Ca200645

Adenosine, NECA, IB-MECA, HEMADO, DPCPX (8-cyclopentyl-1,3-dipropyl-7H-purine-2,6-dione) and MRS 1220 were purchased from Sigma-Aldrich and dissolved in dimethyl-sulphoxide (DMSO). CA200645, a high affinity AR xanthine amine congener (XAC) derivative containing a polyamide linker connected to the BY630 fluorophore, was purchased from HelloBio (Bristol, UK) and dissolved in DMSO. AV039, a highly potent and selective fluorescent antagonist of the human A₃R based on the 1,2,4-Triazolo[4,3-a]quinoxalin-1-one linked to BY630^{39 (link)}, was kindly gifted to us by Stephen Hill and Stephen Briddon (University of Nottingham). PMA was purchased from Sigma-Aldrich. Compounds under investigation were purchased from e-molecules and dissolved in DMSO. The concentration of DMSO was maintained to < 1.5% across all experiments (1.26% for all cAMP assays, 1% for pERK1/2 assays and 1.02% or 1.1% for NanoBRET ligand-binding experiments using CA200645 or AV039, respectively).

CA200645 was from HelloBio (Bristol, UK). ZM 241385 and CGS21680 were from Tocris (Bristol, UK).

Foetal calf serum (FCS) was obtained from PAA Laboratories (Wokingham, UK). Furimazine was purchased from Promega (Southampton, UK). Bicinchoninic acid protein assay kit and white 96-well microplates were obtained from Thermo Fisher Scientific (Waltham, MA, USA). GF/B filter plates and Microscint-O were from PerkinElmer (Groningen, The Netherlands). CA200645 was obtained from HelloBio (Bristol, UK). The synthesis of AV039 was described in Vernall et al. as compound 19 [34 (link)], while the synthesis of XAC-S-ser-S-tyr-X-BY630 (compound 27) and XAC-S-ser-S-tyr-X-BYFL (compound 28) was described in Vernall et al. 2013 [33 (link)]. PSB-11 and MRS1220 were purchased from Tocris Bioscience (Bristol, UK), and NECA was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). [³H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([³H]PSB-11) was kindly donated by Prof. C.E. Müller (University of Bonn, Germany) and its synthesis described in Müller et al. [35 (link)]. 1-Benzyl-8-methoxy-1H,3H-pyrido[2,1-f]purine-2,4-dione (compound 5) synthesis was described in Priego et al. as compound number 3 [36 (link)] and referred in Xia et al. [37 (link)] as compound number 5, while LUF7565 synthesis was described in Xia et al. as compound 27 [30 (link)]. All other chemicals and reagents were obtained from Sigma-Aldrich (Gillingham, UK).