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Substrate-Dependent Inhibition of the Human Organic Cation Transporter OCT2: A Comparison of Metformin with Experimental Substrates.

ABSTRACT: The importance of the organic cation transporter OCT2 in the renal excretion of cationic drugs raises the possibility of drug-drug interactions (DDIs) in which an inhibitor (perpetrator) drug decreases OCT2-dependent renal clearance of a victim (substrate) drug. In fact, there are clinically significant interactions for drugs that are known substrates of OCT2 such as metformin. To identify drugs as inhibitors for OCT2, individual drugs or entire drug libraries have been investigated in vitro by using experimental probe substrates such as 1-methyl-4-phenylpyridinium (MPP+) or 4-4-dimethylaminostyryl-N-methylpyridinium (ASP+). It has been questioned whether the inhibition data obtained with an experimental probe substrate such as MPP+ or ASP+ might be used to predict the inhibition against other, clinical relevant substrates such as metformin. Here we compared the OCT2 inhibition profile data for the substrates metformin, MPP+ and ASP+. We used human embryonic kidney (HEK 293) cells stably overexpressing human OCT2 as the test system to screen 125 frequently prescribed drugs as inhibitors of OCT2-mediated metformin and MPP+ uptake. Data on inhibition of OCT2-mediated ASP+ uptake were obtained from previous literature. A moderate correlation between the inhibition of OCT2-mediated MPP+, ASP+, and metformin uptake was observed (pairwise rs between 0.27 and 0.48, all P < 0.05). Of note, the correlation in the inhibition profile between structurally similar substrates such as MPP+ and ASP+ (Tanimoto similarity T = 0.28) was even lower (rs = 0.27) than the correlation between structurally distinct substrates, such as ASP+ and metformin (T = 0.01; rs = 0.48) or MPP+ and metformin (T = 0.01; rs = 0.40). We identified selective as well as universal OCT2 inhibitors, which inhibited transport by more than 50% of one substrate only or of all substrates, respectively. Our data suggest that the predictive value for drug-drug interactions using experimental substrates rather than the specific victim drug is limited.

SUBMITTER: Hacker K

PROVIDER: S-EPMC4556614 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Substrate-Dependent Inhibition of the Human Organic Cation Transporter OCT2: A Comparison of Metformin with Experimental Substrates.

Hacker Kristina K Maas Renke R Kornhuber Johannes J Fromm Martin F MF Zolk Oliver O

PloS one 20150901 9

The importance of the organic cation transporter OCT2 in the renal excretion of cationic drugs raises the possibility of drug-drug interactions (DDIs) in which an inhibitor (perpetrator) drug decreases OCT2-dependent renal clearance of a victim (substrate) drug. In fact, there are clinically significant interactions for drugs that are known substrates of OCT2 such as metformin. To identify drugs as inhibitors for OCT2, individual drugs or entire drug libraries have been investigated in vitro by ...[more]

PMID: 26327616

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Project description:Introduction: The human organic cation transporter 2 (OCT2) is involved in the transport of endogenous quaternary amines and positively charged drugs across the basolateral membrane of proximal tubular cells. In the absence of a structure, the progress in unraveling the molecular basis of OCT2 substrate specificity is hampered by the unique complexity of OCT2 binding pocket, which seemingly contains multiple allosteric binding sites for different substrates. Here, we used the thermal shift assay (TSA) to better understand the thermodynamics governing OCT2 binding to different ligands. Methods: Molecular modelling and in silico docking of different ligands revealed two distinct binding sites at OCT2 outer part of the cleft. The predicted interactions were assessed by cis-inhibition assay using [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) as a model substrate, or by measuring the uptake of radiolabeled ligands in intact cells. Crude membranes from HEK293 cells harboring human OCT2 (OCT2-HEK293) were solubilized in n-Dodecyl-β-D-Maltopyranoside (DDM), incubated with the ligand, heated over a temperature gradient, and then pelleted to remove heat-induced aggregates. The OCT2 in the supernatant was detected by western blot. Results: Among the compounds tested, cis-inhibition and TSA assays showed partly overlapping results. Gentamicin and methotrexate (MTX) did not inhibit [3H]MPP+ uptake but significantly increased the thermal stabilization of OCT2. Conversely, amiloride completely inhibited [3H]MPP+ uptake but did not affect OCT2 thermal stabilization. [3H]MTX intracellular level was significantly higher in OCT2-HEK293 cells than in wild type cells. The magnitude of the thermal shift (ΔTm) did not provide information on the binding. Ligands with similar affinity showed markedly different ΔTm, indicating different enthalpic and entropic contributions for similar binding affinities. The ΔTm positively correlated with ligand molecular weight/chemical complexity, which typically has high entropic costs, suggesting that large ΔTm reflect a larger displacement of bound water molecules. Discussion: In conclusion, TSA might represent a viable approach to expand our knowledge on OCT2 binding descriptors.

Dataset Information

Substrate-Dependent Inhibition of the Human Organic Cation Transporter OCT2: A Comparison of Metformin with Experimental Substrates.

Publications

Substrate-Dependent Inhibition of the Human Organic Cation Transporter OCT2: A Comparison of Metformin with Experimental Substrates.

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