Fluorescent A and A 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 and AARs 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 AAR were K 144-316 nM for 10, 12, and 19, and at AAR affinity of K 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface AAR 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 AAR characterization in whole cells by flow cytometry (K 11.8 nM), and its bitopic interaction mode with an AAR homology model was predicted. Given its affinity and selectivity (11-fold vs. AAR, ~ 50-fold vs. AAR and AAR) 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 AAR.