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1.
Int J Mol Sci ; 20(21)2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31653114

RESUMO

Renal drug transporters such as the organic cation transporters (OCTs), organic anion transporters (OATs) and multidrug resistance proteins (MRPs) play an important role in the tubular secretion of many drugs influencing their efficacy and safety. However, only little is known about the distinct protein abundance of these transporters in human kidneys, and about the impact of age and gender as potential factors of inter-subject variability in their expression and function. The aim of this study was to determine the protein abundance of MDR1, MRP1-4, BCRP, OAT1-3, OCT2-3, MATE1, PEPT1/2, and ORCTL2 by liquid chromatography-tandem mass spectrometry-based targeted proteomics in a set of 36 human cortex kidney samples (20 males, 16 females; median age 53 and 55 years, respectively). OAT1 and 3, OCT2 and ORCTL2 were found to be most abundant renal SLC transporters while MDR1, MRP1 and MRP4 were the dominating ABC transporters. Only the expression levels of MDR1 and ORCTL2 were significantly higher abundant in older donors. Moreover, we found several significant correlations between different transporters, which may indicate their functional interplay in renal vectorial transport processes. Our data may contribute to a better understanding of the molecular processes determining renal excretion of drugs.


Assuntos
Córtex Renal/metabolismo , Proteoma/análise , Espectrometria de Massas em Tandem , 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 , Adulto , Fatores Etários , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/genética , Transportador 2 de Cátion Orgânico/metabolismo , Fatores Sexuais
2.
J Pharm Sci ; 108(2): 1035-1046, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30267783

RESUMO

Intestinal transporters and metabolizing enzymes are the important factors of the intestinal absorption barrier. Because there is evidence that their expression and function may be affected during inflammatory conditions, we investigated gene expression, protein abundance, and regulation of relevant intestinal transporters and metabolizing enzymes in the intestinal mucosa of patients with ulcerative colitis (UC). Specimens from inflamed and noninflamed tissues of 10 patients with UC as well as colonic control tissues of 10 patients without inflammation were subjected to gene (9 enzymes, 15 transporters, 9 cytokines) and microRNA (N = 54) expression analysis. Protein abundance was quantified by liquid chromatography-tandem mass spectrometry-based targeted proteomics. Gene expression of several metabolizing enzymes (e.g., CYP2C9, UGT1A1) and transporters such as ABCB1 (ABCB1), ABCG2 (ABCG2), and monocarboxylate transporter 1 (MCT1, SLC16A1) were significantly decreased during inflammation and negatively correlated to microRNAs. On contrary, multidrug resistance-protein 4 (MRP4, ABCC4), organic anion-transporting polypeptide 2B1 (OATP2B1, SLCO2B1), and organic cation transporter-like 2 (ORCTL2, SLC22A18) were significantly elevated in inflamed tissue. However, at protein level, these findings could only be confirmed for MCT1. UC is associated with complex changes in the intestinal expression of enzymes, transporters, cytokines, and microRNAs, which may affect efficacy of anti-inflammatory drug therapy or the disease state itself.


Assuntos
Colite Ulcerativa/genética , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Preparações Farmacêuticas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Adulto Jovem
3.
Clin Pharmacol Ther ; 105(5): 1204-1212, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30447067

RESUMO

Bioavailability of orally administered drugs is partly determined by function of drug transporters in the liver and intestine. Therefore, we explored adenosine triphosphate-binding cassette (ABC) and solute carriers family transporters expression (quantitative polymerase chain reaction) and protein abundance (liquid chromatography tandem mass spectrometry (LC-MS/MS)) in human liver and duodenum, jejunum, ileum, and colon in paired tissue specimens from nine organ donors. The transporter proteins were detected in the liver (permeability-glycoprotein (P-gp), multidrug resistance protein (MRP)2, MRP3, breast cancer resistance protein (BCRP), organic anion-transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic cation transporter (OCT)1, OCT3, organic anion transporter 2, Na+-taurocholate cotransporting polypeptide, monocarboxylate transporter (MCT)1, and multidrug and toxin extrusion 1) and the intestine (P-gp, multidrug-resistance protein (MRP)2, MRP3, MRP4, BCRP, OATP2B1, OCT1, apical sodium-bile acid transporter (only ileum), MCT1, and peptide transporter (PEPT1)). Significantly higher hepatic gene expression and protein abundance of ABCC2/MRP2, SLC22A1/OCT1, and SLCO2B1/OATP2B1 were found, as compared to all intestinal segments. No correlations between hepatic and small intestinal protein levels were observed. These observations provide a description of drug transporters distribution without the impact of interindividual variability bias and may help in construction of superior physiologically based pharmacokinetic and humanized animal models.


Assuntos
Disponibilidade Biológica , Hepatócitos/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras , Trifosfato de Adenosina/metabolismo , Administração Oral , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Correlação de Dados , Humanos , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/metabolismo , Taxa de Depuração Metabólica/efeitos dos fármacos , Proteína 2 Associada à Farmacorresistência Múltipla , Espectrometria de Massas em Tandem/métodos , Distribuição Tecidual
4.
Clin Pharmacol Ther ; 104(3): 515-524, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29205295

RESUMO

This work revises and complements existing findings on the distribution of drug-metabolizing enzymes in the first-pass effect organs. We explored gene expression (quantitative polymerase chain reaction) and protein abundance (liquid chromatography/ tandem mass spectrometry) of CYP1A2, CYP2B6, CYP2C8/9/19, CYP2D6, CYP2E1, CYP3A4/5, UGT1A1/3, UGT2B7/15 in the liver, duodenum, jejunum, ileum, and colon in paired tissues from nine organ donors. All proteins were detected in the liver, but in the intestine CYP2C9/19, CYP2D6, CYP3A4/5, UGT1A1/3, and UGT2B7 were found. CYP3A4 showed comparable abundance in the liver and jejunum, whereas other enzymes were markedly higher in the hepatic tissue. Nearly all detected enzymes showed their highest abundance in the jejunum. Significant correlations between mRNA and protein levels in liver or intestine were found for most enzymes. CYP3A4 and CYP3A5 protein abundance, but not other enzymes, were significantly correlated in the liver and the small intestine. Our data may contribute to an improved understanding of hepatic and intestinal drug metabolism.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Intestinos/enzimologia , Fígado/enzimologia , Adulto , Biotransformação , Cromatografia Líquida , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Espectrometria de Massas em Tandem , Adulto Jovem
5.
J Pharm Sci ; 106(9): 2909-2913, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28450237

RESUMO

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.


Assuntos
Preparações Farmacêuticas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Sistemas CRISPR-Cas , Cães , Descoberta de Drogas , Expressão Gênica , Humanos , Células Madin Darby de Rim Canino/metabolismo
6.
J Pharm Sci ; 106(9): 2657-2663, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28408210

RESUMO

The organic anion-transporting polypeptide (OATP) 2B1 which is ubiquitously expressed in the human body is assumed to play an important role in the cellular uptake of many drugs. Although the expression and function of this solute carrier transporter is well characterized in the human liver and other tissues, little is known about its localization and functional relevance in the intestine. Thus, it was the aim of this study to investigate its localization and function in the human jejunum and in the frequently used intestinal Caco-2 cell line. The basolateral membrane of jejunal tissue from 6 individuals showed a significant enrichment of OATP2B1 (17-fold) and the known basolateral proteins ABCC3 and Na/K-ATPase compared to the apical membrane as derived from targeted proteomics analysis. On the contrary, apical localization could be confirmed for ABCB1, ABCC2, and PEPT1. Basolateral localization of OATP2B1 could also be verified in Caco-2 cells. Bidirectional transport studies with established OATP2B1 substrates (sulfasalazine and pravastatin) across freshly exercised human jejunum and Caco-2 cell monolayers demonstrated a markedly higher transport from the basal to the apical compartment than in the opposite direction. Our data provide evidence for a basolateral localization of OATP2B1 which may improve our understanding of intestinal drug absorption.


Assuntos
Membrana Celular/metabolismo , Jejuno/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Peptídeos/metabolismo , Adenosina Trifosfatases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Transporte Biológico , Células CACO-2 , Calibragem , Feminino , Humanos , Absorção Intestinal/fisiologia , Masculino , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Peptídeos/química , Proteômica/métodos
7.
Biol Chem ; 398(2): 175-192, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27611766

RESUMO

Although oral drug administration is currently the favorable route of administration, intestinal drug absorption is challenged by several highly variable and poorly predictable processes such as gastrointestinal motility, intestinal drug solubility and intestinal metabolism. One further determinant identified and characterized during the last two decades is the intestinal drug transport that is mediated by several transmembrane proteins such as P-gp, BCRP, PEPT1 and OATP2B1. It is well-established that intestinal transporters can affect oral absorption of many drugs in a significant manner either by facilitating their cellular uptake or by pumping them back to gut lumen, which limits their oral bioavailability. Their functional relevance becomes even more apparent in cases of unwanted drug-drug interactions when concomitantly given drugs that cause transporter induction or inhibition, which in turn leads to increased or decreased drug exposure. The longitudinal expression of several intestinal transporters is not homogeneous along the human intestine, which may have functional implications on the preferable site of intestinal drug absorption. Besides the knowledge about the expression of pharmacologically relevant transporters in human intestinal tissue, their exact localization on the apical or basolateral membrane of enterocytes is also of interest but in several cases debatable. Finally, there is obviously a coordinative interplay of intestinal transporters (apical-basolateral), intestinal enzymes and transporters as well as intestinal and hepatic transporters. This review aims to give an updated overview about the expression, localization, regulation and function of clinically relevant transporter proteins in the human intestine.


Assuntos
Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Transporte Biológico , Humanos , Proteínas de Membrana Transportadoras/química
8.
Appl Microbiol Biotechnol ; 100(4): 1945-1953, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26671615

RESUMO

As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate.


Assuntos
Biotecnologia/métodos , Peptídeo Hidrolases/metabolismo , Proteínas/isolamento & purificação , Proteínas/genética
9.
Chembiochem ; 16(12): 1791-6, 2015 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-26058745

RESUMO

A few lipases, such as Candida antarctica lipase A (CAL-A), are known to possess acyltransferase activity. This enables the enzyme to synthesize fatty acid esters from natural oils and alcohols even in the presence of bulk water. Unfortunately, fatty acids are still formed in these reactions as undesired side-products. To reduce the amount of fatty acids, several CAL-A variants were rationally designed based on its crystal structure. These variants were expressed in Escherichia coli and Pichia pastoris, purified, and their acyltransferase/hydrolase activities were investigated by various biocatalytic approaches. Among the investigated variants, mutant Asp122Leu showed a significant decrease in the hydrolytic activity, thus reducing the side-product yield during acylation. As desired, this variant retained wild-type process-relevant features like pH profile and thermostability.


Assuntos
Aciltransferases/metabolismo , Candida/enzimologia , Lipase/metabolismo , Aciltransferases/química , Domínio Catalítico , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Estrutura Molecular , Engenharia de Proteínas
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