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ScienceDirect Biased ligands: pathway validation for novel GPCR therapeutics David H Rominger, Conrad L Cowan, William Gowen-MacDonald and Jonathan D Violin G protein-coupled receptors (GPCRs), in recent years, have been shown to signal via multiple distinct pathways. Furthermore, biased ligands for some receptors can differentially stimulate or inhibit these pathways versus unbiased endogenous ligands or drugs. Biased ligands can be used to gain a deeper understanding of the molecular targets and cellular responses associated with a GPCR, and may be developed into therapeutics with improved efficacy, safety and/or tolerability. Here we review examples and approaches to pathway validation that establish the relevance and therapeutic potential of distinct pathways that can be selectively activated or blocked by biased ligands. Addresses Trevena Inc., King of Prussia, PA, USA Corresponding author: Violin, Jonathan D ([email protected])
Current Opinion in Pharmacology 2014, 16:108–115 This review comes from a themed issue on Respiratory Edited by John T Fisher and Julia K Walker
http://dx.doi.org/10.1016/j.coph.2014.04.002 S1471-4892/Published by Elsevier Ltd.
receptor internalization and sequestration of the GPCR [5]. Recently, there has been a growing appreciation of barrestin mediated signaling, independent of G-protein signaling, thus providing an even greater opportunity for unique receptor mediated pharmacology [6–8]. The signaling pathways regulated by b-arrestins include Src family kinases, extracellular signal-regulated kinase 1/2, and phosphatases among others. The many disparate signaling mechanisms by which a GPCR can operate within a cell have led to the recognition that some ligands can stimulate and inhibit signaling of the various pathways differentially, not simply stimulating or inhibiting all pathways to the same extent. This has led to the concept of ligand bias, the ability of compounds to selectively engage or inhibit different signaling pathways. The studies discussed in this review provide evidence for bias between G protein and b-arrestin pathway signaling. However, bias between other signaling pathways such as G protein signaling (e.g. Gaq, Gai and Gas) has also been described [9–11]. These insights have fostered a new paradigm that has expanded opportunities for drug discovery, wherein compounds can be sought to interact with a GPCR to cause unique signaling patterns within the cell which may unmask novel and unappreciated pharmacology or provide greater therapeutic benefit while avoiding on-target adverse effects. This review will focus on pathway validation studies that have differentiated b-arrestin from G protein pathways, where most of the initial validation has been performed, serving as good examples of biased ligand pathway validation.
Introduction One of the most therapeutically relevant drug target classes is GPCRs (also referred to as 7 transmembrane receptors) which account for approximately 40% of all marketed drugs [1]. GPCR function was long thought to be solely mediated through second messenger modulation via G protein coupling to the GPCR. Agonists promote distinct receptor conformations that can activate receptor associated G protein signaling. In fact, receptors may couple multiple subfamilies of G proteins resulting in activation of multiple signaling pathways with various sensitivities in different tissues [2,3]. Agonist binding promotes phosphorylation of the receptor intracellular loops and C terminus by G protein receptor kinases (GRKs) which promote binding of regulatory adapter proteins known as the b-arrestins (b-arrestin1 and b-arrestin2) [4]. b-Arrestins bind phosphorylated receptors, sterically hindering G protein signaling resulting in receptor desensitization. In addition, b-arrestins scaffold accessory proteins, including clathrin, which lead to Current Opinion in Pharmacology 2014, 16:108–115
Pathway validation for biased signaling Target validation, which aims to determine if a target protein is mechanistically relevant to a disease condition, determines if a particular protein (e.g. a GPCR, kinase or ion channel) might be functioning improperly in the disease and if pharmacological manipulation might offer therapeutic benefit. Pathway validation goes further and evaluates the physiologic and potential therapeutic relevance of signaling pathways downstream of the receptor. In order to demonstrate the potentially therapeutic mechanism of a biased ligand, one must identify which downstream signaling pathways associated with a given GPCR are responsible for either a beneficial or detrimental outcome in a disease state. Approaches to target-specific pathway validation include genetic deletion, siRNA, overexpression, and chemical inhibition to test the dependence of multiple endpoints on specific signals (Figure 1). The comparison of biased and unbiased ligand pharmacology offers an additional www.sciencedirect.com
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Figure 1
Signal Based Pathway Validation Receptor mutants: eliminate coupling to specific effector proteins. e.g. loop, tail or phosphoacceptor disruption
Unbiased Agonist G proteins: genetic deletion, RNA silencing or chemical inhibition to disrupt G protein signaling
GRKs G proteins
β-arrestins
Pathway inhibitors: various inhibitors of downstream signal transduction
Pathway 1 Response
PO3
GRK/β-arrestins: genetic deletion and RNA silencing to disrupt β-arrestin recruitment and subsequent signaling
Pathway 2 Response Current Opinion in Pharmacology
Signal based pathway validation requires controlled manipulation of signal transduction events resulting from GPCR activation. Stimulation of intact endogenous GPCR systems with endogenous ligand or unbiased agonist can elicit multiple on-target effects, which can be both beneficial and adverse. Additionally, unbiased agonism can result in b-arrestin mediated desensitization of G protein signaling. Modifying these systems via genetic deletion, RNA silencing, mutation, or chemical inhibition can dissect effector coupling or downstream signal transduction providing differentiated pharmacologic outcomes with altered target-specific therapeutic indices. Pathway validation compares data from intact and modified in vivo model systems to discriminate pathway-specific behaviors or physiologic ontarget effects that can be targeted with biased ligands to unmask novel pharmacology or improve the therapeutic benefit.
approach to specifically validate pathways one would want to target therapeutically (Figure 2). In this review, we highlight several receptors for which pathway validation has served to confirm the pharmacology and potential benefits of biased ligands. We also highlight some key examples that are beginning to demonstrate the translatability of these efforts toward effective therapeutics.
b2-Adrenoceptor: ligand-based pathway validation b-Adrenoceptor (bAR) antagonists have long been a mainstay therapy for hypertension, ischemic heart disease and heart failure. Differences in therapeutic efficacies or outcomes initially were attributed to differences in receptor subtype selectivity profiles. However, as the concept of GPCR signaling through multiple pathways emerged [12], the role of signaling via these different pathways was investigated. Studies by Azzi et al. [13] and Baker et al. [14] were the first to show that compounds classically considered to be b2AR antagonists (e.g. propranolol and ICI 118,551) were able to signal differentially by simultaneously inhibiting G protein mediated cAMP accumulation while stimulating MAPK activity. Through the use of G protein inhibitors, receptor mutants [15], pertussis toxin [14], and Gas knock out (KO) lines [13] the G protein independence of the MAPK www.sciencedirect.com
stimulation was demonstrated. Further studies in cells expressing a b-arrestin mutant and MEF cells lacking b-arrestin1 and b-arrestin2 demonstrated the role of b-arrestins in mediating MAPK activation [15]. Thus, the precedence of G protein independent signaling was established. Subsequent studies explored the signaling profiles of panels of clinically relevant bAR antagonists. Galandrin and Bouvier [16] evaluated eight well characterized bAR ligands, some of which are used clinically, and showed that they differentially signaled between cAMP and MAPK. Of the six compounds that were either neutral or inverse agonists of cAMP accumulation, three stimulated b2AR dependent ERK1/2 activation. Furthering this approach, Wisler et al. [17] employed a number of techniques to validate the relevance of b-arrestin in mediating signaling events evoked by a panel of clinically relevant bAR antagonists. Carvedilol uniquely was an inverse agonist for cAMP accumulation with positive efficacy for stimulatin receptor phosphorylation, barrestin recruitment, receptor internalization, and ERK1/2 phosphorylation. These effects were all modest, but use of b-arrestin2 siRNA and a mutant b2AR incapable of G protein coupling demonstrated the G protein independence and b-arrestin-dependence of these Current Opinion in Pharmacology 2014, 16:108–115
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Figure 2
Ligand Based Pathway Validation (a)
(b)
Unbiased Ligand
G proteins
β-arrestin Biased Ligand
PO3 β-arrestins
Pathway 2 Response
Unbiased ligand • Both beneficial and adverse pharmacology can be associated with activating G protein and βarrestin pathways. • Self-Iimited efficacy resulting from βarrestin mediated desensitization of G protein signal.
G protein Biased Ligand
G proteins
β-arrestins
Pathway 1 Response
(c)
Pathway 2 Response
β-arrestin biased ligand • Beneficial β-arrestin mediated pharmacology without G protein mediated adverse events.
Pathway 1 Response
G protein biased ligand • Beneficial G protein mediated pharmacology without β-arrestin mediated adverse events. • Enhanced efficacy by avoiding βarrestin mediated desensitization of G protein signal
Current Opinion in Pharmacology
Ligand based pathway validation requires well characterized ligands to uncover the divergence of distinct pharmacological responses linked to either G-protein or b-arrestin signal transduction pathways in vivo. Unbiased agonists (a) signaling through intact endogenous GPCR signaling cascades can elicit distinct responses, which can include both beneficial and ‘on-target’ adverse effects. Furthermore, b-arrestin recruitment to the receptor can desensitize the G protein signal, limiting efficacy associated with down-stream G protein mediated signal transduction. Biased ligands can elicit distinct pathway-specific pharmacology. b-arrestin biased ligands (b) can induce beneficial pharmacology compared to unbiased reference agonist if on -target adverse events are linked to G protein signaling cascades. Conversely, if on-target adverse events are associated with b-arrestin signal transduction, G protein biased ligands (c) can generate improved pharmacologic outcomes and can display enhanced efficacy by avoiding the barrestin mediated desensitization of G protein signaling seen with unbiased agonists.
signals, confirming that carvedilol is a b-arrestin biased ligand. Conformational receptor studies demonstrate that biased ligands such as carvedilol can stabilize different receptor conformations and induce distinct receptor phosphorylation patterns compared to unbiased ligands [18,19].
(endothelial growth factor receptor) via b-arrestin, which has been shown to be cardioprotective in a mouse heart failure model [21]. Together these findings suggest that biased ligands with more efficacy for b-arrestin2 at bARs may offer increased therapeutic benefit compared to currently used beta blockers.
In addition to its effects on bAR, carvedilol is an a1adrenoceptor (a1AR) antagonist and antioxidant, thus attributing any clinical differentiation to bias alone is not possible at this point. However, carvedilol has shown potentially superior beneficial effects compared to other bAR blockers after myocardial infarction. It has shown as well, significantly reduced all-cause mortality in systolic heart failure patients compared to atenolol, bisoprolol, metoprolol, and nebivolol in randomized direct comparison trials [20]. One possible explanation for the benefits of b-arrestin stimulation is b1AR transactivation of EGFR
The b2AR also plays a key role in pulmonary function and b2AR agonists (e.g. salbutamol) have long been used to treat asthma. Although b2AR agonists are bronchodilators which relieve acute asthma episodes, there is growing concern that long-term use of b2AR agonists may worsen the disease [22]. b2AR G protein-biased ligands, with little to no b-arrestin activity, have been sought for their use in asthma with the hope that such compounds would offer the bronchodilation of acute b2AR agonism without the tolerance that develops with repeated and prolonged use of this therapy [23]. Recent studies have even linked
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b2AR signaling via b-arrestin with development of allergic asthma. Nguyen et al. [24] found chronic treatment of a murine model of asthma with beta-blockers resulted in reduced inflammation and mucus production, both hallmark symptoms of asthma. Subsequent studies showed similar resistance to development of the asthma symptoms in b2AR KO mice indicating the effects are target mediated [25]. Coupled with an earlier finding by Walker et al. [26] that development of the asthma symptoms was dependent on b-arrestin2, these findings suggest a hypothesis that b2AR via b-arrestin2-dependent signals promote disease progression, while G protein activation is responsible for acute bronchodilation. If confirmed, this hypothesis suggests a G protein-biased ligand that blocks endogenous b2AR b-arrestin stimulation will provide superior long-term management of pulmonary disease (see Walker and Defay, in this issue).
k-Opioid receptor: signal-based pathway validation k-Opioid receptor (k-OR) agonists have long been sought to provide opioid strength analgesia without the side effects associated with m-opioid receptor agonists (e.g. morphine) including respiratory depression, constipation and addiction. However, CNS-active k-OR agonists also are associated with their own development-limiting side effects, most notably dysphoria, an unpleasant state characterized by depersonalization, dissociation, and psychotomimetic effects. Historically, opioid receptors have been thought to mediate their cellular effects through Gai inhibition of cAMP accumulation, G protein-coupled inward-rectifying potassium channels, and voltage-dependent L-type Ca2+ channels modulating membrane potential. More recent studies showed that k-OR agonists also stimulate ERK1/ 2, MAPK, and c-Jun pathways [27]. Studies by Bruchas et al. [28] demonstrated that the k-OR agonist U50,488 also stimulated p38 MAPK and that this stimulation was dependent on phosphorylation of the k-OR tail by G protein-coupled receptor kinase (GRK) 3, thus linking this signaling event to b-arrestin recruitment. The k-OR selective ligand U50,488 produced conditioned place aversion suggesting that treatment with U50,488 caused an unpleasant experience in the mice which they sought to avoid. Pretreatment with the p38 MAPK inhibitor SB203580 blocked the U50,488 conditioned place aversion suggesting that the dysphoric effects of k-OR agonism are mediated via p38 MAPK. Furthermore, mice lacking the receptor phosphorylating GRK3 did not develop conditioned place aversion in response to U50,488. Moreover, they did not show increased p38 MAPK activation after k-OR activation, implicating barrestin recruitment to the receptor in this pathway [29]. By contrast, SB203580 did not block swim stress mediated by k-OR dependent analgesia observed in www.sciencedirect.com
the tail withdrawal model, demonstrating that divergent signaling pathways are associated with k-OR analgesia and dysphoria. These findings suggested that CNS-penetrant k-OR agonists lacking b-arrestin engagement may provide powerful CNS k-OR analgesia without intolerable dysphoria. Several groups have discovered k-OR agonists that inhibit cAMP accumulation via Gai but avoid b-arrestin mediated activation of p38 MAPK [30,31]. One such compound recently described is 60 GNTI, a close analog of the k-OR antagonist 50 GNTI [32]. 60 GNTI is a partial agonist in GTPgS, ERK phosphorylation and cAMP inhibition (37–75% of maximal response) in cells expressing k-OR while in contrast stimulating b-arrestin only very weakly (12% of U69,593 max) [33,34]. In mouse striatal neurons U69,593 stimulates G protein-dependent Akt phosphorylation and b-arrestin-dependent ERK1/2 phosphorylation; 60 GNTI stimulates G protein-dependent Akt phosphorylation to the same extent as U69,593, but does not stimulate ERK1/2 phosphorylation. Despite its G protein-biased profile and partial efficacy, 60 GNTI is analgesic in mouse tail-flick assays following i.t. but not i.c.v. administration [35]. However, the in vivo relevance of bias of this compound has not yet been explored in models of dysphoria (e.g. conditioned place aversion) so the hypothesis that a G protein-biased k-OR agonist could provide analgesia without dysphoria remains untested. Thus, k-OR agonism typifies signal-based pathway validation (Figure 1) for a target that has both desirable and undesirable effects, although cross-validation with biased ligands remains to be demonstrated.
m-Opioid receptor: signal-based pathway validation Opioids such as morphine produce effective analgesia by activating the m-opioid receptor (m-OR) [36]. However, opioids produce adverse events including nausea and vomiting, respiratory depression, constipation, and sedation, which limit their utility. Pathway validation studies using KO mice demonstrated that the beneficial and adverse effects of m-OR may be separable with biased ligands. Mice lacking b-arrestin2 showed greater and more prolonged analgesia to morphine but reduced constipation and respiratory depression [37,38]. Similar effects on analgesia were seen with knockdown of b-arrestin2 in specific brain regions with siRNA [39,40]. These signal based pathway validation data suggest that a G protein-biased m-OR agonist that does not recruit b-arrestins could provide analgesia with reduced dose-limiting adverse effects associated with current opioid therapies. Based on this hypothesis, a drug discovery effort led to the identification and characterization of TRV130, a novel G protein-biased m-OR ligand. TRV130 is a potent agonist of G protein mediated inhibition of cAMP accumulation with comparable intrinsic efficacy to morphine while at same time Current Opinion in Pharmacology 2014, 16:108–115
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recruiting significantly less b-arrestin2 than morphine [41,42]. Consistent with selective activity for the G protein pathway, TRV130 exhibits reduced MOR internalization and phosphorylation compared to strong opioids such as fentanyl and morphine [42]. In mice and rats, the biased in vitro profile of TRV130 translated to potent analgesia with reduced gastrointestinal dysfunction and respiratory depression compared to morphine [42]. The in vivo efficacy of TRV130 demonstrates that a biased ligand may provide superior analgesia. The threat of respiratory depression leads to underdosing of opioids and sub-optimal pain relief; nausea and vomiting can increase patient discomfort and delay oral intake; and sedation can lead to longer time to ambulation and in some cases increasing hospital length of stay and therefore the overall cost of care. If a biased ligand such as TRV130 could more effectively treat postoperative pain while mitigating these adverse effects, patients would be expected to experience better pain relief and progress more efficiently through postoperative recuperation. Such a compound might also be an attractive agent for chronic obstructive pulmonary disease palliation where the risk of respiratory depression limits the use of currently approved opioids [43,44]. In first-time in human studies, intravenously infused TRV130 produced pupil constriction, a well validated marker of m-OR activity in the CNS, at well tolerated doses [45]. The magnitude of pupil constriction for TRV130 was comparable to that elicited by clinically used doses of morphine and fentanyl in other studies [46] however, unlike morphine and fentanyl, TRV130 at several doses caused this robust pharmacology without causing nausea or vomiting. Although comparator studies are required to assess the true differentiation of TRV130, these initial clinical results indicate TRV130 may be better tolerated than unbiased opioids like morphine and that the beneficial therapeutic index seen preclinically for this biased ligand may translate in the clinic [45].
Angiotensin II type 1 receptor: signal-based and ligand-based pathway validation The angiotensin II type 1 receptor (AT1R) was one of the earliest GPCRs for which evidence of biased signaling was shown. Angiotensin II (AngII) regulates cardiovascular functions and increased levels have been linked to hypertension, renal dysfunction and heart failure. AngII binding to the AT1R induces coupling to Gaq, activation of phospholipase C, and mobilization of calcium resulting in vasoconstriction and increased blood pressure [47,48]. AngII also promotes AT1R phosphorylation by GRKs and the recruitment of b-arrestins resulting in receptor internalization and desensitization of G protein signals. Studies over the last decade demonstrated that AT1R mediated signaling can be G protein or b-arrestin dependent and Current Opinion in Pharmacology 2014, 16:108–115
that this differential signaling can be leveraged to optimize pharmacologic activity at the AT1R [8]. Receptor mutagenesis studies revealed that elimination of Gaq coupling did not abate AngII induced phosphorylation, internalization, recruitment and signaling via b-arrestins [49,50]. Knockout studies eliminating AT1R or b-arrestin2 exacerbated cardiac stretch induced apoptosis providing evidence that b-arrestin signaling through the AT1R is cardioprotective [51]. Experiments with the b-arrestin biased AT1R ligand [Sar1, Ile4, Ile8] angiotensin II (SII) complimented these findings. SII binds the AT1R and directs signaling through the b-arrestin pathway while antagonizing G protein signaling [50,52,53]. SII produces AT1R and b-arrestin-dependent reduction in apoptosis, stimulation of chemotaxis, increased cardiac contractility, cell growth and proliferation [52,54–57]. Other work showed that SII weakly activates several G protein complexes but in a pattern that differs from that activated by AngII, consistent with the existence of discrete receptor conformations stabilized by AngII and SII [58]. A number of factors, including assay type, receptor, and signal transducer expression, may account for the additional pathway activity of the b-arrestin biased ligand SII. Separate work demonstrated different receptor conformations associated with G protein-biased, neutral, and b-arrestin-biased ligands by measuring transducer-specific thermodynamics of ligand binding [59]. The in vitro and in vivo signal-based pathway validation studies, as well as subsequent demonstration of ligandbased pathway validation for beneficial b-arrestin-dependent effects at the AT1R, inspired the discovery and development of TRV027, (initially called TRV120027) a selective b-arrestin-biased AT1R ligand with increased potency and b-arrestin efficacy compared to SII [60]. TRV027 competitively binds the AT1R, reduces AngIImediated hypertension, increases contractility of isolated mouse cardiomyocytes and enhances cardiac performance in rodents [60]. This profile translated to beneficial hemodynamic, renal and cardiac effects in a tachypaced canine model of acute heart failure [61,62]. In each of these studies, TRV027 behaved similarly to an angiotensin receptor blocker with respect to renal and vascular effects, but with increased cardiac performance associated with its b-arrestin efficacy. In addition to stimulating cardiac contractility, b-arrestin biased ligands like TRV027 may also be cardioprotective. TRV120023, an analog of TRV027 with similar biased AT1R pharmacology, reduced cell death in a mouse ischemic reperfusion model [63]. Furthermore, the enhanced cardiac contractility induced by TRV120023 is not due to direct calcium mobilization as occurs with classic inotropes and with AngII. Chronic infusion of TRV1200023, but not the full antagonist losartan, to normal rats and rats co-infused with either saline or AngII showed increased calcium sensitivity, likely through post-translational modification of cardiac myofilaments www.sciencedirect.com
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[64]. These findings coincided with a blockade of AngII-mediated cardiac hypertrophy by both TRV120023 and losartan, consistent with the well-established role of Gaq-coupling in the development of cardiac hypertrophy. Together these pathway validation studies suggest a profile of b-arrestin-biased AT1R ligands that may offer unique benefits in treating cardiovascular disease. Because it selectively intercedes at the AT1R to block renal and systemic vasoconstriction while producing enhanced cardiac performance and cardioprotective effects, TRV027 might be an effective novel treatment for acute heart failure. In early clinical trials, TRV027 caused renin-angiotensin system-dependent reductions in mean arterial pressure (MAP) in salt-restricted healthy volunteers [65] and in stable advanced chronic heart failure [65], and is now in Phase 2 clinical trials for treating AHF.
Summary The dissection of different receptor-mediated signal transduction pathways underpins the scientific and potential therapeutic benefits of biased ligands. Pathway validation links specific signaling pathways to different pharmacological outcomes, establishing both the mechanisms of receptor function and opportunities for biased ligands to improve on-target pharmacology. As illustrated in the examples above, pathway validation can be achieved at several levels. It can be studied downstream of the receptor at the level of signal transduction, using a variety of techniques including genetic deletion, siRNA, overexpression, and chemical inhibition to test the dependence of multiple endpoints on specific signals (Figure 1). Pathway validation also can be elucidated at the level of ligands by comparing biased and unbiased ligands for differential effects in cells, tissues, and in vivo (Figure 2). These approaches have been valuable in biased ligand research and are widely applicable to any relevant downstream signaling pathways. In some cases, (e.g. m-OR), signalbased validation fostered biased ligand discovery efforts leading to successful and complementary ligand-level validation. In other cases, such as with the AT1R, the early identification of biased ligands allowed pathway validation to be established across numerous experimental systems in parallel with signal-level pathway validation. Both ligandbased and signal-based validation approaches show promise for establishing pathway-specific pharmacology at the b2AR. For other receptors, such as the k-OR, signal-level pathway validation based on disruption of multiple signal transduction components has not yet been cross-validated at the level of ligand pharmacology. Importantly, though this review focuses on the dissection of G protein and b-arrestin pathways, the same approaches can be used to interrogate selective pathway signaling of other GPCR-associated downstream pathways.
Concluding remarks The number of reported biased ligands has increased over the last decade, as has the number of GPCRs for which www.sciencedirect.com
complex pharmacological responses have been dissected to reveal unique contributions from distinct and separable downstream signaling pathways. This has engendered growing enthusiasm for the prospective discovery and optimization of biased ligands as novel, differentiated therapeutics targeting both old and new GPCR drug targets by improving on-target pharmacology. The receptors discussed here illustrate how different approaches taken to dissect receptor pharmacology can illuminate novel pharmacology, mechanism of action and therapeutic utility of biased ligands. These methods, also discussed elsewhere [8,66], compliment methods of assaying GPCR molecular pharmacology [6] and accurately detecting and quantifying ligand bias [67,68,69]. From the perspective of drug discovery, the integration of these efforts, with particular emphasis on building a strong scientific rationale for why ligand bias might matter at a given receptor, may lead to numerous opportunities to develop safer, more efficacious and better tolerated new medicines targeting GPCRs.
Conflict of interest statement Nothing declared.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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42. DeWire SM et al.: A G protein-biased ligand at the m-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine. J Pharmacol Exp Therap 2013, 344:708-717. This work describes differentiated pharmacologic profiles, both in vitro and in vivo, of the G protein-biased ligand TRV130 compared to morphine. TRV130 displays rapid, potent analgesia in mice and possesses improved therapeutic indices of analgesia to constipation and respiratory suppression relative to morphine. 43. Marciniuk DD et al.: Managing dyspnea in patients with advanced chronic obstructive pulmonary disease: a Canadian Thoracic Society clinical practice guideline. Can Respir J 2011, 18:69-78. 44. Janssen DJ et al.: Symptoms: comorbidities, and health care in advanced chronic obstructive pulmonary disease or chronic heart failure. J Palliat Med 2011, 14:735-743. 45. Soergel DG et al.: First clinical experience with TRV130: pharmacokinetics and pharmacodynamics in healthy volunteers. J Clin Pharmacol 2014, 54:351-357. 46. Saunders DL et al.: Assessment of the relative potency of fentanyl buccal tablet to intravenous morphine in healthy volunteers using a thermally induced hyperalgesia pain model. J Clin Pharmacol 2012, 52:870-879. 47. Fitzsimons JT: Angiotensin, thirst, and sodium appetite: retrospect and prospect. Fed Proc 1978, 37:2669-2675.
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61. Boerrigter G et al.: TRV120027: a novel beta-arrestin biased ligand at the angiotensin II type I receptor, unloads the heart and maintains renal function when added to furosemide in experimental heart failure. Circ Heart Fail 2012, 5:627-634. 62. Boerrigter G et al.: Cardiorenal actions of TRV120027, a novel ss-arrestin-biased ligand at the angiotensin II type I receptor, in healthy and heart failure canines: a novel therapeutic strategy for acute heart failure. Circ Heart Fail 2011, 4:770-778. 63. Kim KS et al.: beta-Arrestin-biased AT1R stimulation promotes cell survival during acute cardiac injury. Am J Physiol Heart Circ Physiol 2012, 303:H1001-H1010. The authors used both ligand-based and signal-based pathway validation approaches to describe the beneficial effects of a b-arrestin-biased AT1R agonist (TRV120023) during acute ischemic injury. Compared to unbiased AT1R blocker losartan, TRV120023 displays distinct properties in vivo by enhancing cardiac contractibility, activating prosurvival signaling, and reducing apoptosis after cardiac injury. These effects were lost in barrestin2 knockout mice, confirming the pathway selectivity of the biased ligand for these responses. 64. Monasky MM et al.: The beta-arrestin-biased ligand TRV120023 inhibits angiotensin II-induced cardiac hypertrophy while preserving enhanced myofilament response to calcium. Am J Physiol Heart Circ Physiol 2013, 305:H856-H866. 65. Soergel D et al.: TRV027, a beta-arrestin biased ligand at the angiotensin 2 type 1 receptor, produces rapid, reversible changes in hemodynamics in patients with stable systolic heart failure. J Am College Cardiol 2013, 61 E683–E683. 66. Raehal KM et al.: Functional selectivity at the mu-opioid receptor: implications for understanding opioid analgesia and tolerance. Pharmacol Rev 2011, 63:1001-1019. 67. Kenakin T, Christopoulos A: Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat Rev Drug Discov 2013, 12:205-216. This review describes in detail methods to quantify ligand bias. In addition, the review highlights key aspects and challenges in biased ligand identification. 68. Kenakin T et al.: A simple method for quantifying functional selectivity and agonist bias. ACS Chem Neurosci 2012, 3: 193-203. 69. Rajagopal S et al.: Quantifying ligand bias at seventransmembrane receptors. Mol Pharmacol 2011, 80(3):367-377.
Current Opinion in Pharmacology 2014, 16:108–115
ScienceDirect Biased ligands: pathway validation for novel GPCR therapeutics David H Rominger, Conrad L Cowan, William Gowen-MacDonald and Jonathan D Violin G protein-coupled receptors (GPCRs), in recent years, have been shown to signal via multiple distinct pathways. Furthermore, biased ligands for some receptors can differentially stimulate or inhibit these pathways versus unbiased endogenous ligands or drugs. Biased ligands can be used to gain a deeper understanding of the molecular targets and cellular responses associated with a GPCR, and may be developed into therapeutics with improved efficacy, safety and/or tolerability. Here we review examples and approaches to pathway validation that establish the relevance and therapeutic potential of distinct pathways that can be selectively activated or blocked by biased ligands. Addresses Trevena Inc., King of Prussia, PA, USA Corresponding author: Violin, Jonathan D ([email protected])
Current Opinion in Pharmacology 2014, 16:108–115 This review comes from a themed issue on Respiratory Edited by John T Fisher and Julia K Walker
http://dx.doi.org/10.1016/j.coph.2014.04.002 S1471-4892/Published by Elsevier Ltd.
receptor internalization and sequestration of the GPCR [5]. Recently, there has been a growing appreciation of barrestin mediated signaling, independent of G-protein signaling, thus providing an even greater opportunity for unique receptor mediated pharmacology [6–8]. The signaling pathways regulated by b-arrestins include Src family kinases, extracellular signal-regulated kinase 1/2, and phosphatases among others. The many disparate signaling mechanisms by which a GPCR can operate within a cell have led to the recognition that some ligands can stimulate and inhibit signaling of the various pathways differentially, not simply stimulating or inhibiting all pathways to the same extent. This has led to the concept of ligand bias, the ability of compounds to selectively engage or inhibit different signaling pathways. The studies discussed in this review provide evidence for bias between G protein and b-arrestin pathway signaling. However, bias between other signaling pathways such as G protein signaling (e.g. Gaq, Gai and Gas) has also been described [9–11]. These insights have fostered a new paradigm that has expanded opportunities for drug discovery, wherein compounds can be sought to interact with a GPCR to cause unique signaling patterns within the cell which may unmask novel and unappreciated pharmacology or provide greater therapeutic benefit while avoiding on-target adverse effects. This review will focus on pathway validation studies that have differentiated b-arrestin from G protein pathways, where most of the initial validation has been performed, serving as good examples of biased ligand pathway validation.
Introduction One of the most therapeutically relevant drug target classes is GPCRs (also referred to as 7 transmembrane receptors) which account for approximately 40% of all marketed drugs [1]. GPCR function was long thought to be solely mediated through second messenger modulation via G protein coupling to the GPCR. Agonists promote distinct receptor conformations that can activate receptor associated G protein signaling. In fact, receptors may couple multiple subfamilies of G proteins resulting in activation of multiple signaling pathways with various sensitivities in different tissues [2,3]. Agonist binding promotes phosphorylation of the receptor intracellular loops and C terminus by G protein receptor kinases (GRKs) which promote binding of regulatory adapter proteins known as the b-arrestins (b-arrestin1 and b-arrestin2) [4]. b-Arrestins bind phosphorylated receptors, sterically hindering G protein signaling resulting in receptor desensitization. In addition, b-arrestins scaffold accessory proteins, including clathrin, which lead to Current Opinion in Pharmacology 2014, 16:108–115
Pathway validation for biased signaling Target validation, which aims to determine if a target protein is mechanistically relevant to a disease condition, determines if a particular protein (e.g. a GPCR, kinase or ion channel) might be functioning improperly in the disease and if pharmacological manipulation might offer therapeutic benefit. Pathway validation goes further and evaluates the physiologic and potential therapeutic relevance of signaling pathways downstream of the receptor. In order to demonstrate the potentially therapeutic mechanism of a biased ligand, one must identify which downstream signaling pathways associated with a given GPCR are responsible for either a beneficial or detrimental outcome in a disease state. Approaches to target-specific pathway validation include genetic deletion, siRNA, overexpression, and chemical inhibition to test the dependence of multiple endpoints on specific signals (Figure 1). The comparison of biased and unbiased ligand pharmacology offers an additional www.sciencedirect.com
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Figure 1
Signal Based Pathway Validation Receptor mutants: eliminate coupling to specific effector proteins. e.g. loop, tail or phosphoacceptor disruption
Unbiased Agonist G proteins: genetic deletion, RNA silencing or chemical inhibition to disrupt G protein signaling
GRKs G proteins
β-arrestins
Pathway inhibitors: various inhibitors of downstream signal transduction
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GRK/β-arrestins: genetic deletion and RNA silencing to disrupt β-arrestin recruitment and subsequent signaling
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Signal based pathway validation requires controlled manipulation of signal transduction events resulting from GPCR activation. Stimulation of intact endogenous GPCR systems with endogenous ligand or unbiased agonist can elicit multiple on-target effects, which can be both beneficial and adverse. Additionally, unbiased agonism can result in b-arrestin mediated desensitization of G protein signaling. Modifying these systems via genetic deletion, RNA silencing, mutation, or chemical inhibition can dissect effector coupling or downstream signal transduction providing differentiated pharmacologic outcomes with altered target-specific therapeutic indices. Pathway validation compares data from intact and modified in vivo model systems to discriminate pathway-specific behaviors or physiologic ontarget effects that can be targeted with biased ligands to unmask novel pharmacology or improve the therapeutic benefit.
approach to specifically validate pathways one would want to target therapeutically (Figure 2). In this review, we highlight several receptors for which pathway validation has served to confirm the pharmacology and potential benefits of biased ligands. We also highlight some key examples that are beginning to demonstrate the translatability of these efforts toward effective therapeutics.
b2-Adrenoceptor: ligand-based pathway validation b-Adrenoceptor (bAR) antagonists have long been a mainstay therapy for hypertension, ischemic heart disease and heart failure. Differences in therapeutic efficacies or outcomes initially were attributed to differences in receptor subtype selectivity profiles. However, as the concept of GPCR signaling through multiple pathways emerged [12], the role of signaling via these different pathways was investigated. Studies by Azzi et al. [13] and Baker et al. [14] were the first to show that compounds classically considered to be b2AR antagonists (e.g. propranolol and ICI 118,551) were able to signal differentially by simultaneously inhibiting G protein mediated cAMP accumulation while stimulating MAPK activity. Through the use of G protein inhibitors, receptor mutants [15], pertussis toxin [14], and Gas knock out (KO) lines [13] the G protein independence of the MAPK www.sciencedirect.com
stimulation was demonstrated. Further studies in cells expressing a b-arrestin mutant and MEF cells lacking b-arrestin1 and b-arrestin2 demonstrated the role of b-arrestins in mediating MAPK activation [15]. Thus, the precedence of G protein independent signaling was established. Subsequent studies explored the signaling profiles of panels of clinically relevant bAR antagonists. Galandrin and Bouvier [16] evaluated eight well characterized bAR ligands, some of which are used clinically, and showed that they differentially signaled between cAMP and MAPK. Of the six compounds that were either neutral or inverse agonists of cAMP accumulation, three stimulated b2AR dependent ERK1/2 activation. Furthering this approach, Wisler et al. [17] employed a number of techniques to validate the relevance of b-arrestin in mediating signaling events evoked by a panel of clinically relevant bAR antagonists. Carvedilol uniquely was an inverse agonist for cAMP accumulation with positive efficacy for stimulatin receptor phosphorylation, barrestin recruitment, receptor internalization, and ERK1/2 phosphorylation. These effects were all modest, but use of b-arrestin2 siRNA and a mutant b2AR incapable of G protein coupling demonstrated the G protein independence and b-arrestin-dependence of these Current Opinion in Pharmacology 2014, 16:108–115
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Figure 2
Ligand Based Pathway Validation (a)
(b)
Unbiased Ligand
G proteins
β-arrestin Biased Ligand
PO3 β-arrestins
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Unbiased ligand • Both beneficial and adverse pharmacology can be associated with activating G protein and βarrestin pathways. • Self-Iimited efficacy resulting from βarrestin mediated desensitization of G protein signal.
G protein Biased Ligand
G proteins
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(c)
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β-arrestin biased ligand • Beneficial β-arrestin mediated pharmacology without G protein mediated adverse events.
Pathway 1 Response
G protein biased ligand • Beneficial G protein mediated pharmacology without β-arrestin mediated adverse events. • Enhanced efficacy by avoiding βarrestin mediated desensitization of G protein signal
Current Opinion in Pharmacology
Ligand based pathway validation requires well characterized ligands to uncover the divergence of distinct pharmacological responses linked to either G-protein or b-arrestin signal transduction pathways in vivo. Unbiased agonists (a) signaling through intact endogenous GPCR signaling cascades can elicit distinct responses, which can include both beneficial and ‘on-target’ adverse effects. Furthermore, b-arrestin recruitment to the receptor can desensitize the G protein signal, limiting efficacy associated with down-stream G protein mediated signal transduction. Biased ligands can elicit distinct pathway-specific pharmacology. b-arrestin biased ligands (b) can induce beneficial pharmacology compared to unbiased reference agonist if on -target adverse events are linked to G protein signaling cascades. Conversely, if on-target adverse events are associated with b-arrestin signal transduction, G protein biased ligands (c) can generate improved pharmacologic outcomes and can display enhanced efficacy by avoiding the barrestin mediated desensitization of G protein signaling seen with unbiased agonists.
signals, confirming that carvedilol is a b-arrestin biased ligand. Conformational receptor studies demonstrate that biased ligands such as carvedilol can stabilize different receptor conformations and induce distinct receptor phosphorylation patterns compared to unbiased ligands [18,19].
(endothelial growth factor receptor) via b-arrestin, which has been shown to be cardioprotective in a mouse heart failure model [21]. Together these findings suggest that biased ligands with more efficacy for b-arrestin2 at bARs may offer increased therapeutic benefit compared to currently used beta blockers.
In addition to its effects on bAR, carvedilol is an a1adrenoceptor (a1AR) antagonist and antioxidant, thus attributing any clinical differentiation to bias alone is not possible at this point. However, carvedilol has shown potentially superior beneficial effects compared to other bAR blockers after myocardial infarction. It has shown as well, significantly reduced all-cause mortality in systolic heart failure patients compared to atenolol, bisoprolol, metoprolol, and nebivolol in randomized direct comparison trials [20]. One possible explanation for the benefits of b-arrestin stimulation is b1AR transactivation of EGFR
The b2AR also plays a key role in pulmonary function and b2AR agonists (e.g. salbutamol) have long been used to treat asthma. Although b2AR agonists are bronchodilators which relieve acute asthma episodes, there is growing concern that long-term use of b2AR agonists may worsen the disease [22]. b2AR G protein-biased ligands, with little to no b-arrestin activity, have been sought for their use in asthma with the hope that such compounds would offer the bronchodilation of acute b2AR agonism without the tolerance that develops with repeated and prolonged use of this therapy [23]. Recent studies have even linked
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b2AR signaling via b-arrestin with development of allergic asthma. Nguyen et al. [24] found chronic treatment of a murine model of asthma with beta-blockers resulted in reduced inflammation and mucus production, both hallmark symptoms of asthma. Subsequent studies showed similar resistance to development of the asthma symptoms in b2AR KO mice indicating the effects are target mediated [25]. Coupled with an earlier finding by Walker et al. [26] that development of the asthma symptoms was dependent on b-arrestin2, these findings suggest a hypothesis that b2AR via b-arrestin2-dependent signals promote disease progression, while G protein activation is responsible for acute bronchodilation. If confirmed, this hypothesis suggests a G protein-biased ligand that blocks endogenous b2AR b-arrestin stimulation will provide superior long-term management of pulmonary disease (see Walker and Defay, in this issue).
k-Opioid receptor: signal-based pathway validation k-Opioid receptor (k-OR) agonists have long been sought to provide opioid strength analgesia without the side effects associated with m-opioid receptor agonists (e.g. morphine) including respiratory depression, constipation and addiction. However, CNS-active k-OR agonists also are associated with their own development-limiting side effects, most notably dysphoria, an unpleasant state characterized by depersonalization, dissociation, and psychotomimetic effects. Historically, opioid receptors have been thought to mediate their cellular effects through Gai inhibition of cAMP accumulation, G protein-coupled inward-rectifying potassium channels, and voltage-dependent L-type Ca2+ channels modulating membrane potential. More recent studies showed that k-OR agonists also stimulate ERK1/ 2, MAPK, and c-Jun pathways [27]. Studies by Bruchas et al. [28] demonstrated that the k-OR agonist U50,488 also stimulated p38 MAPK and that this stimulation was dependent on phosphorylation of the k-OR tail by G protein-coupled receptor kinase (GRK) 3, thus linking this signaling event to b-arrestin recruitment. The k-OR selective ligand U50,488 produced conditioned place aversion suggesting that treatment with U50,488 caused an unpleasant experience in the mice which they sought to avoid. Pretreatment with the p38 MAPK inhibitor SB203580 blocked the U50,488 conditioned place aversion suggesting that the dysphoric effects of k-OR agonism are mediated via p38 MAPK. Furthermore, mice lacking the receptor phosphorylating GRK3 did not develop conditioned place aversion in response to U50,488. Moreover, they did not show increased p38 MAPK activation after k-OR activation, implicating barrestin recruitment to the receptor in this pathway [29]. By contrast, SB203580 did not block swim stress mediated by k-OR dependent analgesia observed in www.sciencedirect.com
the tail withdrawal model, demonstrating that divergent signaling pathways are associated with k-OR analgesia and dysphoria. These findings suggested that CNS-penetrant k-OR agonists lacking b-arrestin engagement may provide powerful CNS k-OR analgesia without intolerable dysphoria. Several groups have discovered k-OR agonists that inhibit cAMP accumulation via Gai but avoid b-arrestin mediated activation of p38 MAPK [30,31]. One such compound recently described is 60 GNTI, a close analog of the k-OR antagonist 50 GNTI [32]. 60 GNTI is a partial agonist in GTPgS, ERK phosphorylation and cAMP inhibition (37–75% of maximal response) in cells expressing k-OR while in contrast stimulating b-arrestin only very weakly (12% of U69,593 max) [33,34]. In mouse striatal neurons U69,593 stimulates G protein-dependent Akt phosphorylation and b-arrestin-dependent ERK1/2 phosphorylation; 60 GNTI stimulates G protein-dependent Akt phosphorylation to the same extent as U69,593, but does not stimulate ERK1/2 phosphorylation. Despite its G protein-biased profile and partial efficacy, 60 GNTI is analgesic in mouse tail-flick assays following i.t. but not i.c.v. administration [35]. However, the in vivo relevance of bias of this compound has not yet been explored in models of dysphoria (e.g. conditioned place aversion) so the hypothesis that a G protein-biased k-OR agonist could provide analgesia without dysphoria remains untested. Thus, k-OR agonism typifies signal-based pathway validation (Figure 1) for a target that has both desirable and undesirable effects, although cross-validation with biased ligands remains to be demonstrated.
m-Opioid receptor: signal-based pathway validation Opioids such as morphine produce effective analgesia by activating the m-opioid receptor (m-OR) [36]. However, opioids produce adverse events including nausea and vomiting, respiratory depression, constipation, and sedation, which limit their utility. Pathway validation studies using KO mice demonstrated that the beneficial and adverse effects of m-OR may be separable with biased ligands. Mice lacking b-arrestin2 showed greater and more prolonged analgesia to morphine but reduced constipation and respiratory depression [37,38]. Similar effects on analgesia were seen with knockdown of b-arrestin2 in specific brain regions with siRNA [39,40]. These signal based pathway validation data suggest that a G protein-biased m-OR agonist that does not recruit b-arrestins could provide analgesia with reduced dose-limiting adverse effects associated with current opioid therapies. Based on this hypothesis, a drug discovery effort led to the identification and characterization of TRV130, a novel G protein-biased m-OR ligand. TRV130 is a potent agonist of G protein mediated inhibition of cAMP accumulation with comparable intrinsic efficacy to morphine while at same time Current Opinion in Pharmacology 2014, 16:108–115
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recruiting significantly less b-arrestin2 than morphine [41,42]. Consistent with selective activity for the G protein pathway, TRV130 exhibits reduced MOR internalization and phosphorylation compared to strong opioids such as fentanyl and morphine [42]. In mice and rats, the biased in vitro profile of TRV130 translated to potent analgesia with reduced gastrointestinal dysfunction and respiratory depression compared to morphine [42]. The in vivo efficacy of TRV130 demonstrates that a biased ligand may provide superior analgesia. The threat of respiratory depression leads to underdosing of opioids and sub-optimal pain relief; nausea and vomiting can increase patient discomfort and delay oral intake; and sedation can lead to longer time to ambulation and in some cases increasing hospital length of stay and therefore the overall cost of care. If a biased ligand such as TRV130 could more effectively treat postoperative pain while mitigating these adverse effects, patients would be expected to experience better pain relief and progress more efficiently through postoperative recuperation. Such a compound might also be an attractive agent for chronic obstructive pulmonary disease palliation where the risk of respiratory depression limits the use of currently approved opioids [43,44]. In first-time in human studies, intravenously infused TRV130 produced pupil constriction, a well validated marker of m-OR activity in the CNS, at well tolerated doses [45]. The magnitude of pupil constriction for TRV130 was comparable to that elicited by clinically used doses of morphine and fentanyl in other studies [46] however, unlike morphine and fentanyl, TRV130 at several doses caused this robust pharmacology without causing nausea or vomiting. Although comparator studies are required to assess the true differentiation of TRV130, these initial clinical results indicate TRV130 may be better tolerated than unbiased opioids like morphine and that the beneficial therapeutic index seen preclinically for this biased ligand may translate in the clinic [45].
Angiotensin II type 1 receptor: signal-based and ligand-based pathway validation The angiotensin II type 1 receptor (AT1R) was one of the earliest GPCRs for which evidence of biased signaling was shown. Angiotensin II (AngII) regulates cardiovascular functions and increased levels have been linked to hypertension, renal dysfunction and heart failure. AngII binding to the AT1R induces coupling to Gaq, activation of phospholipase C, and mobilization of calcium resulting in vasoconstriction and increased blood pressure [47,48]. AngII also promotes AT1R phosphorylation by GRKs and the recruitment of b-arrestins resulting in receptor internalization and desensitization of G protein signals. Studies over the last decade demonstrated that AT1R mediated signaling can be G protein or b-arrestin dependent and Current Opinion in Pharmacology 2014, 16:108–115
that this differential signaling can be leveraged to optimize pharmacologic activity at the AT1R [8]. Receptor mutagenesis studies revealed that elimination of Gaq coupling did not abate AngII induced phosphorylation, internalization, recruitment and signaling via b-arrestins [49,50]. Knockout studies eliminating AT1R or b-arrestin2 exacerbated cardiac stretch induced apoptosis providing evidence that b-arrestin signaling through the AT1R is cardioprotective [51]. Experiments with the b-arrestin biased AT1R ligand [Sar1, Ile4, Ile8] angiotensin II (SII) complimented these findings. SII binds the AT1R and directs signaling through the b-arrestin pathway while antagonizing G protein signaling [50,52,53]. SII produces AT1R and b-arrestin-dependent reduction in apoptosis, stimulation of chemotaxis, increased cardiac contractility, cell growth and proliferation [52,54–57]. Other work showed that SII weakly activates several G protein complexes but in a pattern that differs from that activated by AngII, consistent with the existence of discrete receptor conformations stabilized by AngII and SII [58]. A number of factors, including assay type, receptor, and signal transducer expression, may account for the additional pathway activity of the b-arrestin biased ligand SII. Separate work demonstrated different receptor conformations associated with G protein-biased, neutral, and b-arrestin-biased ligands by measuring transducer-specific thermodynamics of ligand binding [59]. The in vitro and in vivo signal-based pathway validation studies, as well as subsequent demonstration of ligandbased pathway validation for beneficial b-arrestin-dependent effects at the AT1R, inspired the discovery and development of TRV027, (initially called TRV120027) a selective b-arrestin-biased AT1R ligand with increased potency and b-arrestin efficacy compared to SII [60]. TRV027 competitively binds the AT1R, reduces AngIImediated hypertension, increases contractility of isolated mouse cardiomyocytes and enhances cardiac performance in rodents [60]. This profile translated to beneficial hemodynamic, renal and cardiac effects in a tachypaced canine model of acute heart failure [61,62]. In each of these studies, TRV027 behaved similarly to an angiotensin receptor blocker with respect to renal and vascular effects, but with increased cardiac performance associated with its b-arrestin efficacy. In addition to stimulating cardiac contractility, b-arrestin biased ligands like TRV027 may also be cardioprotective. TRV120023, an analog of TRV027 with similar biased AT1R pharmacology, reduced cell death in a mouse ischemic reperfusion model [63]. Furthermore, the enhanced cardiac contractility induced by TRV120023 is not due to direct calcium mobilization as occurs with classic inotropes and with AngII. Chronic infusion of TRV1200023, but not the full antagonist losartan, to normal rats and rats co-infused with either saline or AngII showed increased calcium sensitivity, likely through post-translational modification of cardiac myofilaments www.sciencedirect.com
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[64]. These findings coincided with a blockade of AngII-mediated cardiac hypertrophy by both TRV120023 and losartan, consistent with the well-established role of Gaq-coupling in the development of cardiac hypertrophy. Together these pathway validation studies suggest a profile of b-arrestin-biased AT1R ligands that may offer unique benefits in treating cardiovascular disease. Because it selectively intercedes at the AT1R to block renal and systemic vasoconstriction while producing enhanced cardiac performance and cardioprotective effects, TRV027 might be an effective novel treatment for acute heart failure. In early clinical trials, TRV027 caused renin-angiotensin system-dependent reductions in mean arterial pressure (MAP) in salt-restricted healthy volunteers [65] and in stable advanced chronic heart failure [65], and is now in Phase 2 clinical trials for treating AHF.
Summary The dissection of different receptor-mediated signal transduction pathways underpins the scientific and potential therapeutic benefits of biased ligands. Pathway validation links specific signaling pathways to different pharmacological outcomes, establishing both the mechanisms of receptor function and opportunities for biased ligands to improve on-target pharmacology. As illustrated in the examples above, pathway validation can be achieved at several levels. It can be studied downstream of the receptor at the level of signal transduction, using a variety of techniques including genetic deletion, siRNA, overexpression, and chemical inhibition to test the dependence of multiple endpoints on specific signals (Figure 1). Pathway validation also can be elucidated at the level of ligands by comparing biased and unbiased ligands for differential effects in cells, tissues, and in vivo (Figure 2). These approaches have been valuable in biased ligand research and are widely applicable to any relevant downstream signaling pathways. In some cases, (e.g. m-OR), signalbased validation fostered biased ligand discovery efforts leading to successful and complementary ligand-level validation. In other cases, such as with the AT1R, the early identification of biased ligands allowed pathway validation to be established across numerous experimental systems in parallel with signal-level pathway validation. Both ligandbased and signal-based validation approaches show promise for establishing pathway-specific pharmacology at the b2AR. For other receptors, such as the k-OR, signal-level pathway validation based on disruption of multiple signal transduction components has not yet been cross-validated at the level of ligand pharmacology. Importantly, though this review focuses on the dissection of G protein and b-arrestin pathways, the same approaches can be used to interrogate selective pathway signaling of other GPCR-associated downstream pathways.
Concluding remarks The number of reported biased ligands has increased over the last decade, as has the number of GPCRs for which www.sciencedirect.com
complex pharmacological responses have been dissected to reveal unique contributions from distinct and separable downstream signaling pathways. This has engendered growing enthusiasm for the prospective discovery and optimization of biased ligands as novel, differentiated therapeutics targeting both old and new GPCR drug targets by improving on-target pharmacology. The receptors discussed here illustrate how different approaches taken to dissect receptor pharmacology can illuminate novel pharmacology, mechanism of action and therapeutic utility of biased ligands. These methods, also discussed elsewhere [8,66], compliment methods of assaying GPCR molecular pharmacology [6] and accurately detecting and quantifying ligand bias [67,68,69]. From the perspective of drug discovery, the integration of these efforts, with particular emphasis on building a strong scientific rationale for why ligand bias might matter at a given receptor, may lead to numerous opportunities to develop safer, more efficacious and better tolerated new medicines targeting GPCRs.
Conflict of interest statement Nothing declared.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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