Fluorescent A2A and A3 adenosine receptor antagonists as flow cytometry probes

Development of an Affinity-Based Probe to Profile Endogenous Human Adenosine A3 Receptor Expression

“…Having established the potential of 9 in overexpressing cell systems, we turned to native hA 3 AR expression in flow cytometry experiments in order to cope with expected low levels of observable fluorescence caused by the notoriously low expression levels of GPCRs. Similar usage of fluorescent probes in flow cytometry experiments have thus far led to kinetic studies of ligand binding, , and detection of the hA 3 AR on the HL-60 model cell line . In order to establish an assay setup for primary cells we first used CHO cells as a model system.…”

“…In the past decade, multiple small molecules have been developed as tool compounds to study the hA 3 AR on a molecular level. Most prominently developed are the fluorescent ligands: agonists or antagonists conjugated to a fluorophore. − Noteworthy, one of these fluorescent ligands has been used to study internalization, localization and certain PPIs of the hA 3 AR on hA 3 AR-overexpressing Chinese hamster ovary (CHO) cells as well as activated neutrophils. , Yet, the current use of fluorescent ligands is limited to the type of fluorophore conjugated, a fluorescent read-out in specific assay types, and reversible binding to the receptor. Therefore, in this study, we aimed to develop a clickable affinity-based probe (AfBP) to broaden the current possibilities to measure and detect the receptor.…”

Development of an Affinity-Based Probe to profile endogenous Human Adenosine A3 Receptor Expression

“…Having established the potential of 9 (LUF7960) in overexpressing cell systems, we turned to native hA3AR expression in flow cytometry experiments in order to cope with expected low levels of observable fluorescence after receptor labeling caused by the notoriously low expression levels of GPCRs. Similar usage of fluorescent probes in flow cytometry experiments have thus far led to kinetic studies of ligand binding, [20,27] and detection of the hA3AR on the HL-60 model cell line. [21] In order to establish an assay setup for primary cells we first used CHO cells as a model system.…”

“…Similar click strategies have already been used in the synthesis of fluorescent ligands for the hA3AR. [20,21,26,27,47] These ligands are however all lacking the electrophilic warhead. Additionally, contrary to those studies, we mainly performed the click reaction after binding of the AfBP to the receptor, preventing a loss of affinity due to bulky substituents.…”

“…Most prominently developed are the fluorescent ligands: agonists or antagonists conjugated to a fluorophore. [19][20][21][22][23][24][25][26][27][28] Noteworthy, one of these fluorescent ligands has been used to study internalization, localization and certain PPIs of the hA3AR on hA3AR-overexpressing Chinese hamster ovary (CHO) cells, as well as activated neutrophils. [12,24] Yet, the current use of fluorescent ligands is limited to the type of fluorophore conjugated, a fluorescent read-out in specific assay types and reversible binding to the receptor.…”