Key phosphorylation sites for robust ß-arrestin2 binding at the MOR revisited.

Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, phosphorylation is important for ß-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser is the primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr get subsequently and rapidly phosphorylated. Here we used GRK KO cells, phosphosite specific antibodies and site-directed mutagenesis to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and ß-arrestin2 recruitment. We show that both GRK2/3 and GRK5/6 subfamilies promote phosphorylation of 370Thr and 375Ser. Importantly, only GRK2/3 induce phosphorylation of 376Thr and 379Thr, and we identify these residues as key sites to promote robust ß-arrestin recruitment to the MOR. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of GRK subfamilies plays an important role in determining the tissue responses of opioid agonists.

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.

A high-affinity, cis-on photoswitchable beta blocker to optically control ß2-adrenergic receptors in vitro and in vivo.

This study introduces (S)-Opto-prop-2, a second-generation photoswitchable ligand designed for precise modulation of ß2-adrenoceptor (ß2AR). Synthesised by incorporating an azobenzene moiety with propranolol, (S)-Opto-prop-2 exhibited a high PSScis (photostationary state for cis isomer) percentage (∼90 %) and a favourable half-life (>10 days), facilitating diverse bioassay measurements. In vitro, the cis-isomer displayed substantially higher ß2AR binding affinity than the trans-isomer (1000-fold), making (S)-Opto-prop-2 one of the best photoswitchable GPCR (G protein-coupled receptor) ligands reported so far. Molecular docking of (S)-Opto-prop-2 in the X-ray structure of propranolol-bound ß2AR followed by site-directed mutagenesis studies, identified D1133.32, N3127.39 and F2896.51 as crucial residues that contribute to ligand-receptor interactions at the molecular level. In vivo efficacy was assessed using a rabbit ocular hypertension model, revealing that the cis isomer mimicked propranolol's effects in reducing intraocular pressure, while the trans isomer was inactive. Dynamic optical modulation of ß2AR by (S)-Opto-prop-2 was demonstrated in two different cAMP bioassays and using live-cell confocal imaging, indicating reversible and dynamic control of ß2AR activity using the new photopharmacology tool. In conclusion, (S)-Opto-prop-2 emerges as a promising photoswitchable ligand for precise and reversible ß2AR modulation with light. The new tool shows superior cis-on binding affinity, one of the largest reported differences in affinity (1000-fold) between its two configurations, in vivo efficacy, and dynamic modulation. This study contributes valuable insights into the evolving field of photopharmacology, offering a potential avenue for targeted therapy in ß2AR-associated pathologies.

A time-resolved Förster resonance energy transfer assay to investigate drug and inhibitor binding to ABCG2.

The human ATP-binding cassette (ABC) transporter, ABCG2, is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Förster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps.

Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor.

Small-Molecule Fluorescent Ligands for the CXCR4 Chemokine Receptor.

The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.

Bitter taste sensitivity in domestic dogs (Canis familiaris) and its relevance to bitter deterrents of ingestion.

As the most favoured animal companion of humans, dogs occupy a unique place in society. Understanding the senses of the dog can bring benefits to both the dogs themselves and their owners. In the case of bitter taste, research may provide useful information on sensitivity to, and acceptance of, diets containing bitter tasting materials. It may also help to protect dogs from the accidental ingestion of toxic substances, as in some instances bitter tasting additives are used as deterrents to ingestion. In this study we examined the receptive range of dog bitter taste receptors (Tas2rs). We found that orthologous dog and human receptors do not always share the same receptive ranges using in vitro assays. One bitter chemical often used as a deterrent, denatonium benzoate, is only moderately active against dTas2r4, and is almost completely inactive against other dog Tas2rs, including dTas2r10, a highly sensitive receptor in humans. We substituted amino acids to create chimeric dog-human versions of the Tas2r10 receptor and found the ECL2 region partly determined denatonium sensitivity. We further confirmed the reduced sensitivity of dogs to this compound in vivo. A concentration of 100µM (44.7ppm) denatonium benzoate was effective as a deterrent to dog ingestion in a two-bottle choice test indicating higher concentrations may increase efficacy for dogs. These data can inform the choice and concentration of bitter deterrents added to toxic substances to help reduce the occurrence of accidental dog poisonings.

Advances in the application of fluorescence correlation spectroscopy to study detergent purified and encapsulated membrane proteins.

Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand, substrate and inhibitor binding to membrane proteins in solution. However, the purification of membrane proteins in their active forms is complex, as the lipid bilayer provides stability and its removal often causes the protein to become conformationally unstable. This has limited the application of biophysical techniques such as FCS to study the function of membrane proteins. The recent application of native extraction techniques such as styrene maleic acid lipid particles (SMALPs) has resolved this issue and FCS has emerged as a powerful option for studying proteins extracted in this way. This review will discuss the application of FCS to study purified membrane proteins in detergent micelles, nanodiscs and SMALPs and its potential to be used routinely in membrane protein drug discovery.

Simple methods for quantifying super-resolved cortical actin.

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or 'corrals', in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by Super Resolved Radial Fluctuations (SRRF), Structured Illumination Microscopy (3D-SIM) and Expansion Microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 µm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus, this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET.

Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rß1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rß1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure.

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.

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.

Subtype selective fluorescent ligands based on ICI 118,551 to study the human ß2-adrenoceptor in CRISPR/Cas9 genome-edited HEK293T cells at low expression levels.

Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective ß2 -adrenoceptor (ß2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the ß2 AR (pKD >7.0) with good selectivity over the ß1 AR (pKD <6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; ß2 AR pKD 7.48), 9c (ICI 118,551-ßAla-ßAla-BODIPY-FL-X; ß2 AR pKD 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; ß2 AR pKD 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; ß2 AR pKD 7.42). 9a (ICI 118,551-ßAla-ßAla-BODIPY-X-630/650) had the highest affinity at recombinant ß2 ARs (pKD 7.57), but also exhibited significant binding affinity to the ß1 AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular ß2 ARs in HEK293 T cell expressing exogenous ß2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular ß2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T ß2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.

Analysis of Sequence Divergence in Mammalian ABCGs Predicts a Structural Network of Residues That Underlies Functional Divergence.

The five members of the mammalian G subfamily of ATP-binding cassette transporters differ greatly in their substrate specificity. Four members of the subfamily are important in lipid transport and the wide substrate specificity of one of the members, ABCG2, is of significance due to its role in multidrug resistance. To explore the origin of substrate selectivity in members 1, 2, 4, 5 and 8 of this subfamily, we have analysed the differences in conservation between members in a multiple sequence alignment of ABCG sequences from mammals. Mapping sets of residues with similar patterns of conservation onto the resolved 3D structure of ABCG2 reveals possible explanations for differences in function, via a connected network of residues from the cytoplasmic to transmembrane domains. In ABCG2, this network of residues may confer extra conformational flexibility, enabling it to transport a wider array of substrates.

Efficient G protein coupling is not required for agonist-mediated internalization and membrane reorganization of the adenosine A3 receptor.

Organization of G protein-coupled receptors at the plasma membrane has been the focus of much recent attention. Advanced microscopy techniques have shown that these receptors can be localized to discrete microdomains and reorganization upon ligand activation is crucial in orchestrating their signaling. Here, we have compared the membrane organization and downstream signaling of a mutant (R108A, R3.50A) of the adenosine A3 receptor (A3 AR) to that of the wild-type receptor. Fluorescence Correlation Spectroscopy (FCS) studies with a fluorescent agonist (ABEA-X-BY630) demonstrated that both wild-type and mutant receptors bind agonist with high affinity but in subsequent downstream signaling assays the R108A mutation abolished agonist-mediated inhibition of cAMP production and ERK phosphorylation. In further FCS studies, both A3 AR and A3 AR R108A underwent similar agonist-induced increases in receptor density and molecular brightness which were accompanied by a decrease in membrane diffusion after agonist treatment. Using bimolecular fluorescence complementation, experiments showed that the R108A mutant retained the ability to recruit ß-arrestin and these receptor/arrestin complexes displayed similar membrane diffusion and organization to that observed with wild-type receptors. These data demonstrate that effective G protein signaling is not a prerequisite for agonist-stimulated ß-arrestin recruitment and membrane reorganization of the A3 AR.

The use of fluorescence correlation spectroscopy to monitor cell surface ß2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying ß2 -adrenoceptor (ß2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged ß2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged ß2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface ß2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface ß2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting ß2AR agonists.

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.

Detection of genome-edited and endogenously expressed G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major targets for FDA-approved drugs. The ability to quantify GPCR expression and ligand binding characteristics in different cell types and tissues is therefore important for drug discovery. The advent of genome editing along with developments in fluorescent ligand design offers exciting new possibilities to probe GPCRs in their native environment. This review provides an overview of the recent technical advances employed to study the localisation and ligand binding characteristics of genome-edited and endogenously expressed GPCRs.

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.

Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies.

Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15-kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognizes the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C terminus to the 11-amino-acid HiBiT tag (VUN400-HiBiT) which complements LgBiT protein, forming a full-length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect allosteric interactions with CXCR4. These data demonstrate that the high specificity offered by extracellular targeted nanobodies can be utilized to probe receptor pharmacology.