Τίτλος:
Pharmacological characterisation of novel adenosine A3 receptor antagonists
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
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. © 2020, The Author(s).
Συγγραφείς:
Barkan, K.
Lagarias, P.
Stampelou, M.
Stamatis, D.
Hoare, S.
Safitri, D.
Klotz, K.-N.
Vrontaki, E.
Kolocouris, A.
Ladds, G.
Περιοδικό:
Scientific Reports
Εκδότης:
Institute of Geographic Sciences and Natural Resources Research
Λέξεις-κλειδιά:
adenosine A3 receptor; adenosine A3 receptor antagonist; adenosine receptor; ADORA3 protein, human; cyclic AMP; recombinant protein, animal; binding competition; binding site; chemistry; CHO cell line; Cricetulus; genetics; human; kinetics; metabolism; molecular dynamics; preclinical study; radioassay; rat; site directed mutagenesis; species difference; structure activity relation, 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
DOI:
10.1038/s41598-020-74521-y
