Abstract
The adenosine A1 receptor (A1R) is a class A G protein-coupled receptor (GPCR) that has been implicated in disease pathologies such as neurodegenerative disorders, inflammation, cardiovascular and pain disorders. Although GPCRs were traditionally thought to act as individual signalling units (monomers), there is now mounting evidence that GPCRs can form complexes with another monomer of the same GPCR (homodimers), with other types of GPCRs (heterodimer), and/or even higher order oligomers. Dimerisation of GPCRs could be one mechanism that can regulate GPCR function, and it has also been proposed that the predisposition of different GPCRs to form dimers may be modulated in pathological states.
The A1R has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. The development of bivalent dicovalent ligands for the A1R is an attractive concept whereby a bivalent ligand is designed to be capable of chemoreactive covalent binding to each protomer of a A1R homodimer. This thesis describes the synthesis (Chapter 2) and pharmacological evaluation (Chapter 3) of bivalent dicovalent chemical tools targeted to A1R homodimers, and an attempt to extend this system to “click and fluoresce” ligands that can form bivalent ligands in situ to detect A1R homodimers (Chapter 4).
A series of seven A1R targeted bivalent dicovalent ligands were designed inspired by a recently reported xanthine based A1R and A3R bifunctional chemoreactive, bioorthogonally reactive ligand (UODC14). UODC14 is amenable to a bivalent ligand system wherein the existing bioorthogonally reactive handle of UODC14 was instead reacted with diazide linkers of varying lengths to form pre-assembled bivalent dicovalent ligands (Chapter 2). These bivalent dicovalent ligands contained dual sulfonyl fluoride warheads, to allow covalent binding to each monomer of an A1R homodimer. Bivalent ligands that did not contain a covalent warhead, and monovalent ligands (both with and without a covalent warhead) (five compounds) were also synthesised, to probe the true binding mode of the putative bivalent dicovalent ligands.
Pharmacological evaluation was carried out on the bivalent dicovalent ligands and non-covalent and monovalent ligands (Chapter 3). In forskolin stimulated cAMP assays, the putative bivalent ligands showed a trend of increasing A1R activity with increasing linker length and acted as competitive A1R antagonists. Radioligand binding washout assays provided evidence that the ligands containing at least one covalent sulfonyl fluoride warhead acted as irreversible antagonists at A1R, as they were shown to be wash resistant and therefore covalently bound to the receptor presumably via a tyrosine residue in the orthosteric site of the A1R. Out of the 12 ligands biologically evaluated, the highest affinity bivalent (putative) dicovalent A1R ligand was UOCMP5, (A1R pKi = 6.71 ± 0.26) had a 4-fold increase in affinity for the A1R over the equivalent monovalent ligand, tentative evidence that UOCMP5 contained a linker of sufficient length to allow for bivalent binding. In radioligand binding experiments, bivalent dicovalent ligands UOCMP12 and UOCMP13 with longer linkers than UOCMP5 exhibited Hill Slopes >1, which is indicative of positive cooperativity across an A1R homodimer.
The bivalent dicovalent ligands (Chapters 2 and 3) could be adapted, in that instead of pre-forming the overall ligand in a chemical synthesis, the bivalent ligand could be formed in situ using a fluorogenic reaction as a measure of real-time receptor proximity. It has previously been shown that bifunctional ligands such as UODC14 could be ‘clicked’ to a fluorophore in situ. In this work a pro-fluorophore-ligand conjugate is designed to instead react in a fluorogenic reaction with another ‘ligand-linker’ reaction partner, forming an overall fluorescent bivalent ligand. This fluorogenic reaction would ideally be a bio-orthogonal ‘click’ reaction that requires a trigger to limit background reaction and fluorescence, therefore the copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction was selected for applying to this chemical tool.
A series of four alkyne and azide functionalised coumarin-PEG pro-fluorophores (4.1 – 4.4) were designed and planned to be reacted with complementary pro-fluorophore coupling partners. Unfortunately, due to challenges encountered with decomposition of coumarins, the synthesis of 4.1 – 4.4 was not achieved. Trial “click and fluoresce” reactions were carried out between non-PEG functionalised alkyne and azide coumarin pro-fluorophores and resulted in fluorescent reaction mixtures that were spectroscopically characterised. These preliminary results showed promise that this system could eventually be useful to detect A1R homodimers by assembly of the bivalent ligand in situ using a fluorogenic CuAAC reaction.