Targeting the Angiotensin II Type 1 Receptor in Cerebrovascular Diseases: Biased Signaling Raises New Hopes.

The physiological and pathophysiological relevance of the angiotensin II type 1 (AT1) G protein-coupled receptor no longer needs to be proven in the cardiovascular system. The renin-angiotensin system and the AT1 receptor are the targets of several classes of therapeutics (such as angiotensin converting enzyme inhibitors or angiotensin receptor blockers, ARBs) used as first-line treatments in cardiovascular diseases. The importance of AT1 in the regulation of the cerebrovascular system is also acknowledged. However, despite numerous beneficial effects in preclinical experiments, ARBs do not induce satisfactory curative results in clinical stroke studies. A better understanding of AT1 signaling and the development of biased AT1 agonists, able to selectively activate the ß-arrestin transduction pathway rather than the Gq pathway, have led to new therapeutic strategies to target detrimental effects of AT1 activation. In this paper, we review the involvement of AT1 in cerebrovascular diseases as well as recent advances in the understanding of its molecular dynamics and biased or non-biased signaling. We also describe why these alternative signaling pathways induced by ß-arrestin biased AT1 agonists could be considered as new therapeutic avenues for cerebrovascular diseases.

Identification of a Putative ß-Arrestin Superagonist of the Growth Hormone Secretagogue Receptor (GHSR).

Ghrelin is a pleiotropic feeding hormone which also has a pivotal role in the central nervous system. Upon the activation of its receptor, growth hormone secretagogue receptor (GHSR), the Gαq/11 -mediated and the ß-arrestin-mediated signaling pathways are activated. As the ß-arrestin pathway is a potential drug target, there is a strong need for ß-arrestin-biased GHSR modulators. Activation of the ß-arrestin pathway should inhibit the Gαq/11 -mediated calcium flux through internalization of the receptor. Hence, we used the antagonistic activity in the calcium assay as the first screening for the ß-arrestin activation. By conducting the second screening assay for the ß-arrestin activation based on extracellular signal regulated kinase (ERK) 1/2 phosphorylation, we discovered a putative ß-arrestin-biased superagonist. The activity of the compound was not completely blocked with the competitive antagonist, which implies that the effect is mediated, at least partly, by allosteric binding of the compound.

Signaling diversity of mu- and delta- opioid receptor ligands: Re-evaluating the benefits of ß-arrestin/G protein signaling bias.

Opioid analgesics are elective for treating moderate to severe pain but their use is restricted by severe side effects. Signaling bias has been proposed as a viable means for improving this situation. To exploit this opportunity, continuous efforts are devoted to understand how ligand-specific modulations of receptor functions could mediate the different in vivo effects of opioids. Advances in the field have led to the development of biased agonists based on hypotheses that allocated desired and undesired effects to specific signaling pathways. However, the prevalent hypothesis associating ß-arrestin to opioid side effects was recently challenged and multiple of the newly developed biased drugs may not display the superior side effects profile that was sought. Moreover, biased agonism at opioid receptors is now known to be time- and cell-dependent, which adds a new layer of complexity for bias estimation. Here, we first review the signaling mechanisms underlying desired and undesired effects of opioids. We then describe biased agonism at opioid receptors and discuss the different perspectives that support the desired and undesired effects of opioids in view of exploiting biased signaling for therapeutic purposes. Finally, we explore how signaling kinetics and cellular background can influence the magnitude and directionality of bias at those receptors.

ß-Arrestin-Biased Agonist Targeting the Brain AT1R (Angiotensin II Type 1 Receptor) Increases Aversion to Saline and Lowers Blood Pressure in Deoxycorticosterone Acetate-Salt Hypertension.

Activation of central AT1Rs (angiotensin type 1 receptors) is required for the increased blood pressure, polydipsia, and salt intake in deoxycorticosterone acetate (DOCA)-salt hypertension. TRV120027 (TRV027) is an AT1R-biased agonist that selectively acts through ß-arrestin. We hypothesized that intracerebroventricular administration of TRV027 would ameliorate the effects of DOCA-salt. In a neuronal cell line, TRV027 induced AT1aR internalization through dynamin and clathrin-mediated endocytosis. We next evaluated the effect of chronic intracerebroventricular infusion of TRV027 on fluid intake. We measured the relative intake of water versus various saline solutions using a 2-bottle choice paradigm in mice subjected to DOCA with a concomitant intracerebroventricular infusion of either vehicle, TRV027, or losartan. Sham mice received intracerebroventricular vehicle without DOCA. TRV027 potentiated DOCA-induced water intake in the presence or absence of saline. TRV027 and losartan both increased the aversion for saline-an effect particularly pronounced for highly aversive saline solutions. Intracerebroventricular Ang (angiotensin) II, but not TRV027, increased water and saline intake in the absence of DOCA. In a separate cohort, blood pressure responses to acute intracerebroventricular injection of vehicle, TRV, or losartan were measured by radiotelemetry in mice with established DOCA-salt hypertension. Central administration of intracerebroventricular TRV027 or losartan each caused a significant and similar reduction of blood pressure and heart rate. We conclude that administration of TRV027 a selective ß-arrestin biased agonist directly into the brain increases aversion to saline and lowers blood pressure in a model of salt-sensitive hypertension. These data suggest that selective activation of AT1R ß-arrestin pathways may be exploitable therapeutically.

Molecular mechanisms of fentanyl mediated ß-arrestin biased signaling.

The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or ß-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent ß-arrestin biased agonist, binds the µ-opioid receptor (µOR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. Following an in-silico derived hypothesis we found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce ß-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate an activation mechanism where the n-aniline ring of fentanyl mediates µOR ß-arrestin through a novel M153 "microswitch" by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated ß-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles.

Methods to Monitor the Trafficking of ß-Arrestin/G Protein-Coupled Receptor Complexes Using Enhanced Bystander BRET.

ß-Arrestins are adaptors that regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). Bioluminescence resonance energy transfer (BRET) is a sensitive and versatile method for real-time monitoring of protein-protein interactions and protein kinesis within live cells, such as the recruitment of ß-arrestins to activated receptors at the plasma membrane (PM) and the trafficking of GPCR/ß-arrestin complexes to endosomes. Trafficking of receptor/ß-arrestin complexes can be assessed by BRET through tagging ß-arrestins with the donor luciferase from Renilla reniformis (Rluc) and anchoring the acceptor green fluorescent protein from the same species (rGFP) in distinct cell compartments (e.g., PM or endosomes) to generate highly efficient bystander BRET (referred to as enhanced bystander BRET (EbBRET)) upon re-localization of ß-arrestins to these compartments following receptor activation. Here, we outline the protocol for quantitatively monitoring ß-arrestin recruitment to agonist-activated Angiotensin II type 1 receptor (AT1R) and ß2-adrenergic receptor (ß2AR) at the PM and the trafficking of receptor/ß-arrestin complexes into endosomes using EbBRET-based biosensors.

Discovery of ß-Arrestin Biased Ligands of 5-HT7R.

Though many studies have been published about therapeutic potentials of selective 5-HT7R ligands, there have been few biased ligands of 5-HT7R. The development of potent and selective biased ligands of 5-HT7R would be of great help in understanding the relationship between pharmacological effects and G protein/ß-arrestin signaling pathways of 5-HT7R. In order to identify 5-HT7R ligands with biased agonism, we designed and synthesized a series of tetrahydroazepine derivatives 1 and 2 with arylpyrazolo moiety or arylisoxazolo moiety. Through several biological evaluations such as binding affinity, selectivity profile, and functions in G protein and ß-arrestin signaling pathways, 3-(4-chlorophenyl)-1,4,5,6,7,8-hexahydropyrazolo[3,4- d]azepine 1g was discovered as the ß-arrestin biased ligand of 5-HT7R. In an electroencephalogram (EEG) test, 1g increased total non-rapid eye movement (NREM) sleep time and decreased total rapid eye movement (REM) sleep time.

Ang-(1-7) is an endogenous ß-arrestin-biased agonist of the AT1 receptor with protective action in cardiac hypertrophy.

The renin-angiotensin system (RAS) plays a key role in the control of vasoconstriction as well as sodium and fluid retention mediated mainly by angiotensin (Ang) II acting at the AT1 receptor (AT1R). Ang-(1-7) is another RAS peptide, identified as the endogenous ligand of the Mas receptor and known to counterbalance many of the deleterious effects of AngII. AT1R signaling triggered by ß-arrestin-biased agonists has been associated to cardioprotection. Because position 8 in AngII is important for G protein activation, we hypothesized that Ang-(1-7) could be an endogenous ß-arrestin-biased agonist of the AT1R. Here we show that Ang-(1-7) binds to the AT1R without activating Gq, but triggering ß-arrestins 1 and 2 recruitment and activation. Using an in vivo model of cardiac hypertrophy, we show that Ang-(1-7) significantly attenuates heart hypertrophy by reducing both heart weight and ventricular wall thickness and the increased end-diastolic pressure. Whereas neither the single blockade of AT1 or Mas receptors with their respective antagonists prevented the cardioprotective action of Ang1-7, combination of the two antagonists partially impaired the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) mediates at least part of its cardioprotective effects by acting as an endogenous ß-arrestin-biased agonist at the AT1R.

Long-Term Biased ß-Arrestin Signaling Improves Cardiac Structure and Function in Dilated Cardiomyopathy.

BACKGROUND: Biased agonism of the angiotensin II receptor is known to promote cardiac contractility. Our laboratory indicated that these effects may be attributable to changes at the level of the myofilaments. However, these signaling mechanisms remain unknown. Because a common finding in dilated cardiomyopathy is a reduction in the myofilament-Ca2+ response, we hypothesized that ß-arrestin signaling would increase myofilament-Ca2+ responsiveness in a model of familial dilated cardiomyopathy and improve cardiac function and morphology. METHODS: We treated a dilated cardiomyopathy-linked mouse model expressing a mutant tropomyosin (Tm-E54K) for 3 months with either TRV120067, a ß-arrestin 2-biased ligand of the angiotensin II receptor, or losartan, an angiotensin II receptor blocker. At the end of the treatment protocol, we assessed cardiac function using echocardiography, the myofilament-Ca2+ response of detergent-extracted fiber bundles, and used proteomic approaches to understand changes in posttranslational modifications of proteins that may explain functional changes. We also assessed signaling pathways altered in vivo and by using isolated myocytes. RESULTS: TRV120067- treated Tm-E54K mice showed improved cardiac structure and function, whereas losartan-treated mice had no improvement. Myofilaments of TRV120067-treated Tm-E54K mice had significantly improved myofilament-Ca2+ responsiveness, which was depressed in untreated Tm-E54K mice. We attributed these changes to increased MLC2v and MYPT1/2 phosphorylation seen only in TRV120067-treated mice. We found that the functional changes were attributable to an activation of ERK1/2-RSK3 signaling, mediated through ß-arrestin, which may have a novel role in increasing MLC2v phosphorylation through a previously unrecognized interaction of ß-arrestin localized to the sarcomere. CONCLUSIONS: Long-term ß-arrestin 2-biased agonism of the angiotensin II receptor may be a viable approach to the treatment of dilated cardiomyopathy by not only preventing maladaptive signaling, but also improving cardiac function by altering the myofilament-Ca2+ response via ß-arrestin signaling pathways.

Crosstalk between angiotensin and the nonamyloidogenic pathway of Alzheimer's amyloid precursor protein.

The association between hypertension and an increased risk for Alzheimer's disease (AD) and dementia is well established. Many data suggest that modulation of the renin-angiotensin system may be meaningful for the prevention and therapy of neurodegenerative disorders, in particular AD. Proteolytic cleavage of the amyloid precursor protein (APP) by α-secretase precludes formation of neurotoxic Aß peptides and is expected to counteract the development of AD. An established approach for the up-regulation of α-secretase cleavage is the activation of G protein-coupled receptors (GPCRs). Therefore, our study aimed to analyze whether stimulation of angiotensin AT1 or AT2 receptors stably expressed in HEK cells influence the nonamyloidogenic pathway of APP processing. Treatment of both receptors with angiotensin II clearly showed that only activation of the AT1 receptor increased several fold the α-secretase-mediated shedding of APP. This effect was completely abolished by treatment with the AT1 receptor-specific antagonist telmisartan. Using the BIM-46187 inhibitor, we demonstrate that the Gαq protein-mediated pathway is involved in this stimulation process. Stimulation of AT1 receptors with the ß-arrestin-biased agonist SII was ineffective regarding α-secretase-mediated APP shedding. This result discloses that only the G protein-dependent pathway is involved in the Ang II-induced APP shedding. Blocking of Gßγ subunits by the inhibitor gallein completely prevented constitutive and Ang II-induced APP shedding. Our findings provide evidence that induction of APP shedding via Ang II/AT1 receptor stimulation is effected by G protein activation with Gßγ subunits playing important roles.

ß-arrestin-biased signaling through the ß2-adrenergic receptor promotes cardiomyocyte contraction.

ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.

G-Protein/ß-Arrestin-Linked Fluctuating Network of G-Protein-Coupled Receptors for Predicting Drug Efficacy and Bias Using Short-Term Molecular Dynamics Simulation.

The efficacy and bias of signal transduction induced by a drug at a target protein are closely associated with the benefits and side effects of the drug. In particular, partial agonist activity and G-protein/ß-arrestin-biased agonist activity for the G-protein-coupled receptor (GPCR) family, the family with the most target proteins of launched drugs, are key issues in drug discovery. However, designing GPCR drugs with appropriate efficacy and bias is challenging because the dynamic mechanism of signal transduction induced by ligand-receptor interactions is complicated. Here, we identified the G-protein/ß-arrestin-linked fluctuating network, which initiates large-scale conformational changes, using sub-microsecond molecular dynamics (MD) simulations of the ß2-adrenergic receptor (ß2AR) with a diverse collection of ligands and correlation analysis of their G protein/ß-arrestin efficacy. The G-protein-linked fluctuating network extends from the ligand-binding site to the G-protein-binding site through the connector region, and the ß-arrestin-linked fluctuating network consists of the NPxxY motif and adjacent regions. We confirmed that the averaged values of fluctuation in the fluctuating network detected are good quantitative indexes for explaining G protein/ß-arrestin efficacy. These results indicate that short-term MD simulation is a practical method to predict the efficacy and bias of any compound for GPCRs.