Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells.

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.

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 to 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 ß2 -adrenoceptor (ß2 AR) 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 °C versus 59 °C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and drug discovery screening.

Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors.

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB1R) and 2 (CB2R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB1R and CB2R are Gαi protein-coupled receptors but detecting Gαi protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [35S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gαi protein activity assays. Methods: Here, we describe the development of a membrane-based Gαi signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB1R or CB2R, and components of the Gαi-CASE biosensor. This BRET-based system allows direct detection of Gαi signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the ßγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 µM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC50) and efficacy (Emax) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gαi protein activation assay is applicable to other Gαi-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.

Combined docking and machine learning identify key molecular determinants of ligand pharmacological activity on ß2 adrenoceptor.

G protein-coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands that bind them. Ligands exert their action via the interactions in the ligand binding pocket. We hypothesized that there is a common set of receptor interactions made by ligands of diverse structures that mediate their action and that among a large dataset of different ligands, the functionally important interactions will be over-represented. We computationally docked ~2700 known ß2AR ligands to multiple ß2AR structures, generating ca 75 000 docking poses and predicted all atomic interactions between the receptor and the ligand. We used machine learning (ML) techniques to identify specific interactions that correlate with the agonist or antagonist activity of these ligands. We demonstrate with the application of ML methods that it is possible to identify the key interactions associated with agonism or antagonism of ligands. The most representative interactions for agonist ligands involve K972.68×67 , F194ECL2 , S2035.42×43 , S2045.43×44 , S2075.46×641 , H2966.58×58 , and K3057.32×31 . Meanwhile, the antagonist ligands made interactions with W2866.48×48 and Y3167.43×42 , both residues considered to be important in GPCR activation. The interpretation of ML analysis in human understandable form allowed us to construct an exquisitely detailed structure-activity relationship that identifies small changes to the ligands that invert their pharmacological activity and thus helps to guide the drug discovery process. This approach can be readily applied to any drug target.

Functional solubilization of the ß2-adrenoceptor using diisobutylene maleic acid.

The ß2-adrenoceptor (ß2AR) is a well-established target in asthma and a prototypical G protein-coupled receptor for biophysical studies. Solubilization of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilizes membrane proteins. Use of amphiphilic copolymers is a promising strategy to solubilize membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the ß2AR in the polymer diisobutylene maleic acid (DIBMA). We demonstrate that ß2AR remains functional in the DIBMA lipid particle and shows improved thermal stability compared with the n-dodecyl-ß-D-maltopyranoside detergent-solubilized ß2AR. This unique method of extracting ß2AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilizing mutations and the use of detergents, particularly for functional biophysical studies.

Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells.

To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

GPCR Solubilization and Quality Control.

G protein-coupled receptors (GPCRs) are versatile membrane proteins involved in the regulation of many physiological processes and pathological conditions, making them interesting pharmacological targets. In order to study their structure and function, GPCRs are traditionally extracted from membranes using detergents. However, due to their hydrophobic nature, intrinsic instability in aqueous solutions, and their denaturing effects, the isolation of properly folded and functional GPCRs is not trivial. Therefore, it is of crucial importance to solubilize receptors under mild conditions and control the sample quality subsequently. Here we describe widely used methods for small-scale GPCR solubilization, followed by quality control based on fluorescence size-exclusion chromatography, SDS-PAGE, temperature-induced protein unfolding (CPM dye binding) and fluorescent ligand binding assay. These methods can easily be used to assess the thermostability and functionality of a GPCR sample exposed to different conditions, such as the use of various detergents, addition of lipids and ligands, making them valuable for obtaining an optimal sample quality for structural and functional studies.