Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists

Characterization of Dual-Acting A3 Adenosine Receptor Positive Allosteric Modulators That Preferentially Enhance Adenosine-Induced Gαi3 and GαoA Isoprotein Activation

“…In fact, the EC 50 of Cl-IB-MECA in the presence of 10 μM compound 4 was significantly reduced 5-fold from 10.5 to 2.24 nM ( p < 0.05), while in assays with the wild-type hA 3 AR the EC 50 was increased 5-fold by 10 μM compound 4 (15.1 vs 72.5 nM; p < 0.05). Because the binding affinity of non-nucleoside typical non-nucleoside hA 3 AR antagonist ligands in the mA 3 AR is greatly reduced as compared to the hA 3 AR, we predicted that the enhanced potency of Cl-IB-MECA observed with the M Out /H In chimera is explained by reduced affinity of compound 4 for the orthosteric binding site composed of the mouse sequence. To further test this idea, we examined the 2-cyclopropyl imidazoquinolin-4-amine derivative ( 1 ) in [ 35 S]­GTPγS binding assays with human/mouse chimeric receptors, which lacks efficacy-enhancing allosteric activity but greatly right-shifts the Cl-IB-MECA concentration–response relationship in assays with the hA 3 AR in a manner suggestive of competitive antagonism.…”

“…For other S1 and S2. We next evaluated the two series of modulators in a [ 35 S]GTPγS binding assay using isolated HEK293 cell membranes expressing the hA 3 AR, which detects receptorinduced G protein activation. For these studies, concen- S3.…”

“…Radioactivity trapped in the filters was measured by liquid scintillation counting. Nonspecific binding of [ 35 Bioluminescence Resonance Energy Transfer Assays. To measure A 3 AR-mediated β-arrestin-2 recruitment by BRET 1 , HEK293T cells were cotransfected (1:15) with two constructs (pcDNA3.1) encoding the human A 3 AR with Cterminally fused Renilla Luciferase (Rluc8) and N-terminally fused Venus-tagged β-arrestin-2 in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/ mL streptomycin using TransIT-293 transfection reagent (Mirus Bio, Madison, WI).…”

Effect of Adenosine Receptor Antagonists on Adenosine-Pretreated PC12 Cells Exposed to Paraquat

“… 18 Experiments showed that the other A3R antagonist VUF5574 was responsible for neuroprotective and anti-inflammatory effects and was suitable for potential treatment of glaucoma and asthma. 19 …”

“…18 Experiments showed that the other A3R antagonist VUF5574 was responsible for neuroprotective and antiinflammatory effects and was suitable for potential treatment of glaucoma and asthma. 19 Most previous studies have examined the effects of ADO, whereas the role of AR antagonists has been evaluated alone, and there is a lack of data on what transpires in real-world environments where adenosine exerts its protective effects in response to injury. In this study, we applied PC12 (rat adrenal pheochromocytoma) cells to imitate neuronal cells and test whether antagonists might adjust the PC12 cell injury induced by paraquat in the presence or absence of adenosine pretreatment.…”