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1.
Yakugaku Zasshi ; 144(6): 659-674, 2024.
Artigo em Japonês | MEDLINE | ID: mdl-38825475

RESUMO

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.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Proteínas Facilitadoras de Transporte de Glucose , Hiperuricemia , Proteínas de Neoplasias , Transportadores de Ânions Orgânicos , Ácido Úrico , Xantina Desidrogenase , Humanos , Hiperuricemia/etiologia , Hiperuricemia/metabolismo , Hiperuricemia/genética , Ácido Úrico/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Xantina Desidrogenase/metabolismo , Xantina Desidrogenase/genética , Xantina Desidrogenase/deficiência , Animais , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Erros Inatos do Transporte Tubular Renal/genética , Erros Inatos do Transporte Tubular Renal/etiologia , Erros Inatos do Transporte Tubular Renal/metabolismo , Cálculos Urinários/etiologia , Cálculos Urinários/metabolismo , Cálculos Urinários/genética , Erros Inatos do Metabolismo
2.
PLoS One ; 19(6): e0304512, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38829838

RESUMO

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.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Transporte de Cátions Orgânicos , Humanos , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Simportadores/química , Simportadores/metabolismo , Sítios de Ligação , Ligação Proteica , Ergotioneína/química , Ergotioneína/metabolismo , Sódio/metabolismo , Sódio/química , Simulação por Computador , Acetilcolina/metabolismo , Acetilcolina/química , Ligantes
3.
Stroke ; 55(6): 1650-1659, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38738428

RESUMO

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.


Assuntos
Barreira Hematoencefálica , Isquemia Encefálica , Junções Íntimas , Animais , Camundongos , Humanos , Junções Íntimas/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/genética , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Morte Celular , Masculino , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Camundongos Endogâmicos C57BL , Células Endoteliais/metabolismo
4.
Nat Commun ; 15(1): 4380, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38782905

RESUMO

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.


Assuntos
Primatas , Animais , Humanos , Sequência de Aminoácidos , Estradiol/metabolismo , Células HEK293 , Hominidae/genética , Hominidae/metabolismo , Mutação de Sentido Incorreto , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Primatas/genética , Pseudogenes , Especificidade por Substrato
5.
Biochem Pharmacol ; 223: 116188, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38580166

RESUMO

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.


Assuntos
Aminoácidos , Proteínas de Transporte de Cátions Orgânicos , Humanos , Cátions/metabolismo , Mutagênese , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico
6.
Biomolecules ; 14(4)2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38672410

RESUMO

Inflammation is a physiological condition characterized by a complex interplay between different cells handled by metabolites and specific inflammatory-related molecules. In some pathological situations, inflammation persists underlying and worsening the pathological state. Over the years, two membrane transporters namely OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been shown to play specific roles in inflammation. These transporters form the OCTN subfamily within the larger SLC22 family. The link between these proteins and inflammation has been proposed based on their link to some chronic inflammatory diseases such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA). Moreover, the two transporters show the ability to mediate the transport of several compounds including carnitine, carnitine derivatives, acetylcholine, ergothioneine, and gut microbiota by-products, which have been specifically associated with inflammation for their anti- or proinflammatory action. Therefore, the absorption and distribution of these molecules rely on the presence of OCTN1 and OCTN2, whose expression is modulated by inflammatory cytokines and transcription factors typically activated by inflammation. In the present review, we wish to provide a state of the art on OCTN1 and OCTN2 transport function and regulation in relationships with inflammation and inflammatory diseases focusing on the metabolic signature collected in different body districts and gene polymorphisms related to inflammatory diseases.


Assuntos
Inflamação , Proteínas de Transporte de Cátions Orgânicos , Membro 5 da Família 22 de Carreadores de Soluto , Simportadores , Humanos , Inflamação/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/genética , Animais , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Ergotioneína/metabolismo , Doença de Crohn/metabolismo , Doença de Crohn/genética , Doença de Crohn/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/genética , Microbioma Gastrointestinal , Carnitina/metabolismo , Asma/metabolismo , Asma/genética , Acetilcolina/metabolismo
7.
Eur J Med Chem ; 271: 116407, 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38663283

RESUMO

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are two most widely studied targets involved in production and reabsorption of uric acid, respectively. Marketed drugs almost target XOR or URAT1, but sometimes, single agents might not achieve aim of lowering uric acid to ideal value in clinic. Thus, therapeutic strategies of combining XOR inhibitors with uricosuric drugs were proposed and implemented. Based on our initial work of virtual screening, A and B were potential hits for dual-targeted inhibitors on XOR/URAT1. By docking A/B with XOR/URAT1 respectively, compounds I1-7 were designed to get different degree of inhibition effect on XOR and URAT1, and I7 showed the best inhibitory effect on XOR (IC50 = 0.037 ± 0.001 µM) and URAT1 (IC50 = 546.70 ± 32.60 µM). Further docking research on I7 with XOR/URAT1 led to the design of compounds II with the significantly improved inhibitory activity on XOR and URAT1, such as II11 and II15. Especially, for II15, the IC50 of XOR is 0.006 ± 0.000 µM, superior to that of febuxostat (IC50 = 0.008 ± 0.000 µM), IC50 of URAT1 is 12.90 ± 2.30 µM, superior to that of benzbromarone (IC50 = 27.04 ± 2.55 µM). In acute hyperuricemia mouse model, II15 showed significant uric acid lowering effect. The results suggest that II15 had good inhibitory effect on XOR/URAT1, with the possibility for further investigation in in-vivo models of hyperuricemia.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos , Piridinas , Animais , Piridinas/farmacologia , Piridinas/química , Piridinas/síntese química , Camundongos , Humanos , Relação Estrutura-Atividade , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Simulação de Acoplamento Molecular , Xantina Desidrogenase/antagonistas & inibidores , Xantina Desidrogenase/metabolismo , Relação Dose-Resposta a Droga , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Masculino , Ácido Úrico/metabolismo
8.
Biochem Biophys Res Commun ; 712-713: 149922, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38626531

RESUMO

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.


Assuntos
Proteínas de Transporte de Cátions Orgânicos , Animais , Camundongos , Ratos , Masculino , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Camundongos Knockout , Hepatócitos/metabolismo , Triglicerídeos/metabolismo , Camundongos Transgênicos , Metabolismo dos Lipídeos/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Adiposidade/genética , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Células Cultivadas , Ratos Sprague-Dawley
9.
Bioorg Chem ; 147: 107381, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38669781

RESUMO

The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC50 of 5.34 ± 0.65 µM, which is less potent than benzbromarone (2.01 ± 0.36 µM) but more potent than lesinurad (10.36 ± 1.23 µM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC50 of 1.04 ± 0.23 µM. Its metabolic product, gallic acid, inhibited XOD with an IC50 of 0.91 ± 0.14 µM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug.


Assuntos
Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Hiperuricemia , Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos , Hiperuricemia/tratamento farmacológico , Humanos , Animais , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Urato Oxidase/química , Descoberta de Drogas , Simulação de Acoplamento Molecular , Camundongos , Masculino , Ácido Gálico/química , Ácido Gálico/farmacologia , Ácido Gálico/análogos & derivados , Ratos Sprague-Dawley
10.
Int J Mol Sci ; 25(5)2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38474165

RESUMO

Cisplatin (CDDP) stands out as an effective chemotherapeutic agent; however, its application is linked to the development of significant adverse effects, notably nephro- and ototoxicity. The human organic cation transporter 2 (hOCT2), found in abundance in the basolateral membrane domain of renal proximal tubules and the Corti organ, plays a crucial role in the initiation of nephro- and ototoxicity associated with CDDP by facilitating its uptake in kidney and ear cells. Given its limited presence in cancer cells, hOCT2 emerges as a potential druggable target for mitigating unwanted toxicities associated with CDDP. Potential strategies for mitigating CDDP toxicities include competing with the uptake of CDDP by hOCT2 or inhibiting hOCT2 activity through rapid regulation mediated by specific signaling pathways. This study investigated the interaction between the already approved cationic drugs disopyramide, imipramine, and orphenadrine with hOCT2 that is stably expressed in human embryonic kidney cells. Regarding disopyramide, its influence on CDDP cellular transport by hOCT2 was further characterized through inductively coupled plasma isotope dilution mass spectrometry. Additionally, its potential protective effects against cellular toxicity induced by CDDP were assessed using a cytotoxicity test. Given that hOCT2 is typically expressed in the basolateral membrane of polarized cells, with specific regulatory mechanisms, this work studied the regulation of hOCT2 that is stably expressed in Madin-Darby Canine Kidney (MDCK) cells. These cells were cultured in a matrix to induce the formation of cysts, exposing hOCT2 in the basolateral plasma membrane domain, which was freely accessible to experimental solutions. The study specifically tested the regulation of ASP+ uptake by hOCT2 in MDCK cysts through the inhibition of casein kinase II (CKII), calmodulin, or p56lck tyrosine kinase. Furthermore, the impact of this manipulation on the cellular toxicity induced by CDDP was examined using a cytotoxicity test. All three drugs-disopyramide, imipramine, and orphenadrine-demonstrated inhibition of ASP+ uptake, with IC50 values in the micromolar (µM) range. Notably, disopyramide produced a significant reduction in the CDDP cellular toxicity and platinum cellular accumulation when co-incubated with CDDP. The activity of hOCT2 in MDCK cysts experienced a significant down-regulation under inhibition of CKII, calmodulin, or p56lck tyrosine kinase. Interestingly, only the inhibition of p56lck tyrosine kinase demonstrated the capability to protect the cells against CDDP toxicity. In conclusion, certain interventions targeting hOCT2 have demonstrated the ability to reduce CDDP cytotoxicity, at least in vitro. Further investigations in in vivo systems are warranted to ascertain their potential applicability as co-treatments for mitigating undesired toxicities associated with CDDP in patients.


Assuntos
Cistos , Ototoxicidade , Humanos , Animais , Cães , Transportador 2 de Cátion Orgânico , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Cisplatino/metabolismo , Disopiramida , Calmodulina/metabolismo , Imipramina , Orfenadrina , Células Madin Darby de Rim Canino , Proteínas Tirosina Quinases/metabolismo
11.
Drug Metab Dispos ; 52(4): 312-321, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38307853

RESUMO

Humans are chronically exposed to benzalkonium chlorides (BACs) from environmental sources. The U.S. Food and Drug Administration (FDA) has recently called for additional BAC safety data, as these compounds are cytotoxic and have great potential for biochemical interactions. Biodistribution studies revealed that BACs extensively distribute to many tissues and accumulate at high levels, especially in the kidneys, but the underlying mechanisms are unclear. In this study, we characterized the interactions of BACs of varying alkyl chain length (C8 to C14) with the human organic cation transporters (hOCT1-3) and multidrug and toxin extrusion proteins (hMATE1/2K) with the goal to identify transporters that could be involved in BAC disposition. Using transporter-expressing cell lines, we showed that all BACs are inhibitors of hOCT1-3 and hMATE1/2K (IC50 ranging 0.83-25.8 µM). Further, the short-chain BACs (C8 and C10) were identified as substrates of these transporters. Interestingly, although BAC C8 displayed typical Michaelis-Menten kinetics, C10 demonstrated a more complex substrate-inhibition profile. Transwell studies with transfected Madin-Darby canine kidney cells revealed that intracellular accumulation of basally applied BAC C8 and C10 was substantially higher (8.2- and 3.7-fold, respectively) in hOCT2/hMATE1 double-transfected cells in comparison with vector-transfected cells, supporting a role of these transporters in mediating renal accumulation of these compounds in vivo. Together, our results suggest that BACs interact with hOCT1-3 and hMATE1/2K as both inhibitors and substrates and that these transporters may play important roles in tissue-specific accumulation and potential toxicity of short-chain BACs. Our findings have important implications for understanding human exposure and susceptibility to BACs due to environmental exposure. SIGNIFICANCE STATEMENT: Humans are systemically exposed to benzalkonium chlorides (BACs). These compounds broadly distribute through tissues, and their safety has been questioned by the FDA. Our results demonstrate that hOCT2 and hMATE1 contribute to the renal accumulation of BAC C8 and C10 and that hOCT1 and hOCT3 may be involved in the tissue distribution of these compounds. These findings can improve our understanding of BAC disposition and toxicology in humans, as their accumulation could lead to biochemical interactions and deleterious effects.


Assuntos
Compostos de Benzalcônio , Proteínas de Transporte de Cátions Orgânicos , Animais , Cães , Humanos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Distribuição Tecidual , Linhagem Celular , Células Madin Darby de Rim Canino , Transportador 2 de Cátion Orgânico/metabolismo
12.
Biopharm Drug Dispos ; 45(1): 43-57, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38305087

RESUMO

The renal tubular organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the vectorial elimination of many drugs and toxins from the kidney, and endogenous biomarkers for vectorial transport (OCT2-MATE1) would allow more accurate drug dosing and help to characterize drug-drug interactions and toxicity. Human serum uptake in OCT2-overexpressing cells and metabolomics analysis were carried out. Potential biomarkers were verified in vitro and in vivo. The specificity of biomarkers was validated in renal transporter overexpressing cells and the sensitivity was investigated by Km . The results showed that the uptake of thiamine, histamine, and 5-hydroxytryptamine was significantly increased in OCT2-overexpressing cells. In vitro assays confirmed that thiamine, histamine, and 5-hydroxytryptamine were substrates of both OCT2 and MATE1. In vivo measurements indicated that the serum thiamine level was increased significantly in the presence of the rOCT2 inhibitor cimetidine, and the level in renal tissue was increased significantly by the rMATE1 inhibitor pyrimethamine. There were no significant changes in the uptake or efflux of thiamine in cell lines overexpressed OAT1, OAT2, OAT3, MRP4, organic anion transporting polypeptide 4C1, P-gp, peptide transporter 2, urate transporter 1, and OAT4. The Km for thiamine with OCT2 and MATE1 were 71.2 and 10.8 µM, respectively. In addition, the cumulative excretion of thiamine at 2 and 4 h was strongly correlated with metformin excretion (R2  > 0.6). Thus, thiamine is preferentially secreted by the OCT2 and MATE1 in renal tubules and can provide a reference value for evaluating the function of the renal tubular OCT2-MATE1.


Assuntos
Proteínas de Transporte de Cátions Orgânicos , Transportador 1 de Cátions Orgânicos , Humanos , Transportador 1 de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Histamina/metabolismo , Serotonina/metabolismo , Rim/metabolismo , Tiamina/metabolismo , Células HEK293
13.
Drug Metab Dispos ; 52(4): 296-304, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38326034

RESUMO

Accurate predictions of renal drug-drug interactions (DDIs) mediated by the human organic cation transporter 2 (hOCT2) and multidrug and toxin extrusion proteins (hMATEs) remain challenging. Current DDI evaluation using plasma maximal unbound inhibitor concentrations (Imax,u) and IC50 values determined in single transporter-transfected cells frequently leads to false or overprediction especially for hMATE1. Emerging evidence suggests intracellular unbound inhibitor concentration may be more relevant for hMATE1 inhibition in vivo. However, determination of intrarenal inhibitor concentrations is impractical. Here, we explored the use of hOCT2/hMATE1 double-transfected Madin-Darby canine kidney (MDCK) cells as a new in vitro tool for DDI risk assessment. Our results showed that potent in vitro hMATE1 inhibitors (hydroxychloroquine, brigatinib, and famotidine) failed to inhibit metformin B-to-A flux in the double-transfected system. On the other side, the classic hOCT2/hMATE1 inhibitors, pyrimethamine and cimetidine, dose-dependently inhibited metformin apparent B-to-A permeability (Papp). The different behaviors of these hMATE1 inhibitors in the double-transfected system can be explained by their different ability to gain intracellular access either via passive diffusion or transporter-mediated uptake. A new parameter (IC50,flux) was proposed reflecting the inhibitor's potency on overall hOCT2/hMATE1-mediated tubular secretion. The IC50,flux values significantly differ from the IC50 values determined in single transporter-transfected cells. Importantly, the IC50,flux accurately predicted in vivo DDIs (within 2-fold) when used in a static model. Our data demonstrated that the IC50,flux approach circumvents the need to measure intracellular inhibitor concentrations and more accurately predicted hOCT2/hMATE1-mediated renal DDIs. This system represents a new approach that could be used for improved DDI assessment during drug development. SIGNIFICANCE STATEMENT: This study demonstrated that flux studies in double-transfected MDCK cells and the IC50,flux represents a better approach to assess in vivo DDI potential for the renal organic cation secretion system. This study highlights the importance of inhibitor intracellular accessibility for accurate prediction of hMATE1-mediated renal DDIs. This approach has the potential to identify in vitro hMATE1 inhibitors that are unlikely to result in in vivo DDIs, thus reducing the burden of unnecessary and costly clinical DDI investigations.


Assuntos
Metformina , Proteínas de Transporte de Cátions Orgânicos , Animais , Cães , Humanos , Transportador 2 de Cátion Orgânico/genética , Transportador 2 de Cátion Orgânico/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Metformina/metabolismo , Interações Medicamentosas , Rim/metabolismo
14.
Clin Pharmacol Drug Dev ; 13(5): 465-473, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38174905

RESUMO

Rimegepant is a calcitonin gene-related peptide receptor antagonist approved for migraine treatment. This phase 1, open-label, single-center, fixed-sequence study evaluated the effect of rimegepant on the pharmacokinetics (PK) of metformin. Twenty-eight healthy participants received metformin 500 mg twice daily from Days 1 to 4 and Days 7 to 10, and once daily on Days 5 and 11. Rimegepant, 75 mg tablet, was administered once daily from Days 9 to 12. At pre-specified time points, plasma metformin concentration, serum glucose levels, and safety and tolerability were evaluated. A 16% increase in the area under the plasma metformin concentration-time curve (AUC) for 1 dosing interval (AUC0-τ,ss), a statistically insignificant increase in maximum and minimum steady-state metformin concentration (Cmax,ss and Cmin,ss), and a decrease in metformin renal clearance were observed on Day 11 following metformin-rimegepant coadministration compared with metformin alone; however, the changes were not clinically relevant. Additionally, coadministration of rimegepant with metformin did not induce clinically meaningful change in the maximum observed glucose concentration (Gmax) or AUCgluc compared with metformin alone. Overall, rimegepant and metformin coadministration did not result in clinically relevant changes in metformin PK, renal clearance, or the antihyperglycemic effects of metformin. Rimegepant is considered safe for use with metformin.


Assuntos
Área Sob a Curva , Interações Medicamentosas , Voluntários Saudáveis , Hipoglicemiantes , Metformina , Proteínas de Transporte de Cátions Orgânicos , Transportador 2 de Cátion Orgânico , Piperidinas , Piridinas , Humanos , Metformina/farmacocinética , Metformina/administração & dosagem , Metformina/farmacologia , Masculino , Adulto , Feminino , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Adulto Jovem , Piridinas/farmacocinética , Piridinas/administração & dosagem , Piridinas/farmacologia , Piridinas/efeitos adversos , Piperidinas/farmacocinética , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Piperidinas/efeitos adversos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Transportador 2 de Cátion Orgânico/metabolismo , Pessoa de Meia-Idade , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/administração & dosagem , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/farmacocinética , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/farmacologia , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/efeitos adversos , Transporte Biológico
15.
Pharmacol Ther ; 253: 108574, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38072333

RESUMO

Psychiatric and substance use disorders inflict major public health burdens worldwide. Their widespread burden is compounded by a dearth of effective treatments, underscoring a dire need to uncover novel therapeutic targets. In this review, we summarize the literature implicating organic cation transporters (OCTs), including three subtypes of OCTs (OCT1, OCT2, and OCT3) and the plasma membrane monoamine transporter (PMAT), in the neurobiology of psychiatric and substance use disorders with an emphasis on mood and anxiety disorders, alcohol use disorder, and psychostimulant use disorder. OCTs transport monoamines with a low affinity but high capacity, situating them to play a central role in regulating monoamine homeostasis. Preclinical evidence discussed here suggests that OCTs may serve as promising targets for treatment of psychiatric and substance use disorders and encourage future research into their therapeutic potential.


Assuntos
Proteínas de Transporte de Cátions Orgânicos , Transtornos Relacionados ao Uso de Substâncias , Humanos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Homeostase , Cátions/metabolismo , Transporte Biológico
16.
Biochim Biophys Acta Biomembr ; 1866(2): 184263, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38092232

RESUMO

BACKGROUND: OCTN1 belongs to the SLC22 family, which includes transporters for cationic, zwitterionic, and anionic substrates. OCTN1 function and role in cells are still poorly understood. Not only cations, such as TEA, but also zwitterions, such as carnitine and ergothioneine, figure among transported molecules. METHODS: In this work, we carried out transport assays measuring [14C]-TEA and [3H]-Carnitine in proteoliposomes reconstituted with the recombinant human OCTN1 in the presence of Na+ or other cations. The homology model of OCTN1 was built using the structure of OCT3 as a template for docking analysis. RESULTS: TEA and carnitine did not inhibit each other. Moreover, carnitine uptake was not affected by the presence of Na+ and TEBA, whereas TEA was strongly inhibited by both compounds. Computational data revealed that TEA, Na+, and carnitine can interact with E381 in the OCTN1 substrate site. Differently from TEA, in the presence of Na+, carnitine is still able to interact with the binding site via R469. CONCLUSIONS: The lack of mutual inhibition of the two prototype substrates, the different effect of Na+ and TEBA on their transport reaction, together with the computational analysis supports the existence of two transport pathways for cations and zwitterions. GENERAL SIGNIFICANCE: The results shed new light on the transport mechanisms of OCTN1, helping to get further insights into the structure/function relationships. The described results correlate well with previous and very recent findings on the polyspecificity of the OCT group of transporters belonging to the same family.


Assuntos
Proteínas de Transporte de Cátions Orgânicos , Simportadores , Humanos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Simportadores/metabolismo , Transporte Biológico , Carnitina , Cátions/metabolismo
17.
Drug Metab Dispos ; 52(2): 86-94, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38049999

RESUMO

Tubular secretion is a primary mechanism along with glomerular filtration for renal elimination of drugs and toxicants into urine. Organic cation transporters (OCTs) and multidrug and toxic extrusion (MATE) transporters facilitate the active secretion of cationic substrates, including drugs such as metformin and endogenous cations. We hypothesized that administration of cimetidine, an Oct/Mate inhibitor, will result in increased plasma levels and decreased renal clearance of metformin and endogenous Oct/Mate substrates in rats. A paired rat pharmacokinetic study was carried out in which metformin (5 mg/kg, intravenous) was administered as an exogenous substrate of Oct/Mate transporters to six Sprague-Dawley rats with and without cimetidine (100 mg/kg, intraperitoneal). When co-administered with cimetidine, metformin area under the curve increased significantly by 3.2-fold, and its renal clearance reduced significantly by 73%. Untargeted metabolomics was performed to investigate the effect of cimetidine on endogenous metabolome in the blood and urine samples. Over 8,000 features (metabolites) were detected in the blood, which were shortlisted using optimized criteria, i.e., a significant increase (P value < 0.05) in metabolite peak intensity in the cimetidine-treated group, reproducible retention time, and quality of chromatogram peak. The metabolite hits were classified into three groups that can potentially distinguish inhibition of i) extra-renal uptake transport or catabolism, ii) renal Octs, and iii) renal efflux transporters or metabolite formation. The metabolomics approach identified novel putative endogenous substrates of cationic transporters that could be tested as potential biomarkers to predict Oct/Mate transporter mediated drug-drug interactions in the preclinical stages. SIGNIFICANCE STATEMENT: Endogenous substrates of renal transporters in animal models could be used as potential biomarkers to predict renal drug-drug interactions in early drug development. Here we demonstrated that cimetidine, an inhibitor of organic cation transporters (Oct/Mate), could alter the pharmacokinetics of metformin and endogenous cationic substrates in rats. Several putative endogenous metabolites of Oct/Mate transporters were identified using metabolomics approach, which could be tested as potential transporter biomarkers to predict renal drug-drug interaction of Oct/Mate substrates.


Assuntos
Metformina , Ratos , Animais , Metformina/farmacocinética , Cimetidina/farmacologia , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ratos Sprague-Dawley , Interações Medicamentosas , Preparações Farmacêuticas/metabolismo , Rim/metabolismo , Biomarcadores/metabolismo , Cátions/metabolismo
18.
Aging (Albany NY) ; 15(24): 14803-14829, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-38112574

RESUMO

BACKGROUND: Ischemic stroke (IS) is a fearful disease that can cause a variety of immune events. Nevertheless, precise immune-related mechanisms have yet to be systematically elucidated. This study aimed to identify immune-related signatures using machine learning and to validate them with animal experiments and single cell analysis. METHODS: In this study, we screened 24 differentially expressed genes (DEGs) while identifying immune-related signatures that may play a key role in IS development through a comprehensive strategy between least absolute shrinkage and selection operation (LASSO) regression, support vector machine (SVM) and immune-related genes. In addition, we explored immune infiltration using the CIBERSORT algorithm. Finally, we performed validation in mouse brain tissue and single cell analysis. RESULTS: We identified 24 DEGs for follow-up analysis. ID3 and SLC22A4 were finally identified as the better immune-related signatures through a comprehensive strategy among DEGs, LASSO, SVM and immune-related genes. RT-qPCR, western blot, and immunofluorescence revealed a significant decrease in ID3 and a significant increase in SLC22A4 in the middle cerebral artery occlusion group. Single cell analysis revealed that ID3 was mainly concentrated in endothelial_2 cells and SLC22A4 in astrocytes in the MCAO group. A CIBERSORT finds significantly altered levels of immune infiltration in IS patients. CONCLUSIONS: This study focused on immune-related signatures after stroke and ID3 and SLC22A4 may be new therapeutic targets to promote functional recovery after stroke. Furthermore, the association of ID3 and SLC22A4 with immune cells may be a new direction for post-stroke immunotherapy.


Assuntos
Proteínas Inibidoras de Diferenciação , AVC Isquêmico , Proteínas de Transporte de Cátions Orgânicos , Acidente Vascular Cerebral , Simportadores , Animais , Humanos , Camundongos , Algoritmos , Astrócitos , Western Blotting , Proteínas Inibidoras de Diferenciação/imunologia , Proteínas Inibidoras de Diferenciação/metabolismo , AVC Isquêmico/genética , Proteínas de Neoplasias , Proteínas de Transporte de Cátions Orgânicos/imunologia , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/metabolismo , Simportadores/imunologia , Simportadores/metabolismo
19.
Molecules ; 28(21)2023 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-37959834

RESUMO

Urate transporter 1 (URAT1) is a clinically validated target for the treatment of hyperuricemia and gout. Due to the absence of protein structures, the molecular design of new URAT1 inhibitors generally resorts to ligand-based approaches. Two series of biphenyl carboxylic acids were designed based on the structures of URAT1 inhibitors Epaminurad and Telmisartan via a strategy of pharmacophore fusion. Fifty-one novel compounds were synthesized and most of them showed obvious inhibition against human URAT1. A1 and B21 were identified as the most potent URAT1 inhibitors in series A and B, respectively. They exhibited IC50 values of 0.93 µM and 0.17 µM, which were comparable or superior to the clinical uricosuric drug benzbromarone. The results confirmed the effectiveness of ligand-based approaches in identifying novel and potent URAT1 inhibitors.


Assuntos
Hiperuricemia , Transportadores de Ânions Orgânicos , Humanos , Ácido Úrico/metabolismo , Ligantes , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Hiperuricemia/tratamento farmacológico , Ácidos Carboxílicos/farmacologia
20.
Mol Pharm ; 20(12): 6289-6300, 2023 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-37962560

RESUMO

Many drugs have chiral centers and are therapeutically applied as racemates. Thus, the stereoselectivity in their interactions with membrane transporters needs to be addressed. Here, we studied stereoselectivity in inhibiting organic cation transporters (OCTs) 1, 2, and 3 and the high-affinity monoamine transporters (MATs) NET and SERT. Selectivity by the inhibition of 35 pairs of enantiomers significantly varied among the three closely related OCTs. OCT1 inhibition was nonselective in almost all cases, whereas OCT2 was stereoselectively inhibited by 45% of the analyzed drugs. However, the stereoselectivity of the OCT2 was only moderate with the highest selectivity observed for pramipexole. The (R)-enantiomer inhibited OCT2 4-fold more than the (S)-enantiomer. OCT3 showed the greatest stereoselectivity in its inhibition. (R)-Tolterodine and (S)-zolmitriptan inhibited OCT3 11-fold and 25-fold more than their respective counterparts. Interestingly, in most cases, the pharmacodynamically active enantiomer was also the stronger OCT inhibitor. In addition, stereoselectivity in the OCT inhibition appeared not to depend on the transported substrate. For high-affinity MATs, our data confirmed the stereoselective inhibition of NET and SERT by several antidepressants. However, the stereoselectivity measured here was generally lower than that reported in the literature. Unexpectedly, the high-affinity MATs were not significantly more stereoselectively inhibited than the polyspecific OCTs. Combining our in vitro OCT inhibition data with available stereoselective pharmacokinetic analyses revealed different risks of drug-drug interactions, especially at OCT2. For the tricyclic antidepressant doxepine, only the (E)-isomer showed an increased risk of drug-drug interactions according to guidelines from regulatory authorities for renal transporters. However, most chiral drugs show only minor stereoselectivity in the inhibition of OCTs in vitro, which is unlikely to translate into clinical consequences.


Assuntos
Proteínas de Membrana Transportadoras , Transportador 1 de Cátions Orgânicos , Transportador 1 de Cátions Orgânicos/metabolismo , Transporte Biológico , Interações Medicamentosas , Cátions , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo
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