Bitopic A3 Adenosine Receptor Molecular Probes: Positive Allosteric Modulation and Noncanonical Activation

  • J Med Chem. 2025 Oct 9;68(19):20717-20740. doi: 10.1021/acs.jmedchem.5c01985.
Siva Hariprasad Kurma  1 ,  Matteo Pavan  1 ,  Tina C Wan  2 ,  Balaram Pradhan  1 ,  Marc López-Cano  3  4 ,  Francisco Ciruela  3  4 ,  Zhan-Guo Gao  1 ,  John A Auchampach  2 ,  Kenneth A Jacobson  1
Affiliations
  • 1. Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.
  • 2. Department of Pharmacology & Toxicology and the Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
  • 3. Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, L'Hospitalet de Llobregat 08907, Spain.
  • 4. Neuropharmacology and Pain Group, Neuroscience Program, Bellvitge Biomedical Research Institute, L'Hospitalet de Llobregat 08907, Spain.
PMID: 41026931 DOI: 10.1021/acs.jmedchem.5c01985
Abstract

1H-Imidazo[4,5-c]quinolin-4-amines are lipid-facing, positive allosteric modulators (PAMs) of the Gi-coupled A3 Adenosine Receptor (A3AR). Elongated amino-alkyl chains anchor these bitopic PAMs secondarily to anionic Phospholipids in the membrane's inner leaflet. Varied terminal functionalities and introduced reporter groups, as well as N1-alkylation on the core heterocycle, substantially enhanced human A3AR agonist (Cl-IB-MECA) potency and efficacy in [35S]GTPγS binding and revealed ago-PAM activity. Docking calculations predicted N1-benzylation to reduce undesired orthosteric site binding. Fluorophores, biotin, click moieties, chemically reactive, and photouncaging groups were included. Compound 38 (MRS8435, 9-methylenes, 0.1-10 μM) achieved ∼300% agonist Emax without ago-PAM activity. 4-Methyl 36 and 4-iodo 46 substitution of N1-benzyl increased Cl-IB-MECA potency by 14.7- and 30.5-fold, respectively. 9-Methylene N1-benzyl derivatives 35 and 42 achieved high ago-PAM efficacy (∼77% Cl-IB-MECA Emax). Molecular dynamics simulations detected stable electrostatic phospholipid interactions while maintaining A3AR allosteric binding. Thus, we rationally expanded SAR of bitopic A3AR PAMs, including molecular probes.

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