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Apr 3, 2014 - United States. ‡. Discovery Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United Sta...
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Biased Ligand Modulation of Seven Transmembrane Receptors (7TMRs): Functional Implications for Drug Discovery Miniperspective Craig C. Correll*,† and Brian A. McKittrick*,‡ †

Department of Immunology, Merck Research Laboratories, BMB 10-108, 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States ‡ Discovery Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States ABSTRACT: Seven transmembrane receptors (7TMRs), also known as G-protein-coupled receptors (GPCRs), have proven to be valuable targets for the development of therapeutics. The expansion of our understanding of 7TMR downstream signaling pathways beyond G-proteins has broadened our appreciation of the versatility of these cell surface receptors. In particular, the increased awareness of 7TMR engagement of βarrestin signaling has opened up additional avenues for drug discovery. 7TMRs can adopt different conformations and in response to various ligands can lead to a bias in downstream signaling mechanisms when comparing the overall efficacy between G-protein and β-arrestin dependent pathways. In 2012, we organized a session at the Spring National Meeting of the American Chemical Society on biased signaling in 7TMRs.1−4 Building on that experience, we provide in this Miniperspective some examples that exemplify developments in the area of biased 7TMR signaling and highlight some cautionary notes as well as some of the exciting opportunities for drug discovery.

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INTRODUCTION The therapeutic potential of small molecules that modulate the function of 7TM receptors has been well recognized and realized over the past 3 decades since their initial characterization. The family of 7TMRs is the largest of the cell surface receptor families and comprises over 800 members encoded by the human genome.5 This family of receptors recognizes a wide variety of biologically relevant and diverse set of molecules, such as lipids, peptides, carbohydrates, and neurotransmitters, and have been implicated in various diseases.6 Targeting these cellular receptors has been a fruitful area for the development of small molecule therapy, as developed drugs targeting 7TMRs comprise 63% of the drugs targeting cell surface receptors7 and over one-third of the prescription drug market. A great majority of these have come from engaging the aminergic receptors from the A family of GPCRs.8 7TM receptors were originally recognized primarily as Gprotein-coupled cell surface receptors that induce intracellular signaling cascades via activation and engagement of specific Gproteins from a cellular pool of functionally diverse subset of proteins. The central dogma in cellular pharmacology was that different agonists may have different potencies and efficacies, but a comparable set of signaling proteins would be engaged regardless. The basic premise was that receptors had a single active conformation, and the ability to stay in this active state imparted via a full agonist determined both the potency and efficacy as defined by a measurable end point. By utilization of this simple construct of the receptor as a simple on and off switch, © 2014 American Chemical Society

where the active state conformation of the receptor was directly related to overall efficacy, a number of molecules were identified as either agonists or antagonists and ultimately made into marketable drugs. Indeed, a number of therapeutics, including βblockers, antihistamines, anticholinergics, analgesic opiates, and neuroleptics, were developed prior to either the cloning and molecular characterization or the full understanding of the molecular pharmacology of 7TMRs. Despite the incomplete understanding of these receptors and their structure, early studies were able to elucidate the role of cAMP and its characterization as a second messenger downstream of G-protein engagement.9 On the basis of these findings, the idea of targeting GPCRs took hold and spurred on a whole host of pharmacological characterizations, including additional second messengers such as the generation of inositol phosphates and the coupled release of intracellular calcium. This initial relationship between 7TMRs and G-protein activation helped advance drug development for many years. The only point of inquiry was to discern which type of G-protein was engaged and how it could be characterized and quantified, both biochemically and at a cellular level.

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β-ARRESTIN SIGNALING Additional research into understanding 7TMR signaling led to the discovery of β-arrestins.10,11 Ligand-induced activation of 7TMR resulted in engagement of β-arrestins, which were initially Received: October 29, 2013 Published: April 3, 2014 6887

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constructs overexpressing the receptor or a specific G-protein or quantifying the effects in a primary cell when technically feasible. For quantification, the development of 7TMRs coexpressed with a modified β-arrestin can demonstrate scaffolding of β-arrestins onto a 7TMR within a cellular context.14 The advent of monitoring and quantifying engagement of multiple signaling pathways opened the door of inquiry to uncover differences in both the qualitative and quantitative measurements and the functional consequences of engagement of different downstream signaling pathways. Driven by molecules prepared by medicinal chemists, it has become very apparent that a wide variety of distinct ligands could stabilize different conformations of active receptors resulting in ligand-specific cellular responses that were “biased” for either G-protein mediated or β-arrestin mediated signaling15 (Figure 2). In contrast to the previous two-state

characterized as negative regulators of G-protein function, often mediating receptor desensitization and internalization. β-Arrestins were subsequently found to act as scaffolds, which upon ligand binding transduce their own intracellular signaling cascade.12 Like G-protein signaling, β-arrestin signaling is both receptor and ligand dependent. During the past 10 years, it has become increasingly recognized that in addition to the wellestablished G-protein mediated pathways, β-arrestins are ubiquitous and play a central role in 7TMR biochemical and cellular pharmacology (Figure 1). It is now widely appreciated

Figure 1. 7TMR engagement and downstream signaling. Ligand binding to a 7TMR engages G-protein activation leading to either increases (Gs) or decreases (Gi) in cAMP levels of the cell or activation of ion channels leading to influx of Ca2+ into the cytoplasm. In addition, the activation of the 7TMR leads to phosphorylation of the 7TMR by a family of G-protein-coupled kinases (GRK). This leads to recruitment of various β-arrestins, which results in (1) inhibition of further signaling via the G-protein mediated pathway and (2) activation of both downstream signaling events such as MAPK kinases and desensitization of the response via internalization of the receptor.

that upon ligand engagement, 7TMRs interact initially with Gproteins, followed by phosphorylation of the 7TMR via a family of up to at least seven different G-protein receptor kinases (GRKs).13 This specific phosphorylation of the ligand engaged 7TMR leads to subsequent binding by β-arrestins. The binding of β-arrestin leads to desensitization, via first blocking G-protein mediated signaling as well as targeting the receptor for clathrindependent internalization. Furthermore, the understanding that the β-arrestin−7TM complex can also act as a signaling scaffold has led to an intense interest not only to understand the cellular pharmacology but also to pursue the effects derived from small molecule agonists and characterization of both G-protein signaling and β-arrestin mediated signaling in parallel. A more comprehensive understanding of 7TMR pharmacology has developed utilizing a combined battery of diverse cellular assays. Traditional cellular assays to measure G-protein engagement, Gs (increases in cellular cAMP levels), Gi (inhibition of forskolin-induced cAMP levels, or Gq (transient increases in cytosolic Ca2+) have been used to characterize and quantify 7TMR engagement with its cognate G-protein partner in cellular

Figure 2. 7TMR biasing. Some ligands are known to stabilize different conformations of the 7TMRs. These different conformations can promote biased intracellular interactions whereby either the G-protein (A) or β-arrestin (B) pathways are more efficiently engaged. In the top panel, a ligand is biased toward the G-protein signaling, and in the bottom panel the ligand is biased toward the β-arrestin pathway. A number of examples of both types of either partial or full biased signaling of both agonists and antagonists have been reported.

model of on/off receptor pharmacology, both agonists and antagonists can affect not only the overall efficacy but the inherent functional quality of the transduced downstream signaling pathways. Thus, the simple on/off model of linear 7TMR pharmacology is no longer applicable when analyzed over this broader spectrum.

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7TMR BIASED SIGNALING AND DRUG DISCOVERY The concept of biased signaling in 7TMRs16 was first described by Kenakin in 1995 which has added a further layer of detail to our understanding of 7TMR mediated signaling. In 2003, he 6888

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ligands. Unfortunately, many of the reports of biased GPCR signaling, including ones that are described here, have not provided all of these details. The best paradigms for identifying, quantitating, and progressing biased ligands have recently been the subject of intense interest and discussion and are a currently evolving field of inquiry.18,21−24 An important point of consideration to keep in mind is the fact that the majority of receptor pharmacology done today uses engineered cell lines to maximize the biochemical readout of receptor stimulation. However, while these cell lines may produce a signal, the translatability to an endogenous setting can vary greatly. The differences in receptor density, abundance of relative G proteins, and engagement of other signaling systems can alter the cellular pharmacology of various ligands.18 As the artificial systems utilized for development of medicinal chemistry SAR can differ greatly from the relative pharmacology found under endogenous conditions, the biased ligands developed need to be evaluated in more physiologically relevant systems. Both tissue and whole animal based experiments add a layer of complexity but are clearly needed in order to improve the likelihood of determining the overall desired biological profile. Furthermore, these differences between in vitro cellular pharmacology and primary cellular activity can also be observed in different tissues where receptor expression level for adequate screening and bioassay or relative downstream components differ from endogenous expression and therefore impart different efficacies following small molecule stimulation. The additional in vivo nuance of differing receptor signaling from the same receptor in different tissues provides another layer of complexity to drug development. Appreciation of these different effects of small molecules on receptor activation and distinct signaling pathways and their pharmacological significance makes it essential to adequately define these biased effects for the efficient support of medicinal chemistry in the drug development process. We have selected a small number of examples from a wide variety of receptors for which biased signaling has been reported. They serve to illustrate that the direction of the biased signaling can vary widely. For example, there are agonists of 7TMR Gprotein signaling that at the same time are antagonists of βarrestin signaling or conversely cases where ligands are antagonists of, or neutral toward, 7TMR G-protein signaling while being agonists of the β-arrestin pathway. These are summarized in Table 1 and described below. Four of the six examples included herein have recently been advanced into the

reviewed the emerging data that provided the early signs for the existence of biased signaling.17 Over the past 10 years, the concept of a ligand demonstrating biased signaling through a 7TMR at either a G-protein mediated pathway or a β-arrestin pathway has been well-documented in the scientific literature. The number of scientific publications focused on the 7TMR/ GPCR β-arrestin pathway and 7TMR/GPCR-biased signaling overall has steadily increased over the past 5 years (Figure 3) and

Figure 3. Yearly publications based on SciFinder searches of these terms: (a) GPCR and arrestin; (b) GPCR or arrestin biased signaling (as of Dec 17, 2013).

a number of excellent reviews have recently appeared that have in-depth coverage of different aspects of biased signaling such as quantitative analysis,18 signal transduction,19 and β-arrestin scaffolding partners.20 Biased signaling has evolved into a complicated but exciting area of research as more and more evidence has been presented to illustrate how the action of the “biased ligands” differs from that of the traditional agonists, partial agonists, antagonists, and inverse agonists of G-protein signaling.18 Nevertheless, this is still an emerging field and it has yet to standardize procedures for determining and reporting whether ligands display biased signaling. The lack of understanding and establishment of consistently viable standards has impeded, and will impede, interpretation and use of these data. At a minimum, to determine bias, it is necessary to compare the dose−response curve to evaluate both potency and efficacy for both a G-protein signaling response and the β-arrestin response for a given ligand while keeping as many other experimental conditions such as cell type and constructs and buffers, etc. constant. Additionally, it is useful to compare these data to the endogenous ligands, since that benchmark will help determine whether the biased ligand signaling is distinct or simply mirroring the action of the natural

Table 1. Biased Signaling: Representative Examples That Are Highlighted in this Miniperspective receptor/pathway

biased ligands

in vitro profile

D2R

2 and 325

β-Arrestin biased agonists

GPR109a

MK 035430

G-Protein biased agonist with no β-arrestin activity

H4R

JNJ7777120,37 VUF1021440

β-adrenergic (β1-AR) β2-AR

carvediol67

Selective antagonist of H4R G-protein signaling and nonselective agonist of histamine stimulated β-arrestin activity β-Arrestin biased agonists with no G-protein signaling Biased agonist of Gs

μ-opioid receptor

AT1AR

norepinephrine,75 salmeterol75 TRV-130n,51 herkinorin49 TRV027,61 SII56

Biased agonist for G-protein signaling, no β-arrestin activity Partial agonist of β-arrestin and full antagonist of G-protein signaling

advantages

comments

Better antipsychotic activity in mice Reduced incidence of flushing; better therapeutic ratio in mice JNJ7777120 advanced to human clinical trial; no data yet reported Cardioprotective

Demonstrated value of comparing activity in wild type vs β-arrestin KO animals Concluded that the mechanism by which niacin alters HDL levels is GPR109a independent

Long duration of action

Natural ligand bias; biasing not clearly understood at time of therapeutic development Used β-arrestin-2 KO mice to help establish desired profile of a μOR ligand. G-Protein biased ligands have shown fewer side effects and better efficacy

Less adverse effects such as desensitization and constipation Decreased BP with improved cardiomyocyte contraction

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Comparative study of biased signaling for a number of H4R antagonists has been reported. Activity profile distinguished from a set of other β-blockers

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Figure 4. Compounds 1−3 with the D2R binding data and functional activity in β-arrestin and cAMP.

clinic, while the analyses of β1- and β2-adrenergic receptor agonists are retrospective.

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RECEPTOR SIGNALING CASE STUDIES Dopamine D2 Receptor (D2R). Studies of the D2R have provided some excellent examples of β-arrestin biased agonists as reported by Chen.25 Starting from 1 (Figure 4), which is a nonbiased partial agonist of the dopamine D2 receptor, they developed what they called structure−functional selectivity relationships (SFSRs, which are very similar to structure− activity relationships but with an emphasis on multiple functional readouts). In this case they evaluated structural analogues in a D2R radioligand binding assay, a functional cAMP assay, and a D2R mediated Tango assay to determine the effect on the βarrestin pathway. Structural modification of 1 provided 2, which was shown to be a functionally biased compound as a full agonist in the β arrestin assay and inactive in the cAMP assay. More subtle structural modifications of 1 led to 3 to provide a partial agonist at β-arrestin that was inactive at the cAMP assay. Of additional interest, they evaluated the in vivo antipsychotic effects of these compounds in both wild type mice and β arrestin knockout (KO) animals. Compounds 2 and 3 were significantly less active in the β arrestin KO animals, which suggests that activation of the β arrestin pathway is important for the antipsychotic activity of these compounds. This work underscores two important emerging points to be considered when studying molecules that affect GPCR signaling: (i) monitoring activity at the functional assays that reflect the Gprotein pathway (in this case cAMP) as well as the β-arrestin pathway during development of SAR is important because within a structural series small changes can have a big impact on functional selectivity; (ii) there is a lot of value in comparing the in vivo results in both wild type and β-arrestin KO animals to help establish the translatability and impact of the various pathways on the phenotypic effects. Taken together, these data could be invaluable in a drug discovery setting when trying to characterize and optimize the biased in vitro potency of the series and prioritize compounds for in vivo evaluation. Niacin Receptor (GPR109a). Niacin 4 (Figure 5) is an important human therapeutic agent that has been successfully used since the 1950s to elevate HDL levels despite the fact that cutaneous flushing is a common and uncomfortable side effect that limits patient compliance. The biological target of niacin

Figure 5. GPR109a ligands.

responsible for HDL elevation is unknown, and the exact mechanism of action for niacin is still an area for open debate. Interest in identifying the mechanism of action greatly intensified after the report by Lorenzen in 2001 that suggested that 4 activated a 7TMR in adipocytes.26 Subsequent work by scientists at GSK,27 University of Heidelberg,28 and Yamanouchi29 led to the postulation that GPR109a, a recently identified orphan Gi-protein-coupled receptor, could be the receptor responsible for niacin’s biological effects. A subsequent collaboration between Arena and Merck led to the development of a series of compounds30 that were shown to be agonists of GPR109a as measured by their effect on lowering cAMP levels, but these compounds did not stimulate Erk 1/2 phosphorylation. Furthermore, these compounds were able to block the flushing caused by nicotinic acid. One of the first compounds to progress to the clinic from this work was MK 0354 5.30 As designed, it lowered plasma levels of free fatty acids (FFA) without causing vasodilation in mice (which was the assay being used to monitor flushing potential), nor did it lead to receptor internalization.31 All of these findings suggested that 5 was a biased ligand of GPR109a. This was a significant finding that had the potential to provide a greatly improved therapeutic profile compared to niacin which causes flushing and leads to receptor internalization through the βarrestin and MAPK pathway. 5 was later shown to lower FFA in human clinical trials; however, it did not lead to significant elevation of HDL levels.32 From this work, as well as the work of others that took additional GPR109a agonist such as SCH 900271 633 into the clinic, it was ultimately concluded that niacin’s positive effect on lipid profiles (i.e., raising HDL) is not mediated by its interaction with GPR109a.34−36 Although disappointing, this experience supports the idea that biased ligands can offer new opportunities to improve profiles of existing drugs that target 7TMRs. In this case, biasing a ligand toward GRP109a agonism proved to be 6890

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results may help determine if biased signaling of the H4R offers advantages in the clinic. μ-Opioid Receptor (μOR). The natural product morphine is a potent analgesic that acts by signaling through GPCR opiate receptors and is an agonist of both the G-protein and β-arrestin pathways of the μ-receptor. In 1999, Bohn demonstrated that the analgesic effects of morphine are potentiated in knockout mice lacking β-arrestin-2,43 as measured in the mouse hot plate assay. Furthermore, morphine in these KO animals showed a longer duration of action, as would be expected from less receptor desensitization and internalization. In agreement with this, subsequent SiRNA ablation of β-arrestin demonstrated increased analgesic efficacy of morphine.44,45 This supports the hypothesis that a ligand that was an agonist for the μOR, but was neutral toward, or an antagonist of β-arrestin-2 may lead to a more efficacious therapeutic agent. Additional reports on the in vivo profile of the effect of morphine in these β-arrestin-2 KO mice showed that they had fewer adverse events with regard to the known side effects of constipation and respiratory suppression.46−48 Toward the goal of identifying biased ligands of the μOR, synthetic modification of the natural product salvinorin A provided herkinorin 1049 (Figure 7), the first non-nitrogen

successful in separating the 7TMR mediated G-protein activation from the known side effect of flushing which was linked to the βarrestin pathway. H4-Histamine Receptor (H4R). JNJ7777120 7 (Figure 6) was initially described in 2004 as the first selective antagonist of

Figure 6. Histamine H4 receptor ligands.

the H4R.37 Since that time, it has been extensively studied by numerous laboratories using a variety of in vitro systems where its activity as a selective H4R antagonist or inverse agonist has been firmly established. However, more recent studies have shown that it not only antagonizes the G-protein mediated signaling of H4R (as shown in a GTPγS assay, where it blocked histamine activity [pIC50 = 6.7] but also is an agonist of the H4 mediated β-arrestin pathway38 (as detected by PathHunter βgalactosidase enzyme fragmentation complementation [pEC50 = 7.6]). To further complicate the interpretation of the actions of 7, it was recently reported that the agonism of the β-arrestin pathway by this compound is not selective for H4 over some of the other histamine receptors.39 Seifert has reported on the biased signaling of 7 and pointed out some challenges and potential limitations for the experiments that were used to support the claims of biased signaling. It has been suggested that more investigations may still be needed.39 Additional studies have profiled the activity of 31 compounds from the literature that were reported to be H4 receptor ligands.40 These compounds represented nine structural classes and were assayed in both a cAMP reporter gene assay (CRE) used to measure the Gαi signaling pathway, and an enzyme fragment complementation assay to evaluate β-arrestin-2 recruitment. Four of these compounds were shown to be biased toward the Gαi pathway, while another six compounds were found to be biased β-arrestin-2 agonists. One of the latter compounds, VUF10214 8,40 was determined to be a more efficient biased β-arrestin-2 agonist than 7. These results provide additional evidence that biased signaling is not restricted to a few isolated cases and raises important cautionary notes for anyone trying to alter 7TMR signaling. It is no longer sufficient to characterize the molecules of interest as antagonists, inverse agonists, or agonists for the G-protein signaling without profiling the action with regard to the β-arrestin pathway as well. Furthermore, as discussed above, there is growing evidence that the β-arrestin−7TMR complex serves as a scaffold that binds to many different protein partners to elicit a variety of downstream intracellular events. This scaffolding process has recently been extensively summarized by DeFea, which clearly underscores the increasing complexity of the 7TMR signaling pathways that must now be considered.20 In addition to 7, PF-3893787 9 and UR63325 (structure not disclosed) are two additional H4 receptor antagonists that have entered clinical trials.41,42 Whether or not these compounds are also biased ligands has not been reported. If the signaling profile of these compounds is reported, then a comparison of the clinical

Figure 7. μ-Opioid receptor ligands.

containing μOR agonist. Of great interest was the demonstration that 10 does not activate the β-arrestin-2 pathway or lead to receptor internalization but it is still active in the rat formalin test for analgesia.49,50 These in vivo results illustrate the potential advantages of developing biased ligands. In another effort to capitalize on the biased signaling of μOR, a completely unrelated structure, TRV-130 11 (also a similarly biased μOR ligand), entered phase 1 clinical trials in 2012, and encouraging results were recently reported.51,52 Angiotensin II Receptor (AT1R). Therapeutic intervention in the renin−angiotensin (RAS) pathway has been a clinically effective means of treating hypertension since 1981, with the use of Captopril, an angiotensin converting enzyme (ACE) inhibitor to prevent formation of angiotensin II (ang II). Ang II is a vasoconstrictive peptide that exerts its hypertensive effects by agonizing the AT1 receptor. This stimulates phospholipase C, produces IP3, increases calcium levels, and activates GRKs. This leads to phosphorylation of the AT1R and recruitment of βarrestin and ultimately to AT1R desensitization. Commercialization of AT1 receptor blockers (ARBs), starting with losartan in 1995, has provided another effective treatment for hypertension. Although ARBs are efficacious antihypertensive agents, they have some liabilities because they acutely reduce cardiac output as well. In 2002, Holloway,53 on the basis of the earlier work of Feng54 and Noda,55 studied the SAR for Ang II analogues and surprisingly found that side chain modifications that inhibited the phospholipase C signaling (Gαq pathway) did not inhibit receptor desensitization (β-arrestin), as determined by visual6891

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ization of the internalization of the green fluorescent protein tagged AT1. Subsequent similar studies by Wei56 revealed that completely biased signaling toward the β-arrestin pathway leading to downstream kinase activation without measurable levels of G-protein signaling was observed for the Ang II analogue Sar1Ile4Ile8 (SII). Profiling SII in cardiac myocytes showed that it activates ERK1 and ERK2 by a G-protein independent pathway through activation of β-arrestin.57,58 This is in sharp contrast to the actions of the natural ligand Ang II, which activates both the Gprotein and β-arrestin pathways. These differences lead to different cellular events, and a model for these different signaling events has been proposed wherein SII actions result in ERK activation by β-arrestin binding to ERK and that complex is retained in the cytosol, while the Ang II and Gq activated ERK is able to translocate to the nucleus and affect gene transcription and growth of cardiac myocytes.54−56,59,60 Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH (TRV027) is another biased ligand of AT1R that blocks the G-protein signaling pathway of AT1R but activates the β-arrestin pathway.61 This was shown to reduce blood pressure and improve cardiac performance in rats, while the unbiased ligands losartan and telmisartan also decreased blood pressure but decreased cardiac performance. TRV027 recently completed clinical studies where it lowered blood pressure in healthy individuals on a salt restricted diet62 and showed improved hemodynamics in patients with elevated plasma renin activity.63 On the basis of these encouraging results, further clinical trials are planned. To our knowledge TRV027 is the most clinically advanced, designed biased ligand. The results of these trials will help determine the potential advantages of biased ligands and could further increase interest in this area of research. The indication for TRV027 is likely acute heart failure64,65 with treatment by iv infusion. This is in contrast to the orally dosed ARBs that are used as antihypertensive agents. This illustrates a perhaps obvious but important point that the utility of the biased ligands may be entirely different from that of their unbiased counterparts, and thus, they offer the potential of providing new indications from “old” GCPR targets. β1- and β2-Adrenergic Receptors (β1AR and β2AR). Studies of the adrenergic receptors have provided many of the seminal contributions to our understanding of the structure and function of 7TM receptors.66 A number of recent studies have profiled many of the clinically used β-blockers with regard to potentially biased signaling of either the β1- or β2-adrenergic receptors. Evaluation of 16 clinically relevant β-blockers, which are indicated for the treatment of arrhythmias, revealed that only one of them, carvedilol 12 (Figure 8), is both an antagonist of Gprotein mediated signaling and an agonist of β-arrestin as measured by an increase in the phosphorylation of β2-AR and ERK activation and receptor internalization.67 These effects were unaffected by pertussis toxin, thereby ruling out the involvement of the Gi pathway. It has been hypothesized that this biased signaling is responsible for the improved profile and may help explain why carvedilol is one of the few β-blockers that are also clinically approved for treatment of heart failure.67 A separate study of the action of 20 β-blockers on β1-AR Gproteins and β-arrestin pathways also demonstrated that carvedilol resulted in downstream EGRF stimulation of transactivation and activation of ERK, consistent with biased signaling.68 Examination of the in vivo consequences of biased signaling of β-blockers was subsequently reported from a study of

Figure 8. β-Adrenergic receptor ligands.

metoprolol treated mice.67,69 Metoprolol 13 is a β-blocker that was shown not to activate Gs (consistent with it being an inverse agonist). Results from a bioluminescence resonance energy transfer (BRET) study indicated that upon binding with metoprolol the β1-AR interacts with β-arrestin-2 (although weakly compared to the full agonist isoproterenol). This was demonstrated in HEK293 cells, rat heart myoblast cells (H9C2), and also cardiomyocytes. Mice treated with metoprolol were, however, shown to develop cardiac fibrosis. It was postulated that this adverse event was due to the β-arrestin dependent biased signaling, since similar experiments in mice with a knock-down of GRK5 did not develop cardiac fibrosis.69 Whether these results have any implications for clinical use of metoprolol has not been established, but it does suggest that determining whether or not the ligands are biased is an important part of preclinical evaluation. Carvedilol stands out as having demonstrated ligand bias in blocking G-protein mediated effects while promoting βarrestin activation and superior efficacy to other less biased ligands, which would appear to translate into better clinical efficacy.68,70 However, one must keep in mind that some of these β-blockers have activity at more than one 7TMR and 12−16 are used as racemic mixtures, which make interpretation of the consequences of biased signaling more challenging. A clinically validated therapy for respiratory disease, particularly asthma, is the direct bronchodilation of airway smooth muscle. This is achieved by direct agonism of β2-AR residing in the airway smooth muscle. Effective treatment via these β2-receptor agonists can be demonstrated by either a shortacting or long-acting bronchodilator. These drugs are delivered via an inhaled route, and early observations suggested that direct drug uptake or dissolution into the tissue limited the duration of action of the short-acting bronchodilators to 4−6 h after dosing. Alternatively, long-acting bronchodilators, as the name suggests, can maintain efficacy up to 12 h after dosing. Relaxation of the airway smooth muscle is achieved by β2-AR induced activation of the Gs-mediated pathway.71 The rationale for this longer efficacy of long-acting bronchodilators has been attributed to the ability of the compounds to bind tighter and/or longer to the receptor and/or the ability of the compound to partition into the lipid bilayer and thereby act as a pool reserve of compound.72 However, subsequent to the development of these different therapeutic agents, additional studies in this area suggest that the 6892

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relative level of engagement of β-arrestin may also contribute to the observed duration of action. First, enhanced airway relaxation is observed in β-arrestin2 knockout mice73 as well as transgenic mice expressing β2-AR lacking GRK phosphorylation sites.74 Both of these models would most likely be refractory to βarrestin mediated desensitization of the G-protein mediated relaxation pathway. Second, a β-galactosidase complementation assay to quantitate β-arrestin recruitment to β2-AR revealed that the long-acting bronchodilator salmeterol 14 demonstrated very low efficacy in recruiting β-arrestin when compared to the shortacting bronchodilator salbutamol 15.75 Interestingly, the natural β2-AR ligand, norepinephrine, demonstrates significantly more efficient Gs signaling than β-arrestin, demonstrating a natural signaling bias.76 A further understanding of the biasing toward β-arrestin has also been fundamentally demonstrated at the β2-AR. In studies comparing the unbiased ligand isoproterenol 16 to the β-arrestin biased ligand carvediol, a dramatically different GRK-mediated phosphorylation pattern can be observed on β2-AR, where carvediol engagement results in phosphorylation of 2 of the 13 sites phosphorylated after isoproterenol treatment.77 These studies suggest that a GRK phosphorylation fingerprint can be achieved at the receptor depending upon the ligand. Consistent with this observation, a quantitative mass spectrometry analysis of isotopically labeled β2-AR clearly showed that the receptor can adopt a range of conformations depending on the bound ligand.78 These multiple conformations may lead to multiple potencies and efficacies of the various ligands in regard to either G-protein engagement or β-arrestin functionality. If these findings apply to other 7TMRs, it suggests that multiple modalities may be achieved, but this would still beg the question of how one goes about defining which signaling profile to target to provide the best therapeutic benefit.

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AUTHOR INFORMATION

Corresponding Authors

*C.C.C: phone, 617-992-3101; e-mail, [email protected]. *B.A.M: phone, 908-740-3627; e-mail, brian.mckittrick@merck. com. Notes

The authors declare no competing financial interest. Biographies Craig C. Correll obtained his Ph.D. in Biological Chemistry from University of CaliforniaLos Angeles, conducted postdoctoral studies at the California Institute of Technology, and joined the Allergy group at Schering-Plough in 1999. He has been a contributor to various drug discovery programs encompassing a wide array of molecular targets and therapeutic areas including phosphodiesterases, nuclear hormone receptors, ion channels, and several 7TMRs. Currently at Merck, his responsibilities encompass leading late stage preclinical drug discovery efforts, support to clinical development programs, and interrogation of novel targets within the arena of target identification and validation. His main interests lie in understanding how small molecule modulation of specific targets impacts the overall biology and pharmacology in the development of relevant therapeutics. Brian A. McKittrick received his Ph.D. from Brandeis University, MA, completed postdoctoral research at Cornell University, NY, and joined Schering-Plough in 1986. He was part of the team that discovered Zetia which was approved by the FDA in 2002 for the treatment of hypercholesterolemia. He later focused on using high-throughput synthesis for accelerating the drug discovery process. During this time his group contributed to a number of diverse targets including HCV protease and BACE1 inhibitors. At Merck he leads a group of chemists supporting a wide range of therapeutic areas to provide hit validation and early hit to lead optimization. He is keenly interested in understanding the potential impact of biased signaling on the early stages of drug discovery.

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CONCLUSIONS AND FUTURE DIRECTIONS These examples serve to illustrate that unraveling the consequences of biased signaling is currently an area of great interest in the academic and pharmaceutical communities. In some cases, biased signaling may provide improved therapeutics, while in other cases it may be responsible for untoward effects. Nevertheless, elucidating the details of the downstream signaling from molecules that bind to 7TMR provides an exciting opportunity for drug discovery. To that end, a wide array of assays to discern the potency and efficacy of a ligand−7TMR interaction at both a biochemical and cellular level to develop structure−activity relationships are now routinely available.79 Furthermore, additional insights can be gained from the recent advances over the past 5−10 years in the crystallization of 7TMRs with and without various ligands.80 Also, fluorescence spectroscopy and BRET are additional valuable tools available to gain insight into the distinct conformations that ligands with mechanistically different profiles can induce into a specific 7TMR.81−83 A great number of therapeutically validated and successful drugs were developed with limited tools and technologies prior to our fuller understanding of 7TMR functionality. Given the number and wide range of 7TMRs that exist across cell types and their associations with disease and coupled with new insight into the different signaling profiles that can alter the “druggability” of 7TMRs, it is conceivable that many new or improved drugs, which will exploit these findings, are on the horizon.

ACKNOWLEDGMENTS The authors thank Scott Edmondson and Melanie Kleinschek for their critical reviews of the manuscript and James Tata for helpful discussions. Additionally, the authors extend their appreciation to Laura Bohn, Kathryn DeFea, Graeme Semple, and Jonathan Violin for their insights and contributions to this field, which they presented at the Spring 2012 National Meeting of the American Chemical Society.

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ABBREVIATIONS USED 7TMR, seven transmembrane receptor; GPCR, G-proteincoupled receptor; GRK, G-protein receptor kinase; D2R, dopamine D2 receptor; SFSR, structure−functional selectivity relationship; KO, knockout; H4R, H4-histamine receptor; μOR, μ-opioid receptor; AT1R, angiotensin II receptor; ARB, AT1 receptor blocker; ACE, angiotensin converting enzyme; β1AR, β1-adrenergic receptor; β2AR, β2-adrenergic receptor

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