Scott M. DeWire

The concept of “ligand bias” at G protein coupled receptors has been introduced to describe ligands which preferentially stimulate one intracellular signaling pathway over another. There is growing interest in developing biased G protein coupled receptor ligands to yield safer, better tolerated, and more efficacious drugs. The classical μ opioid morphine elicited increased efficacy and duration of analgesic response with reduced side effects in β-arrestin-2 knockout mice compared to wild-type mice, suggesting that G protein biased μ opioid receptor agonists would be more efficacious with reduced adverse events. Here we describe our efforts to identify a potent, selective, and G protein biased μ opioid receptor agonist, TRV130 ((R)-30). This novel molecule demonstrated an improved therapeutic index (analgesia vs adverse effects) in rodent models and characteristics appropriate for clinical development. It is currently being evaluated in human clinical trials for the treatment of acute severe pain.

Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the “druggable” genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, β-arrestins. Biased agonists, which signal with different efficacies to a receptor9s multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from “balanced agonists.” Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the β2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs.

Drug discovery efforts targeting G-protein-coupled receptors (GPCR) have been immensely successful in creating new cardiovascular medicines. Currently marketed GPCR drugs are broadly classified as either agonists that activate receptors or antagonists that prevent receptor activation by endogenous stimuli. However, GPCR couple to a multitude of intracellular signaling pathways beyond classical G-protein signals, and these signals can be independently activated by biased ligands to vastly expand the potential for new drugs at these classic targets. By selectively engaging only a subset of a receptor's potential intracellular partners, biased ligands may deliver more precise therapeutic benefit with fewer side effects than current GPCR-targeted drugs. In this review, we discuss the history of biased ligand research, the current understanding of how biased ligands exert their unique pharmacology, and how research into GPCR signaling has uncovered previously unappreciated capabilities of receptor pharmacology. We focus on several receptors to illustrate the approaches taken and discoveries made, and how these are steadily illuminating the intricacies of GPCR pharmacology. Discoveries of biased ligands targeting the angiotensin II type 1 receptor and of separable pharmacology suggesting the potential value of biased ligands targeting the β-adrenergic receptors and nicotinic acid receptor GPR109a highlight the powerful clinical promise of this new category of potential therapeutics.

Biased G protein-coupled receptor ligands engage subsets of the receptor signals normally stimulated by unbiased agonists. However, it is unclear whether ligand bias can elicit differentiated pharmacology in vivo. Here, we describe the discovery of a potent, selective β-arrestin biased ligand of the angiotensin II type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-d-Ala-OH) competitively antagonizes angiotensin II-stimulated G protein signaling, but stimulates β-arrestin recruitment and activates several kinase pathways, including p42/44 mitogen-activated protein kinase, Src, and endothelial nitric-oxide synthase phosphorylation via β-arrestin coupling. Consistent with β-arrestin efficacy, and unlike unbiased antagonists, TRV120027 increased cardiomyocyte contractility in vitro. In rats, TRV120027 reduced mean arterial pressure, as did the unbiased antagonists losartan and telmisartan. However, unlike the unbiased antagonists, which decreased cardiac performance, TRV120027 increased cardiac performance and preserved cardiac stroke volume. These striking differences in vivo between unbiased and β-arrestin biased ligands validate the use of biased ligands to selectively target specific receptor functions in drug discovery.

Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and G_i/G_o proteins, here we dissected the roles of G proteins and the adaptor proteins, β-arrestins, in nicotinic acid–induced signaling and physiological responses. In a human cell line–based signaling assay, nicotinic acid stimulation led to pertussis toxin–sensitive lowering of cAMP, recruitment of β-arrestins to the cell membrane, an activating conformational change in β-arrestin, and β-arrestin–dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of β-arrestin1 to activated cytosolic phospholipase A₂ as well as β-arrestin1–dependent activation of cytosolic phospholipase A₂ and release of arachidonate, the precursor of prostaglandin D₂ and the vasodilator responsible for the flushing response. Moreover, β-arrestin1–null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by β-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein–biased ligands that activate GPR109A in a β-arrestin–independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins β-arrestin1 and -2 might regulate this pathological process. Deficiency of β-arrestin2 in ldlr ^−/− mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that β-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, β-arrestin1 ^−/− , and β-arrestin2 ^−/− mice. Neointimal hyperplasia was enhanced in β-arrestin1 ^−/− mice, and diminished in β-arrestin2 ^−/− mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in β-arrestin2 ^−/− mice was not altered by transplantation with either wild-type or β-arrestin2 ^−/− bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in β-arrestin1 ^−/− and decreased in β-arrestin2 ^−/− mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in β-arrestin1 ^−/− SMCs and less in β-arrestin2 ^−/− SMCs. Proliferation was less than wild type in β-arrestin2 ^−/− SMCs but not in β-arrestin2 ^−/− endothelial cells. We conclude that β-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by β-arrestin2 and β-arrestin1. These findings identify inhibition of β-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

For many years, β-adrenergic receptor antagonists (β-blockers or βAR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, β-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different β-blockers varies widely in this condition. One drug, carvedilol, a nonsubtype-selective βAR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant βAR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in β2 adrenergic receptor (β2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating G _s -dependent adenylyl cyclase but, nonetheless, stimulates ( i ) phosphorylation of the receptor's cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; ( ii ) recruitment of β-arrestin to the β2AR; ( iii ) receptor internalization; and ( iv ) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant β2AR ^{T68F,Y132G,Y219A} (β2AR ^TYY ) and abolished by β-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from G _s , is nonetheless able to stimulate β-arrestin-mediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic β2AR ligands.

Upon their discovery, β-arrestins 1 and 2 were named for their capacity to sterically hinder the G protein coupling of agonist-activated seven-transmembrane receptors, ultimately resulting in receptor desensitization. Surprisingly, recent evidence shows that β-arrestins can also function to activate signaling cascades independently of G protein activation. By serving as multiprotein scaffolds, the β-arrestins bring elements of specific signaling pathways into close proximity. β-Arrestin regulation has been demonstrated for an ever-increasing number of signaling molecules, including the mitogen-activated protein kinases ERK, JNK, and p38 as well as Akt, PI3 kinase, and RhoA. In addition, investigators are discovering new roles for β-arrestins in nuclear functions. Here, we review the signaling capacities of these versatile adapter molecules and discuss the possible implications for cellular processes such as chemotaxis and apoptosis.