Fig. 4
Characterisation of ligands targeting A2AR/PDE10A using a NanoBRET-based ligand binding assay. a Kinetic binding curve of CA200645 at Nluc-A2AR expressed HEK293T cells. After 19 minutes association with 40 nM CA200645, CGS21680 was injected to give a final concentration of 10 µM in order to displace the fluorescent probe. The curve was fit into “association then dissociation” model built in Prism 8.4.3. b Competition of CA200645 (300 nM) by reference compounds including CGS21680, NECA, and isoprenaline at equilibrium. c Competitive binding curves of triazoloquinazolines in correspond to of 300 nM CA200645. Both curves (panel B and C) were fitted using the “one-site Ki” equation where KD and concentration of hot ligand were set to 65 nM and 300 nM, respectively. Data points are the mean ± SEM from 3–27 repeats performed in duplicate. (D) The summary of binding affinities (pKi) of tested ligands. pKi values were calculated from inhibition of CA200645 binding at equilibrium to Nluc-A2AR-expressed HEK293T cells. # Cmpd 5 did not fully displace binding of CA200645 under condition tested. Statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) compared to CGS21680 was determined by one-way ANOVA with Dunnett’s post-test