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Targeting Intracellular Allosteric Sites in GPCRs

Exploring the intracellular allosteric site

GPCRs represent one of the largest and most vital families of cell surface receptors, playing a pivotal role in cellular signaling. For this reason, GPCRs have been a focus of drug development for decades due to their role in regulating essential physiological processes.

Conventional GPCR drug discovery has largely focused on orthosteric sites, but recent breakthroughs in understanding allosteric modulation have opened up new avenues for drug development. Recent advances in structural biology and pharmacology have revealed the existence of allosteric sites within GPCRs' intracellular domains. These sites act as molecular switches that can modulate receptor activity, providing an untapped opportunity for drug discovery. Unlike orthosteric ligands that bind directly to the receptor's active site, allosteric modulators target distinct, often less conserved sites on the receptor. This allows for the fine-tuning of receptor activity, leading to more specific and safer drugs. Allosteric binding sites on the receptor are those topographically distinct from (do not exhibit any overlap with) the orthosteric site (Rosenbaum, Rasmussen et al. 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 et al. 2011). Allosteric ligands can be classified as positive allosteric modulators (PAMs), which increase the receptor functional and/or affinity for an orthosteric ligand; and negative allosteric modulators (NAMs), that fully or partially dampen the receptor's functional response to the ligand (Wold, Chen et al. 2019). Alternatively, they can function as neutral allosteric ligands (NALs), binding to a receptor's allosteric site without causing any detectable alterations in the receptor or orthosteric ligand behavior (Lindsley, Emmitte et al. 2016). However, a NAM competes for binding with PAMs and NAMs and thereby blocks the effects of positive and negative allosteric modulators (Rodriguez, Nong et al. 2005). Allosteric modulators targeting GPCRs can display one or more of the following pharmacological characteristics: 1) affinity modulation where the resulting change in conformation can influence the orthosteric binding pocket, potentially altering the rate of association, dissociation, or both, of an orthosteric ligand; 2) efficacy modulation where there are modifications in intracellular responses, consequently affecting intrinsic efficacy of the orthosteric ligand; 3) agonism/inverse agonism, where the allosteric modulator can perturb receptor signaling in a manner that is either positive (agonism) or negative (inverse agonism), regardless of whether an orthosteric ligand is present or absent (May, Leach et al. 2007).

Advantages of targeting GPCRs allosteric sites

Allosteric sites offer a more nuanced approach to modulating GPCR activity, enabling greater specificity and fewer side effects. 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 (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). 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, Gregory 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, Chen et al. 2019).

Biased allosteric mechanisms

Allosteric sites can differentially modulate G-protein and β-arrestin coupling, enabling the development of biased agonists and antagonists with distinct therapeutic potential. Allosteric antagonists are compounds that inhibit GPCR activity by binding to intracellular allosteric sites. G-protein-biased allosteric antagonists are under investigation for several GPCRs, including CCR2, CCR7, CCR9, CXCR2, and β2AR. Interestingly, although having different chemical structures they exhibit strikingly similar binding positions on their respective receptors. On the other hand, positive allosteric modulators that target intracellular allosteric sites can enhance β-arrestin-mediated signaling pathways. SBI-553, an allosteric modulator of NTSR1, provides valuable insights into how allosteric modulation can selectively enhance β-arrestin-mediated signaling. Structural studies at high resolution have illuminated the intricate interactions between NTSR1 and GRK2. Allosteric agonists binding to intracellular sites can also promote G-protein signaling. PCO371 is a G-protein-biased allosteric agonist for the adenosine receptor A1, with the promise to improve cardiac contractility. Recent advances in structural biology have allowed researchers to visualize the binding of allosteric agonists like PCO371 to Gs-proteins, further elucidating biased signaling mechanisms.

The process of designing and evaluating biased ligands for GPCRs is complex but essential for understanding their diverse signaling pathways. Advances in understanding the structural and mechanistic aspects of biased GPCR signaling are crucial for designing effective drug molecules. Additionally, considering conformational kinetics for coupling preference with downstream signaling components, such as β-arrestin and G-protein transducers, is important. Allosteric modulation is gaining prominence in GPCR therapeutics, with the discovery of an intracellular allosteric site common in class A and class B GPCRs, particularly in chemokine receptors. This site allows for the direct modulation of downstream receptor signaling in an allosteric manner. Biased allosteric modulators can enhance the safety and efficacy of GPCR-targeted therapeutics by selectively targeting specific signaling pathways. Notably, SBI-553 and PCO371 have been identified as BAMs with intracellular allosteric binding sites. These findings provide a comprehensive understanding of GPCR biased signaling induced by intracellular allosteric agonists and establish a strong foundation for developing biased drugs targeting GPCRs.

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