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The adenosine A 3 receptor (A 3 R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A 3 R 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 A 3 R, 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 (pA 2 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 A 3 R 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 A 3 R (< 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 A 3 R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A 3 receptor (A 3 AR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A 3 AR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 °C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent A 3 AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A 3 AR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the A 3 AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([ 3 H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [ 3 H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.

LJ-2698, a highly potent human A 3 adenosine receptor antagonist with nucleoside structure, was designed to have a minimal species dependence. For further pre-clinical studies, analytical method for the detection of LJ-2698 in rat plasma was developed by liquid chromatography-tandem mass. Plasma samples were processed by protein precipitation method with acetonitrile, using losartan as the internal standard (IS). Chromatographic separation was carried out using a Kinetex C18 column (100 × 4.6 mm; 100 Å; 2.6 μ) with acetonitrile/water with 0.2% (v/v) formic acid (65:35, v/v) in the isocratic mode at a flow rate of 0.4 mL/min. Mass spectrometric detection in multiple reaction monitoring mode was performed with positive electrospray ionization. The mass transitions of LJ-2698 and IS were m/z 412.3 → 294.1 and m/z 423.1 → 207.2, respectively. The calibration curves were linear in the range 5.00-5000 ng/mL (r 2  ≥ 0.998). The lower limit of quantification was established as 5.00 ng/mL. Within- and between-run precisions were <7.01%, as relative standard deviation; and accuracies were in the range 3.37-3.64%, as relative error. The validated method was successfully applied to its pharmacokinetic evaluation after intravenous and oral administration in rats, and the dose-dependent pharmacokinetic behavior of LJ-2698 was elucidated for the first time.

Purpose: Since the adenosine A3 receptor (A3R) is considered to be of high clinical importance in the diagnosis and treatment of ischaemic conditions (heart and brain), glaucoma, asthma, arthritis, cancer and inflammation, a suitable and selective A3R PET tracer such as [(18)F]FE@SUPPY would be of high clinical value for clinicians as well as patients. A3R was discovered in the late 1990s, but there is still little known regarding its distribution in the CNS and periphery. Hence, in autoradiographic experiments the distribution of A3R in human brain and rat tissues was investigated and the specific binding of the A3R antagonist FE@SUPPY and MRS1523 compared. Immunohistochemical staining (IHC) experiments were also performed to validate the autoradiographic findings., Methods: For autoradiographic competition experiments human post-mortem brain and rat tissues were incubated with [(125)I]AB-MECA and highly selective compounds to block the other adenosine receptor subtypes. Additionally, IHC was performed with an A3 antibody., Results: Specific A3R binding of MRS1523 and FE@SUPPY was found in all rat peripheral tissues examined with the highest amounts in the spleen (44.0% and 46.4%), lung (44.5% and 45.0%), heart (39.9% and 42.9%) and testes (27.4% and 29.5%, respectively). Low amounts of A3R were found in rat brain tissues (5.9% and 5.6%, respectively) and human brain tissues (thalamus 8.0% and 9.1%, putamen 7.8% and 8.2%, cerebellum 6.0% and 7.8%, hippocampus 5.7% and 5.6%, caudate nucleus 4.9% and 6.4%, cortex 4.9% and 6.3%, respectively). The outcome of the A3 antibody staining experiments complemented the results of the autoradiographic experiments., Conclusion: The presence of A3R protein was verified in central and peripheral tissues by autoradiography and IHC. The specificity and selectivity of FE@SUPPY was confirmed by direct comparison with MRS1523, providing further evidence that [(18)F]FE@SUPPY may be a suitable A3 PET tracer for use in humans.