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.

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.

Development of fluorescent peptide G protein-coupled receptor activation biosensors for NanoBRET characterization of intracellular allosteric modulators.

G protein-coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at these receptors offer further potential for exploitation. Intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g., G protein) and inhibit agonist responses noncompetitively. This potentially offers greater selectivity between receptor subtypes compared to classical orthosteric ligands. However, while examples of IAM ligands are well described, a more general methodology for assessing compound interactions at the IAM site is lacking. Here, fluorescent labeled peptides based on the Gα peptide C terminus are developed as novel binding and activation biosensors for the GPCR-IAM site. In TR-FRET binding studies, unlabeled peptides derived from the Gαs subunit were first characterized for their ability to positively modulate agonist affinity at the ß2 -adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labeled tracer was synthesized based on the 19 amino acid Gαs peptide (TMR-Gαs19cha18, where cha = cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-Gαs19cha18 was recruited to Gs coupled ß2 -adrenoceptor and EP2 receptors in an agonist-dependent manner, but not the Gi-coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-Gαs19cha18 enabled direct assessment of the affinity of unlabeled ligands for ß2 -adrenoceptor IAM site. Thus, the NanoBRET platform using fluorescent-labeled G protein peptide mimetics offers novel potential for medium-throughput screens to identify IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation.

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex.

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

Plasma membrane dynamics and tetrameric organisation of ABCG2 transporters in mammalian cells revealed by single particle imaging techniques.

ABCG2 is one of three human ATP binding cassette (ABC) transporters involved in the export from cells of a chemically and structurally diverse range of compounds. This multidrug efflux capability, together with a broad tissue distribution in the body, means that ABCG2 exerts a range of effects on normal physiology such as kidney urate transport, as well as contributing towards the pharmacokinetic profiles of many exogenous drugs. The primary sequence of ABCG2 contains only half the number of domains required for a functioning ABC transporter and so it must oligomerise in order to function, yet its oligomeric state in intact cell membranes remains uncharacterized. We have analysed ABCG2 in living cell membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, and stepwise photobleaching to demonstrate a predominantly tetrameric structure for ABCG2 in the presence or absence of transport substrates. These results provide the essential basis for exploring pharmacological manipulation of oligomeric state as a strategy to modulate ABCG2 activity in future selective therapeutics.

Biased Gs versus Gq proteins and ß-arrestin signaling in the NK1 receptor determined by interactions in the water hydrogen bond network.

X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and ß-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) ß-arrestin only; and 3) Gq and ß-arrestin but not through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways.

Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15.

Traceable truncated Neuropeptide Y (NPY) analogues with Y1 receptor (Y1R) affinity and selectivity are highly desirable tools in studying receptor location, regulation, and biological functions. A range of fluorescently labeled analogues of a reported Y1R/Y4R preferring ligand BVD-15 have been prepared and evaluated using high content imaging techniques. One peptide, [Lys(2)(sCy5), Arg(4)]BVD-15, was characterized as an Y1R antagonist with a pKD of 7.2 measured by saturation analysis using fluorescent imaging. The peptide showed 8-fold lower affinity for Y4R (pKD = 6.2) and was a partial agonist at this receptor. The suitability of [Lys(2)(sCy5), Arg(4)]BVD-15 for Y1R and Y4R competition binding experiments was also demonstrated in intact cells. The nature of the label was shown to be critical with replacement of sCy5 by the more hydrophobic Cy5.5 resulting in a switch from Y1R antagonist to Y1R partial agonist.

A G protein-coupled receptor dimer imaging assay reveals selectively modified pharmacology of neuropeptide Y Y1/Y5 receptor heterodimers.

The ability of G protein-coupled receptors (GPCRs) to form dimers, and particularly heterodimers, offers potential for targeted therapeutics with improved selectivity. However, studying dimer pharmacology is challenging, because of signaling cross-talk or because dimerization may often be transient in nature. Here we develop a system to isolate the pharmacology of precisely defined GPCR dimers, trapped by bimolecular fluorescence complementation (BiFC). Specific effects of agonist activation on such dimers are quantified using automated imaging and analysis of their internalization, controlled for by simultaneous assessment of endocytosis of one coexpressed protomer population. We applied this BiFC system to study example neuropeptide Y (NPY) Y1 receptor dimers. Incorporation of binding-site or phosphorylation-site mutations into just one protomer of a Y1/Y1 BiFC homodimer had no impact on efficient NPY-stimulated endocytosis, demonstrating that single-site agonist occupancy, and one phosphorylated monomer within this dimer, was sufficient. For two Y1 receptor heterodimer combinations (with the Y4 receptor or ß2-adrenoceptor), agonist and antagonist pharmacology was explained by independent actions on the respective orthosteric binding sites. However, Y1/Y5 receptor BiFC dimers, compared with the constituent subtypes, were characterized by reduced potency and efficacy of Y5-selective peptide agonists, the inactivity of Y1-selective antagonists, and a change from surmountable to nonsurmountable antagonism for three unrelated Y5 antagonists. Thus, allosteric interactions between Y1 and Y5 receptors modify the pharmacology of the heterodimer, with implications for potential antiobesity agents that target centrally coexpressed Y1 and Y5 receptors to suppress appetite.

Synthetic routes to the Neuropeptide Y Y1 receptor antagonist 1229U91 and related analogues for SAR studies and cell-based imaging.

The potent Y1 receptor antagonist, 1229U91 has an unusual cyclic dimer structure that makes syntheses of analogue series quite challenging. We have examined three new routes to the synthesis of such peptides that has given access to novel structural variants including heterodimeric compounds, ring size variants and labelled conjugates. These compounds, including a fluorescently labelled analogue VIII show potent antagonism that can be utilised in studying Y1 receptor pharmacology.

High-throughput kinetics in drug discovery.

The importance of a drug's kinetic profile and interplay of structure-kinetic activity with PK/PD has long been appreciated in drug discovery. However, technical challenges have often limited detailed kinetic characterization of compounds to the latter stages of projects. This review highlights the advances that have been made in recent years in techniques, instrumentation, and data analysis to increase the throughput of detailed kinetic and mechanistic characterization, enabling its application earlier in the drug discovery process.

Role of G protein-coupled receptor kinases (GRKs) in ß2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the ß2 -AR, transfection of ßARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the ß2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant ß2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by ß2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and ß2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between ß2 -AR and ß-arrestin2 or between ß2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to ß2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to ß2 -AR agonists occurred in CHO-GLUT4myc cells expressing ß2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type ß2 -AR. However, ß2 -ARs lacking phosphorylation sites failed to recruit ß-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the ß2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

Dually Labeled Neurotensin NTS1R Ligands for Probing Radiochemical and Fluorescence-Based Binding Assays.

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([3H]13, [3H]18) and their nontritiated analogues (13, 18). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

The novel adrenergic agonist ATR-127 targets skeletal muscle and brown adipose tissue to tackle diabesity and steatohepatitis.

OBJECTIVE: Simultaneous activation of ß2- and ß3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of ß1-ARs - and thus inducing cardiovascular complications - are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel ß2-and ß3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models. METHODS: In the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective ß-AR agonist isoprenaline across various rodent ß-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis. RESULTS: Exposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis. CONCLUSIONS: Our results demonstrate that ATR-127 is a highly effective, novel ß2- and ß3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies.

The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the ß2-adrenergic receptor.

Recent high resolution x-ray structures of the ß2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.

Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of ß-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors.

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains of single living cells. However, relating these properties to molecular mechanisms can be challenging. We investigated the influence of ß-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluorescence complementation (BiFC) system also identified Y1 receptor-ß-arrestin complexes. Diffusion co-efficients (D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15 × 10(-9)cm(2)s(-1) respectively. At a concentration which promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFP motility (D 1.48 × 10(-9)cm(2)s(-1)), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced changes in Y1 receptor motility were inhibited by mutations (6A) which prevented ß-arrestin recruitment and internalisation; conversely they became apparent in a Y2 receptor mutant with increased ß-arrestin affinity. NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering, and which were abolished by the 6A mutation. The importance of ß-arrestin recruitment for these effects was illustrated by reduced lateral mobility (D 1.20-1.33 × 10(-9)cm(2)s(-1)) of Y1 receptor-ß-arrestin BiFC complexes. Thus NPY-induced changes in Y receptor motility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect the underlying receptor-ß-arrestin signalling complex.

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1.

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin-proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Differential signaling by splice variants of the human free fatty acid receptor GPR120.

GPR120 is a long-chain fatty acid receptor that stimulates incretin hormone release from colonic endocrine cells and is implicated in macrophage and adipocyte function. The functional consequences of long (L) and short (S) human GPR120 splice variants, which differ by insertion of 16 amino acids in the third intracellular loop, are currently unknown. Here we compare signaling and intracellular trafficking of GPR120S and GPR120L receptors, using calcium mobilization and dynamic mass redistribution (DMR) assays, together with quantitative imaging measurements of ß-arrestin2 association and receptor internalization. FLAG- or SNAP-tagged GPR120S receptors elicited both intracellular calcium mobilization and DMR responses in human embryonic kidney 293 cells, when stimulated with oleic acid, myristic acid, or the agonist 4-[[(3-phenoxyphenyl)methyl]amino]benzenepropanoic acid (GW9508). Responses were insensitive to pertussis toxin, but increases in intracellular calcium were attenuated by 2-aminoethoxydiphenyl borate, an inhibitor of store inositol trisphosphate receptors. Despite equivalent cell surface expression of SNAP-tagged GPR120L receptors, no specific calcium or DMR responses were observed in cells transfected with this isoform. However, agonist-stimulated GPR120S and GPR120L receptors both recruited ß-arrestin2 and underwent robust internalization, with similar agonist potencies in each case. After oleic acid-induced internalization, neither GPR120 isoform recycled rapidly to the cell surface. In both cases, confocal microscopy indicated receptor targeting to lysosomal compartments. Thus, the third intracellular loop insertion in GPR120L prevents G protein-dependent intracellular calcium and DMR responses, but this receptor isoform remains functionally coupled to the ß-arrestin pathway, providing one of the first examples of a native ß-arrestin-biased receptor.

Unique interaction pattern for a functionally biased ghrelin receptor agonist.

Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Gα(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.

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.