Pharmacology of Antagonism of GPCR

Abstract

Agonists are defined as the ligands that activate intracellular signaling and evoke cellular responses. Synthetic and endogenous agonists should bind specific amino acids to activate G protein-coupled receptor (GPCR). Agonists that induce maximal responses are full agonists. Partial agonists cannot induce full responses unlike full agonists. In definition, antagonists inhibit agonist-stimulated responses by binding to orthosteric or allosteric sites. Antagonists modulate agonist-induced responses and are often related with inverse agonist activity. However, the relationship between antagonists and partial agonists is complex. An antagonist behaves as a partial agonist when the constitutive activity of the GPCR is high. In contrast, a partial agonist with very weak intrinsic activity may be classified as an antagonist. Thus, antagonisms of the compounds are influenced by constitutive activity of GPCRs, intrinsic activity and differences in the binding sites of GPCRs. Since “antagonism” has been revealed to have multiple aspects and more complex than previously thought, it may be difficult to classify each compound as simply “agonist” or “antagonist” as before. In this review, we discuss the recent findings and perspectives on the pharmacology of GPCR-binding antagonists, inverse agonists, and signaling.

1. INTRODUCTION

G protein-coupled receptors (GPCRs) are the largest family of human membrane proteins, with about 800 members (of which about 400 are olfactory receptors). The relationship between GPCRs and their roles in pathologic progression has led to many drugs targeting GPCRs.¹⁾ GPCRs transduce extracellular signals into intracellular signals mainly through G proteins. They have seven transmembrane regions as a common structural feature. Agonists bind extracellular regions or transmembrane regions to induce structural rearrangement and conformational changes within their corresponding GPCRs. These changes promote the interaction with G proteins in the cell.²⁾ Recently, the structures of ligand-free GPCRs, ligand-bound forms of GPCRs, and complexes of GPCRs with G proteins are reported. Based on these structural changes, drug discovery and drug design have been conducted.²⁾

G proteins are heterotrimers composed of α, β, and γ subunits.³⁾ After β and γ subunits are synthesized individually, they form a complex and behave as one subunit. Activation of GPCRs promotes guanosine 5'-diphosphate–guanosine 5'-triphosphate exchange of α subunits and dissociation of the complex into α and βγ subunits. Since α subunit binds the intracellular domain of GPCR and thereby activates downstream effector signaling molecules, G proteins are classified into four subfamilies according to the function of the α subunits: Gs, Gi, Gq, and G12. Gs increases cAMP production through activating adenylyl cyclase. By contrast, Gi inhibits adenylyl cyclase. Gq releases inositol 1,4,5-triphosphate from phosphatidylinositol 4,5-diphoisphate and increases the intracellular Ca²⁺ level by activating phospholipase C. G12 activates RhoA by interacting with and activating RhoGEF. Each subfamily consists of several members: 18 members of the α subunit, 5 members for the β subunit, and 12 members of the γ subunit. These subunits combine to form a heterotrimeric G protein. However, it is unclear how the three subunits select each other to form the complex.

Antagonists are defined as inhibitors of agonist-induced responses. Recent findings have revealed that antagonists not only inhibit the action of agonists, but also relate to partial agonists and inverse agonists. Here, we review the recent progress of pharmacology of GPCR-binding antagonists.

2. AGONIST AND ANTAGONIST

GPCRs have a common structure of penetrating the cell membrane seven times and are classified into five main families.⁴⁾ Agonists are ligands that elicit a response and can be divided into full agonists that elicit a maximal response and partial agonists that elicit a partial response.⁵⁾ Agonists bind to transmembrane regions or extracellular domains. By changing the arrangement and movement of the transmembrane region, agonists create a site for G protein binding in the intracellular region.

An antagonist is a ligand that inhibits the action of an agonist (Fig. 1). However, it has become clear that antagonists do not fit within the definition of inhibiting the action of agonists. It is more complex than previous thought. Recent studies demonstrated that compounds previously known as antagonist also activate intracellular signaling pathways in some cases and affect the coupling selectivity with G protein (Table 1). The relationship between antagonists, inverse agonists, and biased signaling will be discussed later.

Fig. 1. Dose Response Curves of Antagonist and Various Agonists

Agonists acting on GPCRs elicit a response that depends on the intrinsic activity of the agonist. When stimulated with a full agonist, which elicits the maximal response, the antagonist decreases the response to basal levels as the concentration is increased.

Table 1. Classification of Antagonists

Property of antagonists	Classification	Comments
Inhibition of response	Inhibition of G protein-mediated pathway	Since antagonists only inhibit the interaction of GPCRs with G proteins, they do not induce the G protein-dependent responses.
		This group of antagonists are inhibitors of G protein-biased signaling.
	Inhibition of β-arrestin-biased pathway	A ligand that interacts preferentially with β-arrestin compared to G protein and induce a response through β-arrestin.
		This group of antagonists are inhibitors of β-arrestin-biased signaling.
Binding site	Orthosteric	Competitive antagonists
	Allosteric	Non-competitive antagonists
		Ligands can enhance (positive allosteric ligands) or decrease (negative allosteric ligands) GPCR-mediated responses.
Interaction with G protein	Inverse agonist	It is not always β-arrestin-biased ligand.
	Partial agonist	Partial agonists exhibit inverse agonist activity depending on constitutive activities of GPCRs or cellular status.
	Neutral antagonist	It inhibits the interaction of GPCR with G proteins and β-arrestins.
		It does not modulate basal responses.

Antagonists are classified by inhibitory activity, binding site and signaling.

3. INVERSE AGONIST, PARTIAL AGONIST, AND ANTAGONIST

Inverse agonists are known as ligands that bind to GPCRs and attenuate basal activity (Fig. 2). Even in the absence of any stimulus, GPCR (R) is in an equilibrium between inactive (R) and activated (R*) states (R⇆R*).^6,7) Even in the resting state, GPCR in active conformation is present but with a small percentage. It is believed that they determine the basal activities (constitutive activity) of GPCRs. However, when the expression levels of intracellular signaling molecules downstream of GPCRs are changed by diseases and other causes, the constitutive activities of GPCRs are greatly affected. Since inverse agonists shift the equilibrium to inactive conformation (R), the number of GPCRs with active conformation are decreased.

Fig. 2. Effects of Constitutive Activity on Partial Agonist Activity

If the basal response is elevated, e.g., due to increased basal activity of GPCRs, and exceeds the original response by the partial agonist, the agonist decreases the response as if it were an inverse agonist.

When an activated GPCR triggers a cellular response, various molecules involved in the signaling cascade within the cell should be activated in an orderly fashion. A partial agonist is defined as a ligand that does not elicit as much response as the maximal response by a full agonist. Even in the absence of agonist stimulation, GPCRs exhibit the basal activity (response) without any stimulation.^8,9) Basal response will be elevated when the expression of GPCRs or signaling molecules in the cells are increased. If the response elicited by the elevated basal activity of GPCR is higher than the response by the partial agonist, stimulation of partial agonist will decrease the cellular responses.¹⁰⁾ In other words, the partial agonist will appear to exhibit the action of the inverse agonist. This peculiar response will be observed by partial agonist with low intrinsic activity. Thus, the relationship between inverse agonists and partial agonists depends on constitutive activity of GPCR and intrinsic activity of agonist. It should be noted that high basal responses are associated with the expression levels of GPCRs and activities of signaling molecules including GPCRs. Partial agonist with very low intrinsic activity is possibly classified as an antagonist or biased agonist with G protein selectivity in the past. Biased agonist will be described in later section.

4. BINDING SITES OF ANTAGONISTS

Endogenous and synthetic agonists must bind to specific amino acids at orthosteric sites and induce conformational changes in GPCRs, leading to activation of G proteins.²⁾ Therefore, there is only one orthosteric site. In contrast to orthosteric sites, allosteric sites are theoretically many, since they are spatially different from orthosteric sites (Table 1). The ligands bound to allosteric sites can modulate the action of agonists bound to orthosteric sites. They can positively enhance or negatively attenuate the effects of agonists.^11,12) If the antagonist inhibits the activation of the GPCR by the agonist, it is not necessary for the antagonist to bind to orthosteric sites, but only to the region where it affects the binding of the agonist. The binding site of the antagonist is an important factor in determining the mechanism of inhibition.

As mentioned, orthosteric sites are the sites occupied by endogenous ligands or synthetic agonists. Therefore, antagonists that bind orthosteric site competitively inhibit action of agonists. If antagonists exert the inhibitory actions in a non-competitive fashion, antagonists covalently bind orthosteric site or non-covalently bind allosteric sites. Allosteric antagonists modulate GPCR functions in positive and negative manners when agonists are bound to the receptors.¹²⁾

GPCR plays important roles in cellular signaling. Almost all GPCRs activate several G proteins. When antagonists bind orthosteric sites, antagonists will inhibit the interaction of GPCRs with all G proteins. Allosteric ligands either potentiate (positive allosteric ligands) or inhibit (negative allosteric ligands) the receptor-mediated responses. Negative allosteric ligands reduce the degree of G protein-GPCR coupling. It has not been examined whether commercially available antagonists have biased activity against G protein coupling. Recently, it was reported that antagonist also exhibits biased activity to inhibit GPCR-stimulated activation of multiple G proteins.¹³⁾

5. ASSAY SYSTEM TO EVALUATE G-PROTEIN AND β-ARRESTIN-MEDIATED RESPONSES

The activation of GPCRs can be monitored by several methods. Among them, two methods have been commonly used. One is the luciferase reporter gene assay, and the another is the imaging of G protein activation or GPCR-protein interaction.^14,15) The reporter gene assay measures transcription factor-dependent luciferase activity downstream of G proteins. cAMP generation by Gs is measured by monitoring cAMP-response element (CRE)-dependent luciferase activity. Activation of Gi is determined by the extent of a decrease in CRE-mediated luciferase activity. The Gq-dependent pathway is characterized by Ca²⁺-sensitive nuclear factor of activated T cells (NFAT)-stimulated luciferase activity; activation of G12 is detected by activation of luciferase activity via or serum response factor-response element (SRF-RE) or serum response element (SRE). However, it should be noted that SRF-RE- or SRE-dependent luciferase activity is also increased by activation of the Gq pathway. p63Rho guanine nucleotide exchange factor (p63RhoGEF), which is activated by Gq, has been reported to mediate Gq-mediated activation of SRF-RE- or SRE-dependent luciferase activity.^16,17) Therefore, when the effect of antagonists on these agonist-stimulated luciferase activities is examined, it is not certain whether the inhibition by antagonists is due to inhibition of G12 or Gq.

Since the expression of signaling molecules involved in intracellular signaling cascades depends on cell type,¹⁸⁾ it is necessary to distinguish how each G protein contributes to the activities of the reporter genes in individual cells. In addition to the well-known pathways, unidentified signaling cascades may contribute to G protein activation. In addition, if G protein-mediated signaling pathways crosstalk with other signaling pathways in the cell, the action of antagonists becomes more complicated.

Responses to GPCR stimulation are mediated by G-proteins or β-arrestins. G-protein-mediated responses are usually brought about by a cascade of signaling molecules that are amplified at each step.¹⁹⁾ Therefore, when the activity of a reporter gene linked to a promoter is used as an indicator of GPCR-G protein coupling, the amplified signal is always detected. As described later, β-arrestins are found to mediate GPCR-stimulated signaling. In contrast to G proteins, β-arrestin does not amplify the signal unlike the case of G-proteins but instead acts as a scaffold protein.³⁾ In addition, second messengers of β-arrestin-mediated responses (such as cAMP and Ca²⁺) have not been established. Since β-arrestin translocates to the plasma membranes by binding the agonist-promoted phosphorylation of GPCRs, translocation of β-arrestin or signaling through β-arrestin have been thought as general properties of GPCRs. Therefore, GPCR-β-arrestin interaction is widely used for the screening of GPCR agonists.

To evaluate the activation of G-proteins without signal amplification, the mini-G proteins can be used. Mini-G proteins bind GPCRs but are not easily dissociated.^20,21) When the membrane translocation patterns of mini-G protein and β-arrestin are plotted on the X and Y axes, both activities from the full agonist to various partial agonists fall on a straight line.¹⁰⁾ Whether a new agonist falls on the straight line of this XY plot can be used to evaluate biased activity of the new agonist.

The interaction of GPCRs with G proteins and the dissociation of G proteins into each subunit is also measured by Bioluminescence Resonance Energy Transfer (BRET) and NanoLuc^® Binary Technology (NanoBiT) assays.^14,22) BRET is the method to monitor the dissociation of G proteins. In the NanoBiT assay, the part of luciferase is attached to the α- and β-subunits. When G proteins dissociate, the distance between the α-subunit and β-subunit increases, resulting in decreased fluorescence. The advantages of each technique will be discussed in other reviews.

6. BIASED AGONIST ACTIVITY OF PARTIAL AGONIST

β-Arrestin is known as a molecule involved in the regulation of GPCRs.²³⁾ Agonist-bound GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs) as a first step in desensitization. The phosphorylated GPCRs facilitate the translocation of β-arrestin to the plasma membrane, where they bind to β-arrestin. The GPCR-bound β-arrestin then inhibits further activation cycle of the GPCR. Recent studies have revealed that cellular responses are elicited not only by G-proteins, but also by β-arrestins^24,25) (Fig. 3). Agonists that selectively activate either G-protein or β-arrestin pathways are called biased agonists (Table 1). When the effects and side effects of drugs can be separated according to the G-protein- and β-arrestin-mediated effects, biased agonists are expected to reduce the side effects of drugs. Signaling of biased agonists are being actively studied.

Fig. 3. GPCR-Mediated Biased Signaling

GPCR-mediated responses are mediated by G proteins or β-arrestins. Agonists that equally activate G proteins and β-arrestins to elicit a response are called balanced agonists. On the other hand, if the agonist selectively activates either G protein- or β-arrestin-mediated signal more strongly, the agonist is called a biased agonist.

Among the four members of the β-arrestin family, β-arrestin 1 and β-arrestin 2 are widely expressed and function as mediators of biased agonists.²⁶⁾ Biased activity refers to the relative ability of agonist to activate either β-arrestin or G protein.²⁴⁾ A partial agonist with β-arrestin-biased activity activates G-proteins, but to a lesser extent than a full agonist. Concept of biased agonist has not been extended to antagonists.

G protein-biased agonists are sometimes difficult to distinguish from partial agonists.¹⁰⁾ Stimulation with partial agonists always amplifies intracellular signals. Then, cellular responses are fully elicited. Since the response as determined by reporter gene assay is an amplified response, partial agonists can fully activate cellular responses downstream of the GPCR. Caution should be paid when G protein-biased agonist is an issue. The μ-opioid receptor is an example. The μ-opioid agonists are used as analgesic. Since the side effects of opioid receptor agonists (i.e., respiratory depression and constipation) were reduced in β-arrestin knockout mice, it was proposed that the β-arrestin-mediated pathway would have side effects and the G protein-mediated pathway have analgesic effects.^27,28) However, the hypothesis is not supported by the recent study.^10,29) Gillis et al. demonstrated that the effect of G protein-biased μ-opioid is explained by the action of partial agonist with low intrinsic activity. They also suggested that the methods to detect the interaction of GPCRs with β-arrestins are less sensitive than that with G proteins. They also suggested that the understanding of partial agonists with a low intrinsic activity has become more clinically significant.¹⁰⁾

7. FUNCTION OF ANTAGONISTIC COMPOUNDS

GPCRs usually activate multiple G proteins rather than a single G protein. GPCR-mediated responses in vivo have been analyzed by G protein knockout mice. However, in mice in which a specific G protein is knocked out, G proteins other than the target G protein may compensate for the function of the target G protein. Compensatory signaling cascades may be activated by G protein knockout. Thus, it remains to be determined whether GPCR-mediated responses require activation of multiple G proteins or whether activation of a single G protein is sufficient for the responses.

Antagonists have applications as drugs to suppress GPCR-mediated responses. Selecting the timing and dosage of antagonist administration can produce results similar to that of time-dependent knockout. However, antagonists to date have been suggested to inhibit the activation of all G-proteins by GPCRs. Angiotensin II receptor type 1 couples to three different G protein subfamilies: Gi, Gq, and G12. In clinical practice, many antagonists are used to inhibit angiotensin II receptor type 1. These antagonists bind to the site occupied by angiotensin II. Therefore, it has been thought that these receptor antagonists inhibit the activation of all G proteins by the receptor.

Biased agonists have been synthesized for many GPCRs, but biased antagonist has only recently been reported. Protease-activated receptor-2 (PAR2) couples to Gq, G12/13 and Gi. PAR2 is involved in inflammatory responses and pain. Avet et al. reported that a compound I-287 is a negative allosteric regulator acting on Gq and G12/13 without affecting Gi and β-arrestin2 signaling.¹³⁾ In complete Freund’s adjuvant (CFA)-induced paw edema model, I-287 reduces inflammatory responses. It does not affect Gi signaling and β-arrestin pathway. Thus, Gi signaling and β-arrestin pathway are not necessary for CFA-induced inflammatory responses.

Biased antagonists are expected to allosterically regulate GPCRs. There are several examples of allosteric antagonists.³⁰⁾ There are five subtypes of muscarinic acetylcholine receptors (mAChRs), M1 to M5. mAChRs play important roles in regulating behaviors such as cognition, movement, and reward. Recently, the allosteric modulators are developed for the treatment of Alzheimer’s disease and schizophrenia. The binding sites of allosteric modulators are different from those of acetylcholine. Among allosteric modulators, positive allosteric modulators (PAMs), which are selective for the M1 subtype, have been reported to improve cognitive function and restore memory impairment in Alzheimer’s disease and schizophrenia. The effects of PAMs are expected to be greater than those of acetylcholinesterase inhibitors. The M4 subtype-selective PAMs have been reported to decrease dopamine release and exhibit antipsychotic-like effects in animal models.³¹⁾ On the other hand, the M5 subtype-selective negative allosteric modulators are reported to effectively treat a variety of substance use disorders (pathological behavioral patterns in which patients continue to use a substance despite experiencing significant problems associated with its use) without reducing the effect on natural rewards.³²⁾ Although these allosteric modulators positively or negatively regulate mAchR function, it is unknown whether they affect G protein selectivity.

Allosteric modulators have been developed for fatty acid receptors, cannabinoid receptors, and dopamine receptors. However, these allosteric modulators of GPCRs are considered as agonists with biased activity and not agonists with altered G protein selectivity. Allosteric modulators of these GPCRs are beyond the scope of this review. Please refer to other reviews.^33–35)

8. FUTURE DIRECTION

Agonists and antagonists have used to be simple concepts. However, inverse agonist activity and biased activity open new avenues for antagonist research. Antagonists have been characterized by inverse agonist activity, orthosteric or allosteric sites, and biased activity. When the constitutive activity of GPCR is high, antagonists as well as partial agonists inhibit the response. It should be noted that partial agonists with low intrinsic activity exhibit the activity of inverse agonists. Antagonists bind to orthosteric or allosteric sites. Antagonists bound to allosteric sites not only inhibit but also enhance the action of the agonist. Binding to allosteric sites can affect the coupling selectivity of GPCRs with G proteins.

Antagonists with biased activity is a new concept. However, such antagonists have not been reported until recently. Antagonists that act on GPCRs are widely used in clinical practice. Biased activity of antagonists may provide a unique property to characterize individual antagonists.

The crystal structures of the allosteric ligand-bound GPCRs are reported. The region of the GPCR that affects the coupling with the G protein will be identified. Therefore, we can design the compounds that affect selectivity of G protein with simulation. Careful design of signal-specific antagonistic compounds can lead to G protein selective inhibitor. Such a compound will enhance or reduce G protein-mediated responses. This compound may also contribute to minimizing drug-induced side effects.

Acknowledgments

This work was supported in part by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (2017K1A1A2004511 to and H.K. and S.G.K.).

Conflict of Interest

The authors declare no conflict of interest.

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