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Characterization of ligand-induced thermal stability of the human organic cation transporter 2 (OCT2)


Maane, Max; Xiu, Fangrui; Bellstedt, Peter; Kullak-Ublick, Gerd A; Visentin, Michele (2023). Characterization of ligand-induced thermal stability of the human organic cation transporter 2 (OCT2). Frontiers in Pharmacology, 14:1154213.

Abstract

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 [$^{3}$H]1-methyl-4-phenylpyridinium ([$^{3}$H]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 [$^{3}$H]MPP$^{+}$ uptake but significantly increased the thermal stabilization of OCT2. Conversely, amiloride completely inhibited [$^{3}$H]MPP$^{+}$ uptake but did not affect OCT2 thermal stabilization. [$^{3}$H]MTX intracellular level was significantly higher in OCT2-HEK293 cells than in wild type cells. The magnitude of the thermal shift (ΔT$_{m}$) did not provide information on the binding. Ligands with similar affinity showed markedly different ΔT$_{m}$, indicating different enthalpic and entropic contributions for similar binding affinities. The ΔT$_{m}$ positively correlated with ligand molecular weight/chemical complexity, which typically has high entropic costs, suggesting that large ΔT$_{m}$ 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.

Abstract

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 [$^{3}$H]1-methyl-4-phenylpyridinium ([$^{3}$H]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 [$^{3}$H]MPP$^{+}$ uptake but significantly increased the thermal stabilization of OCT2. Conversely, amiloride completely inhibited [$^{3}$H]MPP$^{+}$ uptake but did not affect OCT2 thermal stabilization. [$^{3}$H]MTX intracellular level was significantly higher in OCT2-HEK293 cells than in wild type cells. The magnitude of the thermal shift (ΔT$_{m}$) did not provide information on the binding. Ligands with similar affinity showed markedly different ΔT$_{m}$, indicating different enthalpic and entropic contributions for similar binding affinities. The ΔT$_{m}$ positively correlated with ligand molecular weight/chemical complexity, which typically has high entropic costs, suggesting that large ΔT$_{m}$ 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.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic for Clinical Pharmacology and Toxicology
04 Faculty of Medicine > University Hospital Zurich > Institute of Clinical Chemistry
Dewey Decimal Classification:610 Medicine & health
Uncontrolled Keywords:Pharmacology (medical), Pharmacology
Language:English
Date:16 March 2023
Deposited On:13 Apr 2023 13:23
Last Modified:29 Jun 2024 01:35
Publisher:Frontiers Research Foundation
ISSN:1663-9812
OA Status:Gold
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.3389/fphar.2023.1154213
PubMed ID:37007010
Project Information:
  • : FunderSchweizerischer Nationalfonds Zur Förderung der Wissenschaftlichen Forschung
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  • : FunderChinese Government Scholarship
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  • Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)