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Pharmacological characterisation of novel adenosine A 3 receptor antagonists

Published version
Peer-reviewed

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Authors

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

Abstract

Abstract: 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.

Description

Funder: Endowment fund for education, Ministry of Finance Republic of Indonesia

Keywords

Article, /631/154/436/2387, /631/535/1267, /631/80/86/820, article

Journal Title

Scientific Reports

Conference Name

Journal ISSN

2045-2322

Volume Title

10

Publisher

Nature Publishing Group UK
Sponsorship
Leverhulme Trust,United Kingdom (RPG-2017-255)
Chiesi Hellas (SARG No 10354)
State Scholarships Foundation (IKY) (MIS5000432)
GRNET (pr005010)
Biotechnology and Biological Sciences Research Council (BB/M00015X/2)