Development of an Affinity-Based Probe to Profile Endogenous Human Adenosine A

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• The adenosine A3 receptor (A3AR), a G protein-coupled receptor (GPCR), plays a role in immune responses, inflammation, and cancer.
• Previous studies on A3AR activation have produced conflicting results, highlighting the need for improved research tools.
• Existing probes for A3AR, such as fluorescent ligands and antibodies, have limitations including reversible binding, large size, and low specificity.

Purpose of the Study:

• To develop a novel, clickable, and covalent affinity-based probe (AfBP) for targeting the human A3AR.
• To validate the utility of the newly synthesized AfBP for studying A3AR expression and function.

Main Methods:

• Synthesis of a clickable and covalent affinity-based probe (AfBP) targeting the human A3AR.
• Validation experiments including radioligand displacement assays, SDS-PAGE, and confocal microscopy.
• Application of the AfBP for detecting endogenous A3AR expression using flow cytometry.

Main Results:

• The synthesized AfBP successfully targeted the human A3AR.
• Validation experiments confirmed the probe's efficacy in various assays.
• The AfBP enabled the detection of endogenous A3AR expression in flow cytometry.

Conclusions:

• The developed clickable covalent AfBP is a valuable new tool for A3AR research.
• This probe overcomes limitations of previous reagents, offering improved specificity and binding characteristics.
• The AfBP will facilitate future investigations into the role of A3AR in various pathologies.