Characterization of ligand-induced thermal stability of the human organic cation transporter 2 (OCT2)
Open Access
- 16 March 2023
- journal article
- research article
- Published by Frontiers Media SA in Frontiers in Pharmacology
- Vol. 14, 1154213
- https://doi.org/10.3389/fphar.2023.1154213
Abstract
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 basies 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. 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. 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. In conclusion, TSA might represent a viable approach to expand our knowledge on OCT2 binding descriptors.Funding Information
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- Chinese Government Scholarship
This publication has 38 references indexed in Scilit:
- Multiple mechanisms of ligand interaction with the human organic cation transporter, OCT2American Journal of Physiology-Renal Physiology, 2013
- The structural basis of secondary active transport mechanismsBiochimica et Biophysica Acta (BBA) - Bioenergetics, 2011
- The use of differential scanning fluorimetry to detect ligand interactions that promote protein stabilityNature Protocols, 2007
- Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein−Ligand ComplexesJournal of Medicinal Chemistry, 2006
- Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluorBiochemistry, 2005
- Understanding Noncovalent Interactions: Ligand Binding Energy and Catalytic Efficiency from Ligand‐Induced Reductions in Motion within Receptors and EnzymesAngewandte Chemie, 2004
- Evaluation of fluorescence-based thermal shift assays for hit identification in drug discoveryAnalytical Biochemistry, 2004
- Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database ScreeningJournal of Medicinal Chemistry, 2004
- Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking AccuracyJournal of Medicinal Chemistry, 2004
- Can thermodynamic measurements of receptor binding yield information on drug affinity and efficacy?Biochemical Pharmacology, 2000