Classification of Allosteric G-Protein-Coupled Receptors Regulators: Comparison
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Allosteric regulation is critical for the functioning of G-protein-coupled receptors (GPCRs) and their signaling pathways. Endogenous allosteric regulators of GPCRs are simple ions, various biomolecules (lipids, amino acids, polypeptides, hormonal agents, etc.), and the peptide components of GPCR signaling. According to the ability to influence the basal and orthosteric/allosteric agonist-stimulated activity, the ligands of GPCR allosteric sites can be divided into the positive (PAM), negative (NAM), and silent (SAM) allosteric modulators, the allosteric full agonists, inverse agonists and neutral antagonists, as well as the allosteric regulators with the combined activity (ago-PAM, ago-NAM). 

  • G protein-coupled receptor
  • allosteric site
  • allosteric modulator
  • pepducin
  • heterotrimeric G protein
  • allosteric agonist

1. Introduction

G protein-coupled receptors (GPCRs), located in the plasma membrane, are the largest superfamily of receptor (sensory) proteins in multicellular eukaryotes. GPCRs have been found in fungi [1[1][2][3],2,3], plants [4], and in all studied invertebrates and vertebrates [5[5][6][7][8][9],6,7,8,9], including trypanosomes [10] and ciliates [11]. At the same time, the yeast Saccharomyces cerevisiae has only 3 genes encoding GPCRs [12], the slime mold Dictyostelium discoideum has 55 such genes [13], while in the human genome there are more than 800 genes for GPCRs [14]. Prototypes of the structural domains of both GPCRs and the adapter and regulatory proteins that interact with them appeared at the earliest stages of evolution, already at the level of prokaryotes and unicellular eukaryotes [2,9,15][2][9][15]. During the early evolution of GPCRs, different structural models of these receptors existed, including hybrid constructs that consisted of an N-terminal GPCR-like molecule and a C-terminal catalytic phosphatidylinositol phosphate kinase, which were identified in some representatives of lower eukaryotes [11,16,17][11][16][17].
Through GPCRs, various extracellular signals, including photons, protons, hormones, neurotransmitters, growth factors, nutrients, metabolites, and odorants, exert their regulatory effects on target cells. The result of the interaction of the GPCR with them is its transition to an active conformation and triggering of intracellular signaling cascades, which, through genomic and non-genomic mechanisms, regulate fundamental cellular processes, such as growth, metabolism, differentiation, apoptosis, and autophagy. The fact that the therapeutic effect of about a third of pharmacological drugs used in medicine is due to their influence on GPCRs and their signaling pathways [18,19][18][19] is the basis for the great importance of studying the molecular mechanisms of GPCR regulation.

2. Classification of Allosteric GPCR Regulators

According to the ability to influence the basal and orthosteric/allosteric agonist-stimulated activity, the ligands of GPCR allosteric sites can be divided into allosteric modulators that have no intrinsic activity and allosteric regulators that affect GPCR activity in the absence of orthosteric agonists [58][20]. In the case of allosteric modulators, the ligand, by binding to the allosteric site, changes or retains the affinity of the orthosteric agonist to GPCR and/or its ability to activate the receptor, which is assessed by its maximum stimulating effect but has no intrinsic activity (Table 1). Allosteric ligands that have their intrinsic activity can function as full agonists, inverse agonists, and neutral antagonists, and their action is independent of orthosteric site occupancy (Table 1). Such independence of the action of allosteric ligands can be realized only when the orthosteric and allosteric sites do not overlap and do not interact through ligand-induced conformational rearrangements [58][20]. When an allosteric ligand acts as a full agonist and affects the affinity and/or potency of an orthosteric agonist, it is classified as ago-PAM or ago-NAM (Table 1). In the case when allosteric ligand reduces the effectiveness of an orthosteric agonist but increases its affinity to GPCR, it is classified as a PAM-antagonist [77][21].
Table 1.
The classification of allosteric regulators of GPCRs.
Allosteric Ligand Pharmacological Characteristics α β τ
Positive allosteric modulator (PAM) It increases the affinity and/or efficacy of an orthosteric agonist but has no intrinsic activity. >1 >1 1 *
Negative allosteric modulator (NAM) It reduces the affinity and/or efficacy of an orthosteric agonist but has no intrinsic activity. <1 <1 1
Silent allosteric modulator (SAM) It does not affect the affinity and efficacy of an orthosteric agonist but is able to change some other characteristics of its interaction with the GPCR (selectivity for different types of G proteins and β-arrestins; specific activation of intracellular target proteins; etc.). 1 1 1
Full allosteric agonist It stimulates the GPCR in the absence of an orthosteric agonist and does not affect the affinity and efficacy of an orthosteric agonist. 1 1 >1
Full allosteric agonist/PAM (ago-PAM) It functions as a full agonist and at the same time enhances the affinity and/or efficacy of an orthosteric agonist. >1 >1 >1
Full allosteric agonist/NAM (ago-NAM) It functions as a full agonist and at the same time reduces the affinity and/or efficacy of an orthosteric agonist. <1 <1 >1
Inverse allosteric agonist It reduces the intrinsic activity of the GPCR (basal or constitutively active) in the absence of an orthosteric agonist. 1 1 <1
Neutral allosteric antagonist It prevents the activation of the GPCR by an orthosteric agonist, including due to stabilization of its inactive state. 1 1 <1
Neutral allosteric antagonist/PAM It reduces the effectiveness of an orthosteric agonist, acting as an antagonist, but at the same time increases the affinity of an orthosteric agonist to the receptor functioning as a PAM. >1 <1 1
Notes: α, the factor of binding cooperativity between the orthosteric agonist and allosteric modulator; β, the operational factor of cooperativity for quantitative evaluation of the effects of allosteric modulator on operational efficacy of orthosteric agonist (receptor activation); τ, operational efficacy for the complex of GPCR with allosteric ligand. Values of binding cooperativity α and operational cooperativity β greater than 1 denote positive cooperativity, and corresponding values below 1 denote negative cooperativity. * The value “one” for operational efficacy τ is normalized since the absolute τ value may vary depending on the used calculated parameters.
Regulatory influences caused by the specific binding of ligands to the orthosteric and allosteric sites of the GPCRs are reciprocal. Just as binding of a ligand to an allosteric site can change the binding characteristics and activation pattern of an orthosteric site, binding of a ligand to an orthosteric site can change the accessibility and regulatory properties of one or more allosteric sites. Given the multiplicity of allosteric sites, the mechanisms of such relationships between receptor sites can be quite complex. Moreover, depending on the nature and binding characteristics of orthosteric ligands, the same allosteric regulator can influence the affinity and efficiency of these ligands in different ways, functioning as a PAM, NAM, or SAM [58,78,79][20][22][23]. Thus, the assignment of a ligand to a certain group of allosteric regulators or modulators is relative. In each specific case, the pharmacological profile of the allosteric ligand largely depends on the nature of the receptor-bound orthosteric agonist, the structural and functional characteristics of the receptor (phosphorylation, N-glycosylation, and other post-translational modifications), the formation of homo- or hetero-oligomeric complexes, the microenvironment (multicomponent complexes with transducer, adapter, and regulatory proteins), as well as on the physicochemical properties of the plasma membrane, ionic composition, and ionic strength and acidity of the extracellular and intracellular environment. Given the variety of regulatory influences of allosteric ligands on the interaction of orthosteric agonists with the GPCR, in the recent years a model has been widely used that describes a ternary complex that includes GPCR and receptor-bound allosteric and orthosteric ligands. The formation of a ternary complex is described by equations A + R + B ⟷ AR + B (KA) ⟷ ARB (KB/α) in the case when, at the first stage, the GPCR forms a complex with the orthosteric agonist A, and by equations A + R + B ⟷ A + RB (KB) ⟷ ARB (KA/α), when, at the first stage, the receptor forms a complex with the allosteric ligand B (KA and KB, the equilibrium dissociation constants for the GPCR-orthosteric agonist and GPCR-allosteric ligand complexes, respectively; α, the factor of binding cooperativity between the orthosteric agonist A and allosteric ligand B) [80][24]. Therefore, the effect of an allosteric ligand on the affinity of an orthosteric agonist is described by the factor α. In turn, its effect on the efficacy (maximum regulatory effect) of an orthosteric agonist is described by the factor β. When the allosteric ligand increases the affinity and efficacy of orthosteric agonist, the factors α and β are above 1 (PAM) (Table 1). If the influence of the allosteric ligand is the opposite, then these factors have values below 1 (NAM). In the absence of a significant effect, the α and β are equal to 1 (SAM). To assess the intrinsic activity of allosteric ligands, the factor τ is used, which for full agonists has values above 1 (full agonist, ago-PAM, and ago-NAM), and for an inverse agonist or neutral antagonist it has values below 1. For “pure” allosteric modulators (PAM, NAM, SAM, and PAM-antagonist), the factor τ is equal to 1 (Table 1). However, for each specific case and for each specific “allosteric ligand–orthosteric ligand” pair, the values of α, β, and τ may vary. A separate group consists of compounds that are able to simultaneously interact with both orthosteric and allosteric sites, which are classified as bitopic GPCR ligands [79,81,82,83][23][25][26][27]. They have two pharmacophores: one which binds with the orthosteric site, and the other binds with the allosteric site. If these sites are spatially separated in the receptor, then the pharmacophores in the bitopic ligand must be connected with a flexible linker, the length of which exactly corresponds to the distance between the orthosteric and allosteric sites. At the same time, it is important that the linker does not significantly affect the conformational rearrangements in the receptor induced by its activation by orthosteric and (or) allosteric agonists [79,82][23][26].

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