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

Abstract

The adenosine A 3 receptor (A 3 AR) is a G protein-coupled receptor (GPCR) that exerts immunomodulatory effects in pathophysiological conditions such as inflammation and cancer. Thus far, studies toward the downstream effects of A 3 AR activation have yielded contradictory results, thereby motivating the need for further investigations. Various chemical and biological tools have been developed for this purpose, ranging from fluorescent ligands to antibodies. Nevertheless, these probes are limited by their reversible mode of binding, relatively large size, and often low specificity. Therefore, in this work, we have developed a clickable and covalent affinity-based probe (AfBP) to target the human A 3 AR. Herein, we show validation of the synthesized AfBP in radioligand displacement, SDS-PAGE, and confocal microscopy experiments as well as utilization of the AfBP for the detection of endogenous A 3 AR expression in flow cytometry experiments. Ultimately, this AfBP will aid future studies toward the expression and function of the A 3 AR in pathologies.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study

“…Ligand-directed probes 4a and 4b labeled multiple proteins at concentrations ≥ 10 nM (Figure A), while no concentration-dependent increase in labeling was observed for control compounds 3a and 3b (Figure B), indicating that the cyano substitution is necessary to enhance the electrophilicity of the N -acyl group. In previous experiments on the adenosine A 1 and A 3 receptors, we also observed labeling of multiple proteins upon using electrophilic probes in membrane-derived samples, presumably due to a combination of the electrophilic nature of the warhead with the high overall density of proteins (1 mg mL –1 ) used in the experiments. , In these experiments, clearer labeling of the target GPCR was achieved when the experiment was performed on live cells. We therefore moved toward cellular assays in an attempt to observe more pronounced labeling of the A 2B AR.…”

“…In previous experiments on the adenosine A 1 and A 3 receptors, we also observed labeling of multiple proteins upon using electrophilic probes in membrane-derived samples, presumably due to a combination of the electrophilic nature of the warhead with the high overall density of proteins (1 mg mL –1 ) used in the experiments. 33 , 34 In these experiments, clearer labeling of the target GPCR was achieved when the experiment was performed on live cells. We therefore moved toward cellular assays in an attempt to observe more pronounced labeling of the A 2B AR.…”

“…Such concentrations of protein cause a decrease in signal-to-noise ratio and therefore a higher prevalence of non-selectively labeled proteins. In previous studies using the fluorosulfonyl warhead, a less reactive electrophile, we also observed that expression levels greatly influence the detection of labeled GPCRs. , Therefore, the NASA electrophile might not have the right “balanced” reactivity to selectively label GPCRs at low expression levels, which is the case for most endogenous GPCRs.…”