/function studies on WT and mutant proteins.
Asunto(s)
Regulación de la Expresión Génica , Miembro 5 de la Familia 22 de Transportadores de Solutos , Humanos , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , Miembro 5 de la Familia 22 de Transportadores de Solutos/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/química , Conformación Proteica , Simportadores/genética , Simportadores/metabolismo , Simportadores/químicaRESUMEN
High serum urate levels are the major risk factor for gout. URAT1, the primary transporter for urate absorption in the kidneys, is well known as an anti-hyperuricemia drug target. However, the clinical application of URAT1-targeted drugs is limited because of their low specificity and severe side effects. The lack of structural information impedes elucidation of the transport mechanism and the development of new drugs. Here, we present the cryoelectron microscopy (cryo-EM) structures of human URAT1(R477S), its complex with urate, and its closely related homolog OAT4. URAT1(R477S) and OAT4 exhibit major facilitator superfamily (MFS) folds with outward- and inward-open conformations, respectively. Structural comparison reveals a 30° rotation between the N-terminal and C-terminal domains, supporting an alternating access mechanism. A conserved arginine (OAT4-Arg473/URAT1-Arg477) is found to be essential for chloride-mediated inhibition. The URAT1(R477S)-urate complex reveals the specificity of urate recognition. Taken together, our study promotes our understanding of the transport mechanism and substrate selection of URAT1.
Asunto(s)
Microscopía por Crioelectrón , Transportadores de Anión Orgánico , Proteínas de Transporte de Catión Orgánico , Ácido Úrico , Humanos , Ácido Úrico/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/química , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/química , Especificidad por Sustrato , Células HEK293 , Transporte Biológico , Modelos Moleculares , Transportadores de Anión Orgánico Sodio-IndependienteRESUMEN
Plantaginis semen is the dried ripe seed of Plantago asiatica L. or Plantago depressa Willd., which has a long history in alleviating hyperuricemia (HUA) and chronic kidney diseases. While the major chemical ingredients and mechanism remained to be illustrated. Therefore, this work aimed to elucidate the chemicals and working mechanisms of PS for HUA. UPLC-QE-Orbitrap-MS was applied to identify the main components of PS in vitro and in vivo. RNA sequencing (RNA-seq) was conducted to explore the gene expression profile, and the genes involved were further confirmed by real-time quantitative PCR (RT-qPCR). A total of 39 components were identified from PS, and 13 of them were detected in the rat serum after treating the rat with PS. The kidney tissue injury and serum uric acid (UA), xanthine oxidase (XOD), and cytokine levels were reversed by PS. Meanwhile, renal urate anion transporter 1 (Urat1) and glucose transporter 9 (Glut9) levels were reversed with PS treatment. RNA-seq analysis showed that the PPAR signaling pathway; glycine, serine, and threonine metabolism signaling pathway; and fatty acid metabolism signaling pathway were significantly modified by PS treatment. Further, the gene expression of Slc7a8, Pck1, Mgll, and Bhmt were significantly elevated, and Fkbp5 was downregulated, consistent with RNA-seq results. The PPAR signaling pathway involved Pparα, Pparγ, Lpl, Plin5, Atgl, and Hsl were elevated by PS treatment. URAT1 and PPARα proteins levels were confirmed by Western blotting. In conclusion, this study elucidates the chemical profile and working mechanisms of PS for prevention and therapy of HUA and provides a promising traditional Chinese medicine agency for HUA prophylaxis.
Asunto(s)
Hiperuricemia , Ácido Oxónico , Plantago , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/metabolismo , Animales , Ratas , Ácido Oxónico/efectos adversos , Masculino , Plantago/química , Ácido Úrico/sangre , Extractos Vegetales/farmacología , Riñón/metabolismo , Riñón/efectos de los fármacos , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/genética , Xantina Oxidasa/metabolismoRESUMEN
The main uric acid-lowering agents in clinical use for hyperuricemia and gout are xanthine oxidase (XO) inhibitors or urate transporter 1 (URAT1) inhibitors. While these therapies can partially control the disease, they have various limitations. The development of XO/URAT1 dual inhibitors offers the potential to enhance therapeutic potency and reduce toxicity compared with single-target inhibitors. Through scaffold hopping from the XO inhibitor febuxostat (2) and the URAT1 inhibitor probenecid (3), followed by structure-activity relationship (SAR) studies, we identified compound 27 as a potent dual inhibitor of XO and URAT1. Compound 27 demonstrated significant dual inhibition in vitro (XO IC50 = 35 nM; URAT1 IC50 = 31 nM) and exhibited favorable pharmacology and pharmacokinetic (PK) profiles in multiple species including monkeys. Furthermore, toxicity studies in rats and monkeys revealed general safety profiles, supporting that compound 27 emerges as a promising novel drug candidate with potent XO/URAT1 dual inhibition for the treatment of gout.
Asunto(s)
Gota , Hiperuricemia , Transportadores de Anión Orgánico , Proteínas de Transporte de Catión Orgánico , Xantina Oxidasa , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/metabolismo , Hiperuricemia/tratamiento farmacológico , Animales , Gota/tratamiento farmacológico , Relación Estructura-Actividad , Humanos , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Ratas , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/metabolismo , Administración Oral , Ratas Sprague-Dawley , Masculino , Macaca fascicularis , Febuxostat/farmacología , Febuxostat/farmacocinética , Febuxostat/uso terapéutico , Febuxostat/química , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Supresores de la Gota/farmacocinética , Supresores de la Gota/farmacología , Supresores de la Gota/uso terapéutico , Supresores de la Gota/química , Supresores de la Gota/síntesis química , Disponibilidad Biológica , Probenecid/farmacologíaRESUMEN
Acute kidney injury (AKI) is a worldwide public health problem with high morbidity and mortality. Cisplatin is a widely used chemotherapeutic agent for treating solid tumors, but the induction of AKI restricts its clinical application. In this study, the effect of cisplatin on the expression of organic ion transporters was investigated through in vivo and in vitro experiments. Targeted metabolomics techniques were used to measure the levels of selected endogenous substances in serum. Transmission electron microscopy was used to observe the microstructure of renal tubular epithelial cells. Our results show that the toxicity of cisplatin on HK-2 cells or HEK-293 cells was time- and dose-dependent. Administration of cisplatin decreased the expression of OAT1/3 and OCT2 and increased the expression of MRP2/4. Mitochondrial damage induced by cisplatin lead to renal tubular epithelial cell injury. In addition, administration of cisplatin resulted in significant changes in endogenous substance levels in serum, including amino acids, carnitine, and fatty acids. These serum amino acids and metabolites (α-aminobutyric acid, proline, and alanine), carnitines (tradecanoylcarnitine, hexanylcarnitine, octanoylcarnitine, 2-methylbutyroylcarnitine, palmitoylcarnitine, and linoleylcarnitine) and fatty acids (9E-tetradecenoic acid) represent endogenous substances with diagnostic potential for cisplatin-induced AKI.
Asunto(s)
Lesión Renal Aguda , Cisplatino , Cisplatino/toxicidad , Humanos , Animales , Células HEK293 , Masculino , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Antineoplásicos/toxicidad , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/genética , Transportador 2 de Cátion Orgánico/metabolismo , Transportador 2 de Cátion Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Carnitina/análogos & derivados , Carnitina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
The identification of substrates for solute carriers (SLCs) handling drugs is an important challenge, owing to the major implication of these plasma membrane transporters in pharmacokinetics and drug-drug interactions. In this context, the competitive counterflow (CCF) assay has been proposed as a practical and less expensive approach than the reference functional uptake assays for discriminating SLC substrates and non-substrates. The present article was designed to summarize and discuss key-findings about the CCF assay, including its principle, applications, challenges and limits, and perspectives. The CCF assay is based on the decrease of the steady-state accumulation of a tracer substrate in SLC-positive cells, caused by candidate substrates. Reviewed data highlight the fact that the CCF assay has been used to identify substrates and non-substrates for organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs). The performance values of the CCF assay, calculated from available CCF study data compared with reference functional uptake assay data, are, however, rather mitigated, indicating that the predictability of the CCF method for assessing SLC-mediated transportability of drugs is currently not optimal. Further studies, notably aimed at standardizing the CCF assay and developing CCF-based high-throughput approaches, are therefore required in order to fully precise the interest and relevance of the CCF assay for identifying substrates and non-substrates of SLCs.
Asunto(s)
Transportadores de Anión Orgánico , Humanos , Transporte Biológico , Preparaciones Farmacéuticas/metabolismo , Transportadores de Anión Orgánico/metabolismo , Animales , Interacciones Farmacológicas , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Bioensayo/métodosRESUMEN
BACKGROUND AND OBJECTIVES: Trifluridine/tipiracil, registered for the treatment of patients with metastatic gastric and colorectal cancer, is a substrate and inhibitor for the organic cation transporter 2 (OCT2) and the multidrug and toxin extrusion protein 1 (MATE1), which raises the potential for drug-drug interactions with other OCT2/MATE1 modulators. Therefore, we prospectively examined the effect of an OCT2/MATE1 inhibitor (cimetidine) and substrate (metformin) on the pharmacokinetics of trifluridine. METHODS: In this three-phase crossover study, patients with metastatic colorectal or gastric cancer were sequentially treated with trifluridine/tipiracil alone (phase A), trifluridine/tipiracil concomitant with metformin (phase B) and trifluridine/tipiracil concomitant with cimetidine (phase C). The primary endpoint was the relative difference in exposure of trifluridine assessed by the area under the curve from timepoint zero to infinity. A > 30% change in exposure was considered clinically relevant. A p-value of < 0.025 was considered significant because of a Bonferroni correction. RESULTS: Eighteen patients were included in the analysis. Metformin did not significantly alter the exposure to trifluridine (- 12.6%; 97.5% confidence interval - 25.0, 1.8; p = 0.045). Cimetidine did alter the exposure to trifluridine significantly (+ 18.0%; 97.5% confidence interval 4.5, 33.3; p = 0.004), but this increase did not meet our threshold for clinical relevance. Metformin trough concentrations were not influenced by trifluridine/tipiracil. CONCLUSIONS: Our result suggests that the OCT2/MATE1 modulators cimetidine and metformin can be co-administered with trifluridine/tipiracil without clinically relevant effects on drug exposure. CLINICAL TRIAL REGISTRATION: NL8067 (registered 04-10-2019).
Asunto(s)
Cimetidina , Estudios Cruzados , Interacciones Farmacológicas , Metformina , Proteínas de Transporte de Catión Orgánico , Trifluridina , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cimetidina/farmacocinética , Cimetidina/farmacología , Cimetidina/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Combinación de Medicamentos , Metformina/farmacocinética , Metformina/administración & dosificación , Metformina/farmacología , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Transportador 2 de Cátion Orgánico/metabolismo , Estudios Prospectivos , Pirrolidinas/farmacocinética , Pirrolidinas/administración & dosificación , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Timina , Trifluridina/farmacocinética , Trifluridina/administración & dosificaciónRESUMEN
Fostemsavir is an approved gp120-directed attachment inhibitor and prodrug for the treatment of human immunodeficiency virus type 1 infection in combination with other antiretrovirals (ARVs) in heavily treatment-experienced adults with multi-drug resistance, intolerance, or safety concerns with their current ARV regimen. Initial in vitro studies indicated that temsavir, the active moiety of fostemsavir, and its metabolites, inhibited organic cation transporter (OCT)1, OCT2, and multidrug and toxin extrusion transporters (MATEs) at tested concentration of 100 uM, although risk assessment based on the current Food and Drug Administration in vitro drug-drug interaction (DDI) guidance using the mechanistic static model did not reveal any clinically relevant inhibition on OCTs and MATEs. However, a DDI risk was flagged with EMA static model predictions. Hence, a physiologically based pharmacokinetic (PBPK) model of fostemsavir/temsavir was developed to further assess the DDI risk potential of OCT and MATEs inhibition by temsavir and predict changes in metformin (a sensitive OCT and MATEs substrate) exposure. No clinically relevant impact on metformin concentrations across a wide range of temsavir concentrations was predicted; therefore, no dose adjustment is recommended for metformin when co-administered with fostemsavir.
Asunto(s)
Interacciones Farmacológicas , Metformina , Proteínas de Transporte de Catión Orgánico , Transportador 2 de Cátion Orgánico , Organofosfatos , Metformina/farmacocinética , Metformina/administración & dosificación , Humanos , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Transportador 2 de Cátion Orgánico/metabolismo , Organofosfatos/administración & dosificación , Organofosfatos/farmacocinética , Modelos Biológicos , Animales , Transportador 1 de Catión Orgánico/metabolismo , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , PiperazinasRESUMEN
5-HT clearance, commonly mediated by transporters in the uptake-1 and uptake-2 families, has been linked to 5-HT1B receptor's action on behaviors. Since no specific transporters identified yet, effects of serotonin transporter (SERT) and organic cation transporter (OCTs) on 5-HT1B-elicited immobility phenotype, and 5-HT and HIS uptake were then investigated. Intraperitoneal injections of SERT inhibitor fluoxetine (FLX) and/or OCTs inhibitor decynium (D22) were used prior to local perfusion of 5-HT1B agonist CP93129 into the ventral hippocampus to measure immobility times in the FST and TST, to measure 5-HT uptake efficiencies and HIS uptake efficiencies derived from linear regressions using the transient no-net-flux quantitative microdialysis in C57BL/6 mice. Exogenous 5-HT and HIS uptake were measured following incubation of FLX and/or D22 with CP93129 in the RBL-2H3 cells. Moreover, surface membrane levels of SERT and OCT were detected in response to CP93129. Local CP93129 prolonged immobility times, which were attenuated following pretreatment of either inhibitor. Local CP93129 lowered the slopes obtained from the lineal regressions for 5-HT and HIS (slope is reciprocal to uptake efficiency), which were then weakened following pretreatment of either inhibitor. Similar findings were obtained following CP93129 incubation, and co-incubation of CP93129 with either inhibitor in the RBL-2H3. Moreover, CP93129 dose-dependently moved SERT and OCT3 in the cytosol to the surface membrane. Both SERT and OCT are the target effectors mediating 5-HT1B regulation of immobility time and 5-HT uptake, OCT mediates 5-HT1B regulation of HIS uptake. Their underlying signal transductions need to be further explored.
Asunto(s)
Ratones Endogámicos C57BL , Receptor de Serotonina 5-HT1B , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Serotonina , Animales , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Receptor de Serotonina 5-HT1B/metabolismo , Masculino , Ratones , Conducta Animal/efectos de los fármacos , Fluoxetina/farmacología , Proteínas de Transporte de Catión Orgánico/metabolismo , Ratas , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Hipocampo/metabolismo , Hipocampo/efectos de los fármacosRESUMEN
Serum urate levels are determined by the balance between uric acid production and uric acid excretion capacity from the kidneys and intestinal tract. Dysuricemia, including hyperuricemia and hypouricemia, develops when the balance shifts towards an increase or a decrease in the uric acid pool. Hyperuricemia is mostly a multifactorial genetic disorder involving several disease susceptibility genes and environmental factors. Hypouricemia, on the other hand, is caused by genetic abnormalities. The main genes involved in dysuricemia are xanthine oxidoreductase, an enzyme that produces uric acid, and the urate transporters urate transporter 1/solute carrier family 22 member 12 (URAT1/SLC22A12), glucose transporter 9/solute carrier family 2 member 9 (GLUT9/SLC2A9) and ATP binding cassette subfamily G member 2 (ABCG2). Deficiency of xanthine oxidoreductase results in xanthinuria, a rare disease with marked hypouricemia. Xanthinuria can be due to a single deficiency of xanthine oxidoreductase or in combination with aldehyde oxidase deficiency as well. The latter is caused by a deficiency in molybdenum cofactor sulfurase, which is responsible for adding sulphur atoms to the molybdenum cofactor required for xanthine oxidoreductase and aldehyde oxidase to exert their action. URAT1/SLC22A12 and GLUT9/SLC2A9 are involved in urate reabsorption and their deficiency leads to renal hypouricemia, a condition that is common in Japanese due to URAT1/SLC22A12 deficiency. On the other hand, ABCG2 is involved in the secretion of urate, and many Japanese have single nucleotide polymorphisms that result in its reduced function, leading to hyperuricemia. In particular, severe dysfunction of ABCG2 leads to hyperuricemia with reduced extrarenal excretion.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Proteínas Facilitadoras del Transporte de la Glucosa , Hiperuricemia , Proteínas de Neoplasias , Transportadores de Anión Orgánico , Ácido Úrico , Xantina Deshidrogenasa , Humanos , Hiperuricemia/etiología , Hiperuricemia/metabolismo , Hiperuricemia/genética , Ácido Úrico/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 , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Xantina Deshidrogenasa/metabolismo , Xantina Deshidrogenasa/genética , Xantina Deshidrogenasa/deficiencia , Animales , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Defectos Congénitos del Transporte Tubular Renal/genética , Defectos Congénitos del Transporte Tubular Renal/etiología , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Cálculos Urinarios/etiología , Cálculos Urinarios/metabolismo , Cálculos Urinarios/genética , Errores Innatos del MetabolismoRESUMEN
The polarised expression of specific transporters in proximal tubular epithelial cells is important for the renal clearance of many endogenous and exogenous compounds. Thus, ideally, the in vitro tools utilised for predictions would have a similar expression of apical and basolateral xenobiotic transporters as in vivo. Here, we assessed the functionality of organic cation and anion transporters in proximal tubular-like cells (PTL) differentiated from human induced pluripotent stem cells (iPSC), primary human proximal tubular epithelial cells (PTEC), and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1). Organic cation and anion transport were studied using the fluorescent substrates 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) and 6-carboxyfluorescein (6-CF), respectively. The level and rate of intracellular ASP accumulation in PTL following basolateral application were slightly lower but within a 3-fold range compared to primary PTEC and RPTEC/TERT1 cells. The basolateral uptake of ASP and its subsequent apical efflux could be inhibited by basolateral exposure to quinidine in all models. Of the three models, only PTL showed a modest preferential basolateral-to-apical 6-CF transfer. These results show that organic cation transport could be demonstrated in all three models, but more research is needed to improve and optimise organic anion transporter expression and functionality.
Asunto(s)
Células Epiteliales , Túbulos Renales Proximales , Humanos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/citología , Células Epiteliales/metabolismo , Modelos Biológicos , Compuestos de Piridinio/metabolismo , Aniones/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Transporte Biológico , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/genética , Línea Celular , Cationes/metabolismo , Fluoresceínas/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genéticaRESUMEN
The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de Transporte de Catión Orgánico , Humanos , Proteínas de Transporte de Catión Orgánico/química , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Simportadores/química , Simportadores/metabolismo , Sitios de Unión , Unión Proteica , Ergotioneína/química , Ergotioneína/metabolismo , Sodio/metabolismo , Sodio/química , Simulación por Computador , Acetilcolina/metabolismo , Acetilcolina/química , LigandosRESUMEN
Mitochondrial dysfunction is implicated in a wide range of human disorders including many neurodegenerative and cardiovascular diseases, metabolic diseases, cancers, and respiratory disorders. Studies have suggested the potential of l-ergothioneine (ET), a unique dietary thione, to prevent mitochondrial damage and improve disease outcome. Despite this, no studies have definitively demonstrated uptake of ET into mitochondria. Moreover, the expression of the known ET transporter, OCTN1, on the mitochondria remains controversial. In this study, we utilise mass spectrometry to demonstrate direct ET uptake in isolated mitochondria as well as its presence in mitochondria isolated from ET-treated cells and animals. Mitochondria isolated from OCTN1 knockout mice tissues, have impaired but still detectable ET uptake, raising the possibility of alternative transporter(s) which may facilitate ET uptake into the mitochondria. Our data confirm that ET can enter mitochondria, providing a basis for further work on ET in the prevention of mitochondrial dysfunction in human disease.
Asunto(s)
Ergotioneína , Ratones Noqueados , Mitocondrias , Ergotioneína/metabolismo , Ergotioneína/farmacología , Animales , Mitocondrias/metabolismo , Humanos , Ratones , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Simportadores/metabolismo , Simportadores/genéticaRESUMEN
Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-µM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter.
Asunto(s)
Proteínas Arqueales , Pyrococcus furiosus , Pyrococcus furiosus/metabolismo , Proteínas Arqueales/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Unión Proteica , Especificidad por Sustrato , Sitios de Unión , Modelos Moleculares , Protones , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/química , Proteínas de Transporte de Catión Orgánico/genética , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
Multidrug and toxin extrusion protein 1 (MATE1), an efflux transporter mainly expressed in renal proximal tubules, mediates the renal secretion of organic cationic drugs. The inhibition of MATE1 will impair the excretion of drugs into the tubular lumen, leading to the accumulation of nephrotoxic drugs in the kidney and consequently potentiating nephrotoxicity. Screening and identifying potent MATE1 inhibitors can predict or minimize the risk of drug-induced kidney injury. Flavonoids, a group of polyphenols commonly found in foodstuffs and herbal products, have been reported to cause transporter-mediated food/herb-drug interactions. Our objective was to investigate the inhibitory effects of flavonoids on MATE1 in vitro and in vivo and to assess the effects of flavonoids on cisplatin-induced kidney injury. Thirteen flavonoids exhibited significant transport activity inhibition (>50%) on MATE1 in MATE1-MDCK cells. Among them, the six strongest flavonoid inhibitors, including irisflorentin, silymarin, isosilybin, sinensetin, tangeretin, and nobiletin, markedly increased cisplatin cytotoxicity in these cells. In cisplatin-induced in vivo renal injury models, irisflorentin, isosilybin, and sinensetin also increased serum creatinine and blood urea nitrogen levels to different degrees, especially irisflorentin, which exhibited the most potent nephrotoxicity with cisplatin. The pharmacophore model indicated that the hydrogen bond acceptors at the 3, 5, and 7 positions may play a critical role in the inhibitory effect of flavonoids on MATE1. Our findings provide helpful information for predicting the potential risks of flavonoid-containing food/herb-drug interactions and avoiding the exacerbation of drug-induced kidney injury via MATE1 mediation.
Asunto(s)
Cisplatino , Flavonoides , Proteínas de Transporte de Catión Orgánico , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Animales , Flavonoides/farmacología , Cisplatino/toxicidad , Cisplatino/efectos adversos , Interacciones de Hierba-Droga , Masculino , Perros , Células de Riñón Canino Madin Darby , Ratones , Riñón/efectos de los fármacos , Riñón/metabolismo , Interacciones Alimento-Droga , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/prevención & control , Lesión Renal Aguda/metabolismoRESUMEN
SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17ß-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.
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Primates , Animales , Humanos , Secuencia de Aminoácidos , Estradiol/metabolismo , Células HEK293 , Hominidae/genética , Hominidae/metabolismo , Mutación Missense , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Primates/genética , Seudogenes , Especificidad por SustratoRESUMEN
BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.
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Barrera Hematoencefálica , Isquemia Encefálica , Uniones Estrechas , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Isquemia Encefálica/genética , Muerte Celular , Células Endoteliales/metabolismo , Ratones Endogámicos C57BL , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Uniones Estrechas/metabolismoRESUMEN
Recently published cryo-EM structures of human organic cation transporters of the SLC22 family revealed seven, sequentially arranged glutamic and aspartic acid residues, which may be relevant for interactions with positively charged substrates. We analyzed the functional consequences of removing those negative charges by creating D155N, E232Q, D382N, E390Q, E451Q, E459Q, and D478N mutants of OCT3. E232Q, E459Q, and D478N resulted in a lack of localization in the outer cell membrane and no relevant uptake activity. However, D155N and E451Q showed a substrate-specific loss of transport activity, whereas E390Q had no remaining activity despite correct membrane localization. In contrast, D382N showed almost wild-type-like uptake. D155 is located at the entrance to the substrate binding pocket and could, therefore be involved in guiding cationic substrates towards the inside of the binding pocket. For E390, we confirm its critical function for transporter function as it was recently shown for the corresponding position in OCT1. Interestingly, E451 seems to be located at the bottom of the binding pocket in the outward-open confirmation of the transporter. Substrate-specific loss of transport activity of the E451Q variant suggests an essential role in the transport cycle of specific substances as part of an opportunistic binding site. In general, our study highlights the impact of the cryo-EM structures in guiding mutagenesis studies to understand the molecular level of transporter-ligand interactions, and it also confirms the importance of testing multiple substrates in mutagenesis studies of polyspecific OCTs.