Lin, Chia-Yang
(2025)
Optimisation of Ligand-Directed Labelling Probes for A1 Adenosine Receptors in Whole Cells: Development and Evaluation.
PhD thesis, University of Nottingham.
Abstract
Adenosine receptors (ARs) are widely distributed throughout the human body and exist in four subtypes: A1, A2A, A2B, and A3. The endogenous ligand adenosine modulates numerous physiological responses across various tissues and organs, including the cardiovascular system and both the central and peripheral nervous systems, through activation of these receptors. Among them, the A1 adenosine receptor (A1 AR) has been extensively investigated as a therapeutic target for conditions such as arrhythmia, heart failure, neuropathic pain, and diabetes. However, no A1 AR-targeting candidate has successfully completed clinical trials and reached the market. Challenges in drug development stem not only from the complexity of the A1 AR system but also from an incomplete understanding of its interactions with other proteins and its diverse cellular responses.
Dr. Comeo E. developed ligand-directed (LD) covalent labelling probes for A1 AR. One probe directly transfers a sulfo-Cy5 fluorophore to A1 AR, while the other transfers a trans-cyclooctene (TCO) handle. The covalent sulfo-Cy5 tag enables real-time visualisation of receptor localisation and trafficking. Additionally, protein–protein interactions involving A1 AR can be studied using techniques such as BRET, FRET, and FCCS. The TCO handle allows broader applications via inverse electron demand Diels–Alder (IEDDA) reactions with tetrazine-conjugated reporters, which may include fluorophores, biotin, or radiolabels, depending on experimental design. However, both subtype selectivity and labelling efficiency require further optimisation.
This thesis presents the design, synthesis, and pharmacological evaluation of novel LD probes based on Comeo’s templates. Probe 5-8, a sulfo-Cy5 transferring LD probe, improved A1/ A2A selectivity from 5.9-fold (Comeo’s original probe) to 40-fold, while maintaining selectivity over other AR subtypes. Probe 4-5, a TCO-transferring LD probe, enhanced A1/ A2B selectivity from 60-fold to 210-fold. Parameters such as the click reaction pair, buffer or medium composition, probe concentration, and incubation time were systematically investigated to optimise labelling efficiency.
In addition to using agonist-induced internalisation to assess orthosteric binding pocket accessibility post-labelling, a BRET-based assay was conducted with probe 4-5. FRET between the AF488 tag (introduced via probe 4-5 and a tetrazine-AF488), along with a red fluorescent reversible ligand 3-29 (BODIPY630/650), confirmed the availability of the orthosteric binding site. These findings were further validated using FLIM-FRET experiments.
The novel LD probes 5-8 (sulfo-Cy5) and 4-5 (TCO), with improved subtype selectivity, represent valuable tools for advancing the study of A1 AR biology. These tools are expected to facilitate deeper exploration of A1 AR pharmacology and contribute to expanding our understanding of its therapeutic potential.
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