Novel complementation biosensors to investigate G protein-coupled receptor kinetics and signalling bias

G protein coupled receptors (GPCRs) signal through a complex cellular network, with a range of temporally variable signalling responses generated by GPCR activation. Characterisation of GPCR ligands is generally taken from the ligand “affinity”, the ability to bind the receptor, and the ligand “efficacy”, the ability to activate signalling from the receptor. Often single timepoints are used to quantify ligand affinity and efficacy by being applied to pharmacological models. Biased ligands, which have been suggested to preferentially activate one signalling pathway over another, are often identified from comparisons of concentration response data of an agonist between single timepoints of assays measuring the activation of different signalling proteins. Existing pharmacological models, which estimate ligand potencies, efficacies and ligand bias, such as the Black and Leff operational model, from functional data assume system equilibrium, yet often concentration response data at early timepoints are taken from hemi-equilibrium conditions. More recently, appreciation of agonist binding kinetics and signalling kinetics at GPCRs has increased, enhancing the extent of ligand characterisation and used to investigate discrepancies in ligand bias between different experimental systems. The origin of ligand bias has been suggested to be conformational, where the agonist binds the receptor to allosterically promote the formation of a conformation which preferentially activates one signalling pathway over another. It has also been suggested that bias originates from different receptor binding kinetics, which produce different profiles of signalling and can confound comparisons of bias – especially when bias is compared from a single timepoint. In this thesis we investigate how signalling kinetics and binding kinetics can influence measures of ligand affinities, efficacy and bias using experimental system monitoring dynamic protein:protein and ligand-receptor interactions. Firstly, we established a NanoBiT complementation assay to monitor agonist-stimulated recruitment of effector proteins, either β-arrestin2 or a synthetic mini Gαs protein, at β adrenoceptor subtypes. Recruitment profiles of β-arrestin2 recruitment produced a transient “rise and fall to plateau” profile, peaking at 3 minutes, whilst the mini Gαs protein was recruited to the receptor rapidly in the first 1-5 minutes and then plateaued and/or steadily increased over the timecourse. This experimental system allowed for dynamic agonist-stimulated recruitment profiles to be monitored over time and concentration response data to be gathered from multiple timepoints. Concentration response data from each timepoint was then applied to the Black and Leff operational models to compare how direct comparisons of ligand bias can change in magnitude and direction over time. Full kinetic signalling profiles were then applied to a kinetic operational model, which takes hemi-equilibrium conditions into account, to estimate agonist affinities and efficacies from β-arrestin2 recruitment data. Agonist affinities estimated from the kinetic operational model were strongly correlated with values obtained in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assays, highlighting the model’s value in obtaining drug potency and affinity from a single functional recruitment assay. Secondly, we investigated the value of the NanoBiT complementation assays in monitoring receptor antagonism and how the kinetic context may misrepresent the mechanism of action of the antagonist ligands: propranolol, ICI-118,551 and CGP-12177. Schild-Gaddum based experiments were applied and monitored over a 61 minute timecourse, at β2 adrenoceptors. All antagonists at early timepoints (3 minutes) produced insurmountable antagonism of the formoterol response, with only propranolol demonstrating surmountable antagonism at 61 minutes. The extent of insurmountability correlated with a decreased dissociation rate (kOFF) of the antagonist, with kOFF estimated from a kinetic TR-FRET assay. The signalling profiles of antagonism of mini Gαs protein recruitment at the β2 adrenoceptor were applied to an antagonist form of the kinetic operational model, to provide similar estimates of antagonist affinity in comparison to values obtained in Schild-based and TR-FRET assays. The kinetic operational model also provided estimates of antagonist association (kON) and kOFF, though these were not comparable to estimates from the kinetic TR-FRET assay and highlight the need for model modification for accurate estimates of kinetic values. Lastly, this thesis investigated how the conformation of the receptor can influence binding properties of β2 adrenoceptor ligands. High affinity variants of the NanoBiT complementation fragments (LgBiT and HiBiT) were used to stabilise the β2 adrenoceptor in complex with an effector protein to form effector driven receptor conformations. Initially, high affinity NanoBiT fragments were genetically tagged to the β2 adrenoceptor and either β-arrestin2 or mini Gαs and expressed in dual-expression cell lines. However, unbalanced expression of the proteins resulted in insufficient formation of NanoBiT stabilised receptor-effector conformations. To resolve this issue, the HiBiT-tagged mini Gαs protein was bacterially expressed and purified and added to membranes expressing LgBiT-tagged β2 adrenoceptors, saturating the receptor population. The complimented NanoBiT fragments were used as the donor species to excite an extracellular fluorescent ligand bound to the receptor, to establish a novel transmembrane bioluminescence resonance energy transfer (TM-BRET) assay. In TM-BRET assays, β2 adrenoceptor-mini Gαs protein “high affinity” conformations were shown to have increase affinity for agonist ligands, compared to the receptor alone, whilst antagonist ligands were unchanged. Kinetic TR-FRET assays were used to monitor the kinetic binding properties of β2 adrenoceptor ligands and demonstrated that the increase in agonist affinity at receptor-mini Gαs protein complexes was driven by a decrease in agonist kOFF and that the extent of the decrease in kOFF rate was correlated with ligand efficacy. The findings presented in this thesis highlight the value of kinetic signalling data and binding kinetics in assessing ligand pharmacology, including measures of ligand bias. This thesis went onto to highlight how differences in receptor conformation drive changes in agonist kOFF, combining conformational and kinetic explanations of ligand bias

Optical control of the ?2-adrenergic receptor with opto-prop-2: A cis-active azobenzene analog of propranolol

In this study, we synthesized and evaluated new photoswitchable ligands for the beta-adrenergic receptors ?1-AR and ?2-AR, applying an azologization strategy to the first-generation beta-blocker propranolol. The resulting compounds (Opto-prop-1, -2, -3) have good photochemical properties with high levels of light-induced trans-cis isomerization (>94%) and good thermal stability (t1/2 > 10 days) of the resulting cis-isomer in an aqueous buffer. Upon illumination with 360-nm light to PSScis, large differences in binding affinities were observed for photoswitchable compounds at ?1-AR as well as ?2-AR. Notably, Opto-prop-2 (VUF17062) showed one of the largest optical shifts in binding affinities at the ?2-AR (587-fold, cis-active), as recorded so far for photoswitches of G protein-coupled receptors. We finally show the broad utility of Opto-prop-2 as a light-dependent competitive antagonist of the ?2-AR as shown with a conformational ?2-AR sensor, by the recruitment of downstream effector proteins and functional modulation of isolated adult rat cardiomyocytes

Novel complementation biosensors to investigate G protein-coupled receptor kinetics and signalling bias

G protein coupled receptors (GPCRs) signal through a complex cellular network, with a range of temporally variable signalling responses generated by GPCR activation. Characterisation of GPCR ligands is generally taken from the ligand “affinity”, the ability to bind the receptor, and the ligand “efficacy”, the ability to activate signalling from the receptor. Often single timepoints are used to quantify ligand affinity and efficacy by being applied to pharmacological models. Biased ligands, which have been suggested to preferentially activate one signalling pathway over another, are often identified from comparisons of concentration response data of an agonist between single timepoints of assays measuring the activation of different signalling proteins. Existing pharmacological models, which estimate ligand potencies, efficacies and ligand bias, such as the Black and Leff operational model, from functional data assume system equilibrium, yet often concentration response data at early timepoints are taken from hemi-equilibrium conditions. More recently, appreciation of agonist binding kinetics and signalling kinetics at GPCRs has increased, enhancing the extent of ligand characterisation and used to investigate discrepancies in ligand bias between different experimental systems. The origin of ligand bias has been suggested to be conformational, where the agonist binds the receptor to allosterically promote the formation of a conformation which preferentially activates one signalling pathway over another. It has also been suggested that bias originates from different receptor binding kinetics, which produce different profiles of signalling and can confound comparisons of bias – especially when bias is compared from a single timepoint. In this thesis we investigate how signalling kinetics and binding kinetics can influence measures of ligand affinities, efficacy and bias using experimental system monitoring dynamic protein:protein and ligand-receptor interactions. Firstly, we established a NanoBiT complementation assay to monitor agonist-stimulated recruitment of effector proteins, either β-arrestin2 or a synthetic mini Gαs protein, at β adrenoceptor subtypes. Recruitment profiles of β-arrestin2 recruitment produced a transient “rise and fall to plateau” profile, peaking at 3 minutes, whilst the mini Gαs protein was recruited to the receptor rapidly in the first 1-5 minutes and then plateaued and/or steadily increased over the timecourse. This experimental system allowed for dynamic agonist-stimulated recruitment profiles to be monitored over time and concentration response data to be gathered from multiple timepoints. Concentration response data from each timepoint was then applied to the Black and Leff operational models to compare how direct comparisons of ligand bias can change in magnitude and direction over time. Full kinetic signalling profiles were then applied to a kinetic operational model, which takes hemi-equilibrium conditions into account, to estimate agonist affinities and efficacies from β-arrestin2 recruitment data. Agonist affinities estimated from the kinetic operational model were strongly correlated with values obtained in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assays, highlighting the model’s value in obtaining drug potency and affinity from a single functional recruitment assay. Secondly, we investigated the value of the NanoBiT complementation assays in monitoring receptor antagonism and how the kinetic context may misrepresent the mechanism of action of the antagonist ligands: propranolol, ICI-118,551 and CGP-12177. Schild-Gaddum based experiments were applied and monitored over a 61 minute timecourse, at β2 adrenoceptors. All antagonists at early timepoints (3 minutes) produced insurmountable antagonism of the formoterol response, with only propranolol demonstrating surmountable antagonism at 61 minutes. The extent of insurmountability correlated with a decreased dissociation rate (kOFF) of the antagonist, with kOFF estimated from a kinetic TR-FRET assay. The signalling profiles of antagonism of mini Gαs protein recruitment at the β2 adrenoceptor were applied to an antagonist form of the kinetic operational model, to provide similar estimates of antagonist affinity in comparison to values obtained in Schild-based and TR-FRET assays. The kinetic operational model also provided estimates of antagonist association (kON) and kOFF, though these were not comparable to estimates from the kinetic TR-FRET assay and highlight the need for model modification for accurate estimates of kinetic values. Lastly, this thesis investigated how the conformation of the receptor can influence binding properties of β2 adrenoceptor ligands. High affinity variants of the NanoBiT complementation fragments (LgBiT and HiBiT) were used to stabilise the β2 adrenoceptor in complex with an effector protein to form effector driven receptor conformations. Initially, high affinity NanoBiT fragments were genetically tagged to the β2 adrenoceptor and either β-arrestin2 or mini Gαs and expressed in dual-expression cell lines. However, unbalanced expression of the proteins resulted in insufficient formation of NanoBiT stabilised receptor-effector conformations. To resolve this issue, the HiBiT-tagged mini Gαs protein was bacterially expressed and purified and added to membranes expressing LgBiT-tagged β2 adrenoceptors, saturating the receptor population. The complimented NanoBiT fragments were used as the donor species to excite an extracellular fluorescent ligand bound to the receptor, to establish a novel transmembrane bioluminescence resonance energy transfer (TM-BRET) assay. In TM-BRET assays, β2 adrenoceptor-mini Gαs protein “high affinity” conformations were shown to have increase affinity for agonist ligands, compared to the receptor alone, whilst antagonist ligands were unchanged. Kinetic TR-FRET assays were used to monitor the kinetic binding properties of β2 adrenoceptor ligands and demonstrated that the increase in agonist affinity at receptor-mini Gαs protein complexes was driven by a decrease in agonist kOFF and that the extent of the decrease in kOFF rate was correlated with ligand efficacy. The findings presented in this thesis highlight the value of kinetic signalling data and binding kinetics in assessing ligand pharmacology, including measures of ligand bias. This thesis went onto to highlight how differences in receptor conformation drive changes in agonist kOFF, combining conformational and kinetic explanations of ligand bias

ThermoBRET: A Ligand‐Engagement Nanoscale Thermostability Assay Applied to GPCRs

Measurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre‐existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence‐detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable for high‐throughput screening. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2) and the b2‐adrenoceptor (b2AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non‐purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol‐reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc‐fused GPCRs can be determined in non‐purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split‐Nluc (Tm of 87 ⁰C versus 59 ⁰C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and for drug discovery screening