Fluorescent A_2A and A₃ adenosine receptor antagonists as flow cytometry probes

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A_2A and A₃ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled to eight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A_2AAR were K_i 144-316 nM for 10, 12, and 19, and at A₃AR affinity of K_i 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A_2AAR was imaged using confocal microscopy. Compound 19 MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A₃AR characterization in whole cells by flow cytometry (K_d 11.8 nM), and its bitopic interaction mode with an A₃AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A_2AAR, ~ 50-fold vs. A₁AR and A_2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A₃AR.