Allostery and Biased Agonism at Class B G Protein-Coupled

Review pubs.acs.org/CR

Allostery and Biased Agonism at Class B G Protein-Coupled Receptors Denise Wootten,*,† Laurence J. Miller,*,‡ Cassandra Koole,†,§ Arthur Christopoulos,† and Patrick M. Sexton*,† †

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia ‡ Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, United States § Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065, United States ABSTRACT: Class B G protein-coupled receptors (GPCRs) respond to paracrine or endocrine peptide hormones involved in control of bone homeostasis, glucose regulation, satiety, and gastro-intestinal function, as well as pain transmission. These receptors are targets for existing drugs that treat osteoporosis, hypercalcaemia, Paget’s disease, type II diabetes, and obesity and are being actively pursued as targets for numerous other diseases. Exploitation of class B receptors has been limited by difficulties with small molecule drug discovery and development and an under appreciation of factors governing optimal therapeutic efficacy. Recently, there has been increasing awareness of novel attributes of GPCR function that offer new opportunity for drug development. These include the presence of allosteric binding sites on the receptor that can be exploited as drug binding pockets and the ability of individual drugs to enrich subpopulations of receptor conformations to selectively control signaling, a phenomenon termed biased agonism. In this review, current knowledge of biased signaling and small molecule allostery within class B GPCRs is discussed, highlighting areas that have progressed significantly over the past decade, in addition to those that remain largely unexplored with respect to these phenomena.

CONTENTS 1. Introduction 2. Allostery and Biased Agonism 2.1. Allostery 2.2. Biased Agonism 2.3. Intersection between Bias and Allostery 2.4. Quantification of Allostery and Bias 3. Class B GPCRs 3.1. Structural Classification 3.2. Signaling Pleiotropy 3.3. Receptor Dimerization 4. Class B GPCR Biased Agonism 4.1. Secretin Receptor 4.2. Receptors for PTH 4.3. Receptors for PACAP and VIP 4.4. Receptors for CT, amylin, CGRP, and adrenomedullin 4.5. Receptors for Glucagon, GLP-1, GLP-2, and GIP 4.5.1. GLP-1 Receptor 4.5.2. Nonpeptidic-Biased Agonism at the GLP-1 Receptor 4.5.3. GLP-2 Receptor 4.5.4. GIP Receptor 4.5.5. Glucagon Receptor 4.6. GHRH Receptor

© XXXX American Chemical Society

4.7. CRF Receptors 5. Mechanistic Studies of Biased Agonism 6. Allosteric Modulation of Class B GPCRs 7. Summary/Conclusion/Future Directions Author Information Corresponding Authors Notes Biographies Acknowledgments References

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1. INTRODUCTION Class B G protein-coupled receptors (GPCRs) respond to paracrine or endocrine peptide hormones involved in the physiology or pathophysiology of bone homeostasis, glucose regulation, satiety and gastro-intestinal function, as well as pain transmission. As a result, these receptors are targets for existing drugs that treat osteoporosis, hypercalcaemia, Paget’s disease, type II diabetes, and obesity and are being actively pursued as targets for migraine, depression and anxiety, irritable bowel syndrome/Crohn’s disease, cancer and pancreatic cancer

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Special Issue: G-Protein Coupled Receptors Received: January 19, 2016

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DOI: 10.1021/acs.chemrev.6b00049 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Overview of allosteric modulation and biased signaling. (A) Allosteric interactions. (Left-hand panel) Orthosteric (Ortho) and allosteric (Allo) ligands can bind to the free receptor and each may have its own intrinsic efficacy (EO and EA, respectively). However, as these bind to topographically distinct sites, they can also bind concomitantly to yield cooperative effects (depicted by the rheostat). Allosteric interactions can be positive (positive allosteric modulator, PAM), negative (negative allosteric modulator, NAM), or neutral (neutral allosteric ligand, NAL). (A, righthand panel) The concept of probe dependence. As individual ligands bind with different chemical contacts (even if they share an overlapping binding site), there is the potential for the same allosteric modulator to have an orthosteric-ligand specific effect (and vice versa). Depicted are 2 orthosteric ligands (Ortho 1, Ortho 2) with probe-dependent effects of a positive modulator (PAM). In this example, the PAM increases the efficacy of Ortho 1 (E01) to a greater extent than it does for Ortho 2 (E02); the potential for opposite effects on orthosteric ligand efficacy (i.e., PAM vs NAM) also exists. (B) Biased agonism. (Left-hand panel) Two chemically distinct orthosteric agonists, Ortho 1 and Ortho 2, can bind to the receptor in a competitive manner. A comparison of two signaling pathways, P1 and P2, reveals that Ortho 1 has greater efficacy in stimulating P1, relative to P2, whereas Ortho 2 displays the opposite pattern of effect. The differential stimulation of the two pathways can result in distinct cellular function. (Right-hand panel) An example of biased allosteric modulation, with interaction between the allosteric modulator and Ortho 1 changing the quality of the signal, increasing signaling from P2, while decreasing signaling from P1.

diagnostics.1−7 These diseases represent major global health burdens; however, the exploitation of class B receptors has been limited by difficulties with small molecule drug discovery and development and an under appreciation of factors governing optimal therapeutic efficacy. Recently, there has been increasing awareness of novel attributes of GPCR function that offer new opportunity for drug development. These include the presence of allosteric (topographically distinct) binding sites on the receptor, in addition to the classic orthosteric (endogenous ligand) binding site, which can be exploited as drug binding pockets, and the ability of individual drugs to enrich subpopulations of receptor conformations to selectively control signaling, a phenomenon termed biased agonism.8−11 These two phenomena, summarized in Figure 1, are particularly relevant to peptide hormone class B GPCR drug discovery as these receptors are pleiotropically coupled (Table 1) and have broad, diffuse pharmacophores that are not readily mimicked by small molecule drugs. In this review, the present knowledge of biased signaling and small molecule allostery within class B GPCRs is discussed, highlighting areas that have progressed significantly over the past decade, in addition to those that remain largely unexplored with respect to these phenomena.

receptor.12 For many class A GPCRs, including those for the biogenic amines, this site is located within the transmembrane (TM) core of the receptor, however, the location of the orthosteric site can vary greatly between different subclasses of GPCRs and has been localized to different positions within the TM bundle, within the extracellular domain of the receptor or in the interface between these domains. Indeed, the nature of the access and egress from the orthosteric site can vary; while some pockets are accessible via free diffusion, others are located deeper within the receptor core and entry to these may be via the lipid bilayer or via an extracellular vestibule that forms an intermediate binding site. Allosteric binding sites are spatially distinct from the orthosteric ligand-binding site, such that the potential exists for orthosteric and allosteric ligands to bind simultaneously. Each of these ligands can interact with the free receptor and will have characteristic properties when bound, including an affinity for the receptor and varying degrees of efficacy for activation of effector proteins or recruitment of regulatory proteins (Figure 1A). However, the ability of an orthosteric and an allosteric ligand to cobind allows each of the ligands to influence the behavior of the other, and this effect is termed cooperativity.8,10,13 This cooperative effect can be on the affinity of the ligand or on the efficacy of the ligand to alter signaling of the receptor. Moreover, effects on binding are reciprocal between the orthosteric and allosteric ligands. In effect, the binding of an allosteric ligand changes the conformational landscape of the receptor such that it can be considered a unique receptor. Positive allosteric modulators (PAMs) produce a net enhanced effect on the receptor function, while PAMs with intrinsic activity are termed PAM-agos; negative allosteric modulators (NAMs) reduce receptor function, while neutral allosteric ligands (NALs) bind the allosteric site on the receptor but do not alter receptor function.13

2. ALLOSTERY AND BIASED AGONISM 2.1. Allostery

GPCRs are natural allosteric proteins that translate extracellular binding events to activation of intracellular effectors via conformational rearrangements, enabling the engagement of effectors, classically G proteins. As such, modulation of the conformational landscape, be it via additional protein−protein interactions or via interaction with drugs, can alter the way the receptor responds to its endogenous ligand(s). The orthosteric binding site of a protein is defined as the canonical binding site of the recognized natural ligand of the B


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Table 1. Summary of Class B GPCRs. Adapted from Bortolato et al.,1 Culhane et al.,7 Hoare et al.,96 and Archbold et al.283

parathyroid hormone (PTH) and PTH-related protein (PTHrP) for the PTH1 receptor, as examples. As such, the response of an allosteric drug will depend not only on the intrinsic affinity and efficacy of the drug but also on the nature of cooperativity of the drug with each of the endogenous ligands and the concentrations of ligands at the site of action. Furthermore, the quality of the orthosteric drug response can change with the cooperative effects often varying across different effector and regulatory pathways, with the exception of the simplest cases where cooperativity follows the “two-state” characteristics of the classic Monod, Wyman, and Changeux model.14 These latter properties create significant challenges for allosteric drug discovery but nonetheless provide an unprecedented opportunity to sculpt cellular response for therapeutic benefit.

In the context of drug discovery, allosteric ligands offer both opportunities and challenges. These have been reviewed in detail elsewhere,8,13−17 so they are only briefly summarized below. As allosteric sites are generally less conserved than orthosteric sites, there is the potential for these to present novel pockets for drug development, often with greater subtype selectivity than drugs targeting the conserved orthosteric binding site. Allosteric drugs can also provide mechanismbased safety in overdose, as the magnitude of the on-target drug effect is limited by the cooperativity, regardless of drug concentration. However, the effect of an allosteric drug will be specific to the orthosteric ligand present, a phenomenon termed “probe dependence”.16 This is of particular importance for class B GPCRs, where many receptors respond to multiple endogenous ligands, including, glucagon-like peptide-1 (GLP1), oxyntomodulin, and glucagon for the GLP-1 receptor, and C


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Figure 2. Web of bias illustrating distinctions in the pattern of signaling of GLP-1 receptor agonists. (A) High affinity peptide agonists, (B) lowaffinity peptide agonists, (C) nonpeptidic agonists interacting with the extracellular face of the receptor, and (D) nonpeptidic agonists interacting with the intracellular face of the receptor. The “web of bias” plots ΔΔτ/KA values on a logarithmic scale for each ligand and for each signaling pathway tested. Determination of these values requires normalization to a reference ligand (GLP-17−36 amide in this example) and a reference pathway (cAMP accumulation). The plots do not provide information on absolute potency but on relative efficacy for signaling of individual pathways in comparison with that for cAMP. Data are from refs 83, 159, and 170. Data points plotted as ● indicate statistically significant bias relative to GLP-1 and cAMP, whereas data plotted as ▲ (at a value of −100) indicate that no significant signal could be detected for a particular pathway.

ellular-signal regulated kinase 1/2 (ERK1/2).24 Biased agonism is currently a major paradigm in GPCR drug discovery, with multiple companies seeking to separate on-target therapeutic efficacy from on-target side effects. Examples of this include biased angiotensin peptides, biased opioids, and biased adenosine receptor compounds.20,25 The increasing evidence for biased agonism has arisen as investigators have realized that most, if not all, GPCRs can couple to multiple effectors, including multiple G proteins, arrestins, as well as other scaffolding and regulatory proteins. This has led to concurrent investigation of multiple signaling end points, the most widely studied end point being recruitment of arrestin proteins, in addition to the canonical signaling pathways, revealing distinct ligand responses.20,26,27

2.2. Biased Agonism

It is increasingly recognized that the binding of individual ligands to a GPCR can elicit a distinct spectra of responses, even when acting via a common binding pocket (Figure 1B). As each ligand is chemically distinct, they can form unique contacts or combinations of contacts with the receptor, and this is true for peptide ligands with differing amino acid sequences or small molecule drugs. These distinctive ligand interaction patterns govern both the kinetics of binding and how binding events are propagated through the GPCR and the conformational ensembles available to interact with signaling and regulatory proteins.9,18,19 Differences in ligand responses may occur via differential recruitment of signaling proteins, including G proteins, or they may alter the interaction with regulatory or scaffolding proteins. The latter events can change receptor trafficking and compartmentalization that may in turn alter the nature of cellular signaling. Similarly, individual ligands can drive differential phosphorylation of the receptor via GPCR kinases (GRKs) or second messenger kinases to engender specific interactions with arrestins and other proteins.9,20−23 This phenomenon of biased agonism (also known as liganddirected signaling bias or functional selectivity) is believed to underlie differences in therapeutic efficacy of existing drugs, including β-adrenoceptor antagonists that inhibit canonical Gαs-mediated cAMP formation but induce selective effects on mitogen-activated protein (MAP) kinases, including extrac-

2.3. Intersection between Bias and Allostery

Both biased agonism and allosteric modulation of a receptor are mediated by changes to the conformational ensemble sampled by the receptor in the presence of individual agonists or by allosteric drugs or proteins. As allosteric agonists bind to topographically distinct sites, it is not surprising that these types of ligands are often observed to display distinct signaling/ regulatory bias relative to the natural, orthosteric ligand. Similarly, as allosteric drugs alter the conformational landscape of the receptor, they can change the signaling profile of the endogenous ligand(s), and this is true for both pure modulators D


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and “PAM-Agos” (Figure 1B). The exceptions to this are PAMs that only alter the affinity of the orthosteric ligand but do not alter efficacy.

receptor. The 7 transmembrane (TM) domain core shares < 10% homology with class A or class C GPCRs and ∼30% conservation within the class B subfamily. Two X-ray crystal structures have been solved for the TM domain of class B receptors, the glucagon receptor53 and the CRF-1 receptor.54 Consistent with the differences in amino acid sequence between class B GPCRs and other receptor classes, the TM domain has a distinct structural organization with a wide-open extracellular face, at least in the apo/inactive form that presumably favors interaction of the receptor core with the N-terminal domain of peptide agonists. Similarities and differences between the TM domains between different GPCR subclasses have been reviewed extensively elsewhere.1,7,55,56 To enable direct comparison of amino acids within the TM core of class B receptors, a numbering scheme has been developed57 based on that utilized by Ballesteros and Weinstein58 for class A receptors. In this scheme, the most conserved residue within each TM helix is designated the number “50” with numbering of other TM residues sequentially from this residue. Thus, the most conserved residue in TM2 will be numbered “2.50”, those preceding it 2.49, 2.48 etc., and those following it 2.51, 2.52, etc. This numbering convention allows direct comparison of functional importance of individual residues across the class B subfamily. Throughout this review, superscripted Wootten numbering57 for residues within the TM domain will follow receptor amino acid numbers.

2.4. Quantification of Allostery and Bias

The exploitation of both biased agonism and allostery for therapeutic development requires quantitative frameworks to define key parameters of drug action. In the case of biased agonism, this needs to be distinguished from system-dependent parameters, including partial agonism. Similarly, although gross changes in signal bias can be recognized as a reversal of efficacy or potency,9,15,28 more subtle effects require a quantitative framework to identify true differences in ligand response. While a range of quantitative models exist,11,29−32 the most robust method to quantify efficacy is the operational model;11 here the transduction ratio of τ/Ka is used to define strength of signaling of an individual ligand for a specific pathway, and this ratio can be determined from classic concentration−response experiments.31 τ is the operational term for efficacy, relating receptor occupancy to response, while Ka is the functional affinity (expressed as an equilibrium dissociation constant) for the receptor-effector complex driving signaling. Normalizing responses to a reference ligand (normally the main endogenous agonist) enables differences in signaling profiles between agonists to be calculated. Further normalization to a reference pathway enables broad signaling profiles to be readily displayed for multiple ligands (Figure 2). Although mechanistic models describing allosteric interactions can be developed, these are often too complex to derive meaningful information on key parameters for drug development from routine experiments.33 As such, we have adopted an operational model of allostery that can parsimoniously describe the key parameters of drug action. These are Kb, the affinity (dissociation constant) of the allosteric ligand for the free receptor, α, the cooperative effect on affinity, β, the modulatory effect on efficacy (that needs to be independently derived for each signaling pathway), and intrinsic efficacy (τ) for each pathway.

3.2. Signaling Pleiotropy

The canonical signaling pathway for class B GPCRs is Gαsmediated production of cAMP. However, all receptors couple promiscuously to other effectors, including other G proteins, scaffolding proteins, including arrestins, and various regulatory and trafficking proteins,3−7 leading to activation of a diverse array of downstream effectors including multiple second messenger kinases [e.g., protein kinase (PK) A, PKC, PKB], MAP kinases (e.g., ERK1/2, p38), transactivation of tyrosine kinase receptors, opening of ion channels, as well as other effectors controlling gene transcription. This leads to a great diversity in cellular responses that are often context/tissue dependent and may include distinct signaling from different cellular compartments (e.g., lipid rafts or endosomal compartments).59,60 It is the cumulative sum of all these pathways that controls specific cellular responses. Clear roles for individual signaling pathways have been described for specific cell types, including requirement for cAMP for GLP-1-mediated insulin secretion, PTH-mediated Ca2+ and phosphate mobilization, and vasoactive intestinal peptide (VIP)-mediated smooth muscle relaxation. The great diversity in cellular response provides fertile ground for potential drug development for the selective control of cellular function. However, in many/most cases the link between signaling and physiological effect is still poorly or only partially resolved and requires better understanding for effective exploitation of biased ligands. Nonetheless, the realization that biased agonism is likely to be commonplace has led to a redesign of experimental approaches to drug development such that measurement of multiple end points is now routinely incorporated into many drug discovery programs, leading to an increase in the number of tools (potential biased ligands, genetically and chemogenetically modified animals) to probe how different signaling profiles are linked to cellular function. This is discussed below in the context of specific class B receptors.

3. CLASS B GPCRS 3.1. Structural Classification

Class B receptors are a small subfamily of 15 GPCRs (20 recognized phenotypes with multiple novel phenotypes arising from GPCR/receptor activity modifying protein (RAMP) heterodimerization) that bind physiologically important peptides (Table 1). Structurally, class B receptors range in length from approximately 450 to 600 amino acids in humans. They have a relatively large N-terminal extracellular domain (ECD) of 120 to 200 amino acids that include six conserved disulfidebonded cysteines. While amino acid homology between receptors is relatively limited within this domain, all the ECDs display similar tertiary structure with a conserved internal fold, termed a “sushi domain” that is supported by the disulfide-bonded cysteines. The structure of the ECD has been solved at high resolution for the glucagon receptor,34,35 GLP-1 receptor,36,37 gastric inhibitory polypeptide (GIP) receptor,38,39 PTH-1 receptor,40−42 corticotropin releasing factor (CRF)-1 and CRF-2 receptors,43−47 pituitary adenylate cyclase (PAC)-1 receptor,48,49 and the calcitonin receptor-like receptor (CLR), the latter in complex with RAMP1 or RAMP2 to yield the ECD of the calcitonin gene-related peptide (CGRP) and adrenomedullin (AM)-1 receptors, respectively.50−52 Most of these structures have been cocrystallized with C-terminal fragments of the cognate peptides for the E


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3.3. Receptor Dimerization

biased agonism where the latter is observed. The impact of class B GPCR homodimerization on recruitment of scaffolding and regulatory proteins has yet to be studied. Interestingly, disruption of the GLP-1 receptor dimer interface abolishes cAMP responses of the allosteric agonists, Novo Nordisk’s 6,7dichloro2-methylsulfonyl-3-tert-butylaminoquinoxaline (NN compound 2) and 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6(trifluoromethyl) pyrimidine (BETP/compound B), while ERK1/2 phosphorylation is relatively preserved. These small molecule ligands act via the intracellular face of the receptor, forming a covalent attachment to Cys347 (intracellular loop (ICL) 3)72 and thus are mechanistically distinct in their mode of receptor activation, requiring the homodimeric form of the receptor for efficient Gαs coupling. Nonetheless, despite abolition of their intrinsic activity under monomeric conditions, the allosteric modulation of peptide-mediated cAMP formation is preserved,71 indicating that the allosteric effect is likely occurring in cis and that potentiation of cAMP signaling can occur under conditions of diminished homodimerization. This may be an important consideration for future studies, as the absolute stoichiometry of receptor protomers to G proteins and the structural basis underpinning their interaction has not yet been experimentally defined. In addition to forming homodimeric complexes, heteroreceptor complexes involving more than one member of the class B receptor family have also been reported.64−66,73−75 Use of resonance transfer techniques and coimmunoprecipitation has provided evidence that most class B receptors can coassociate with each other. In one study, the secretin receptor demonstrated associations with VIP receptor (VPAC)-1, VPAC2, growth hormone releasing hormone (GHRH) receptor, GLP-1 receptor, GLP-2 receptor, CLR, PTH1 receptor, and PTH2 receptor, but not the CTR.64 On the basis of our understanding of the lipid-exposed face of TM4 of several of the class B GPCRs being the predominant determinant of homodimerization, it is likely that the same interface is active for class B GPCR heterodimerization events, although this has not been directly established. Inconsistencies with respect to the ligand binding on these complexes impacts data interpretation; it is likely that ligand-induced changes in conformation of the component receptors have produced at least some of these observations. The impact of heteroreceptor association within this family on signaling is unclear, as no major changes have been reported for agonist-stimulated signaling events. With all class B GPCRs known to signal via similar pathways, where Gαs coupling and cAMP generation is most prominent, this is perhaps not surprising. The most obvious functional impact of receptor heteroassociation has been related to receptor trafficking, exemplified by an intracellularly trapped GIP receptor mutant lacking glycosylation sites that can be functionally restored by coexpression with the GLP-1 receptor.75 While agonist occupation typically stimulates internalization of class B GPCRs, the impact of occupation of one receptor in a heterocomplex with another has been inconsistent, sometimes resulting in cross-receptor internalization and other times in the absence of effect. This may, in part, reflect the stoichiometry of coexpression and the presence of heterogeneity in the system (a conglomerate of monomers, homo-, and heterodimers). Nonetheless, despite the importance of homodimerization observed for class B receptor signaling, heterodimer formation should not be overlooked, as it has the propensity to significantly impact receptor function.

The association of GPCRs to form oligomeric complexes within the plasma membrane is now well-recognized and accepted, although the structural basis, stability, and functional implications of such complexes are quite varied across this superfamily.61 Dimerization of class C GPCRs is pervasive and most stable, often required for ligand recognition, selectivity, and signaling, with some complexes even defined by covalent disulfide bonds through amino-terminal domains of involved receptor protomers.62 Currently, there are no recognized rules for the association of class A GPCRs, with all combinations of occurrence, functional impact, and stability having been described for different family members, and with these complexes often oligomeric rather than dimeric, and involving multiple potential interfaces.61 The theme for class B GPCR association seems to be intermediate between the other more dominant classes of GPCRs. All members of the class B family have been described to form homodimeric complexes, with many also capable of forming heterodimeric complexes.63−66 Of note, the complexes that have been structurally defined dimerize along the lipid face of TM467 and, as expected from a single defined interface, these do not form higher-order oligomeric complexes.68 While ligand binding does not appear to affect the formation or stability of these complexes,65,67 they seem to be more stable than the often transient complexes that may be formed for class A GPCRS and appear to be less stable than the dimeric complexes of class C GPCRs. The stability of these complexes varies among the members studied, and the functional impact has also varied quantitatively. Where it has been studied, via disruption of the dimer interface by either mutation of key residues in TM4 or via competition for the dimeric interface with TM4 peptides, homodimeric complexes of class B GPCRs are required to facilitate high affinity binding and potency of natural agonists.67 This has been particularly well-studied for the secretin receptor, where disruption of the dimeric interface causes nearly a 100-fold decrease in secretin potency for cAMP formation.67 Cysteine substitution and cross-linking of external facing TM4 amino acids revealed key residues that are involved in high affinity secretin binding, although functional studies could not be performed.69 Nonetheless, dimerization is also observed to be important for Gαsmediated cAMP formation via the calcitonin receptor (CTR)70 and GLP-1 receptor,71 albeit that the magnitude of effect was less (∼10-fold) than that seen for the secretin receptor.67 As such, dimerization of class B receptors appears to have an important contribution to efficacy of Gαs-mediated signaling. There has been no systematic evaluation of the impact of class B GPCR dimerization on the association with specific G proteins, although as a family, Gαs association is typically most stable and dominant after agonist occupation. Gαi and Gαq association is also characteristic of this family, although typically much higher concentrations of the natural agonist are necessary to stabilize such complexes. There has only been a single report of the impact of the dimerization of members of this family on the function of orthosteric and allosteric agonist ligands across different signaling end points.71 In this study, following mutational disruption of the dimeric TM4 interface, there was preferential loss of intracellular Ca2+ (iCa2+) mobilization in response to peptide agonists of the GLP-1 receptor, including GLP-1(7−36)NH2, exendin-4, and oxyntomodulin, over that for cAMP formation,71 albeit that the latter was also decreased, as noted above. This suggests dimerization may also contribute to differential coupling of the receptor and thus to F


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the PTH2 receptor is activated by a precursor-derived PTH peptide (TIP39) and PTH but poorly activated by PTHrP. Of note, TIP39 is a weak antagonist of the PTH1 receptor. All of these naturally occurring peptides couple most strongly to cAMP responses (acting through Gαs) and are much less potent agonists of iCa2+ (Gαq). Gαs association results in increased cAMP and activation of PKA that is known to activate MAP kinases, β-catenin, and cAMP reponse elementbinding protein (CREB). This pathway is believed to be responsible for both anabolic and catabolic actions on bone, regulation of phosphate excretion in renal proximal tubules, and regulation of cytokine secretion at both of the most prominent target organs, bone, and kidney.6,90−92 Gαq association results in phospholipase (PL) C activation and increased iCa2+, that also results in activation of MAP kinases and ERK1/2. The Gαq-coupled pathway is also believed to be responsible for activation of L-type voltage-gated Ca2+ channels. A direct interaction of low-density lipoprotein-related protein-6 (LRP6)93 and disheveled94 has been observed with the PTH receptor, the latter providing another regulatory input into the Wnt pathway affecting β-catenin. Agonists of these receptors also stimulate β-arrestin translocation95 to mediate both receptor desensitization and regulate independent signaling events. The naturally occurring PTH peptides are thought to signal similarly to each other and, therefore, by definition, are not biased, although this may be due to limited examination of signaling end points to date. However, many analogues have been studied to examine structure−activity relationships. Like most natural peptide ligands of receptors in this family, the critical determinants of biological activity are predominantly located in the amino terminus of the ligands, a region believed to interact with the receptor core helical bundle.96 The midregion and carboxy-terminal portions of the ligands tend to form alpha helices with a hydrophobic face that docks within a cleft in the receptor amino terminus, providing much of the binding energy and binding affinity. Like most natural ligands for receptors in this family, progressive truncation of the amino terminus of PTH peptides gradually decreases and ultimately eliminates biological activity.97 Modifications of the amino terminus are described to preferentially activate (bias) signaling through Gαs over signaling through Gαq, but this may also reflect the loss in potency of the ligands and sensitivity of the assays. A particularly interesting analogue of PTH has been reported as the best current candidate for therapeutically relevant bias.98 This peptide, [(D-Trp12, Tyr34)bPTH(7−34)], is reported to act as an inverse agonist of PTH-stimulated cAMP responses, while stimulating the translocation of β-arrestin and stimulating ERK1/2 in osteoblasts. In mice, this peptide increases lumbar spine bone mineral density, trabecular volume, thickness, and number but does not initiate osteoclast recruitment.98 These effects were absent in β-arrestin-2-null mice, consistent with a biased mode of action. However, this apparent biased response may be limited to a particular cellular environment, with no activation of the pathways attributed to β-arrestin observed in transfected HEK-293 or CHO cells or in the human osteoblastic cell line, U2OS.91,99 Nonetheless, the pattern of β-arrestin bias observed at this receptor in a physiological setting has a theoretical advantage in osteoporosis, since this has been suggested to mediate the anabolic bone effect of PTH, while reducing bone resorption associated with a PTH-like full agonist.100

GPCR oligomeric complexes are not limited to the involvement of structurally related protomers but have also been documented across classes.76,77 The association of the class B secretin receptor with the class A angiotensin receptor has been shown to have physiological significance for drinking behavior. In this context, angiotensin appears to predominantly signal via cAMP, rather than stimulating Ca2+ mobilization, suggesting that heterodimerization with the secretin receptor could introduce bias into the system.77 In addition to receptor−receptor interactions, many class B receptors can heterodimerize with RAMPs. This family of three single transmembrane spanning proteins was originally identified as requisite partners of the CLR to form receptors for CGRP (CLR/RAMP1) and adrenomedullin (AM1, CLR/ RAMP2; AM2, CLR/RAMP3).78 They have subsequently been shown to interact with the CTR to generate three forms of amylin receptor (AMY1, CT/RAMP1; AMY2, CT/RAMP2; and AMY3, CT/RAMP3).79,80 Currently, at least nine class B receptors are reported to interact with at least one of the RAMPs, as determined by the ability of receptors to translocate RAMPs to the cell surface: CLR, CTR, CRF-1, VPAC1, VPAC2, secretin, PTH1, PTH2, and the glucagon receptor (Table 1).81 With the exception of CLR and CTR, in the majority of cases, the functional importance of RAMP/class B GPCR heterodimers is unclear, although there is accumulating evidence for a role in signaling initiated by peptides (see below) and as such could contribute to observed bias in some systems.

4. CLASS B GPCR BIASED AGONISM As noted above (Table 1), many class B receptors can respond to multiple endogenous ligands and thus there is significant potential for signaling bias between these different ligands to contribute to control of physiological responses. One of the first published cases of biased agonism was on the class B, PAC1 receptor, where differential responses to PACAP-27 and PACAP-38 were observed.82 More recently, biased agonism of endogenous orthosteric ligands has also been observed for the GLP-1 receptor.83 These are discussed in more detail below. 4.1. Secretin Receptor

To date, only a single form of the secretin receptor84 and a single endogenous ligand for this receptor85 have been recognized and described. The secretin receptor was the first member of this family identified and is prototypic of the family. The secretin receptor signals principally through Gαs coupling, while high concentrations of natural agonist also mediate Gαq coupling. The most potent signaling responses involve cAMP and its recognized impact on other cellular signaling components, while the iCa2+ response occurs with approximately 100-fold lower potency. Systematic evaluation of G protein-coupling through receptor mutation found determinants in the cytosolic face that differentially affect coupling with Gαs and Gαq.86 In addition, secretin receptor agonists are known to stimulate receptor phosphorylation, β-arrestin translocation, and subsequent receptor internalization.87,88 To date, no small molecule ligands acting allosterically have been described for this receptor. As noted above, the complex of secretin and angiotensin receptors can have a disproportionate impact on cAMP over Ca2+ signaling.77 4.2. Receptors for PTH

There are two structurally related class B GPCRs that bind and are activated by parathyroid PTH and related peptides.89 The PTH1 receptor responds similarly to PTH and PTHrP, while G


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changing the selectivity for PACAP-27 and PACAP-38 and their ability to stimulate a Ca2+ response.117,118 Another variant involves sequence changes in TM2 and TM4, described as having no effect on cAMP or Ca2+ responses to PACAP but resulting in influx of Ca2+ through L-type voltage-sensitive Ca2+ channels not normally observed.119 Of the naturally occurring peptide ligands for these receptors, there are differences in binding affinities and potencies to stimulate signaling. The PAC1 receptor responds much better to PACAP peptides than to VIP. pKi values range from 8.8 to 9.0 for PACAP-38, 8.5 for PACAP-27 and 6.0−6.3 for VIP. Of note, the short form of PAC1 recognizes VIP much better than PACAP peptides, evident by a pKi of 8.4 for VIP. VPAC1 recognizes all of these peptides quite well, with pKi values of 8.5−9.8 for VIP, 8.9 for PACAP-27, and 8.2 for PACAP-38. PHI, PHM, and PHV also recognize this receptor with pKi in the 6 range. VPAC2 recognizes VIP better than PACAP peptides, with pKi values of 7.8−8.8 for VIP, 7.7−9.3 for PACAP-38, and 7.6−8.0 for PACAP-27. PHI and PHV recognize this receptor better than the other receptors, with pKi values of 7.5 and 8.8, respectively. Modification of these natural peptide ligands, as well as other unrelated peptides, yields more selective agonists of this group of receptors. Of the analogues widely studied, the most selective peptide ligand for the PAC1 receptor is maxadilan, isolated from sand fly salivary gland with no sequence homology to either PACAP or VIP, yet with a pEC50 value of 9.2.120 A VIP analogue with Ala in positions 11, 22, and 28 is currently the most selective agonist for the VPAC1 receptor,121 while Ro 25−1553, a cyclic analogue of VIP,122 and Bay 55−9837 [(Lys15, Arg16, Leu27)VIP(1−7)/GRF(8−27)]123 are the most selective ligands reported for the VPAC2 receptor. The relative abilities of these agonists to stimulate the various signaling pathways as a measure of possible signaling bias have not yet been explored. Recently, an extensive systematic structure−activity series including 46 conformationally restrained analogues was reported, with each peptide evaluated for binding, cAMP, and inositol phosphate responses.124 Fifteen of these analogues had increased selectivity for the PAC1 receptor over the VPAC1 receptor, improving binding up to 778-fold and 13 of these were agonists. The most selective agonist analogue was a PACAP(1−38) variant with Iac1 (4-imidazole acrylic acid), Ala16, Ala17, and D-Lys38 modifications. Some analogues also markedly reduced the inositol phosphate responses relative to the cAMP responses by up to 103-fold. Twenty-nine of these analogues had increased selectivity for the PAC1 receptor over the VPAC2 receptor, the most selective increasing 806-fold [Ala22(PACAP(1−38)]. This provides highly useful new insight into the determinants for selectivity and biological activity among these peptides. In addition to differences in signaling arising from individual peptides activating the receptor, for at least one of these receptors, VPAC1, an additional level of control can occur via the interaction of this receptor with select members of the RAMP family. VPAC1 receptors interact with all three RAMPs,125 and while the significance of the interaction with RAMP1 and RAMP3 has yet to be identified, coexpression of RAMP2 and VPAC1, at least in recombinant systems, selectively augments VIP-mediated phosphoinositide hydrolysis, without altering cAMP signaling, suggesting that RAMPs can alter the bias of the receptor.

Differences in spatial and temporal aspects of signaling have recently been recognized as another explanation for apparent differences in functional profiles stimulated by a given agonist. The PTH receptor is one in which endosomal signaling has been postulated to exist.101,102 It is not clear whether the responses from an intracellular compartment might differ from those originating at the plasma membrane, but this could provide another mechanism for apparent signaling bias. This might be particularly relevant to the long-acting PTH analogues developed in efforts to improve therapeutic efficacy. Currently, two forms of PTH are approved for clinical use, recombinant human PTH(1−84) and the amino-terminal fragment PTH(1−34). While no particular bias has been reported between these two forms of this hormone, it is possible that future allosteric modulators could have differential impact on biological effects of these two forms of PTH. Additionally, a number of PTH receptor agonists are in the drug development pipeline and may reach clinical use,103 including PTH analogues that can be administered orally, transdermally, or via inhalation. Examples of such analogs include the PTH(1−31) fragment that has been studied for both oral and subcutaneous administration.104 Additionally, a PTH(1−31) analogue with Leu27 and cyclo(Glu22-Lys26) modification is also being studied for inhalation-based therapy. Other injectable forms include Pro-Pro[Arg11]PTH(1−34)Pro-Pro-Asp, a PTH(1−34) analogue with Leu8, Asp10, Lys11, Ala16, Gln18, Thr33, and Ala34 modifications, and a cyclic analogue of PTH(1−17) with AcC1, Aib3, Leu8, Gln10, Har11, Ala12, Trp14, and Asp17 modifications.105−107 Although already used therapeutically, PTH(1−34) continues to be studied, with recent progress using a transdermal patch showing promise,108 while the β-arrestin-biased analogue described above, [(DTrp12, Tyr34)bPTH(7−34)], is being evaluated in preclinical studies. In addition to PTH analogues, fragments of PTHrP, are also undergoing preclinical studies and clinical trials. These include amino-terminal fragments, PTHrP(1−34), PTHrP(1−36), PTHrP(1−74), and PTHrP(1−84),109,110 as well as RS66271, an analogue of PTHrP(1−34) in which a model amphipathic α-helical decapeptide is inserted in place of residues 22−31, reducing affinity and traditional activity (presumably by reducing G protein coupling), while enhancing anabolic activity.111,112 Furthermore, a carboxyl-terminal fragment, PTHrP(107−111), is also being studied.104 4.3. Receptors for PACAP and VIP

The endogenous peptide ligands for this group of receptors include PACAP-38 and PACAP-27 encoded by the ADCYAP1R1 gene, and VIP, peptide His Ile/Met (PHI/PHM), and peptide His Val (PHV) encoded by the VIP gene.4,113 Three receptors bind and respond to PACAP, the PAC1, VPAC1, and VPAC2 receptors, with the latter two also responding to VIP.4,113 Several splice variants of PAC1 have been described in different species, leading to variation in sequence of the ICL3, with a short (S) variant and hip or hop variants that include cassettes of 27 or 28 residues. One short variant has been reported to respond to both PACAP-38 and PACAP-27, with strong cAMP responses, yet only PACAP-38 stimulates IP3 responses at this form of the receptor.82,114,115 The inclusion of the hip cassette has been reported to be associated with reduced cAMP responses and absent Ca2+ responses, likely mediated by affecting G protein-coupling.116 A shortened amino-terminal tail variant has also been described, H


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4.4. Receptors for CT, amylin, CGRP, and adrenomedullin

and AMY3 receptors, compared with CTRa alone, for formation of cAMP is paralleled by a similar increase in amylin binding affinity.138 In contrast, only a 2- to 5-fold induction of amylin potency was seen for iCa2+ mobilization or activation of ERK1/2. Additionally, in COS-7 cells, the increase in amylin potency for Ca2+ mobilization was 2-fold greater for AMY3 receptors over that induced at AMY1 receptors and this paralleled the relative capacity of overexpression of Gαq proteins to augment induction of high affinity 125I-amylin binding. Collectively, these data are consistent with RAMPcomplexed receptors having a different signaling profile to CTR expressed in the absence of RAMPs, and this is likely due to effects of the RAMP on G protein-coupling efficiency. A direct involvement of RAMPs in G protein-coupling efficiency of CTcomplexed receptors is supported by RAMP C-terminal deletion or exchange chimera experiments. Deletion of the short RAMP C-terminus reduces induction of AMY phenotype and CTR-mediated translocation to the cell surface;139 these effects could be partially reversed by overexpression of Gαs, suggesting the RAMP C-terminus directly contributes to G protein-coupling efficiency of AMY receptors. Interestingly, exchange of the C-terminus between RAMP1 and RAMP2 altered ligand specificity, with CGRP potency linked to the presence of the RAMP1 C-terminus, providing evidence of long-range allosteric interactions between the intracellular face of the CTR/RAMP complex and the peptide-binding site that involve interaction with intracellular effectors.140 Unlike the CTR, CLR cannot function independently of RAMPs, forming receptors for CGRP and adrenomedullin only when coexpressed with RAMP1 and RAMP2 or RAMP3, respectively.78,81,126 These receptor heteromers also interact with an additional protein, receptor component protein (RCP), critical for efficient coupling of the receptor to Gαs.141,142 As such, the expression of RCP is likely to affect signaling via this subfamily of receptors. Both CGRP and AM receptors exhibit pleiotropic coupling that can arise from both G proteindependent and -independent effector coupling, in turn contributing to a diverse range of physiological functions, including regulation of pain and vascular tone (CGRP), lymphogenesis, cardiac, and neuroprotection (adrenomedullin).143−146 However, the link between specific proximal effector coupling events and physiological signaling is not fully resolved. The CGRP receptor has high affinity for αCGRP (also known as CGRP1) and β-CGRP (CGRP2) peptides but lower affinity for adrenomedullin, adrenomedullin2, and weak affinity/potency for other peptides. To date, a detailed, multipathway comparison of signaling has not been performed for different endogenous ligands of the CGRP or AM receptors, and as such, no data on potential for biased agonism for natural ligands is available. Considerable work has been performed analyzing structure−function relationships of CGRP peptide analogues;147 however, these have been mostly described in the context of the canonical cAMP pathway, with limited work examining effects across different pathways. An intriguing study that looked at amino acid substitutions within the N-terminus of CGRP provided evidence for differential effects on cAMP formation versus β-arrestin translocation, with disparate effects on potency and/or Emax responses for the two pathways, which appeared greatest for amino acids 5 or 6.148 However, these responses were measured in different cell types (SK-N-MC for cAMP formation; CHO-K1 for β-arrestin translocation), making clear conclusions about introduction of bias difficult. Nonetheless, the data support the potential for

The calcitonin (CT) family of peptides bind to two class B GPCRs, alone (CTR) or in combination with one of the RAMPs (CTR and CLR) (Table 1).81,126 Each of these receptors can bind other members of the peptide family to varying extents. CTR alone responds with high affinity to CT but lower affinity to amylin and CGRP, with limited affinity for adrenomedullin or adrenomedullin-2 (also known as intermedin). The human CTR has two major splice variants that differ by 16 amino acids in ICL1 (CTRa, insert negative; CTRb, insert positive), altering signaling and regulatory responses of the receptor.127,128 In addition, the receptor is subject to a major single nucleotide polymorphism encoding either a Leu or Pro at amino acid 447 in the insert negative form of the receptor.129,130 The vast majority of studies have been conducted with the CTRa. Expressed alone, the CTR has high affinity for CT but lower affinity for related peptides.79,126 The CTR can activate a broad array of pathways across a wide variety of cell types,131−133 although the direct link to proximal effectors is not well-resolved in most cases. In osteoclasts, both Gαs/PKA and Gαq/PKC pathways have been implicated in inhibition of cellular function, in a species-dependent manner,134 but it is likely that other signaling effectors are also involved. There has been very limited assessment on whether biased agonism occurs at the CTR. In an intriguing study, Watson and colleagues135 examined the impact of overexpression of different G protein α-subunits on responses of different species of CT, CGRP, or amylin peptides. In that study, overexpression of Gαs led to host cell-dependent shifts in iCa2+ potency ratios, between different peptides (e.g., rat CT and rat amylin or porcine CT and rat amylin), indicative of agonist selective arrays of conformational states that differentially interact with G proteins. In a recent study, Andreassen et al.136 demonstrated distinct responses to human CT (hCT) and the clinically used salmon CT (sCT). sCT undergoes a slow transition to a pseudo irreversible state137 and thus has a distinct kinetic profile of interaction with the CTR to that of hCT. In short-term assays of cAMP signaling or β-arrestin recruitment, hCT and sCT had similar responses, but while hCT responses were lost relatively rapidly, sCT responses persisted for up to 72 h.136 While some of the persistent response was related to maintenance of receptor occupancy at the cell surface, acid washing did not fully abrogate sCT responses, indicating that some signaling arose from intracellular compartments. These data support agonist-specific differences in signaling that are at least partially related to differences in the binding kinetics of the ligands, and to differences in agonist-induced receptor trafficking and signaling over time. The importance of time in the measurement and quantification of biased agonism has recently been addressed in the context of dopamine receptor signaling.19 In the presence of RAMPs, the CTR forms amylin receptors. AMY1 (CT/RAMP1) has high affinity for amylin and CGRP and lower affinity for hCT, while AMY2 (CT/RAMP2) and AMY3 (CT/RAMP3) have the highest affinity for amylin but lower affinity for CGRP and hCT.79,126 While biased agonism at AMY receptors has not been widely investigated, earlier experiments suggest that RAMPs, in addition to altering ligand specificity, change the signaling preference of the receptors. Comparison of the signaling between different pathways indicates that RAMP interaction with CTRa engenders selective modulation of signaling pathways activated by the receptor complex. A 20- to 30-fold increase in amylin potency at AMY1 I


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study,83 GLP-1(1−36)NH2 was also biased, with a relative bias toward ERK1/2 but a loss of iCa2+ signaling compared to the N-terminally processed form of the peptide. Thus, this work revealed that the GLP-1 receptor exhibits peptide-mediated biased agonism, though no significant bias was observed between nonamidated and amidated forms of the processed peptide or between GLP-1(7−36)NH2 and exendin-4 for the three signaling pathways studied.83 Broader analysis of receptor function, including recruitment of arrestins, identified further evidence of biased signaling, with both exendin-4 and oxyntomodulin displaying stronger recruitment of β-arrestin-1 and β-arrestin-2, relative to GLP-1(7−36)NH2, while GLP1(1−36)NH2 did not recruit arrestins even at micromolar concentrations (Figure 2).159 In contrast, BMS21, a synthetic 11mer peptide, while much less potent than GLP-1(7− 36)NH2, displayed a similar profile of activation for ERK1/2 phosphorylation, cAMP production, and iCa2+ mobilization, but did not recruit arrestins.159 Of note, the main metabolite of GLP-1, GLP-1(9−36)NH2, exhibits a relative preservation of ERK1/2 signaling,160,161 despite abrogation of cAMP production and the ability to promote insulin secretion.160 This indicates that the metabolite is heavily biased toward ERK1/2 and could promote physiologically relevant signaling via this pathway. Further evidence for peptide-mediated biased agonism has been observed using a yeast assay of chimeric G protein activation.162 In this system, exendin-4, oxyntomodulin and glucagon, when compared to signaling by GLP-1(7− 36)NH2, displayed relative bias toward the GPA1/Gαi chimera over the GPA1/Gαs chimera. In contrast, liraglutide was not biased away from GLP-1(7−36)NH2 across these two pathways.162 Thus, biased agonism is observed even at the most proximal measures of receptor activation and is consistent with the stabilization of distinct ensembles of receptor conformations by individual peptides. There has been widespread interest in the development of novel peptides as therapeutic agonists of the GLP-1 receptor. This investigation has concentrated on modifications to the peptides to improve plasma half-life, through resistance to protease degradation (exendin-4, dulaglutide, GLP-1 amino acid substitutions),155−157,163 increased binding to plasma proteins (e.g., liraglutide),155,156 or to enhance bioavailability (stabilized 11-mer peptides).164,165 Others have been designed to interact with both GLP-1 and glucagon, or GLP-1 and GIP receptors.166−168 To date, there has been only limited investigation into whether these modifications alter signaling bias of the ligands. As noted above, broad analysis of signaling/ regulatory protein interaction has revealed bias for the clinically approved exendin-4,159 and there is also preliminary evidence of potential biased agonism of liraglutide in driving GLP-1 receptor internalization, although this has not been directly quantified. The potency for liraglutide-mediated receptor internalization was 10-fold lower than the potencies for internalization of exendin-4 and GLP-1(7−36)NH2, despite similar kinetics of internalization for all three peptides.169 While biased agonism by other peptides has not been directly assessed, the BMS21 11-mer peptide displays marked bias compared to GLP-1(7−36)NH2,159 suggesting that distinct patterns of signaling response are also likely with other 11-mer peptides. Similarly, given the strong bias of oxyntomodulin,83,159,170 which is a dual agonist of the GLP-1 and glucagon receptors, it seems probable that other designed dual agonists or mixed agonist/antagonists will also have altered interactions with the GLP-1 receptor.

engineering of peptides to alter the signaling bias at the CGRP receptor. To date, very limited studies have been performed with modified adrenomedullin peptides. Full-length human adrenomedullin is 52 amino acids; however, many studies utilize the 13−52 fragment, as this has similar affinity and potency for generation of cAMP. Adrenomedullin-2 is a 53 amino acid peptide that can undergo further, N-terminal processing to yield 47 and 40 amino acid peptides. Adrenomedullin has high affinity for both the AM1 (CLR/RAMP2) and AM2 (CLR/ RAMP3) receptors, while adrenomedullin-2 is reported to have higher affinity/potency at AM2 receptors.144−146 Both AM1 and AM2 have low affinity/cAMP potency for other related CT family peptides.144 The actions of adrenomedullin-2 generally resemble those of adrenomedullin, although in some systems it is reportedly more potent, or appears to have unique actions.149,150 Whether these reported differences could be due to biased signaling or to activation of distinct populations of receptors has not been investigated. As adrenomedullin has only a short plasma half-life, the peptide has been modified by N-terminal conjugation to polyethylene glycol, leading to a 7to 8-fold increase in half-life.151 This peptide has lower potency than parental adrenomedullin in cAMP assays, but no multipathway analysis has been performed to date, so it is unclear if this changes the signaling/regulatory profile of the peptide/receptor complex. 4.5. Receptors for Glucagon, GLP-1, GLP-2, and GIP

4.5.1. GLP-1 Receptor. The best-studied class B receptor with respect to biased agonism is the GLP-1 receptor. The GLP-1 receptor is a key incretin receptor with a wide range of physiologically beneficial actions, including glucose-dependent insulin secretion, promotion of insulin synthesis, inhibition of glucagon release, inhibition of gastric acid emptying, reduced feeding, and cardio and neuroprotection.5,152−154 Multiple endogenous peptides interact with and activate the GLP-1 receptor that include the fully processed GLP-1(7−37) and GLP-1(7−36)NH2 peptides, extended GLP-1(1−37) and GLP1(1−36)NH2 forms as well as oxyntomodulin, and to a lesser extent glucagon. The search for more stable forms of GLP-1 for therapeutic use in type II diabetes and obesity has also led to synthesis of mimetic peptides, including exendin-4 and modified forms of GLP-1.155−157 Early characterization of the pharmacology of ligands for the GLP-1 receptor was mostly limited to measurement of the canonically coupled cAMP, critical for the incretin effect in pancreatic β-cells. Thus, it is only relatively recently, following the adoption of multipathway analysis of peptide signaling, that evidence for ligand-directed signaling has emerged. A study by Koole et al.83 utilized heterologously expressed GLP-1 receptor to more broadly examine and quantify ligandmediated signaling, focusing on three important signaling pathways linked to physiological function in β-cells, explicitly, cAMP accumulation, phosphorylation of ERK1/2, and iCa2+ mobilization. Even with this relatively limited assessment of signaling, the first quantitative evidence for peptide-mediated biased agonism was observed. Oxyntomodulin, an endogenous ligand for the GLP-1 receptor, demonstrated a relative bias toward ERK1/2 phosphorylation over cAMP and iCa 2+ signaling compared to either GLP-1(7−36)NH2 or exendin4,83 and this supported earlier observations of differences between oxyntomodulin and GLP-1(7−36)NH2 in cAMP signaling versus β-arrestin recruitment.158 In the Koole et al. J


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Figure 3. In vivo actions of the G protein-biased agonist, P5, in models of obesity. A modified form of exendin-4 was identified from combinatorial peptide libraries. P5 demonstrated minimal loss of G protein-mediated signaling in CHO-hGLP-1R cells but marked attenuation of arrestin recruitment, thus this was biased toward G protein signaling relative to the parental exendin-4 peptide. In Ob/Ob or DIO models of diabetes and obesity, P5 demonstrated similar glucose lowering effect to that seen with exendin-4, but insulin response was markedly attenuated compared to the parental peptide. P5, but not exendin-4, increased adipose number but reduced adipose size, while both reduced hepatic steatosis in DIO mice. These data are consistent with differential in vivo effects from biased GLP-1 mimetic peptides. Images for this figure were reproduced from ref 171 under the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). Copyright 2015 Nature Publishing Group.

to fully understand the extent of biased signaling, the data suggest that biased GLP-1 receptor agonists could be therapeutically useful. 4.5.2. Nonpeptidic-Biased Agonism at the GLP-1 Receptor. There is significant interest in the identification and development of nonpeptidic ligands for the GLP-1 receptor as agonists with enhanced bioavailability, particularly oral absorption.172−174 While such compounds are routinely assessed in cAMP accumulation assays and as insulin secretagogues, a number of these ligands have more recently been assessed across multiple signaling/regulatory end points, enabling evaluation of bias relative to native GLP-1 signaling, at least in the context of recombinant expression systems. The most extensively studied are NN compound 2 and the Eli Lilly compound BETP (also known as compound B);83,159,170,174−176 however, there is now data comparing signaling/regulatory profiles for Transtech Pharma compound TT15 and Boc5159 (Figure 2). Although all these ligands have low potency for cAMP production relative to GLP-1(7− 36)NH2, they also display distinct profiles of second messenger activation and recruitment of regulatory proteins.83,159 TT15 and Boc5 elicit similar signaling patterns to GLP-1(7−36)NH2 for canonical pathways (cAMP production, ERK1/2, iCa2+ mobilization), but display attenuated recruitment of arrestin proteins. In contrast, BETP and NN compound 2 exhibit a relatively enhanced capacity to recruit arrestins but have distinct effects on ERK1/2 phosphorylation and iCa 2+ mobilization. BETP trends toward higher iCa2+ mobilization

Although clinically used peptides such as exendin-4 (exenatide) or liraglutide are likely to have at least some subtle biased signaling/regulation compared to native GLP-1 peptides, the pharmacological/therapeutic consequences of such bias are difficult to assess in vivo due to other differences in the behavior of the peptides, in particular differences in the biodistribution and pharmacokinetics of the peptides. Highly biased and receptor selective ligands, as well as other tools such as mouse models of genetically modified (biased) receptors, are required to address the importance of biased agonism at the GLP-1 receptor. In this vein, a recent publication by Zhang and colleagues171 may provide some insight into the in vivo pharmacological consequence of G protein/arrestin bias (Figure 3). These authors used a combinatorial approach to generate novel peptides from an exendin-4 template that had modified N-termini (7−10 amino acids). One of the peptides, ELVDNAVGG-(9−39-exendin-4) (denoted P5), demonstrated preservation of cAMP and iCa2+ signaling, but a markedly attenuated recruitment of β-arrestins in in vitro cellular assays. In varying mouse models of type II diabetes, the authors demonstrated that P5 was a weak insulin secretagogue, but that chronic treatment of diabetic mice with P5 increased adipogenesis, reduced inflammation of adipose tissue, as well as hepatic steatosis, and was significantly better at correcting hyperglycaemia and decreasing hemoglobin A1c levels when compared to exendin-4.171 While additional work is required, including comparative assessment of peptide pharmacokinetics, and more broad assessment of parameters of receptor function K


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performed; however, given the extremely biased profile of small molecule agonists at the GLP-1 receptor, it may be expected that these compounds would also be biased agonists of the GLP-2 receptor. 4.5.4. GIP Receptor. GIP, the endogenous agonist for the GIP receptor, is an incretin hormone with a principal role in promoting glucose-dependent insulin secretion, as well as pancreatic β-cell proliferation and survival. The receptor has also been implicated in control of body weight and appetite. Activation of the GIP receptor is coupled to increases in cAMP and iCa2+ levels, in addition to activation of PI3-kinase, PKA, Akt, p38 MAP kinases, and phospholipase A2. GIP receptor agonists also induce rapid internalization and reversible desensitization that involves regulator of G protein signaling 2 (RGS-2), G protein-coupled receptor kinase (GRK) 2, and βarrestin-1. Like other incretins, GIP is subject to proteolytic cleavage that has driven the development of novel degradation-resistant C-terminally truncated analogues of GIP. One such analogue, 2 D-Ala -GIP (1−30), robustly improved glucose homeostasis in rats, reduced β-cell apoptosis in isolated islets, and preserved βcell mass in rodents.188 However, it is not as potent as full length GIP at inducing lipoprotein lipase in 3T3L1 cells, suggestive that there may be some biased agonism with this analogue, although direct comparisons across multiple end points will need to be performed to conclude this. GIP receptor antagonists have also been developed, by either truncation of both the N- and C-terminal region of the peptides [GIP(6−30) and GIP(7−30)] or a Pro3 substitution in native GIP.189,190 These analogues are not reported to promote intracellular signaling (cAMP or insulin secretion), and antagonize the actions of GIP in vitro and in vivo.189,191 However, sustained chronic administration of [Pro3]-GIP into obese diabetic mice engendered improvements in glucose levels, glycated hemoglobin, and pancreatic insulin,192 effects that are also reported with GIP. Interestingly, this analogue also reduces body weight, although GIP does not have any significant effects on food intake or body weight, despite significantly improving metabolic profiles.193 Long-acting GIP analogues GIP(Lys37MYR), N-AcGIP(Lys37MYR), and NAcGIP(Lys37PAL) also have no effect on body weight or food intake.194,195 This suggests that either [Pro3]-GIP is a partial agonist of the GIP receptor, has altered interaction with related receptors, or potentially displays biased agonism compared to GIP, but direct comparative studies profiling related receptors across multiple signaling and physiological end points will be required to confirm this. 4.5.5. Glucagon Receptor. The glucagon receptor plays an essential role in regulating blood glucose levels through controlling the rate of hepatic glucose production, promoting glycogen hydrolysis and gluconeogenesis, and regulating pathways controlling hepatic lipid oxidation and lipid secretion.196 The glucagon receptor is activated by glucagon, thus glucagon has a counter regulatory role to insulin in controlling glucose homeostasis. The glucagon receptor is also expressed in extrahepatic tissues including the central and peripheral nervous system, blood vessels and heart, pancreas, adipose tissue, kidney, and smooth muscle cells in the gastrointestinal tract where it has multiple physiological roles.197 Activation of the glucagon receptor promotes cAMP formation via Gαs coupling in multiple cell types and backgrounds. Glucagon also activates iCa2+ mobilization via

but reduced ERK1/2 phosphorylation, whereas NN compound 2 has the reverse profile (compared to the signaling profile of GLP-1 and the reference cAMP pathway).159,170 Other differences in the pharmacological behavior of BETP and/or NN compound 2 have also been noted by other investigators;175−177 however, the lack of a quantitative framework for these analyses has limited the extent to which these differences can be attributed to true biased agonism. That nonpeptidic ligands have distinct signaling profiles from peptide agonists, and other nonpeptidic compounds is unsurprising as their chemical diversity will engender quite distinct interactions with the receptor. Indeed, BETP and NN compound 2 interact, via covalent modification, with Cys347 in the ICL3 of the GLP1 receptor.72 4.5.3. GLP-2 Receptor. GLP-2 receptor expression is restricted predominantly to the gastrointestinal tract and CNS, primarily regulating nutrient absorption and energy homeostasis.178 There are very few ligands identified to date that activate this receptor. GLP-2 is found endogenously and promotes signaling via interaction with the GLP-2 receptor coupling it to Gαs and formation of cAMP.179,180 Profiling of additional signaling pathways has only been conducted in limited cell lines including BHK fibroblasts that overexpress the GLP-2 receptor181 and in murine subepithelial myofibroblasts.182 These revealed pleiotropic signaling from activation of the GLP-2 receptor, including increases in levels of iCa2+, early gene expression of c-fos, c-jun, junB, and zif268, transient increases in p70 S6 kinase, inhibition of ERK1/2 and coupling to AP-1-dependent transcriptional activity that was dependent on PKA.180,181,183 One of the principal actions of GLP-2 is to stimulate epithelial cell proliferation and inhibition of enterocyte apoptosis.178 While cell proliferation was enhanced by GLP-2 in BHK fibroblasts, this was not dependent on cAMP.181 Studies in the subepithelial myofibroblasts revealed these effects may be dependent on the PI-3 kinase/Akt pathway, with no cAMP, ERK1/2 or iCa2+ signaling observed at low concentrations of GLP-2.182 There are also a number of identified GLP-2 receptor agonists and partial agonists, some of which have been developed for clinical/preclinical trials for various gastrointestinal diseases. However, to date there has been no detailed multipathway analysis of these ligands; therefore, there are no reported biased ligands of this receptor. Like most natural ligands of class B GPCRs, truncated metabolites of GLP-2 [GLP-2(2−33), GLP-2(3−33)] are low-affinity partial agonists for GLP-2 receptor-mediated cAMP;180,184 however, as the GLP-2 receptor is pleiotropically coupled, there is the potential that these metabolites are biased ligands similar to studies that imply biased agonism from metabolites that activate the related GLP-1 receptor.185 Teduglutide is a modified form of GLP-2 that differs by just a single amino acid to GLP-2 and has an improve half-life through resistance to protease cleavage.186 This is reported to be a full agonist and is used clinically to treat short bowel syndrome; however, whether it exerts any biased agonism in relation to GLP-2 is not known. A series of analogues derived from the compound methyl 2{[(2Z)-2-(2,5-dichlorothiophen-3-yl)-2-(hydroxyimino)ethyl]sulfanyl}benzoate (compound 1) are the only GLP-2 receptor small molecule agonists reported to date.187 These compounds are positive allosteric modulators of GLP-2 signaling but also act as agonists for cAMP production. Further profiling of these compounds in other signaling pathways has not been L


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implies that TH-glucagon may be a biased glucagon receptor agonist when compared to glucagon. The glucagon receptor can also be regulated by interaction with RAMP2, resulting in changes in both ligand selectivity and G protein-coupling preference that can bias the signaling profile of the receptor.125,214 In recombinant cells, coexpression of RAMP2 with the glucagon receptor potentiated both glucagonand oxyntomodulin-mediated cAMP production; however, the mechanism by which this occurred was different for the two ligands. Use of a yeast assay of chimeric G protein activation revealed that RAMP2 decreased glucagon-mediated activation of Gαi proteins while enhancing Gαs, whereas modulation of oxyntomodulin responses occurred solely through enhanced Gαs. In addition, RAMP2 also abolished the partial activity observed by GLP-1 and liraglutide at the glucagon receptor.214 RAMP expression levels vary in different tissues and in disease and therefore may contribute to distinct signaling profiles that lead to discrete physiological functions of glucagon receptor activation.

the inositol phosphate/PLC pathway that have been linked to cell type-dependent Gαs, Gαq, and/or Gαi signaling. These pathways are believed to be responsible for the majority of the physiological and pharmacological effects of glucagon. Glucagon-mediated production of cAMP stimulates glucose output in the liver,198,199 lipolysis in adipose tissue,200 insulin secretion in pancreatic islets, and inotropy and chronotropy in the heart.201−203 In recombinant cells lines, glucagon receptor stimulation enhances ERK1/2 phosphorylation via cAMPmediated PKA; however, iCa2+ mobilization is required for PKA to maximally activate this pathway.204 PKA-mediated ERK1/2 phosphorylation is responsible for activation of K-type ATP channels in the CNS that are stimulated through the dorsal vagal complex to lower hepatic glucose production.205 Increased cAMP and activation of PKA also activates Wnt/βcatenin signaling, consistent with reports linking activation of this pathway by cAMP/PKA in the PTH-1 receptor.94,206 A direct interaction of the glucagon receptor with low-density lipoprotein-5 Lrp5 enhances glucagon receptor-mediated βcatenin signaling that is believed to contribute to the metabolic phenotypes of Lrp5 mutations associated with metabolic syndrome.207 However, despite glucagon promoting cAMP production, there are multiple lines of evidence to support cAMP-independent actions of glucagon on adipose tissue, liver, and heart.208 In the liver, glucagon can promote glycogenolysis, gluconeogenesis, and glucose output at very low concentrations without activating adenylate cyclase.209 This occurs through rapid inhibition of pyruvate kinase mediated by inositol phosphate-Ca2+-dependent signaling. In the heart, activation of PI3-kinase/Akt at concentrations of glucagon that do not activate cAMP promotes myocardial glycolysis,210 while activation of p38 MAP kinase promotes cardiomyocyte apoptosis, reducing survival in mouse models of post myocardial infarction remodeling and heart failure.211 In addition to glucagon, there are further endogenous ligands for this receptor, although their importance and role is less wellunderstood. Oxyntomodulin is a low-affinity agonist with dual actions at both the glucagon receptor and the GLP-1 receptor. Mini-glucagon, the C-terminal 19−29 fragment processed from glucagon, is present in pancreatic cells and is a potent inhibitor of glucagon- and GLP-1-mediated insulin secretion; however, these effects may be independent of the glucagon receptor itself.212,213 The GLP-1 receptor agonists, GLP-1 and liraglutide are also agonists at the glucagon receptor, albeit with lower efficacy than at the GLP-1 receptor.214 To date, there is limited information around biased agonism at the glucagon receptor, as most efforts in ligand development have focused on development of antagonists that are normally profiled in inhibition of either glucagon binding or glucagonmediated cAMP. Detailed multipathway profiling for agonists at this receptor has not been performed. However, early studies by Houslay and colleagues209,215 reported an interesting analogue (1-N-α-trinitrophenylhistidine,12-homoarginine)glucagon (TH-glucagon) that may be a biased glucagon receptor agonist. These studies showed that TH-glucagon did not exert any increase in cAMP in hepatocytes, yet could stimulate production of inositol phosphates and could fully stimulate glycogenolysis, gluconeogenesis, and urea synthesis in vivo, further supporting the hypothesis that these physiological functions occur independent of cAMP. These effects at the time of publication were hypothesized to arise due to two different glucagon receptors, but the most likely explanation for these data is biased agonism. Although not quantified, this study

4.6. GHRH Receptor

When activated by GHRH, the GHRH receptor stimulates growth hormone production and release that is required for normal postnatal growth with roles in bone growth and regulatory effects on protein, carbohydrate, and lipid metabolism.216 The receptor is primarily located in the anterior pituitary and when activated promotes both cAMP-dependent signaling by coupling to Gαs and inositol phosphate/DAG/ PLC-mediated iCa2+ release. Growth hormone production is stimulated via cAMP/PKA-mediated phosphorylation of CREB, whereas both GHRH-mediated cAMP-dependent and -independent pathways are required for influx of extracellular Ca2+, leading to the release of growth hormone secretory granules that result in the rapid rise in circulating growth hormone. Recent work has also identified a role for the GHRH receptor in proliferation of cardiac stem cells with agonists of this receptor promoting survival through mechanisms involving ERK1/2 and Akt.217 Interestingly, while GHRH receptor expression is predominantly limited to the pituitary, a number of splice variants for the receptor have been identified in various nonpituitary tissues and in human tumor cell lines.218−221 The most common of these splice variants, SV1, lacks only a portion of the extracellular domain of the full length receptor but is still activated by GHRH to promote cAMP signaling and stimulate cell proliferation.222 However, SV1 also promotes ligandindependent constitutive proliferative effects on cells. In addition, GHRH antagonists produce antiproliferative effects in tumor cell lines expressing GHRH receptor splice variants.219−222 These studies suggest that these splice variants are biased receptors relative to full length GHRH, with different basal receptor conformational landscapes that promote ligandindependent signaling and may have roles in development of malignancies. In addition to splice variants, there are also a number of confirmed GHRH receptor polymorphic variants that can alter the signaling capacity of the receptor, some of which have been linked to disease.223−225 Consistent with glucagon/GLP-1/GLP-2 receptor ligands, substitution of D-Ala at position 2 of the GHRH peptide ligand provides resistance to proteolytic degradation. Interestingly, this analogue has significantly enhanced potency for growth hormone release compared to GHRH.226 Another series of GHRH analogues generated around the scaffold [Dat1,Gln8, M


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Orn12,21,Abu15,Nle27,Asp28,Agm29]hGHRH(1−29), also display higher potency than GHRH for growth hormone release in vivo.227 However, to date, no detailed multipathway profiling of any of these peptides has been performed, and therefore, it is unknown if any of these compounds display biased agonism or whether these effects are due to increased affinity/stability of the modified peptides for the receptor.

-8, -9, -10, -13; Nal-6, -9, -13, -15) were identified that promoted Gαs activation similar to the native peptide but were inactive at coupling to Gαi activation in HEK-293 cells overexpressing the CRF1 receptor.246 This reveals the potential for different agonists to alter receptor conformation resulting in differential effector coupling at these receptors; however, to date, no comprehensive multipathway comparisons have been performed between endogenous agonists of CRF receptors. Interestingly, gender differences in CRF receptor signaling have been reported, with CRF being a more potent electrophysiological activator of rat LC neurons (the primary target of CRF during stress) in females compared with males. Furthermore, this activation is almost completely prevented by a PKA antagonist in females, but only 50% inhibition was observed in males.247 CRF1 receptors isolated from the cortex of unstressed female mice were associated with similar levels of Gαs to stressed males and significantly more Gαs than unstressed males. In addition, CRF1 receptors in LC neurons of female animals do not recruit β-arrestin-2 following stress; however, after exposure of male animals to stress, there was enhanced association of CRF1 receptors with β-arrestin-2. These differences in receptor association with signaling/ regulatory effectors observed between the two genders indicate differences in the way the receptor signals. Additionally, these differences were also coupled with distinctive localization of receptors, both before and after exposure to stress.247 CRF1 receptors were located at the plasma membrane and cytosolically in unstressed males with an increase in cytoplasmic localization, indicative of internalization, following stress. In contrast, in unstressed female LC neurons, the receptor was predominantly localized intracellularly, with stress shifting the distribution to the plasma membrane. Differences in spatial and temporal aspects of signaling are now well-recognized for influencing bias profiles of agonist ligands. Females are more vulnerable to certain stress-related disease and this could be attributed in part to differential signaling/trafficking profiles of CRF receptors in males and females due to their ability to adapt to the excessive CRF that is predicted to be present in diseases related to severe or chronic stress. This may have therapeutic implications for biased CRF1 receptor agonists that can shift the bias of signaling toward β-arrestin-2 in female LC neurons. An additional level of signal regulation has been reported for the CRF1 receptor via the interaction of this receptor with RAMP2.248 This results in selective augmentation of Ca2+ signaling but occurs in a ligand-dependent manner, with augmentation of CRF and urocortin-1 responses, yet no effect on sauvagine-induced Ca2+ mobilization. Of note, in RAMP2± mice, CRF responses are diminished, suggesting that these RAMP2-mediated effects are physiologically important.248 Intriguingly, RAMP2 also interacts with the VPAC1 receptor to selectively augment inositol phosphate signaling.125 Inositol triphosphate is a key initiator of iCa2+ mobilization, suggesting that the effect of RAMP2 on CRF1 and VPAC1 receptor signaling may be mechanistically similar. To date, there are no reports assessing the influence of RAMPs on CRF2 receptor function.

4.7. CRF Receptors

The related receptors CRF1 and CRF2 bind the endogenous peptide CRF and the urocortin family of peptides.228 These receptors have approximately 70% similarity at the amino acid level but differ principally in their N-terminal domains, which is responsible for their distinct pharmacological properties and agonist selectivity. CRF1 receptors bind both CRF and urocortin-1 with equal affinity but do not bind the related peptides urocortin-2 and urocortin-3. In contrast, CRF2 receptors bind CRF and all three urocortin peptides; however, the urocortin peptides display higher affinity than CRF.229−231 Both receptors also bind the related peptide agonists from fish (urotensin-1) and amphibians (sauvagine). CRF and urocortins acting at CRF receptors are important for mediating central and peripheral stress responses.232 Studies in native tissues, recombinant cell lines, and in yeast expressing different Gα chimeric proteins demonstrate that CRF receptors are highly promiscuous in their G protein-coupling, with evidence for coupling to Gαs, Gαq/11, Gαo, and Gαi1/2.233−236 CRF1 and 2 receptors predominantly activate cAMP via Gαs coupling and transient Ca2+ mobilization by activation of PLC and PKC through a combination of Gαs, Gαq, and Gαi coupling.237−239 CRF receptors are internalized following agonist binding in a process that is dependent on β-arrestin-2. There are nine CRF1 variants, with CRF1a the main functional receptor and the one most characterized. The CRF1b isoform has a 29 amino acid insertion in ICL1, impairing agonist activity.240 Most other isoforms (CRF1c‑h) have exon 6 spliced out and cannot signal but may play a modulatory role in CRF1a function as expression of e and h isoforms in COS7 cells either decreased or amplified urocortin1-mediated cAMP at the CRF1a receptor, respectively.241,242 CRF1i is characterized by a deletion of exon 4 but is functional in promoting ERK1/2 phosphorylation in transfected HEK-293 cells, although no further assessment of signaling has been performed.243 For CRF2 receptors, there are three reported biologically important splice variants (CRF2a‑c) that differ predominantly in their N-terminal domain. The predominately expressed and most well-characterized of these is CRF2a, and while differential pharmacological or biological properties of these splice variants have not been reported, there are apparent effects with respect to tissue distribution.244 Most of the physiological functions of CRF receptors in the CNS and periphery have been linked to Gαs coupling. However, in the placenta, these receptors are unable to activate Gαs proteins, while still activating MAP kinases and iCa2+ pathways.245 In contrast, in SK-N-MC neuroblastoma cells, CRF receptor activation induces robust Gαs-mediated cAMP with no iCa2+ mobilization.238 This reveals distinct signaling profiles by the same ligands acting at the same receptors in discrete tissues, although the physiological basis for this signaling specificity is not clear. Ligand selective signaling is also evident at CRF receptors. Ten analogues of urocortin-1 involving single amino acids substitutions with either a Bpa group or a Nal group (Bpa-6, -7,

5. MECHANISTIC STUDIES OF BIASED AGONISM There have been relatively limited studies addressing the mechanism underlying differential signaling of peptides from individual class B receptors. Conformational changes promoting activation transition involve reorganization of polar, hydrogen bond networks, and intrahelical packing that drive N


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Figure 4. A central, polar, hydrogen-bonded network controls biased agonism at the GLP-1 receptor. Homology modeling of the inactive GLP-1 receptor predicts a stable interaction network comprising R2.60, N3.43, H6.52, E6.53 and Q7.49. (A) Mutational analysis reveals differential usage of the amino acids within this network by individual peptides, in a pathway-specific manner; amino acids are depicted in x-stick, with unaffected amino acids colored by side chain; red, mutation decreases efficacy; green, mutation increases efficacy. (B) Apo (blue) and GLP-1-bound (orange) models of the GLP-1 receptor. The expanded region illustrates the network in each of the models with amino acids in x-stick format, colored by side chain. The network is predicted to be disrupted in the peptide-bound model, with a predicted key interaction between R2.60 and the glutamic acid at position 9 in the GLP-1(7−36)NH2 sequence when this peptide is present. (C) Inactive model with top down (left-hand panel) and side (righthand panel) views illustrating the position of the central network residues (red space fill) and also small amino acids (blue) that are globally important (all ligands) for signaling through individual pathways. Individual panels reproduced with permission from ref 254. Copyright 2016 ASPET.

reordering of the intracellular surface of GPCRs to engender interaction with effector proteins.249−252 Although best studied for class A receptors, these polar networks are conserved within GPCR subfamilies, suggesting evolutionary preservation of the molecular mechanisms controlling activation transition.253 It is anticipated that such conserved polar residues within class B GPCRs play an equally important structural role and contribute to the mechanism of biased agonism. A major fulcrum for activation transition in the class B GLP-1 receptor is located in the core of the TM domain bundle at the point of convergence of the splayed extracellular TM helices.

Mutagenesis of polar residues conserved across class B receptors revealed a cluster of residues, comprising Arg1902.60, Asn2403.43, His3636.52, and Gln3947.49 that alter the pattern of peptide-mediated signaling for cAMP formation, pERK1/2, and iCa2+ mobilization in a peptide and amino acid specific manner.57,254 Molecular modeling, based on the recently solved inactive crystal structure of the glucagon receptor,53 predict that these residues form a hydrogen-bonded network inclusive of Glu3646.53, a less conserved amino acid within the B subclass.254 Differences in the profile of responses were particularly notable between GLP-1(7−36)NH2 and O


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mediated bias. Nonetheless, this has been investigated for canonical signaling of the amidated GLP-1 peptides, exendin-4 and oxyntomodulin via Ala scanning mutagenesis of ECL2.260,261 Lys288, Cys296, Trp297, and Asn300 had important roles in controlling signal bias of the receptor but were also globally significant for peptide signaling. Nonetheless, peptide-selective effects on relative efficacy and signal bias were most frequently seen for residues 301−305, although the mutation of Arg299Ala also led to distinct effects for individual peptides. Met303 played a greater role for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Interestingly, ECL2 mutation was overall more disadvantageous to exendin-4-mediated iCa2+ mobilization than GLP-1(7− 36)NH2, providing further support for subtle variances in receptor activation by these peptides. Collectively, this provides initial insight into the early engagement of peptides with the receptor and how conformational propagation is differentially controlled.

oxyntomodulin, with the predicted interaction between Arg1902.60 and Glu3646.53 important for GLP-1 action but not oxytomodulin signaling.254 However, selective differences between GLP-1(7−36)NH2 and exendin-4, in the effect of mutation, were also observed. Collectively, this provides molecular evidence for mechanistic differences in receptor activation between the three peptides through the central polar network (Figure 4). Analysis of the equivalent central network residues in the glucagon53 and CRF154 receptor structures revealed conservation in relative orientation of side chains,254 despite TM6 of the CRF1 receptor having distinct amino acids, namely Thr at 6.52 and Tyr at 6.53. In the glucagon receptor, maintenance of the central network was predicted to be partially coordinated by water-mediated H-bonding and likely to exist in a highly constrained state in the inactive form of the receptor.254 The structural consistency in side chain position in the solved class B crystal structures, combined with conservation of key residues in the network and its fulcrum position in the crystal structures, is consistent with a critical role of the network in signaling for this subfamily. There are supporting data from other class B GPCRs of a key role of this network in receptor activation, with interaction of Arg1882.60, Asn2293.43, and Gln3807.49 predicted from mutagenesis and modeling studies of the VPAC1 receptor.255−257 Interestingly, Arg1882.60 is predicted to engage, via a salt-bridge, with Asp3 of VIP, with this interaction contributing to receptor activation;256 in GLP-1(7−36)NH2 and exendin-4, the equivalent amino acid is Glu, whereas in oxyntomodulin it is a Gln. Molecular modeling of GLP-1(7−36)NH2 docking to the full-length receptor254 has predicted a direct, salt-bridge interaction between peptide Glu9 and Arg1902.60 (Figure 4), and it is speculated that the lack of an acidic residue at the third amino acid of oxyntomodulin may underlie the lack of engagement of the Arg1902.60/Glu3646.53 interaction in receptor activation, thus contributing to the biased agonism observed for this peptide. The extent to which the central network contributes to biased agonism of other class B peptides has yet to be investigated. In addition to the fulcrum amino acids that exhibit ligandspecific effects, a series of Ser residues (Ser1551.50, Ser1862.56, and Ser3927.47) in the GLP-1 receptor have been identified to contribute to control of receptor-dependent signal bias, where mutation of these amino acids globally affects GLP-1, exendin4, and oxyntomodulin signaling.57 These amino acids are situated at the interface between either TM1 and TM7 (Ser1551.50 and Ser3927.47) or between TM2 and TM3 (Ser1862.56), and likely contribute to tight packing of these TM helices. There is an additional polar Thr1491.44 in TM1 that is the site of a naturally occurring Thr/Met polymorphism, although the Thr is not broadly conserved in class B receptors.258 Met at this position leads to a marked attenuation of peptide-mediated cAMP production 258,259 and iCa 2+ mobilization, while ERK1/2 phosphorylation is relatively preserved,259 and like the Ser residues in TM1, this was a global effect for peptide agonists.259 In homology-based molecular models of the GLP-1 receptor TM domain, the smaller polar residues that are globally important for signaling are located external to the central polar network that is involved in ligand-dependent signaling (Figure 4). While there is an emerging appreciation of how peptides can selectively alter key hydrogen bonding networks to control signaling, there is relatively limited information on how amino acids in the extracellular loops (ECLs) contribute to ligand-

6. ALLOSTERIC MODULATION OF CLASS B GPCRS Allosteric modulation of GPCRs is now a well-established paradigm that provides both challenges and advantages for drug discovery over classic orthosteric ligands.8 As noted above, peptide ligands engage class B receptors via a diffuse pharmacophore including critical interactions for affinity with the N-terminal extracellular domain and less well-defined interactions with the ECLs and TM domain core that initiate and propagate receptor activation. Nonpeptidic ligands have an alternate mode of binding, often involving topographically distinct, allosteric sites from those of the native peptides, allowing cooperative interactions on binding or efficacy to occur.8,10,16,33,262 Nonetheless, nonpeptidic ligands for class B GPCRs have been notoriously difficult to discover and to develop, and for those that have been identified, full molecular and pharmacological characterization of their mode of binding and action is often absent. Small molecule ligands fall principally into two categories: those whose binding is topographically distinct and those that have at least partial overlap with the binding site of orthosteric peptides. As peptide binding to class B receptors is multimodal with both a receptor N-terminal (N-domain) and juxtamembrane/TM core interaction (J-domain),96,263 it is possible for the latter class of ligands to exhibit allosteric inhibitor properties (concomitant binding, changes to ligand kinetics), while inhibiting receptor function by competitive interaction with the peptide N-terminus that binds to the receptor core. As discussed by Hoare,263 the explicit description of this phenomenon is the “Charniere” effect,264 and a key expectation of this type of interaction is that peptide interaction with small molecule (where this is the reporter) should appear fully competitive, without any effect on small molecule ligand dissociation.263 The paucity of high affinity, labeled small molecule ligands limits testing for this type of interaction. Nonetheless, other approaches can provide insight into potential mechanism of action. One example is small molecule ligands of the PTH1 receptor, AH-3960 and SW106, initially described as agonist and antagonist, respectively,265−267 though the latter may be a very weak partial agonist.268 ECD truncation receptor studies support binding of these ligands to the TM core of the PTH1 receptor. Both small molecules alter the dissociation kinetics of PTH(1−34) that binds according to the classic two domain model; however, they do not alter the kinetics of a modified PTH(1−11) peptide agonist that binds P


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interactions is “probe-dependence” that describes the differential effects occurring dependent upon the orthosteric and allosteric ligand combination (Figure 1). This is clearly observed for ligands of the GLP-1 receptor. While NN compound 2 has very little effect on GLP-1(7−36)NH2induced cAMP production, it engenders a ∼30-fold enhancement of oxyntomodulin signaling via this pathway.83,159,170 Probe-dependence is also observed for effects on the extended GLP-1 peptides and exendin-4, where very limited effect on cAMP production is observed. A similar profile of effect is seen for the Eli Lilly compound BETP (“compound B”) in that BETP enhances oxyntomodulin-mediated cAMP production but has minimal effect on GLP-1(7−36)NH2-, exendin-4-, or GLP-1(1−36)NH2-mediated signaling via this pathway.159,170 Limited augmentation of NN compound 2-mediated cAMP accumulation (or surrogate measures of cAMP such as CREluciferase) has also been noted for interaction of this ligand with truncated forms of exendin-4, including exendin-4(5−39), exendin-4(7−39), and exendin-4(9−39), even where no intrinsic activity was measurable.175,176 Remarkably, NN compound 2 and BETP dramatically augment cAMP production elicited by the principal GLP-1 metabolite, GLP-1(9−36)NH2; ∼ 400-fold for NN compound 2.160,161 Parallel enhancement of insulin secretion was observed in isolated rat islets and in vivo when rats were stimulated with pharmacological levels of the metabolite together with subthreshold levels of BETP.160 The increase in signaling was principally limited to cAMP production, although weak potentiation of ERK1/2 phosphorylation and iCa2+ mobilization has been observed in INS1E cells and HEK-293 cells recombinantly expressing the GLP-1 receptor,161 but not in CHO cells expressing the receptor,160 providing additional evidence that allosteric modulators have the ability to change the signal bias of the receptor. Nonetheless, a lack of effect on the extended, amidated GLP-1 peptide suggests that the magnitude of cooperativity of NN compound 2 and BETP is not manifested solely by the intrinsic efficacy of the activating ligand. NN compound 2 and BETP are highly electrophilic and are capable of forming adducts with free cysteine residues,272 and indeed covalent interaction of the compounds with Cys347 in ICL3 is critical for both the intrinsic efficacy and the allosteric cooperative effect.72 Nonetheless, this covalent interaction is insufficient to fully explain the activity of these compounds, as NN compound 2 and BETP and have distinct profiles of intrinsic efficacy (see above; Figure 2) and display differences in their cooperative effect.159 Intriguingly, the naturally occurring human GLP-1 receptor polymorphism Ser/Cys333 selectively attenuates NN compound 2-mediated cAMP formation and the positive cooperativity between NN compound 2 and oxyntomodulin for this pathway, without altering orthosteric peptide agonist response.259,273 These data are consistent with the environment surrounding Cys347 contributing to the selective actions of NN compound 2. In contrast to the effect of position 333 polymorphism, the Met1491.44 polymorphic variant does not impact on the intrinsic efficacy of NN compound 2; however, as discussed above, it engenders a strong abrogation of peptide-mediated cAMP production with >100-fold loss of exendin-4 and GLP-1(7−36)NH2 potency. At this mutant, NN compound 2 led to recovery of the potency of both GLP-1(7−36)NH2 and exendin-4 to that of the T1491.44 polymorphic variant of the receptor.259 Thus, allosteric

only within the TM core.268 Nonetheless, SW106 displays competitive inhibition of the PTH(1−11)-mediated cAMP production elicited from both full-length and the N-terminally deleted PTH1 receptor.268 Similar behavior has been observed for small molecule inhibitors of the CGRP receptor that partially overlap the binding of peptides to the N-terminal complex of CLR/RAMP1.50,52 Likewise, Boc5, an agonist of the GLP-1 receptor, is reported to interact via the N-terminal ECD with apparent competitive interaction with peptide ligands.172 In contrast, at least one class of CRF1 inhibitor acts via classic allosteric inhibition. This is exemplified by the crystal structure of the CRF1 receptor in complex with CP-376395.54 The cocrystallized small molecule inhibitor binds deep within the TM core toward the intracellular face of the receptor;54 comparative mapping of the location of drug binding sites in current published crystal structures indicates this is the deepest structurally characterized binding pocket identified to date1 and is located away from the peptide binding pocket, which is predicted to reside within the extracellular face of the receptor. Although structural detail on the site of interaction is not yet available, the low-affinity small molecule inhibitors of the VPAC2 receptor (termed “compound 1” and “compound 2”) bind in the TM core of the receptor and demonstrate noncompetitive inhibition of VIP signaling,269 consistent with an allosteric mode of action. T-0632, an inhibitor of GLP-1 receptor signaling, is reported to bind to the receptor ECD in a manner that is dependent upon the presence of Trp33, as substitution of this residue with Ser (the equivalent amino acid in the rat receptor) led to a 100-fold loss in affinity.270 Further investigation is required to determine whether this could lead to partial occlusion of peptide binding or whether the ligand acts in a fully allosteric manner. Beyond CRF1 receptors, the most active inhibitor drug discovery programs have targeted the glucagon receptor, as a potential antidiabetic therapy.271 These are predicted to bind within the TM bundle of the glucagon receptor, and indeed one of these inhibitors, NNC0640, was required for the successful crystallization of the TM domain of the glucagon receptor.53 Unfortunately, there was insufficient electron density resolved within the solved crystal structure to localize the site of small molecule binding. In addition to small molecule allosteric antagonists, antibodies that recognize the extracellular domain of the glucagon receptor can allosterically regulate its activity. The antibody mAb7 behaves as a negative allosteric modulator by binding to two regions within the N-terminal domain distinct to that of the glucagon binding cleft, inhibiting glucagon binding via an allosteric mechanism.35 In contrast, the antibody mAb23 blocks glucagon binding by directly occluding the hormone binding cleft. However, this antibody is an inverse agonist, inhibiting basal activity of the receptor by removing interactions of the Nterminal domain with ECL3, interactions that are required to allosterically regulate ligand-independent receptor activity.34 While small molecule drug discovery for all class B receptors has been difficult, this is particularly true for agonists or positive allosteric modulators. The best-studied examples of PAMs are modulators of the GLP-1 receptor. GLP-1 receptor PAMs were first identified by Novo Nordisk and are exemplified by NN compound 2, an allosteric agonist of the receptor for cAMP production. In addition to its intrinsic efficacy, this series of compounds augmented the binding of radiolabeled GLP-1, indicating that it could act as a PAM.173 Nonetheless, there was only limited impact on the efficacy of GLP-1(7−36)NH2 for cAMP signaling.83 As described above, a hallmark of allosteric Q


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As discussed above, the physiological and/or therapeutic implications of biased signaling are largely unknown, and this is particularly true for allosteric small molecule agonists/PAMs. Despite the distinct signaling/regulatory profiles of ligands such as Boc5 or TT15, these compounds (or related analogues) modulate both ex vivo and in vivo insulin secretion.172,280 Allosteric compounds such as NN compound 2 or BETP can also augment insulin secretion, albeit that mechanistic interpretation of such responses is complicated by endogenous circulating peptides. Pharmacologically, both NN compound 2 and BETP enhance select responses of oxyntomodulin and the GLP-1 metabolite, GLP-1(9−36)NH2, and this is linked to higher in vivo insulin secretion, at least in the context of threshold doses of the orthosteric and allosteric ligands.160,170 Broader, in vitro investigation of the effect of the modulators on the signaling profile of oxyntomodulin or GLP-1(9−36)NH2 revealed that the most prominent modulatory effect was augmentation of cAMP production, implying that, at least in the context of the baseline signaling of the peptides/ modulators, this alone may be sufficient to improve insulin secretion. The significance of allosterically driven bias for GLP1 receptor function outside of insulin secretion is even more ambiguous, due to lack of investigation of the impact of modulation on these other functions, and none controlled for the influence of biased signaling. Unfortunately, most nonpeptidic compounds have low potency, nonfavorable pharmacokinetic profiles, or unknown interactions with other targets, limiting their utility.

modulators can alleviate loss of function of the GLP-1 receptor arising from disease of genetic origin. While electrophilic compounds such as NN compound 2 and BETP are the most widely studied allosteric modulators of the GLP-1 receptor, other modulators of peptide response have been reported. One example is the flavonol, 3,3′,4′,5,7pentahydroxyflavone (quercetin) that has no inherent intrinsic activity but specifically enhances iCa2+ mobilization elicited by efficacious peptides such as GLP-1(7−36)NH2, GLP-1(7−37), and exendin-4, albeit with a bell-shaped effect, inhibiting responses observed at high concentrations of the flavonol.83,274 This amplification of iCa2+ mobilization required the presence of a 3-hydroxyl group on the flavone backbone and was greater if a 3′4′-dihydroxyl was present.274 Other compounds that selectively increased iCa2+ signaling were recently reported following identification by high-throughput screening.275 In contrast to the flavonols, this compound series had significant intrinsic efficacy for iCa2+ mobilization. A prototypical ligand from this series, termed (S)-9b, was reported to also augment exendin-4-mediated insulin secretion in primary mouse islets as well as liraglutide-mediated GLP-1 receptor internalization in recombinant cells.275 However, the insulin secretagogue effect occurred in both high and low glucose conditions, which would be therapeutically problematic but may also indicate a requirement for further investigation to determine if this is truly a GLP-1 receptor-mediated event. In addition to the insulinotropic effects, (S)-9b was also reported to reduce haloperidol-induced catalepsy in rats,275 but again, confirmation that this is mediated via the GLP-1 receptor is required. In other work using virtual screening against a molecular model of the glucagon receptor, de Graaf and colleagues276 identified compounds with both glucagon and GLP-1 receptor activity. This included weak inhibitors of both receptors but also one compound that displayed weak positive modulation of GLP-1mediated cAMP production via the GLP-1 receptor, but was an inhibitor of glucagon-mediated cAMP signaling via the glucagon receptor.276 Outside of the GLP-1 receptor, and the single example from the PTH1 receptor (described above), there are very few reports of small molecule agonists/PAMs for class B receptors. Yamazaki and colleagues187 identified a series of 2-([(2Z)-2(2,5-dichlorothiophen-3-yl)-2-(hydroxyimino)ethyl]sulfanyl)benzoate-based compounds as PAM-agos of the GLP-2 receptor that potentiated the placental alkaline phosphatase activity of various GLP-2 peptides in recombinant HEK-293 cells expressing the GLP-2 receptor. It is unclear whether the sulfanyl group could also be reactive in a similar manner to the covalent GLP-1 receptor PAM-agos described. The only other receptor for which small molecule agonists have been described is the CTR. SUN B8155, a small molecule agonist of the CTR identified by Katayama and colleagues277 induced cAMP production in a manner that was inhibited by the peptide antagonist sCT(8−32). However, this compound did not compete for 125I-hCT binding. The mode of binding of this compound has not been investigated further. More recently, a series of pyrazolopyridine-based CTR agonists were described.278 Chimeric and mutagenesis studies indicate that these compounds bind to the CTR at the TM1/ECD interface and that amino acids 150/151 are critical for their activity.279 The compounds exhibit incomplete inhibition of 125I-hCT binding and weakly potentiate cAMP production by hCT,279 suggesting a pure allosteric mode of interaction.

7. SUMMARY/CONCLUSION/FUTURE DIRECTIONS Class B GPCRs are physiologically important receptors that are potential high value targets for the treatment of both acute and chronic disease. These receptors are pleiotropically coupled and can regulate signaling from distinct compartments in a cell specific manner, making them ideal candidates for exploitation by biased and/or allosteric drugs. Peptide-based therapies are well-advanced for many of these receptors; however, the current lack of detailed understanding of both the signaling properties of these peptides and the relationship between select signaling pathways and cellular response may limit optimal therapeutic development as these parameters are critical for successful application of superior, biased drug therapies. This is an area that requires urgent investigation. Small molecule and allosteric drug development have been difficult areas for class B GPCRs. One novel approach to drug development that is being actively pursued for other (nonclass B) peptide GPCRs is that of pepducins. These lipid-modified peptides, based on ICL or helix 8 sequences of receptors, allosterically modulate target receptors.281,282 Positive, negative, and biased pepducin modulators have been described for other classes of GPCRs, and these could likely also be developed for class B receptors. Nonetheless, recent advances in structural understanding of this family of receptors offers new insight for directed drug design, both for structure-based design of biased peptides and for the identification and development of small molecule compounds. AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. R


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boundaries through elucidation of fundamental biology and the intersection of this with drug-receptor interactions. He has authored over 230 publications, is a 2014 Thomson Reuters highly cited researcher in Pharmacology and Toxicology, and the recipient of a 2015 Thomson Reuters Citation Award (Biology and Biochemistry). He is an elected Fellow of the British Pharmacological Society, an Associate Editor for Pharmacological Reviews, and is on the scientific advisory board of the Chinese National Centre for Drug Screening.

The authors declare no competing financial interest. Biographies Dr. Denise Wootten is a Career Development Fellow of National Health and Medical Research Council of Australia and Research Fellow working at the Monash Institute of Pharmaceutical Sciences in Melbourne, Australia. Her expertise is in the study of G proteincoupled receptors (GPCRs), particularly the Class B subfamily. The principal interest of her research is directed toward understanding the modes of their signaling and regulation in an effort to identify novel approaches for drug discovery. Her research efforts encompass differential signaling, interaction of receptors with regulatory accessory proteins, allosterism, and the structure and mechanism by which these GPCRs are activated. More recently her work has focused on addressing the link between in vitro pharmacology and signal transduction with the physiological effects elicited in vivo following activation of these receptors.

ACKNOWLEDGMENTS This work was supported by National Health and Medical Research Council of Australia (NHMRC) project Grants [1061044] and [1065410], and NHMRC program Grant [1055134]. P.M.S. is a NHMRC Principal Research Fellow. A.C. is a NHMRC Senior Principal Research Fellow. D.W. is a NHMRC Career Development Fellow. C.K. is a NHMRC CJ Martin Postdoctoral Fellow. REFERENCES

Laurence J. Miller is Professor of Medicine, Biochemistry & Molecular Biology, and Pharmacology at Mayo Clinic College of Medicine, located at Mayo Clinic in Scottsdale, Arizona. He trained as a physician at Jefferson Medical College and Mayo Clinic and received his scientific training in the Cell Biology Department at Yale University School of Medicine. His scientific interests focus on the molecular basis of drug action at G protein-coupled receptors.

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AB


Allostery and Biased Agonism at Class B G Protein-Coupled

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