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Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations

  • Margarita Stampelou
    Margarita Stampelou
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
  • Anna Suchankova
    Anna Suchankova
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
  • Efpraxia Tzortzini
    Efpraxia Tzortzini
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
  • Lakshiv Dhingra
    Lakshiv Dhingra
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
  • Kerry Barkan
    Kerry Barkan
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
    More by Kerry Barkan
  • Nikolaos Lougiakis
    Nikolaos Lougiakis
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
  • Panagiotis Marakos
    Panagiotis Marakos
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
  • Nicole Pouli*
    Nicole Pouli
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    *Email: [email protected]
    More by Nicole Pouli
  • Graham Ladds*
    Graham Ladds
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
    *Email: [email protected]. Phone: +44 (0) 12023 334020.
    More by Graham Ladds
  • , and 
  • Antonios Kolocouris*
    Antonios Kolocouris
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    *Email: [email protected]. Phone: 210-727-4834.
Cite this: J. Med. Chem. 2022, 65, 19, 13305–13327
Publication Date (Web):September 29, 2022
https://doi.org/10.1021/acs.jmedchem.2c01123
Copyright © 2022 American Chemical Society

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    Supporting Info (14)»

    Abstract

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    Drugs targeting adenosine receptors (AR) can provide treatment for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]pyridine (A17) displaying the highest affinity at both receptors with a long residence time of binding, as determined using a NanoBRET-based assay. Two binding orientations of A17 produce stable complexes inside the orthosteric binding area of A1R in molecular dynamics (MD) simulations, and we selected the most plausible orientation based on the agreement with alanine mutagenesis supported by affinity experiments. Interestingly, for drug design purposes, the mutation of L2506.51 to alanine increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against A1R using alchemical binding free energy calculations with the thermodynamic integration coupled with the MD simulation (TI/MD) method, applied on the whole G-protein-coupled receptor–membrane system, which showed a good agreement (r = 0.73) between calculated and experimental relative binding free energies.

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01123.

    • Pharmacological experiments, simulation methods, MD simulation results; chemical structures of compounds from our in-house library; functional activities for A15, A17, and A27 against A2AR and A2BR and for L2–L10, L12, L15, and L21 against A2AR and A2BR; pIC50 of NECA in the presence of DMSO and in the presence of a potential antagonist in A3R and A1R Flp-In CHO cells; pKds measured through BRET and the rmsd values of the ligand and protein Ca carbons at A1R; binding affinities for A26 measured using NanoBRET against WT and mutant A1Rs; characterization of A15/A17 and A26 analogues’ selectivity, at the A2AR and A2BR; frames, rmsd plots, and frequency interaction plots for A1R in complex with antagonists not shown in the article; sequence alignment of the residues surrounding the binding site of A1R, A3R; complex of a cyanopyridine ring in complex with three waters stabilized through hydrogen bonding interactions; two docking poses 1 or 2 of A17; and representative frames and plots of Y2717.36A A1Rs with the docking poses 1 and 2 of A17 or T913.36A, H2516.52A, S2677.32A, and A1Rs with docking pose 1 of A17 from 100 ns MD simulations (PDF)

    • SMILES biological activity (CSV)

    • MD of A17 (PDB)

    • MD of A26 (PDB)

    • MD of L2 (PDB)

    • MD of L3 (PDB)

    • MD of L4 (PDB)

    • MD of L5 (PDB)

    • MD of L6 (PDB)

    • MD of L7 (PDB)

    • MD of L8 (PDB)

    • MD of L9 (PDB)

    • MD of L10 (PDB)

    • MD of L12 (PDB)

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    Cited By

    This article is cited by 1 publications.

    1. Margarita Stampelou, Graham Ladds, Antonios Kolocouris. Computational Workflow for Refining AlphaFold Models in Drug Design Using Kinetic and Thermodynamic Binding Calculations: A Case Study for the Unresolved Inactive Human Adenosine A3 Receptor. The Journal of Physical Chemistry B 2024, 128 (4) , 914-936. https://doi.org/10.1021/acs.jpcb.3c05986

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