Translating in vitro ligand bias into in vivo efficacy

Abstract

It is increasingly apparent that ligand structure influences both the efficiency with which G protein-coupled receptors (GPCRs) engage their downstream effectors and the manner in which they are activated. Thus, 'biased' agonists, synthetic ligands whose intrinsic efficacy differs from the native ligand, afford a strategy for manipulating GPCR signaling in ways that promote beneficial signals while blocking potentially deleterious ones. Still, there are significant challenges in relating in vitro ligand efficacy, which is typically measured in heterologous expression systems, to the biological response in vivo, where the ligand is acting on natively expressed receptors and in the presence of the endogenous ligand. This is particularly true of arrestin pathway-selective 'biased' agonists. The type 1 parathyroid hormone receptor (PTH₁R) is a case in point. Parathyroid hormone (PTH) is the principal physiological regulator of calcium homeostasis, and PTH₁R expressed on cells of the osteoblast lineage are an established therapeutic target in osteoporosis. In vitro, PTH₁R signaling is highly sensitive to ligand structure, and PTH analogs that affect the selectivity/kinetics of G protein coupling or that engage arrestin-dependent signaling mechanisms without activating heterotrimeric G proteins have been identified. In vivo, intermittent administration of conventional PTH analogs accelerates the rate of osteoblastic bone formation, largely through known cAMP-dependent mechanisms. Paradoxically, both intermittent and continuous administration of an arrestin pathway-selective PTH analog, which in vivo would be expected to antagonize endogenous PTH₁R-cAMP signaling, also increases bone mass. Transcriptomic analysis of tissue from treated animals suggests that conventional and arrestin pathway-selective PTH1R ligands act in largely different ways, with the latter principally affecting pathways involved in the regulation of cell cycle, survival, and migration/cytoskeletal dynamics. Such multi-dimensional in vitro and in vivo analyses of ligand bias may provide insights into the physiological roles of non-canonical arrestin-mediated signaling pathways in vivo, and provide a conceptual framework for translating arrestin pathway-selective ligands into viable therapeutics.

Keywords: Arrestin; G protein-coupled receptor; Osteoblast; Osteoporosis; Parathyroid hormone; Pharmacology.

Fig. 1

Conceptual representation of biased agonism viewed from multiple levels within a biological system. At the initial point of contact, ligand binding changes the distribution of receptor conformations leading to stabilization of one or more active states. As signals radiate outward from the ligand-receptor complex, receptors engage proximal effectors like G proteins and arrestins (seconds), second messenger and kinase cascades are activated, and global changes in downstream protein phosphorylation occur (seconds-minutes), leading to changes in cell behavior, e.g. proliferation, migration or apoptosis (hours-days). When assayed in vitro, biased ligands appear to affect a subset of the pathways activated by the native ligand (represented as Venn diagrams). But when the in vivo effects of native and biased agonists are compared, data from microarray analysis and tissue phenotyping suggest that biased agonists may have unpredictable effects arising from ‘unbalanced’ GPCR activation. This ‘disconnect’ between the in vitro and in vivo efficacy of biased ligands is a potential barrier to the rational development of biased GPCR therapeutics.

Fig. 2

PTH₁R ligand bias in vitro. A. Multiaxial plot of PTH analog efficacy in cAMP, calcium and ERK1/2 assays performed using HEK293 cells overexpressing human PTH₁R. Estimated RA_i values for each ligand are plotted to represent the magnitude and direction of effect in each signaling response. Figure adapted from Appleton et al. (2013) Methods Enzymol 522: 229–262 [52]. B. Effects of hPTH(1–34) and [D-Trp¹²,Tyr³⁴]-bPTH(7–34) [bPTH(7–34)] on cAMP production (left) and ERK1/2 activation (right) in 10 day-old cultures of differentiating primary calvarial osteoblasts isolated from wild type (WT) and β-arrestin2−/− mice. Figure adapted from Gesty-Palmer et al. (2009) Science – Transl Med 1ra1 [33]. C. Effects of hPTH (1–34) and [D-Trp¹²,Tyr³⁴]-bPTH(7–34) on the survival and random migration of primary calvarial osteoblasts isolated from wild type and β-arrestin2−/− mice. Anti-apoptotic effects (left) were measured in 10 day-old differentiating osteoblast cultures subjected to serum-withdrawal or exposed to etoposide in the presence or absence of hPTH(1–34) or [D-Trp¹²,Tyr³⁴]-bPTH(7–34). Random cell migration (right) was measured by scratch assay performed on 3-day old confluent monolayers of primary preosteoblasts. Figure adapted from Gesty-Palmer et al. Mol Endocrinol 27:296–314 [55].

Fig. 3

Comparison of the in vivo efficacy of hPTH(1–34) and [D-Trp¹²,Tyr³⁴]-bPTH(7–34) at the tissue and transcriptome levels. A. Representative quantitative computed tomography (qCT) images of the trabecular compartment of proximal tibia from wild type male C57BL/6 mice treated for 8 weeks with daily injection of vehicle, hPTH(1–34) or [D-Trp¹²,Tyr³⁴]-bPTH (7–34) [bPTH(7–34)] (top). The table below summarizes the actions of hPTH(1–34) and [D-Trp¹²,Tyr³⁴]-bPTH(7–34) in vitro and in vivo. qCT of trabecular and cortical bone, histomorphometric analysis of osteoblast and osteoclast numbers, serum osteoid surface and mineral apposition rate, and assays of bone turnover markers were performed on serum, urine and tissue samples from wild type and β-arrestin2−/− mice after 8 weeks treatment with vehicle, hPTH(1–34) or [D-Trp¹²,Tyr³⁴]-bPTH(7–34). ‘+’ to ‘+++’ denotes magnitude of increase relative to vehicle-treated controls. ‘-’ denotes decrease relative to vehicle-treated. ‘NS’ denotes no significant change. The table summarizes data originally published in Gesty-Palmer et al. (2009) Science – Transl Med 1ra1 [33]. B. Transcriptomic signatures from calvarial bone of mice treated with hPTH(1–34) and [D-Trp¹²,Tyr³⁴]-bPTH(7–34). Global changes in mRNA abundance in wild type and male mice treated with vehicle, hPTH(1–34) or [D-Trp¹²,Tyr³⁴]-bPTH(7–34) were depicted using the vector based graphic program Omnimorph [71]. C. Parametric geneset enrichment analysis of signaling pathways and biological processes affected by hPTH(1–34) and [D-Trp¹²,Tyr³⁴]-bPTH(7–34) in vivo. Analyses were performed using microarray genesets consisting of calvarial transcripts with significantly different expression between wild type (WT) mice treated with vehicle (NS), hPTH(1–34) or [D-Trp¹²,Tyr³⁴]-bPTH (7–34). The left panel compares signaling pathway gene clusters identified using the WT bPTH(7–34) vs WT NS (red bars) and WT hPTH(1–34) vs WT NS (blue bars) genesets. Signaling pathways corresponding to signal transduction, growth factor signaling, nuclear receptor signaling and cell cycle control are shown. The right panel depicts an identical comparison derived by querying the Gene Ontology biological processes (GObp) database. In each panel, hybrid scores (−log₁₀(p) × pathway enrichment ratio) reflect the statistical probability that the observed differences did not occur by chance. All signaling pathway and GObp terms shown exceed a minimal threshold significance of p ≤0.05. Figure adapted from Gesty-Palmer et al. (2013) Mol Endocrinol 27:296–314 [55].