Ligand-biased Signaling Research Articles

Ligand-directed biased agonism at human histamine H3 receptor isoforms across Gαi/o- and β-arrestin2-mediated pathways

The histamine H3 receptor (H3R) is a neurotransmitter receptor that is primarily found in the brain, where it controls the release and synthesis of histamine, as well as the release of other neurotransmitters (e.g. dopamine, serotonin). Notably, 20 H3R isoforms are differentially expressed in the human brain as a consequence of alternative gene splicing. The hH3R-445, -415, -365 and -329 isoforms contain the prototypical GPCR (7TM) structure, yet exhibit deletions in the third intracellular loop, a structural domain that is pivotal for G protein-coupling, signaling and regulation. To date, the physiological relevance underlying the individual and combinatorial function of hH3R isoforms remains poorly understood. Nevertheless, given their significant implication in physiological processes (e.g. cognition, homeostasis) and neurological disorders (e.g. Alzheimer’s and Parkinson’s disease, schizophrenia), widespread targeting of hH3R isoforms by drugs may lead to on-target side effects in brain regions that are unaffected by disease. To this end, isoform- and/or pathway-selective targeting of hH3R isoforms by biased agonists could be of therapeutic relevance for the development of region- and disease-specific drugs. Hence, we have evaluated ligand biased signaling at the hH3R-445, -415, -365 and -329 isoforms across various Gαi/o-mediated (i.e. [35S]GTPγS accumulation, cAMP inhibition, pERK1/2 activation, pAKT T308/S473 activation) and non Gαi/o-mediated (i.e. β-arrestin2 recruitment) endpoints that are relevant to neurological diseases. Our findings indicate that H3R agonists display significantly altered patterns in their degree of ligand bias, in a pathway- and isoform-dependent manner, underlining the significance to investigate GPCRs with multiple isoforms to improve development of selective drugs. Subject categoryNeuropharmacology.

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

Structure, Function, and Pharmaceutical Ligands of 5-Hydroxytryptamine 2B Receptor.

Since the first characterization of the 5-hydroxytryptamine 2B receptor (5-HT2BR) in 1992, significant progress has been made in 5-HT2BR research. Herein, we summarize the biological function, structure, and small-molecule pharmaceutical ligands of the 5-HT2BR. Emerging evidence has suggested that the 5-HT2BR is implicated in the regulation of the cardiovascular system, fibrosis disorders, cancer, the gastrointestinal (GI) tract, and the nervous system. Eight crystal complex structures of the 5-HT2BR bound with different ligands provided great insights into ligand recognition, activation mechanism, and biased signaling. Numerous 5-HT2BR antagonists have been discovered and developed, and several of them have advanced to clinical trials. It is expected that the novel 5-HT2BR antagonists with high potency and selectivity will lead to the development of first-in-class drugs in various therapeutic areas.

Bitter-Tasting Amino Acids l-Arginine and l-Isoleucine Differentially Regulate Proton Secretion via T2R1 Signaling in Human Parietal Cells in Culture.

This study aimed at identifying whether the bitter-tasting amino acids l-arginine (l-ARG) and l-isoleucine (l-ILE) differentially regulate mechanisms of gastric acid secretion in human parietal cells (HGT-1 cells) via activation of bitter taste sensing receptors (T2Rs). In a first set of experiments, involvement of T2Rs in l-ARG and l-ILE-modulated proton secretion was demonstrated by co-treatment of HGT-1 cells with T2R antagonists. Subsequent whole genome screenings by means of cDNA arrays revealed T2R1 as a prominent target for both amino acids. Next, the functional role of T2R1 was verified by means of a T2R1 CRISPR-Cas9 knock-out approach. Here, the effect of l-ARG on proton secretion decreased by 65.7 ± 21.9% and the effect of l-ILE increased by 93.2 ± 24.1% in HGT-1 T2R1 ko versus HGT-1 wt cells (p < 0.05). Overall, our results indicate differential effects of l-ARG and l-ILE on proton secretion in HGT-1 cells and our molecular docking studies predict distinct binding for these amino acids in the binding site of T2R1. Further studies will elucidate whether the mechanism of differential effects involves structure-specific ligand-biased signaling of T2R1 or additional cellular targets.

5894The biased angiotensin receptor 1-ligand TRV023 attenuates atherosclerosis through selective inhibition of Gq-mediated signaling

Abstract Introduction Antagonists of the angiotensin II receptor type 1 (AT1) belong to the most successful treatments of cardiovascular disease. Besides G-protein-independent β-arrestin-mediated signaling, AT1 preferencially signals via G-proteins of the families Gq and G13. Conventional AT1-antagonists equally inhibit all of these pathways, even though the beneficial effects are generally attributed to the inhibition of Gq only. While data on β-arrestin signaling in the vascular disease context are conflicting, we found an excessive pathological remodeling and dramatic exacerbation of atherosclerosis upon interference with G13-mediated signaling. Against this background, selective inhibition of the Gq-pathway seems desirable. Thus, based on the novel concept of ligand-biased signaling, we aimed to identify biased AT1-antagonists that selectively inhibit the Gq-pathway, while allowing for continued signaling through G13. Methods and results We systematically profiled known AT1-ligands and identified the angiotensin analogue Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH (TRV023) as a Gq-selective AT1-antagonist: TRV023 has recently been described as a β-arrestin-biased ligand; however its signaling properties regarding distinct G-protein isoforms have never been defined. Indeed, using bioluminescence reporter assays, we found that TRV023 inhibits Gq-signaling at least as potently as losartan and telmisartan, but unlike the conventional antagonists, it does not impair β-arrestin-signaling (Fig. 1A). Intriguingly, while the conventional antagonists effectively inhibited induction of a G13 luciferase reporter, TRV023 did not significantly interfere with G13-mediated signaling, indicating Gq-selective antagonistic effects. According to our hypothesis, these G-protein-biased signaling properties could render TRV023 superior to conventional antagonists in the treatment of vascular disease. In order to test this hypothesis, we exposed ApoE-deficient mice to one of two different atherosclerosis models: A novel model for accelerated atherosclosis based on partial carotid ligation and western diet, or a conventional atherosclerosis model based on 12 weeks of western diet. Using osmotic pumps, mice were continuously treated with vehicle control, TRV023 or losartan in doses that proved equipotent with respect to blood pressure lowering effects. In line with our hypothesis, TRV023 but not losartan significantly attenuated plaque development in both atherosclerosis models (Fig. 1B). Figure 1 Conclusion Our studies have identified TRV023 as a Gq-biased AT1-antagonist and indicate that selective inhibition of AT1-dependent Gq-mediated signaling by biased ligands may be a promising approach for the treatment of atherosclerosis. Hence, TRV023 qualifies as a drug candidate, particularly since it has already proven to be safe and well-tolerable in clinical phase I and II trials in the context of acute heart failure. Acknowledgement/Funding Else Kröner-Fresenius-Foundation

Innovative Bioluminescence Resonance Energy Transfer Assay Reveals Differential Agonist-Induced D2 Receptor Intracellular Trafficking and Arrestin-3 Recruitment.

The dopamine D2 receptor (D2R) mediates ligand-biased signaling with potential therapeutic implications. However, internalization, choice of endocytic routes, and degradation of the D2R in lysosomes may also participate in agonist-directed trafficking. We developed bioluminescence resonance energy transfer (BRET) assays that measure relative distances between Renilla luciferase8-tagged D2R and green fluorescent protein 2 (GFP2)-tagged K-Ras (plasma membrane marker), and between luciferase8-tagged D2R and GFP2-Rab5 (early), GFP2-Rab4 (recycling), or GFP2-Rab7 (late) endosomal markers. The BRET signal between D2R-Luc and GFP2-K-Ras was robustly diminished after receptor internalization induced by dopamine, with subsequent BRET signals increasing when luciferase8-tagged D2R approached GFP2-Rab proteins in endosomal compartments. All BRET signals were blocked by the selective D2R antagonist haloperidol and were decreased by low temperature and high sucrose blocks, two parameters interfering with internalization. Some antipsychotic drugs, such as aripiprazole, are less efficacious in internalizing D2R than most of the antiparkinsonian agents. However, antipsychotics were nearly as efficacious as antiparkinsonians in directing the D2R toward early and recycling endosomes. The Rab7 marker for the late endosome/lysosome route was also capable of discriminating between D2R compounds. We could show that some drugs engaged the D2R either to interact preferentially with arrestin-3 or to internalize. Our study revealed that D2R trafficking in cells was differentially regulated by antipsychotic and antiparkinsonian drugs. Taken together, the BRET assays reported here could further help decipher D2R ligand-induced arrestin-3 recruitment and trafficking, with potentially more selective therapeutic profiles and fewer undesired side effects.

Function-related conformational dynamics of G protein-coupled receptors revealed by NMR.

G protein-coupled receptors (GPCRs) function as receptors for various neurotransmitters, hormones, cytokines, and metabolites. GPCR ligands impart differing degrees of signaling in the G protein and arrestin pathways, in phenomena called biased signaling, and each ligand for a given GPCR has a characteristic level of ability to activate or deactivate its target, which is referred to as its efficacy. The ligand efficacies and biased signaling of GPCRs remarkably affect the therapeutic properties of the ligands. However, these features of GPCRs can only be partially understood from the crystallography data, although numerous GPCR structures have been solved. NMR analyses have revealed that GPCRs have multiple interconverting substates, exchanging on various timescales, and that the exchange rates are related to the ligand efficacies and biased signaling. In addition, NMR analyses of GPCRs in the lipid bilayer environment of rHDLs revealed that the exchange rates are modulated by the lipid bilayer environment, highlighting the importance of the function-related dynamics in the lipid bilayer. In this review, we will describe several solution NMR studies that have clarified the conformational dynamics related to the ligand efficacy and biased signaling of GPCRs.

Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches.

Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.

Exploiting agonist biased signaling of chemokines to target cancer.

As knowledge of growth-independent functions of cancer cells is expanding, exploration into the role of chemokines in modulating cancer pathogenesis, particularly metastasis, continues to develop. However, more study into the mechanisms whereby chemokines direct the migration of cancer cells is needed before specific therapies can be generated to target metastasis. Herein, we draw attention to the longstanding conundrum in the field of chemokine biology that chemokines stimulate migration in a biphasic manner; and explore this phenomenon's impact on chemokine function in the context of cancer. Typically, low concentrations of chemokines lead to chemotactic migration and higher concentrations halt migration. The signaling mechanisms that govern this phenomenon remain unclear. Over the last decade, we have defined a novel signaling mechanism for regulation of chemokine migration through ligand oligomerization and biased agonist signaling. We provide insight into this new paradigm for chemokine signaling and discuss how it will impact future exploration into chemokine function and biology. In the pursuit of producing more novel cancer therapies, we suggest a framework for pharmaceutical application of the principles of chemokine oligomerization and biased agonist signaling in cancer. © 2016 Wiley Periodicals, Inc.

GPCR Dynamics: Structures in Motion.

The function of G protein-coupled receptors (GPCRs)-which represent the largest class of both human membrane proteins and drug targets-depends critically on their ability to change shape, transitioning among distinct conformations. Determining the structural dynamics of GPCRs is thus essential both for understanding the physiology of these receptors and for the rational design of GPCR-targeted drugs. Here we review what is currently known about the flexibility and dynamics of GPCRs, as determined through crystallography, spectroscopy, and computer simulations. We first provide an overview of the types of motion exhibited by a GPCR and then discuss GPCR dynamics in the context of ligand binding, activation, allosteric modulation, and biased signaling. Finally, we discuss the implications of GPCR conformational plasticity for drug design.

Tachykinin-Related Peptides Share a G Protein-Coupled Receptor with Ion Transport Peptide-Like in the Silkworm Bombyx mori.

Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR)-A24 as an ion transport peptide-like (ITPL) receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs), we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1–5). In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling.

Identification and Preclinical Pharmacology of BMS-986104: A Differentiated S1P1 Receptor Modulator in Clinical Trials

Clinical validation of S1P receptor modulation therapy was achieved with the approval of fingolimod (Gilenya, 1) as the first oral therapy for relapsing remitting multiple sclerosis. However, 1 causes a dose-dependent reduction in the heart rate (bradycardia), which occurs within hours after first dose. We disclose the identification of clinical compound BMS-986104 (3d), a novel S1P1 receptor modulator, which demonstrates ligand-biased signaling and differentiates from 1 in terms of cardiovascular and pulmonary safety based on preclinical pharmacology while showing equivalent efficacy in a T-cell transfer colitis model.

Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor.

CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ(8)-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced [(3)H]SR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased "fingerprints" that may ultimately allow the development of improved CB1R-targeted therapies.

Designing BRET-based conformational biosensors for G protein-coupled receptors

Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.

Boronic Acids as Probes for Investigation of Allosteric Modulation of the Chemokine Receptor CXCR3

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

Hallucinogens induce a specific barcode of phosphorylation on the serotonin2A receptor that underlies a weaker receptor desensitization and internalization

The serotonin (5-Hydroxytryptamine, 5-HT) 2A receptor represents one of the most striking examples where functional selectivity (or ligand-biased signaling) is transduced in distinct behaviours. This receptor is the primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline and psilocybin, which reproduce some of the core symptoms of schizophrenia and are often used to probe the disease. Why only some 5-HT 2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and agonist activity (e.g. lisuride and ergotamine) lack such a psychoactive activity remains an incompletely resolved paradox. In a recent paper published in Molecular and Cellular Proteomics (doi: 10.1074/mcp.M113.036558) we demonstrated a biased phosphorylation of the 5-HT 2A receptor in response to hallucinogenic versus non-hallucinogenic agonists that leads to a weaker receptor desensitization and internalization by hallucinogens.

ChemInform Abstract: GPCR Structures in Drug Design, Emerging Opportunities with New Structures

AbstractReview: 48 refs

A conformation-equilibrium model captures ligand-ligand interactions and ligand-biased signalling by G-protein coupled receptors.

G-protein coupled receptors (GPCRs) are a versatile, important class of cell-surface receptors. GPCRs occur in different conformations that exist in a dynamic ligand-sensitive equilibrium. These conformations vary in their affinities for intracellular signalling proteins and initiate signalling via different intracellular routes. The binding of extracellular ligands and allosteric ligand-ligand interactions shift conformation equilibria to cause biased signalling. Here, we present a mathematical model that describes the effects of ligands on the conformation equilibria of GPCRs. Our extended Monod-Wyman-Changeux model describes the receptor as shifting between active and inactive receptor conformations under the influence of extracellular ligands. For each receptor conformation, the intracellular domain of the receptor can attain alternative domain conformations that differ in their affinity for intracellular signalling proteins. At the extracellular domain, the model can accommodate different mechanisms for allosteric ligand-ligand interactions that induce shifts in receptor and domain conformation equilibria. We use the model to study ligand-biased signalling and how ligand affinity, ligand sensitivity and maximal signalling output depend on allosteric ligand-ligand interactions.

Allosteric and biased g protein-coupled receptor signaling regulation: potentials for new therapeutics.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that participate in many aspects of the endocrine function and are important targets for drug development. They transduce signals mainly, but not exclusively, via hetero-trimeric G proteins, leading to a diversity of intracellular signaling cascades. Ligands binding at the hormone orthosteric sites of receptors have been classified as agonists, antagonists, and/or inverse agonists based on their ability to mainly modulate G protein signaling. Accumulating evidence also indicates that such ligands, alone or in combination with other ones such as those acting outside the orthosteric hormone binding sites (e.g., allosteric modulators), have the ability to selectively engage subsets of signaling responses as compared to the natural endogenous ligands. Such modes of functioning have been variously referred to as “functional selectivity” or “ligand-biased signaling.” In this review, we provide an overview of the current knowledge regarding GPCR-biased signaling and their functional regulation with a focus on the evolving concept that receptor domains can also be targeted to allosterically bias signaling, and discuss the usefulness of such modes of regulation for the design of more efficient therapeutics.

Quantification of Ligand Bias for Clinically Relevant β2-Adrenergic Receptor Ligands: Implications for Drug Taxonomy

The concepts of functional selectivity and ligand bias are becoming increasingly appreciated in modern drug discovery programs, necessitating more informed approaches to compound classification and, ultimately, therapeutic candidate selection. Using the β2-adrenergic receptor as a model, we present a proof of concept study that assessed the bias of 19 β-adrenergic ligands, including many clinically used compounds, across four pathways [cAMP production, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, calcium mobilization, and receptor endocytosis] in the same cell background (human embryonic kidney 293S cells). Efficacy-based clustering placed the ligands into five distinct groups with respect to signaling signatures. In some cases, apparent functional selectivity originated from off-target effects on other endogenously expressed adrenergic receptors, highlighting the importance of thoroughly assessing selectivity of the responses before concluding receptor-specific ligand-biased signaling. Eliminating the nonselective compounds did not change the clustering of the 10 remaining compounds. Some ligands exhibited large differences in potency for the different pathways, suggesting that the nature of the receptor-effector complexes influences the relative affinity of the compounds for specific receptor conformations. Calculation of relative effectiveness (within pathway) and bias factors (between pathways) for each of the compounds, using an operational model of agonism, revealed a global signaling signature for all of the compounds relative to isoproterenol. Most compounds were biased toward ERK1/2 activation over the other pathways, consistent with the notion that many proximal effectors converge on this pathway. Overall, we demonstrate a higher level of ligand texture than previously anticipated, opening perspectives for the establishment of pluridimensional correlations between signaling profiles, drug classification, therapeutic efficacy, and safety.