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Pharmacological characterisation of novel adenosine A3 receptor antagonists.

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Barkan, Kerry 
Lagarias, Panagiotis 
Stampelou, Margarita 
Stamatis, Dimitrios 
Hoare, Sam 


The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.



Adenosine A3 Receptor Antagonists, Animals, Binding Sites, Binding, Competitive, CHO Cells, Cricetulus, Cyclic AMP, Drug Evaluation, Preclinical, Humans, Kinetics, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Radioligand Assay, Rats, Receptor, Adenosine A3, Receptors, Purinergic P1, Recombinant Proteins, Species Specificity, Structure-Activity Relationship

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Springer Science and Business Media LLC


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Biotechnology and Biological Sciences Research Council (BB/M00015X/2)
Leverhulme Trust (via University of Essex) (DBG3000)
We gratefully acknowledge the support of the Leverhulme Trust (RPG-2017-255) (KB and GL) and the BBSRC (BB/M00015X/2) (GL). We acknowledge the support of Endowment Fund for education from Ministry of Finance Republic of Indonesia (DSa). This research represents part of the Ph.D work of P.L. We thank Chiesi Hellas which supported this research (SARG No 10354) and the State Scholarships Foundation (IKY) for providing a Ph.D fellowship to P.L. (MIS 5000432, NSRF 2014-2020). The work of E.V. is implemented through IKY scholarships programme and co-financed by the European Union (European Social Fund - ESF) and Greek national funds through the action entitled “Reinforcement of Postdoctoral Researchers”, in the framework of the Operational Program “Human Resources Development Program, Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) 2014 – 2020. This work was supported by computational time granted from the Greek Research & Technology Network (GRNET) in the National HPC facility - ARIS - under project IDs pr005010.