Investigating the nature of the secondary binding site of the human β1-adrenoceptor using fluorescent ligands and confocal microscopy

CGP 12177 is a high affinity β-blocker that antagonises agonist responses mediated through the catecholamine binding site of the human β1-adrenoceptor (β1AR). However, CGP 12177 also exerts agonist activity through a secondary, low affinity “CGP 12177” binding site/conformational state of the β1AR. In this thesis, we aimed to further our understanding of the nature of the secondary “CGP 12177” site by investigating ligand-receptor interactions at this site at the single cell level, using fluorescent derivatives of CGP 12177 (BODIPY-TMR-CGP, BY-CGP) and propranolol (BODIPY630/650-S-PEG8-propranolol, BY-PROP) in confocal microscopy studies. Initial studies demonstrated that both fluorescent β-adrenoceptor ligands displayed similar pharmacology at the human β1AR to their respective parent compounds, and that both ligands allowed visualisation of β1AR expressed in CHO cells. Using BY-CGP in a live cell fluorescence-based automated screening assay revealed two-phase antagonist displacement binding curves. In subsequent kinetic binding studies performed on a confocal perfusion system, we used infinite dilution conditions to determine dissociation rates of BY-CGP in the absence and presence of unlabelled ligands at the single cell level. BY-CGP dissociation rates were enhanced in the presence of unlabelled ligands, thus highlighting an allosteric mechanism of action of CGP 12177 at the human β1AR. Preliminary data using bimolecular fluorescence complementation suggested that these co-operative interactions between the two β1-adrenoceptor binding sites were mediated across a β1-adrenoceptor homodimer interface

Investigating the nature of the secondary binding site of the human β1-adrenoceptor using fluorescent ligands and confocal microscopy

CGP 12177 is a high affinity β-blocker that antagonises agonist responses mediated through the catecholamine binding site of the human β1-adrenoceptor (β1AR). However, CGP 12177 also exerts agonist activity through a secondary, low affinity “CGP 12177” binding site/conformational state of the β1AR. In this thesis, we aimed to further our understanding of the nature of the secondary “CGP 12177” site by investigating ligand-receptor interactions at this site at the single cell level, using fluorescent derivatives of CGP 12177 (BODIPY-TMR-CGP, BY-CGP) and propranolol (BODIPY630/650-S-PEG8-propranolol, BY-PROP) in confocal microscopy studies. Initial studies demonstrated that both fluorescent β-adrenoceptor ligands displayed similar pharmacology at the human β1AR to their respective parent compounds, and that both ligands allowed visualisation of β1AR expressed in CHO cells. Using BY-CGP in a live cell fluorescence-based automated screening assay revealed two-phase antagonist displacement binding curves. In subsequent kinetic binding studies performed on a confocal perfusion system, we used infinite dilution conditions to determine dissociation rates of BY-CGP in the absence and presence of unlabelled ligands at the single cell level. BY-CGP dissociation rates were enhanced in the presence of unlabelled ligands, thus highlighting an allosteric mechanism of action of CGP 12177 at the human β1AR. Preliminary data using bimolecular fluorescence complementation suggested that these co-operative interactions between the two β1-adrenoceptor binding sites were mediated across a β1-adrenoceptor homodimer interface

Negative cooperativity across 1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 1-adrenoceptor binding conformation

At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min−1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformatio

Long receptor residence time of C26 contributes to super agonist activity at the human β2 adrenoceptor

Super agonists produce greater functional responses than endogenous agonists in the same assay, and their unique pharmacology is the subject of increasing interest and debate. We propose that receptor residence time and the duration of receptor signaling contribute to the pharmacology of super agonism. We have further characterized the novel β2 adrenoceptor agonist C26 (7-[(R)-2-((1R,2R)-2-benzyloxycyclopentylamino)-1-hydroxyethyl]-4-hydroxybenzothiazolone), which displays higher intrinsic activity than the endogenous ligand adrenaline in cAMP accumulation, β-arrestin-2 recruitment, and receptor internalization assays. C26 recruited β-arrestin-2, and internalized the Green Fluorescent Protein (GFP)-taggedβ2 adrenoceptor at a slow rate, with half-life (t1/2) values of 0.78 ± 0.1 and 0.78 ± 0.04 hours, respectively. This was compared with 0.31 ± 0.04 and 0.34 ± 0.01 hours for adrenaline-mediated β-arrestin-2 recruitment and GFP-β2 internalization, respectively. The slower rate for C26 resulted in levels of β-arrestin-2 recruitment increasing up to 4-hour agonist incubation, at which point the intrinsic activity was determined to be 124.3 ± 0.77% of the adrenaline response. In addition to slow functional kinetics, C26 displayed high affinity with extremely slow receptor dissociation kinetics, giving a receptor residence half-life of 32.7 minutes at 37°C, which represents the slowest dissociation rate we have observed for any β2 adrenoceptor agonist tested to date. In conclusion, we propose that the gradual accumulation of long-lived active receptor complexes contributes to the increased intrinsic activity of C26 over time. This highlights the need to consider the temporal aspects of agonist binding and signaling when characterizing ligands as super agonists

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer’s disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including ‘epileptic-like’ seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD

A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A3 receptor (A3AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A1 receptor, and β1 and β2 adrenoceptors (β1AR and β2AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A3AR for a range of classical adenosine receptor antagonists were consistent with A3AR pharmacology and correlated well (R2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β1AR was potently inhibited by low concentrations of the β1-selective antagonist CGP 20712A (pKi 9.68) but not by the β2-selective antagonist ICI 118551(pKi 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β2AR this affinity order was reversed with ICI 118551 showing the highest affinity (pKi 8.73) and CGP20712A (pKi 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A3AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A3AR (pKi ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A3AR. These were K114 (pKi 6.43), retinoic acid p-hydroxyanilide (pKi 6.13) and SU 6556 (pKi 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A3AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors