Illuminating the life of the adenosine receptors: development and application of subtype-selective fluorescent probes for the study of the human A1 and A2A adenosine receptors in living cells

Comeo, Eleonora (2022) Illuminating the life of the adenosine receptors: development and application of subtype-selective fluorescent probes for the study of the human A1 and A2A adenosine receptors in living cells. PhD thesis, University of Nottingham.

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Adenosine is an important regulatory endogenous molecule which can modulate all aspects of tissue functions upon engagement with four adenosine receptor (AR) subtypes namely A1AR, A2AAR, A2BAR and A3AR, respectively. These membrane proteins are distributed throughout the human body and, owing to their prominent role in modulating many physiological and pathological processes, they provide important therapeutic opportunities for a number of conditions including cardiovascular, inflammatory diseases, neuropathic pain, CNS disorders and cancer. Yet, translation of adenosine ligands from the bench to the clinic proved challenging. This suggests that better understanding of the molecular and cellular mechanisms of action underlying specific AR pharmacology in different tissues, cell types and subcellular compartments is required for the development of new AR therapeutics. Accordingly, subtype-selective fluorescent probes represent valuable tools to provide this knowledge as they allow real-time monitoring of receptor-specific pharmacology at the single-cell level, in its native, non-genetically modified environment.

This Thesis describes the rational design, synthesis and pharmacological validation of novel subtype-selective fluorescent antagonists targeting two AR subtypes, namely the A1AR and A2AAR, respectively. These probes allowed quantification of equilibrium and kinetic ligand binding parameters using biophysical methods such as NanoBRET and TR-FRET, in addition to allow visualisation of specific receptor localisation and distribution patterns in living cells by confocal microscopy and total internal reflection microscopy (TIRF-M). Moreover, to the best of our knowledge, probes 4.44b and 4.46a represent the first examples of subtype-selective fluorescent hA1AR antagonists reported to date.

As an extension of the fluorescent ligand technology, this Thesis also reports the rational design of a Ligand-Directed (LD) chemistry approach, combined with an Inverse-Electron Demand Diels-Alder (IEDDA) click reaction, which enabled fluorescent labelling of hA1ARs expressed in living cells. This method offers a novel, non-invasive, bioorthogonal approach to study A1AR function and dynamics in its native cellular environment.

Together, the tools developed in this Thesis may support the discovery of new sites and mechanisms for therapeutic intervention within the adenosine receptors research field, as well as facilitate the design of AR ligands with improved selectivity and safer mechanism of action.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Kellam, Barrie
Hill, Stephen J.
Scammells, Peter J.
May, Lauren T.
Halls, Michelle L.
Keywords: Fluorescent probes, GPCR, Adenosine receptors, Imaging, Antagonists, Fluorophores
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 67321
Depositing User: Comeo, Eleonora
Date Deposited: 31 Jul 2022 04:40
Last Modified: 31 Jul 2022 04:40

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