GPCR activation typically occurs through the binding of agonists which stabilize receptor conformations that recruit and ultimately activate intracellular transducers.
GPCRs are present in a range of conformations, and the binding of a ligand, as well as interactions with signaling molecules like G proteins, can selectively stabilize specific conformations (Gether 2000). In terms of pharmacology, GPCRs have been identified to exist in at least two distinct states: an active state characterized by high affinity for agonists when coupled to G proteins, and an inactive state in which their affinity for agonists diminishes in the absence of G proteins (Gether 2000), with numerous intermediate sub-states in between (Vauquelin and Van Liefde 2005; Yao et al. 2009). However, GPCRs are not simple on-off switches (Rosenbaum, Rasmussen, and Kobilka 2009), since they are able to adopt an active conformation without agonist engagement referred to as receptor constitutive or basal activity. The initial demonstration of spontaneous or "constitutive" receptor activity was reported for the β2-adrenergic receptor in 1984 (Cerione et al. 1984), where reconstitution of purified β2-adrenergic receptors from guinea pig lung in conjunction with purified Gαs from human erythrocytes led to an elevation in GTPase activity of Gαs without the presence of a ligand. Subsequently, opioid receptors were shown to constitutively activate Gi proteins in a membrane preparation without the need for an agonist (Costa and Herz 1989).
GPCR ligands pharmacology
The impact of a ligand on a receptor's structure and biophysical attributes, and consequently on the biological response, is referred to as ligand efficacy. Natural and synthetic ligands can be categorized into four distinct efficacy classes: 1) full agonists produce the maximal response and can differ in intrinsic efficacy; 2) partial agonists are incapable of inducing maximal activity even when they are present at saturating concentrations; 3) neutral antagonists have null intrinsic efficacy, thus not affecting receptor signaling activity, and can compete with agonist ligands; 4) and inverse agonists decrease the level of receptor constitutive activity. In the same way that agonists possess intrinsic efficacy, inverse agonists also exhibit varying degrees of negative intrinsic efficacy, leading to the presence of both robust and less potent (partial) inverse agonism (Rosenbaum, Rasmussen, and Kobilka 2009).
GPCR targeting drugs: orthosteric vs allosteric sites
Most of the drugs targeting GPCRs bind to the orthosteric site of the receptor, eliciting various effects, such as receptor activation (e.g., agonists or partial agonists), inhibition of activation (e.g., antagonists), or produce an inversion of the functional response (e.g., inverse agonists).
Despite the existence of numerous drugs designed for this primary binding site, there are significant challenges in creating ligands that are both safe and effective. Those include the risk of unintended off-target effects, limited selectivity due to closely related receptor binding sites, the inability to target large and diffuse binding sites activated by peptides or proteins, and the disruption of natural spatial and temporal signaling patterns regulated by physiological systems (Wold and Zhou 2018). An alternative and promising approach is allosteric modulation.
Allosteric binding sites on the receptor are those topographically distinct from (do not exhibit any overlap with) the orthosteric site (Rosenbaum, Rasmussen, and Kobilka 2009). Over the past decade, there has been a notable growth in the discovery of allosteric modulators for GPCRs which possess the capability to modulate and fine-tune the affinity and/or efficacy of orthosteric ligands. While this has the potential to enhance GPCR subtype-selectivity, it also presents a significant challenge when it comes to detecting and confirming allosteric behaviors (Keov, Sexton, and Christopoulos 2011). Compounds employing an allosteric mechanism of action can theoretically offer several advantages compared to orthosteric ligands when considered as potential therapeutic agents. Allosteric modulators that do not exhibit agonistic properties remain inactive in the absence of endogenous orthosteric activity, therefore having the potential to maintain the temporal and spatial aspects of natural physiological signaling. The significance of spatio-temporal attributes in signaling is demonstrated in processes like neurotransmission and chemokine signaling, extending to numerous other GPCR-regulated systems such as free fatty acid receptors (FFARs), now regarded as targets for therapeutic intervention for metabolic diseases such as liver disease, obesity and diabetes (Secor et al. 2021; Wold and Zhou 2018). Moreover, they have the potential to enhance target selectivity, which can arise from greater sequence variation in allosteric sites among receptor subtypes when compared to the conserved orthosteric region, or from selective cooperativity with a particular subtype while excluding others (Christopoulos 2002; Lazareno et al. 2004). This is particularly important when dealing with receptor subtypes that exhibit significant similarity in their orthosteric binding sites, such as chemokine receptors. On the other hand, selectivity could be achieved by merging both orthosteric and allosteric pharmacophores within a single compound, resulting in a novel category of GPCR ligands referred to as 'bitopic' (Valant et al. 2008). A third advantage is that they offer the prospect of reducing the risk of overdose, given that their activity is dependent on the concentration of the orthosteric ligand. In this context, allosteric modulators exhibiting constrained positive or negative cooperativity are characterized by having an upper limit on the extent of their allosteric influence, an attribute that offers a considerable degree of adjustability in terms of pharmacological effects, allowing for the administration of substantial doses of allosteric modulators with a diminished risk of target-related toxicity (Wold et al. 2018).
GPCRs functional selectivity
Recent research has revealed a more intricate understanding of GPCR behavior, extending beyond the traditional G-protein and second messenger activation pathways. Agonist binding to GPCRs doesn't always trigger all associated events sequentially. Depending on the specific functional outcome measured (e.g. activation, interaction with accessory proteins, dimerization, phosphorylation, internalization, or desensitization), the same drug acting on the same receptor in the same cellular context can produce a range of effects, from full activation to partial activation to inverse agonism. This variability in drug effects is due to different ligands inducing unique GPCR conformations that selectively activate specific cellular outcomes linked to that GPCR. This phenomenon is termed "stimulus-trafficking," "biased agonism," or "functional selectivity" (Urban et al. 2007).
Overall, GPCRs are intriguing molecules that deviate from typical textbook proteins. They do not have a binary active or inactive state but instead can adopt numerous distinct conformational states, each of which triggers a distinct array of physiological effects. They can be compared to molecular rheostats, capable of sampling a spectrum of conformations with relatively small energy differences (Ma, Lee, and Vaidehi 2020). The ligand alters the balance of these conformations, increasing the prevalence of some infrequent ones, preventing access to others, or allowing previously inaccessible conformations. To understand how ligands or drugs affect GPCRs is crucial to grasp the dynamics of these conformational shifts and identify the most relevant forms.
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