Biased Allosteric Modulators: New Frontiers in GPCR Drug Discovery

Abstract

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in the genome and the most successful family of targets of FDA-approved drugs. New frontiers in GPCR drug discovery remain, however, as achieving receptor subtype selectivity and controlling off- and on-target side effects are not always possible with classic agonist and antagonist ligands. These challenges may be overcome by focusing development efforts on allosteric ligands that confer signaling bias. Biased allosteric modulators (BAMs) are an emerging class of GPCR ligands that engage less well-conserved regulatory motifs outside the orthosteric pocket and exert pathway-specific effects on receptor signaling. The unique ways that BAMs texturize receptor signaling present opportunities to fine-tune physiology and develop safer, more selective therapeutics. Here, we provide a conceptual framework for understanding the pharmacology of BAMs, explore their therapeutic potential, and discuss strategies for their discovery.

Keywords: G protein-coupled receptor (GPCR); allosteric modulation; drug development; functional selectivity; signaling bias; β-arrestin.

Figure 1, Key Figure.. The molecular basis of biased allosteric modulation and its potential therapeutic advantages.

(A) Depiction of the molecular basis of biased allosteric modulation, based on the empirically supported conformational hypothesis of biased signaling. When a GPCR is activated by a balanced, orthosteric agonist (left), the receptor samples multiple active conformations, which permit receptor association with distinct effector proteins, including Gα family proteins (e.g., Gαs, Gαi/o, Gαq), G protein-receptor kinases and β-arrestins. These effectors give rise to multiple downstream signaling pathways (e.g., changes in cAMP levels, Ca²⁺ mobilization, ERK activation), which, in turn, lead to distinct cellular and physiological outcomes. Biased allosteric modulators (BAMs, right) confer more restricted pharmacological action. In the presence of a BAM, a GPCR samples only a subset of potential active conformations, leading to recruitment of only a subset of potential effectors. Consequently, signaling proceeds through a subset of potential pathways, producing a more limited repertoire of cellular and physiological effects. (B) Representation of the potential therapeutic advantages of BAMs. Balanced, orthosteric agonists (left) disrupt rhythms of endogenous ligands by blocking endogenous ligand binding and produce both on- and off-target side effects. By contrast, BAMs (right) do not prevent endogenous ligand binding, gain receptor subtype selectivity by binding to less well-conserved allosteric motifs and gain pathway selectivity by activating only a subset of signaling pathways.

Figure 2.. Analysis of endogenous receptor-ligand interactions suggests the need to look beyond the orthosteric pocket to develop biased GPCR ligands.

An analysis of non-odorant, rhodopsin family GPCRs was completed to determine inherent informational entropy. For each receptor family, the number of endogenous ligands was divided by the number of receptor variants to yield a “Ligands/Receptor” value indicative of informational entropy. The percent of total receptor families with a given Ligands/Receptor value is shown. Results were fit to a sum of gaussians, indicating that receptors analyzed fall into two main categories with different mean entropies. Mean ± SD for peak 1 and peak 2 are 0.97 ± 0.55 and 3.93 ± 1.6 ligands/receptor, respectively.

Figure 3.. Effects of BAMs on receptor signaling.

GPCRs (green) are bound by an orthosteric agonist (gold ball) and an allosteric modulator (blue triangle). Binding of the agonist to the receptor results in signaling through multiple pathways - labeled by ‘a’, ‘b’ and ‘c’ (arrow thickness and pathway labels indicate the strength of the signaling outcome). Dose-response curves (below) show the effect of agonist on a given signaling outcome in the presence of increasing allosteric modulator concentrations, indicated by increasing blue color saturation. Newest among the classes of allosteric modulators are the BAM and ago-BAM. BAMs are pure modulators that lack intrinsic activity (τ=0) and may or may not alter the binding affinity (α) of the agonist. Characteristically, BAMs enhance signaling (β>1) through some pathways but exert no effect (β=1) or antagonize signaling (β<1) through other pathway subsets. Ago-BAMs act as biasing modulators to exert pathway-specific effects on agonist signaling and act alone as biased allosteric agonists, that is, τ is greater than 0 for some pathways, but not others. (A) This hypothetical BAM potentiates agonist-induced signaling through pathway ‘a’ while antagonizing agonist-induced signaling through pathways ‘b’ and ‘c’. (B) This hypothetical ago-BAM stimulates signaling through pathway ‘a’ when applied alone but has no effect on pathways ‘b’ or ‘c’. In combination with an orthosteric agonist, this ago-BAM enhances agonist signaling efficacy through pathway ‘a’ while antagonizing signaling through ‘b’ and ‘c’. (C-E) Application of Eq. 2, a symmetrical version of the extended operational model, to the agonist ago-BAM pair of neurotensin (NTS) and SBI-553 at the NTSR1. (C) NTS alone acts as a balanced, full orthosteric agonist, activating both Gαq- and β-arrestin-mediated cellular responses. (D) SBI-553 alone acts as a biased allosteric agonist, activating β-arrestin- but not Gαq-mediated cellular responses. (E) SBI-553 confers β-arrestin bias to NTS by concurrently enhancing NTS-induced β-arrestin recruitment and antagonizing NTS-induced Gαq activation. The qualitative behavior depicted here has been directly observed with the action on SBI-553 on NTSR1 [40, 41].

Box 1, Figure I.. Optimizing the extended operational model.

While both Eq. 1 (left) and Eq. 2 (right) capture the qualitative effects of positive allosteric modulation, they produce distinct effects on agonist-modulator dose-response curve Top shifts. The most appropriate model may depend on the characteristics of the allosteric modulator, receptor system and assay platform. The horizontal lines represent the Top values of the corresponding colored response curves.