RT Journal Article SR Electronic T1 Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists JF bioRxiv FD Cold Spring Harbor Laboratory SP 693796 DO 10.1101/693796 A1 Kerry Barkan A1 Panagiotis Lagarias A1 Eleni Vrontaki A1 Dimitrios Stamatis A1 Sam Hoare A1 Karl-Norbert Klotz A1 Antonios Kolocouris A1 Graham Ladds YR 2019 UL http://biorxiv.org/content/early/2019/07/10/693796.abstract AB Background and Purpose 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 ARs orthosteric binding sites, obtaining highly selective receptor antagonists is a challenging but critical task.Experimental approach 39 potential A3R, antagonists were screened 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. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamics (MD) simulations and consistent calculated binding free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies.Key Results 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. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies.Conclusions and Implications These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode.