Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

Chen, Eugene C.; Khuri, Natalia; Liang, Xiaomin; Stecula, Adrian; Chien, Huan‐Chieh; Yee, Sook Wah; Huang, Yong; Šali, Andrej; Giacomini, Kathleen M.

doi:10.1021/acs.jmedchem.6b01317

Cited by 59 publications

(88 citation statements)

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“…pdf). Currently, novel molecular entities (NMEs) are tested for interaction with hOCT1 and hOCT2 expressed in epithelial cells to determine whether they inhibit uptake of a cationic model substrate that is applied at a micromolar concentration (Ahlin et al, 2008(Ahlin et al, , 2011Chen et al, 2017). This procedure has turned out to be insufficient because it was observed that the efficacy of inhibitors was dependent on the molecular structure of the employed substrate and was different when substrate concentrations far below their respective Michaelis-Menten constant (K m ) values were used for uptake measurements (Belzer et al, 2013;Thévenod et al, 2013;Yin et al, 2016;Minuesa et al, 2017;Gorboulev et al, 2018;Sandoval et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Rat Organic Cation Transporter 1 Contains Three Binding Sites for Substrate 1-Methyl-4-phenylpyridinium per Monomer

et al. 2018

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Organic cation transporters OCT1 (SLC22A1) and OCT2 (SLC22A2) are critically involved in absorption and excretion of diverse cationic drugs. Because drug-drug interactions at these transporters may induce adverse drug effects in patients, in vitro testing during drug development for interaction with the human transporters is mandatory. Recent data performed with rat OCT1 (rOCT1) suggest that currently performed in vitro tests assuming one polyspecific binding site are insufficient. Here we measured the binding and transport of model substrate 1-methyl-4-phenylpyridinium 1 (MPP 1 ) by cell-free-expressed fusion proteins of rOCT1 and rOCT1 mutants with green fluorescent protein that had been reconstituted into nanodiscs or proteoliposomes. The nanodiscs were formed with major scaffold protein (MSP) and different phospholipids, whereas the proteoliposomes were formed with a mixture of cholesterol, phosphatidylserine, and phosphatidylcholine. In nanodiscs formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or cholesterol, phosphatidylserine, and phosphatidylcholine, two low-affinity MPP 1 binding sites and one high-affinity MPP 1 binding site per transporter monomer were determined. Mutagenesis revealed that tryptophan 218 and aspartate 475 in neighboring positions in the modeled outward-open cleft contribute to one low-affinity binding site, whereas arginine 440 located distantly in the cleft is critical for MPP 1 binding to another low-affinity site. Comparing MPP 1 binding with MPP 1 transport suggests that the low-affinity sites are involved in MPP 1 transport, whereas high-affinity MPP 1 binding influences transport allosterically. The data will be helpful in the interpretation of future crystal structures and provides a rationale for future in vitro testing that is more sophisticated and reliable, leading to the generation of pharmacophore models with high predictive power. This work was supported by the Deutsche Forschungsgemeinschaft (KO 862/6-1). https://doi.org/10.1124/mol.118.113498. s This article has supplemental material available at molpharm. aspetjournals.org.

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Section: Introductionmentioning

confidence: 99%

Rat Organic Cation Transporter 1 Contains Three Binding Sites for Substrate 1-Methyl-4-phenylpyridinium per Monomer

et al. 2018

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“…Organic cation transporters (OCTs) OCT1, OCT2, and OCT3 (SLC22A1-3) of the major facilitator superfamily MFS play pivotal roles in the absorption, excretion, and tissue distribution of many cationic drugs including psychopharmaca and cytostatics (Koepsell et al, 2007;Ahlin et al, 2008;Nies et al, 2011;Koepsell, 2013;Lin et al, 2015;Chen et al, 2017). The polyspecificity of OCTs explains their predominant role for drug bioavailability and the high probability for drugdrug interactions at the transporter level.…”

Section: Introductionmentioning

confidence: 99%

“…For example, in vitro testing of human OCT1 (hOCT1) for drug-drug interactions was recommended in 2015 by the European Medicines Agency. Although methods to identify new substrates and inhibitors of OCTs have been established, so far no satisfying strategies for in silico and/or in vitro distinction between substrates and inhibitors and for elucidation of biomedically relevant effects of transporter polymorphisms have been found (Koepsell, 2015;Chen et al, 2017). The reasons are that the transport mechanisms of OCTs are not fully understood, no crystal structures are available, and our molecular understanding of cation binding and translocation based on mutagenesis is very limited.…”

Section: Introductionmentioning

confidence: 99%

Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake

et al. 2018

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The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium (MPP) and tetraethylammonium (TEA) or inhibition of MPP uptake by the nontransported inhibitors tetrabutylammonium (TBuA), tetrapentylammonium (TPeA), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant () values, different IC values, and varying effects of mutations were determined. In addition, varying IC values for the inhibition of MPP uptake and varying effects of mutations were obtained when different MPP concentrations far below the apparent value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1M MPP for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent values for TEA and/or MPP Mutation of Trp218 and Asp475 led to altered IC values for TBuA, TPeA, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.

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“…The organic cation transporter 1 (OCT1) mediates hepatic uptake of typically cationic substances with a molecular weight below 400 Dalton . In humans, OCT1 shows strong expression in the sinusoidal membrane of hepatocytes and only minor, if any, expression in other organs .…”

mentioning

confidence: 99%

“…Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research.The organic cation transporter 1 (OCT1) mediates hepatic uptake of typically cationic substances with a molecular weight below 400 Dalton. [1][2][3][4] In humans, OCT1 shows strong expression in the sinusoidal membrane of hepatocytes 5 and only minor, if any, expression in other organs. 6,7 OCT1 can be relevant for the hepatic uptake and pharmacokinetics of numerous drugs, including metformin, morphine, O-desmethyltramadol, sumatriptan, fenoterol, trospium, and ranitidine.…”

mentioning

confidence: 99%

Variability and Heritability of Thiamine Pharmacokinetics With Focus on OCT1 Effects on Membrane Transport and Pharmacokinetics in Humans

Jensen

Matthaei

Blome

et al. 2019

Clin Pharma and Therapeutics

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Thiamine is substrate of the hepatic uptake transporter organic cation transporter 1 (OCT1), and pathological lipid metabolism was associated with OCT1‐dependent thiamine transport. However, it is unknown whether clinical pharmacokinetics of thiamine is modulated by OCT1 genotype. We analyzed thiamine transport in vitro, thiamine blood concentrations after high‐dose and low‐dose (nutritional) intake, and heritability of thiamine and thiamine‐phosphate blood concentrations. The variant OCT1*2 had reduced and OCT1*3 to OCT1*6 had deficient thiamine uptake activity. However, pharmacokinetics of thiamine did not differ depending on OCT1 genotype. Further studies in primary human hepatocytes indicated that several cation transporters, including OCT1, OCT3, and THTR‐2, contribute to hepatic uptake of thiamine. As much as 54% of the variation in thiamine and 75% in variation of thiamine monophosphate plasma concentrations was determined by heritable factors. Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research.

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Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

Cited by 59 publications

References 58 publications

Rat Organic Cation Transporter 1 Contains Three Binding Sites for Substrate 1-Methyl-4-phenylpyridinium per Monomer

Rat Organic Cation Transporter 1 Contains Three Binding Sites for Substrate 1-Methyl-4-phenylpyridinium per Monomer

Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake

Variability and Heritability of Thiamine Pharmacokinetics With Focus on OCT1 Effects on Membrane Transport and Pharmacokinetics in Humans

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