Protein-biased Agonist Research Articles

A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase.

G protein-biased μ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased μ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced μ receptor desensitisation was probed using kinase inhibitors. Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased μ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular μ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid μ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Compound 1 is a novel G protein-biased μ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Development of Diphenethylamines as Selective Kappa Opioid Receptor Ligands and Their Pharmacological Activities.

Among the opioid receptors, the kappa opioid receptor (KOR) has been gaining substantial attention as a promising molecular target for the treatment of numerous human disorders, including pain, pruritus, affective disorders (i.e., depression and anxiety), drug addiction, and neurological diseases (i.e., epilepsy). Particularly, the knowledge that activation of the KOR, opposite to the mu opioid receptor (MOR), does not produce euphoria or leads to respiratory depression or overdose, has stimulated the interest in discovering ligands targeting the KOR as novel pharmacotherapeutics. However, the KOR mediates the negative side effects of dysphoria/aversion, sedation, and psychotomimesis, with the therapeutic promise of biased agonism (i.e., selective activation of beneficial over deleterious signaling pathways) for designing safer KOR therapeutics without the liabilities of conventional KOR agonists. In this review, the development of new KOR ligands from the class of diphenethylamines is presented. Specifically, we describe the design strategies, synthesis, and pharmacological activities of differently substituted diphenethylamines, where structure–activity relationships have been extensively studied. Ligands with distinct profiles as potent and selective agonists, G protein-biased agonists, and selective antagonists, and their potential use as therapeutic agents (i.e., pain treatment) and research tools are described.

Discovery of a G Protein-Biased Radioligand for the Histamine H2 Receptor with Reversible Binding Properties.

Currently employed histamine H2 receptor (H2R) radioligands possess several drawbacks, for example, high non-specificity, insurmountable binding, or short half-life. We report the synthesis and the chemical and pharmacological characterization of the highly stable carbamoylguanidine-type radioligand [3H]UR-KAT479 ([3H]23), a subtype selective histamine H2 receptor G protein-biased agonist. [3H]23 was characterized by saturation, kinetic, and competition binding assays at the human, guinea pig, and mouse H2 receptors (co-)expressed in HEK293(T) cells. [3H]23 reversibly bound to the respective H2Rs with moderate to high affinity (human/guinea pig/mouse Kd: 24/28/94 nM). In order to investigate the applicability of carbamoylguanidine-type ligands in animal studies elucidating the role of the H2R in the brain, we performed a preliminary partitioning experiment in the whole human/mouse blood, which indicated a low binding of [3H]23 to red blood cells. These properties turn [3H]23 into a powerful tool for the determination of binding affinities and demonstrate the promising pharmacokinetic profile of carbamoylguanidine-type ligands.

Biased Agonism at Nociceptin/Orphanin FQ Receptors: A Structure Activity Study on N/OFQ(1-13)-NH2.

Nociceptin/orphanin FQ (N/OFQ) controls different biological functions via selective stimulation of the N/OFQ peptide (NOP) receptor. The pleiotropic actions of N/OFQ may limit the development of NOP ligands as innovative drugs in different therapeutic areas. The pharmacological concept of functional selectivity (aka biased agonism) might be useful for amplifying beneficial actions and/or counteracting side effects. Thus, molecules with large bias factors toward G protein or β arrestin are required for investigating the translational value of NOP biased modulation. Herein, the biased behavior of a heterogeneous library of NOP-targeting peptide derivatives was evaluated in vitro with the aim to provide possible insights into the structural determinants that govern the selective activation of G protein versus β-arrestin. Our results demonstrate that lipidation of N/OFQ(1–13)-NH2 is a useful strategy for obtaining G protein biased agonists for the NOP receptor.

Identification and characterization of plant-derived alkaloids, corydine and corydaline, as novel mu opioid receptor agonists

Pain remains a key therapeutic area with intensive efforts directed toward finding effective and safer analgesics in light of the ongoing opioid crisis. Amongst the neurotransmitter systems involved in pain perception and modulation, the mu-opioid receptor (MOR), a G protein-coupled receptor, represents one of the most important targets for achieving effective pain relief. Most clinically used opioid analgesics are agonists to the MOR, but they can also cause severe side effects. Medicinal plants represent important sources of new drug candidates, with morphine and its semisynthetic analogues as well-known examples as analgesic drugs. In this study, combining in silico (pharmacophore-based virtual screening and docking) and pharmacological (in vitro binding and functional assays, and behavioral tests) approaches, we report on the discovery of two naturally occurring plant alkaloids, corydine and corydaline, as new MOR agonists that produce antinociceptive effects in mice after subcutaneous administration via a MOR-dependent mechanism. Furthermore, corydine and corydaline were identified as G protein-biased agonists to the MOR without inducing β-arrestin2 recruitment upon receptor activation. Thus, these new scaffolds represent valuable starting points for future chemical optimization towards the development of novel opioid analgesics, which may exhibit improved therapeutic profiles.

Molecular Dynamics Simulations to Investigate How PZM21 Affects the Conformational State of the μ-Opioid Receptor Upon Activation.

Opioid analgesics such as morphine have indispensable roles in analgesia. However, morphine use can elicit side effects such as respiratory depression and constipation. It has been reported that G protein-biased agonists as substitutes for classic opioid agonists can alleviate (or even eliminate) these side effects. The compounds PZM21 and TRV130 could be such alternatives. Nevertheless, there are controversies regarding the efficacy and G protein-biased ability of PZM21. To demonstrate a rationale for the reduced biasing agonism of PZM21 compared with that of TRV130 at the molecular level, we undertook a long-term molecular dynamics simulation of the μ-opioid receptor (MOR) upon the binding of three ligands: morphine, TRV130, and PZM21. We found that the delayed movement of the W2936.48 (Ballesteros–Weinstein numbering) side chain was a factor determining the dose-dependent agonism of PZM21. Differences in conformational changes of W3187.35, Y3267.43, and Y3367.53 in PZM21 and TRV130 explained the observed differences in bias between these ligands. The extent of water movements across the receptor channel was correlated with analgesic effects. Taken together, these data suggest that the observed differences in conformational changes of the studied MOR–ligand complexes point to the low-potency and lower bias effects of PZM21 compared with the other two ligands, and they lay the foundation for the development of G protein-biased agonists.

Differential activity and selectivity of N-terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors.

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors' TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2-4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

Reduce efficacy, reduce side effects

Nociception The μ-opioid receptor is an important target for pain management, but serious side effects limit the use of drugs that target this G protein–coupled receptor, which signals through both G proteins and β-arrestins. G protein–biased agonists have been developed to promote pain relief without causing β-arrestin–associated side effects. Gillis et al. noted that the observed reductions in side effects of some G protein–biased agonists of the μ-opioid receptor could be explained by their low intrinsic efficacy rather than by their signaling bias per se. Sci. Signal. 13 , eaaz3140 (2020 ).

G Protein Biased Signaling by Non‐catechol Dopamine D1 Receptor Agonists

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and working memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, until very recently all previously developed D1R selective agonists have a catechol moiety and catechol agonists have poor pharmacokinetic properties. We recently discovered the first non‐catechol D1R selective agonists with drug‐like properties and unexpectedly these ligands showed G protein biased signaling. Here, we define a novel mechanism of action for the non‐catechol D1R agonists involving impaired receptor endocytosis. First, HEK293 cells that have beta‐arrestin1/2 knocked out using CRISPR/Cas9 demonstrated that beta‐arrestins are required for agonist‐induced D1R endocytosis. Beta‐arrestin1/2 knockout significantly reduced agonist‐induced D1R endocytosis and re‐expressing beta‐arrestin1 or 2 rescued endocytosis in imaging and cell surface ELISA assays. Next, catechol and non‐catechol D1R agonists were tested in cAMP Glosensor and beta‐arrestin Tango assays to investigate potential biased signaling. The unbiased catechol D1R full agonist SKF‐81297 was used as the reference compound in all studies. The non‐catechol D1R agonists dose‐dependently increased cAMP production in HEK293 cells similar to the full agonist SKF‐81297 (Emax 100%), but did not engage beta‐arrestin. Interestingly, one non‐catechol agonist (PW0441) robustly activated both cAMP (Emax = 92%, EC50 = 4.4 nM) and also fully recruited beta‐arrestin (Emax = 100%, EC50 = 100 nM). The catechol agonist A‐77636 dose‐dependently increased full cAMP production (Emax = 104%, EC50 = 3.1 nM) but was a super agonist for beta‐arrestin recruitment (Emax = 130%, EC50 = 35nM). To determine the effect of G protein biased agonists on D1R endocytosis, the catechol and non‐catechol D1R agonists were tested in imaging and cell surface ELISA assays. The non‐catechol G protein biased agonists all induced significantly less total D1R endocytosis than the catechol agonist SKF‐81297. The pure G protein biased agonists PF‐1119 and PW0464 maximally induced 5% and 11% loss of cell surface D1R, respectively. In contrast, the catechol A‐77636 maximally induced 47% loss of cell surface D1R and induced significantly more total endocytosis than SKF‐81297. Finally, to elucidate the effect of G protein bias downstream of cAMP/PKA, HEK293 cells were treated from 0–360 min to assess CREB phosphorylation (pCREB) by western blotting. Preliminary results indicate G protein biased agonists have increased and prolonged pCREB levels compared to unbiased agonists. Together, these results indicate that G protein biased D1R agonists do not induce endocytosis and prolong G protein‐mediated signaling. Furthermore, these results suggest that G protein biased D1R agonists may signal longer in the G protein pathway due to reduced agonist‐induced receptor endocytosis. These results report a novel unbiased non‐catechol D1R agonist (PW0441), a D1R beta‐arrestin super agonist (A‐77636) and also elucidate a mechanism of action for G protein biased agonists in which G protein signaling is potentiated while beta‐arrestin mediated endocytosis is impaired.Support or Funding InformationSupported by NIH DA047643 and institutional funding from UTMB to JAA

Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform

Fentanyl and morphine are agonists of the Mu opioid receptor (MOR), which is a member of the GPCR family. Their analgesic effects are associated with unwanted side effects. On a signaling level downstream from MOR, it has been hypothesized that analgesia may be mediated through the G protein pathway, whereas the undesirable effects of opioids have been linked to the β-arrestin (βarr) pathway. Despite being an increasingly debated subject, little is known about a potential ‘bias’ (i.e. the preferential activation of one pathway over the other) of the novel synthetic opioids (NSO) – including fentanyl analogs – that have emerged on the illegal drug market. We have therefore developed and applied a novel, robust bio-assay platform to study the activity of 21 NSO, to evaluate to what extent these MOR agonists show biased agonism and to investigate the potential correlation with their structure. In addition, we evaluated the functional selectivity of TRV130, a purported G protein-biased agonist. We applied newly established stable bio-assays in HEK293T cells, based on the principle of functional complementation of a split nanoluciferase, to assess MOR activation via recruitment of a mini-Gi protein (GTPase domain of Gαi subunit) or βarr2. All but two of the tested NSO demonstrated a concentration-dependent response at MOR in both bio-assays. The developed bio-assays allow to gain insight into the βarr2 or G protein recruitment potential of NSO, which may eventually help to better understand why certain opioids are associated with higher toxicity. Adding to the recent discussion about the relevance of the biased agonism concept for opioids, we did not observe a significant bias for any of the evaluated compounds, including TRV130.

A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity.

Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein-biased agonist of the D2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y1995.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y1995.38A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

Atomic-Level Characterization of the Distinct Methadone-Induced Conformational Sampling and Activation Kinetics of the µ-Opioid Receptor by Molecular Simulations

Although commonly associated with the treatment of opioid addiction, and structurally different from morphine, methadone is a synthetic opioid agonist of the µ-opioid receptor (MOR) with demonstrated efficiency in the management of chronic pain owing to its unique pharmacokinetics and pharmacodynamics properties. A recent biophysical study suggests that methadone's unique pharmacological features lie in its ability to stabilize distinct MOR active conformations from those induced by classical opioid drugs or G protein-biased agonists. However, the specific effects of methadone on the conformational sampling and activation kinetics of MOR remain largely uncharacterized at the atomic level. To shed light upon this matter, we explored the methadone-induced conformational space of MOR in an explicit lipid-water environment using ∼0.3 ms of enhanced molecular dynamics (MD) simulations, and compared it to the receptor conformational sampling of previously published ∼0.5 ms of high-throughput MD simulations of MOR bound to the classical opioid drug morphine or the G protein-biased ligand TRV-130. Markov State Models (MSMs) built using these large datasets revealed distinct ligand-induced conformational dynamics and activation kinetics of MOR. Predicted equilibrium probabilities from these MSMs suggest that the three explored ligand-bound MOR complexes differentially populate active, inactive, and metastable regions along their respective activation pathways. Finally, transfer entropy estimates based on information theory were used to identify the distinct structural features that most contribute to the activation-induced conformational changes in the three ligand-bound MOR complexes.

P5995A novel biased S1P1 agonist improves renal and cardiac functions in ZSF1 rats, a model of metabolic syndrome-associated Heart Failure with preserved Ejection Fraction

Abstract Background/Introduction Heart Failure with preserved ejection fraction (HFpEF) is a major cause of death worldwide with currently no approved treatment. Diastolic dysfunction, dyspnea, intolerance to effort, high cardiac filling pressure, and lung congestion coexist with normal ejection fraction in this clinical syndrome. Ageing, obesity, type 2 diabetes, hypertension and renal dysfunction are the main comorbidities found in this heterogeneous group of patients. Microvascular endothelial dysfunction, driven by these risk factors, may be a common link with other aspects of the HFpEF pathogenesis that include oxidative stress, inflammation, cardiomyocyte stiffness/hypertrophy, and myofibroblast accumulation. Sphingosine-1-phosphate type-1 receptor (S1P1), a G protein–coupled receptor highly expressed in endothelial cells, regulates vascular integrity, vascular development and immune cell trafficking. Compound A is a novel G protein-biased S1P1 agonist that lacks functional antagonism and has endothelial-protective properties. Purpose S1P1 activation could promote phosphorylation of endothelial nitric oxide synthase, restoration of endothelial structure and function, and thus diminish cardiac and vascular stiffness, hypertrophy and fibrosis. The aim of this study was to investigate if compound A could improve renal and cardiac functions in a rat model of HFpEF with metabolic syndrome. Methods 65-week-old obese ZSF1 rats were fed a chow diet containing compound A (8 mg/kg/day) or no compound for 4 weeks. Lean ZSF1 and Wistar rats were included in the study as control groups. Urinary protein/creatinine ratio was measured as an index of glomerular injury. Cardiac hypertrophy and function were assessed by two-dimensional and Doppler echocardiography. Total cardiac and atrial weights and pulmonary edema were assessed. Results The obese ZSF1 rat was confirmed as a relevant model of HFpEF with advanced renal dysfunction. These rats showed severe glomerular filtration impairment, left ventricular and atrial hypertrophy and pulmonary edema. Cardiac systolic function, cardiac output and chamber volumes were preserved, diastolic function was impaired, and left ventricular posterior walls and septal thicknesses were increased compared to control groups. Four weeks of compound A treatment reduced urinary protein/creatinine ratio, blunted cardiac and atrial hypertrophy, and partially restored diastolic function. Circulating lymphocytes were not reduced by compound A, confirming that these pharmacological effects were not associated with S1P1 desensitization. Conclusion Compound A, a novel S1P1 agonist with endothelial properties, improves cardiac and renal functions in a rat model of metabolic syndrome-associated HFpEF. Sustained S1P1 activation with compound A may be a promising strategy for HFpEF treatment.

Discovery of Novel Biased Opioid Receptor Ligands through Structure-Based Pharmacophore Virtual Screening and Experiment.

Gi -protein-biased agonists with minimal β-arrestin recruitment represent opportunities to overcome the serious adverse effects of human mu opioid receptor (μ-OR) agonists and developing alternative and safe treatments for pain. In order to discover novel non-morphinan opioid receptor agonists, we applied hierarchical virtual screening of our in-house database against a pharmacophore based on modeling the active conformations of opioid receptors. We discovered an initial hit compound, a novel μ-OR agonist with a pyrazoloisoquinoline scaffold. We applied computational R-group screening to this compound and synthesized 14 derivatives predicted to be the best. Of these, a new Gi -protein-biased compound, 1-{5-(3-chlorophenyl)-7,8-dimethoxy-3-[4-(methylsulfonyl)benzyl]-3H-pyrazolo[3,4-c]isoquinolin-1-yl}-N,N-dimethylmethanamine, showed an EC50 value of 179 nm against the μ-OR. This resulted in significant pain relief for mice in the phase II period of formalin response tests. This study provides a new strategy to identify diverse sets of promising compounds that might prove useful for the development of drugs that target other G-protein-coupled receptors.

A Dual GLP-1/GIP Receptor Agonist Does Not Antagonize Glucagon at Its Receptor but May Act as a Biased Agonist at the GLP-1 Receptor.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism, making their receptors (GLP-1R and GIPR) attractive targets in the treatment of type 2 diabetes mellitus (T2DM). GLP-1R agonists are used clinically to treat T2DM but the use of GIPR agonists remains controversial. Recent studies suggest that simultaneous activation of GLP-1R and GIPR with a single peptide provides superior glycemic control with fewer adverse effects than activation of GLP-1R alone. We investigated the signaling properties of a recently reported dual-incretin receptor agonist (P18). GLP-1R, GIPR, and the closely related glucagon receptor (GCGR) were expressed in HEK-293 cells. Activation of adenylate cyclase via Gαs was monitored using a luciferase-linked reporter gene (CRE-Luc) assay. Arrestin recruitment was monitored using a bioluminescence resonance energy transfer (BRET) assay. GLP-1, GIP, and glucagon displayed exquisite selectivity for their receptors in the CRE-Luc assay. P18 activated GLP-1R with similar potency to GLP-1 and GIPR with higher potency than GIP. Interestingly, P18 was less effective than GLP-1 at recruiting arrestin to GLP-1R and was inactive at GCGR. These data suggest that P18 can act as both a dual-incretin receptor agonist, and as a G protein-biased agonist at GLP-1R.

BPR1M97, a dual mu opioid receptor/nociceptin-orphanin FQ peptide receptor agonist, produces potent antinociceptive effects with safer properties than morphine

There is unmet need to design an analgesic with fewer side effects for severe pain management. Although traditional opioids are the most effective painkillers, they are accompanied by severe adverse responses, such as respiratory depression, constipation symptoms, tolerance, withdrawal, and addiction. We indicated BPR1M97 as a dual mu opioid receptor (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor full agonist and investigated the pharmacology of BPR1M97 in multiple animal models. In vitro studies on BPR1M97 were assessed using cyclic-adenosine monophosphate production, β-arrestin, internalization, and membrane potential assays. In vivo studies were characterized using the tail-flick, tail-clip, lung functional, heart functional, acetone drop, von Frey hair, charcoal meal, glass bead, locomotor activity, conditioned place preference (CPP) and naloxone precipitation tests. BPR1M97 elicited full agonist properties for all cell-based assays tested in MOP-expressing cells. However, it acted as a G protein-biased agonist for NOP. BPR1M97 initiated faster antinociceptive effects at 10 min after subcutaneous injection and elicited better analgesia in cancer-induced pain than morphine. Unlike morphine, BPR1M97 caused less respiratory, cardiovascular, and gastrointestinal dysfunction. In addition, BPR1M97 decreased global activity and induced less withdrawal jumping precipitated by naloxone. Thus, BPR1M97 could serve as a novel small molecule dual receptor agonist for antinociception with fewer side effects than morphine.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

A Biased View of μ-Opioid Receptors?

The field of biased agonism has grown substantially in recent years and the μ-opioid receptor has been one of the most intensively studied receptor targets for developing biased agonists. Yet, despite extensive research efforts, the development of analgesics with reduced adverse effects remains a significant challenge. In this review we discuss the evidence to support the prevailing hypothesis that a G protein-biased agonist at the μ-opioid receptor would be an effective analgesic without the accompanying adverse effects associated with conventional μ-opioid agonists. We also assess the current status of established and novel μ-opioid-receptor ligands that are proposed to be biased ligands. SIGNIFICANCE STATEMENT: The idea that biased agonists at the μ-opioid receptor might provide a therapeutic advantage in terms of producing effective analgesia with fewer adverse effects has driven the design of novel G protein-biased agonists. However, is the desirability of G protein-biased agonists at μ-opioid receptor substantiated by what we know of the physiology and pharmacology of the receptor? Also, do any of the novel biased agonists live up to their initial promise? Here we address these issues by critically examining the evidence that G protein bias really is desirable and also by discussing whether the ligands so far developed are clearly biased in vitro and whether this produces responses in vivo that might be commensurate with such bias.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Defining Structure-Functional Selectivity Relationships (SFSR) for a Class of Non-Catechol Dopamine D1 Receptor Agonists.

G protein-coupled receptors (GPCRs) are capable of downstream signaling through distinct noncanonical pathways such as β-arrestins in addition to the canonical G protein-dependent pathways. GPCR ligands that differentially activate the downstream signaling pathways are termed functionally selective or biased ligands. A class of novel non-catechol G protein-biased agonists of the dopamine D1 receptor (D1R) was recently disclosed. We conducted the first comprehensive structure-functional selectivity relationship study measuring GS and β-arrestin2 recruitment activities focused on four regions of this scaffold, resulting in over 50 analogs with diverse functional selectivity profiles. Some compounds became potent full agonists of β-arrestin2 recruitment, while others displayed enhanced GS bias compared to the starting compound. Pharmacokinetic testing of an analog with an altered functional selectivity profile demonstrated excellent blood-brain barrier penetration. This study provides novel tools for studying ligand bias at D1R and paves the way for developing the next generation of biased D1R ligands.

Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B2 Receptor Antagonist Icatibant Are G Protein Biased Agonists for MRGPRX2 in Mast Cells.

AG-30/5C is an angiogenic host defense peptide that activates human mast cells (MC) via an unknown mechanism. Using short hairpin RNA-silenced human MC line LAD2 and stably transfected RBL-2H3 cells, we demonstrate that AG-30/5C induces MC degranulation via Mas-related G protein-coupled receptor X2 (MRGPRX2). Most G protein-coupled receptors signal via parallel and independent pathways mediated by G proteins and β-arrestins. AG-30/5C and compound 48/80 induced similar maximal MC degranulation via MRGPRX2, which was abolished by pertussis toxin. However, compound 48/80 induced a robust β-arrestin activation as determined by transcriptional activation following arrestin translocation (Tango), but AG-30/5C did not. Overnight culture of MC with compound 48/80 resulted in reduced cell surface MRGPRX2 expression, and this was associated with a significant decrease in subsequent MC degranulation in response to compound 48/80 or AG-30/5C. However, AG-30/5C pretreatment had no effect on cell surface MRGPRX2 expression or degranulation in response to compound 48/80 or AG-30/5C. Icatibant, a bradykinin B2 receptor antagonist, promotes MC degranulation via MRGPRX2 and causes pseudoallergic drug reaction. Icatibant caused MC degranulation via a pertussis toxin-sensitive G protein but did not activate β-arrestin. A screen of the National Institutes of Health Clinical Collection library led to the identification of resveratrol as an inhibitor of MRGPRX2. Resveratrol inhibited compound 48/80-induced Tango and MC degranulation in response to compound 48/80, AG-30/5C, and Icatibant. This study demonstrates the novel finding that AG-30/5C and Icatibant serve as G protein-biased agonists for MRGPRX2, but compound 48/80 signals via both G protein and β-arrestin with distinct differences in receptor regulation.