Quantification of Ligand Bias for Clinically Relevant β₂-Adrenergic Receptor Ligands: Implications for Drug Taxonomy

Abstract

The concepts of functional selectivity and ligand bias are becoming increasingly appreciated in modern drug discovery programs, necessitating more informed approaches to compound classification and, ultimately, therapeutic candidate selection. Using the b2-adrenergic receptor as a model, we present a proof of concept study that assessed the bias of 19 b-adrenergic ligands, including many clinically used compounds, across four pathways [cAMP production, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, calcium mobilization, and receptor endocytosis] in the same cell background (human embryonic kidney 293S cells). Efficacy-based clustering placed the ligands into five distinct groups with respect to signaling signatures. In some cases, apparent functional selectivity originated from offtarget effects on other endogenously expressed adrenergic receptors, highlighting the importance of thoroughly assessing selectivity of the responses before concluding receptor-specific ligand-biased signaling. Eliminating the nonselective compounds did not change the clustering of the 10 remaining compounds. Some ligands exhibited large differences in potency for the different pathways, suggesting that the nature of the receptoreffector complexes influences the relative affinity of the compounds for specific receptor conformations. Calculation of relative effectiveness (within pathway) and bias factors (between pathways) for each of the compounds, using an operational model of agonism, revealed a global signaling signature for all of the compounds relative to isoproterenol. Most compounds were biased toward ERK1/2 activation over the other pathways, consistent with the notion that many proximal effectors converge on this pathway. Overall, we demonstrate a higher level of ligand texture than previously anticipated, opening perspectives for the establishment of pluridimensional correlations between signaling profiles, drug classification, therapeutic efficacy, and safety.

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*

“…5B). The EC 50 value for ICL3-9-promoted ␤ 2 AR-G s interaction was 3.3 Ϯ 0.4 M (Fig. 5C), which is comparable with the EC 50 value observed for cAMP production (Fig.…”

“…This ␤-arrestin-biased profile was attributed to an agonist-promoted increased rate of GRK phosphorylation of the receptor (49). van der Westhuizen et al (50) continued the "taxonomy" of ␤ 2 AR ligands by screening a number of full and partial ␤-agonists, neutral antagonists, and inverse agonists for their ability to modulate cAMP, Ca 2ϩ mobilization, ERK1/2 phosphorylation, and ␤ 2 AR internalization. Their findings revealed surprising diversity in the signaling profiles of closely related receptor ligands and further support the functional basis of ligand-biased signaling.…”

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

“…Although the b2-AR is ubiquitously expressed and controls a range of physiologic processes, Iso and Sal are classically administered to target its effects in the lung (van der Westhuizen et al, 2014). Quantitative PCR analysis of the transcriptional responses induced by the two ligands in human airway smooth muscle cells reveals comparable upregulation of the expression of two adrenoceptor target genes, PDE4D and CGA (Fig.…”

Human Neuropeptide S Receptor Is Activated via a Gαq Protein-biased Signaling Cascade by a Human Neuropeptide S Analog Lacking the C-terminal 10 Residues

“…The challenge of hNPS-(1-10) induced an increase in luciferase activity but with significantly less potency as compared with wild type NPS, having an EC 50 value of 64.26 Ϯ 2.38 M. Our data indicate that there are interesting differences in agonist activity for hNPS-(1-10) between G␣ q and G␣ s coupling. Therefore, we next performed a quantitative analysis of hNPS-(1-10) bias based on functional parameters using an operational model as described previously (25,27,28). As shown in Table 1, hNPS-(1-10) activation of NPSR revealed a significant bias (Ͼ80-fold) for G␣ q activation over G␣ s .…”