Biased ligands at G-protein-coupled receptors: promise and progress.

TIPS-1135; No. of Pages 9

Review

Biased ligands at G-protein-coupled receptors: promise and progress Jonathan D. Violin, Aimee L. Crombie, David G. Soergel, and Michael W. Lark Trevena Inc., 1018 West 8th Avenue, Suite A, King of Prussia, PA 19406, USA

Drug discovery targeting G protein-coupled receptors (GPCRs) is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. In particular, the concept of ligand bias, whereby a ligand stabilizes subsets of receptor conformations to engender novel pharmacological profiles, has recently gained increasing prominence. This review discusses how biased ligands may deliver safer, better tolerated, and more efficacious drugs, and highlights several biased ligands that are in clinical development. Biased ligands targeting the angiotensin II type 1 receptor and the m opioid receptor illustrate the translation of the biased ligand concept from basic biology to clinical drug development. New frontiers for G protein-coupled receptors G protein-coupled receptors (GPCRs) have long occupied a central role in pharmacology. They are the targets of many of the most widely prescribed drugs in central nervous system (CNS), cardiovascular, pulmonary, gastrointestinal, and many other disease areas. Originally conceived as theoretical entities to explain the drug-specific responses of tissues, GPCRs are now known to be a superfamily of membrane proteins that bind hormones, neurotransmitters, and other extracellular cues to transmit information about the external world to the interior machinery of cells [1]. In the simplest conceptual model, GPCRs operate as switches, normally residing in an inactive state, and are only activated to engender intracellular response when bound by agonists. Antagonists in this paradigm bind the receptor and prevent activation by preventing agonist binding. This light-switch model dominated basic research and drug discovery efforts for many years. However, receptors are in fact capable of much more than simple binary signals. Illumination of these more subtle mechanisms has unveiled new opportunities for research and drug discovery targeting GPCRs. This review summarizes these possibilities, with an emphasis on the class of biased ligands, which provide a previously unattainable level of pharmacological selectivity by targeting not just specific receptors, but specific signaling pathways downstream of those receptors.

Pleuripotency of signaling: pathway validation Classically, agonist-occupied GPCRs mediate cellular responses by binding heterotrimeric G proteins, which initiate cascades of second-messenger responses to alter metabolism, cytoskeletal structure, transcription and translation, and tissue-specific responses such as myocyte contractility and neuron polarization among many others. Almost every GPCR couples to one or more G protein classes, which dictates different cellular responses. Almost every receptor also engages a parallel set of regulatory mechanisms initiated by recruitment of GPCR kinases (GRKs), which phosphorylate agonist-occupied receptor and other targets, and mediate a variety of regulatory protein–protein interactions [2]. Following phosphorylation, GPCRs engage b-arrestins, scaffolding proteins expressed as two isoforms that directly bind GPCRs by recognizing both the agonist-occupied receptor conformation and phosphorylated receptor regions [3]. Typically, b-arrestin recruitment inhibits further G protein coupling, promotes receptor internalization by coupling receptors to endocytic machinery, and triggers signaling by recruiting scaffolded signaling proteins to the receptor. Thus, b-arrestins both regulate G protein signaling and initiate G protein-independent signals. The dual pathways of G protein and b-arrestin coupling are ubiquitous and generic, and have been described in a wide variety of in vitro and in vivo systems [3,4], but GPCRs can also engage many other mechanisms of signal transduction that translate to a diversity of molecular and cellular responses [5]. Several approaches have been used to delineate the contributions of G proteins and b-arrestins to GPCR function, including targeted genetic deletion of GRKs or barrestins, RNA silencing of G protein and b-arrestin pathway components, and application of small-molecule inhibitors of specific signal transduction pathways. These tools have been invaluable in dissecting the pharmacology of specific receptors, and in some cases, described below, have uncovered previously unappreciated signal transduction paradigms. The different experimental strategies taken for such pathway validation are reviewed elsewhere [6,7] and can be thought of as complementary to traditional target validation in assessing potential drug discovery efforts.

Corresponding author: Violin, J.D. ([email protected]). Keywords: angiotensin; opioid; b-arrestin; functional selectivity; TRV027; TRV130. 0165-6147/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2014.04.007

Concept of ligand bias Classically, agonists were thought to entrain the entire signal repertoire of a receptor, and thus pharmacological Trends in Pharmacological Sciences xx (2014) 1–9

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Review selectivity was specified as selectivity of a drug, hormone, or neurotransmitter for a particular receptor type. Receptors were either ‘on’ or ‘off’ as envisioned in the classic twostate model of receptor function [8]. The discovery of partial agonists and inverse agonists revealed new levels of pharmacological properties, often differentiating these agonists from full agonists and neutral antagonists, but still consistent with the two-state model. Thus, it was envisioned that receptors adopt two states, and ligand binding preferentially stabilizes the inactive state (for antagonists and inverse agonists) or the active state (weakly for partial agonists, more strongly for full agonists). This paradigm dominated receptor theory and drug discovery for several decades, and afforded a level of simplicity for investigators: one assay of receptor function was sufficient to predict pharmacological responses in other systems. However, several examples were found that did not fit this paradigm: compound sets yielded different relative potencies in different assays, which could not be explained by off-target pharmacology [9,10]. These findings, controversial at first, were slowly replicated for other receptors, challenging the notion that receptors are only capable of a single spectrum of responses. As these examples have proliferated and been replicated, contemporary pharmacological theory has been revised to incorporate the possibility that receptors engage distinct subsets of their full signaling repertoire. Receptors are now envisioned as occupying myriad discrete conformations, and nonoverlapping sets of these conformations are associated with signaling mechanisms such as G protein coupling, b-arrestin recruitment, receptor trafficking, and other signals. When conformations linked to different signals are distinct, ligands that selectively stabilize these conformations will promote pharmacological responses that differ not just quantitatively but also qualitatively from responses to traditional agonists. These compounds are thus biased towards subsets of receptor function relative to a reference ligand. By convention, the reference ligand is defined as unbiased or balanced; this reference is usually an endogenous agonist or clinically validated drug, but examples of endogenous biased ligands have been described [11,12]. Different receptor conformations permitting ligand bias were first inferred as a parsimonious explanation for measurement of signals downstream of receptors [9,10,13,14], but biophysical techniques have since demonstrated different receptor states for different ligands [15,16]. In addition, several studies of GPCR crystal structures have shown potential structural bases for ligand bias [17,18]. However, there is not yet direct structural evidence for distinct receptor conformations linked to specific signals such as b-arrestin recruitment or distinct G protein classes. Future studies comparing crystal structures of a receptor bound to biased and unbiased ligands may establish these relationships. Definition of ligand bias versus functional selectivity: not just semantics The notion of selective signaling in pharmacology has a long history predating the isolation of receptors, beginning with classification of drugs according to differential 2

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pharmacology in different tissues. As molecular biology and genetics have uncovered the many mechanisms by which compounds can exert distinct pharmacology, pharmacological nomenclature has evolved, so that drugs are now described by their efficacy (agonist, antagonist, partial agonist, or inverse agonist) and target (receptor type and subtype). For ligand bias the nomenclature is not yet settled, and the phenomenon has been described as ligand bias, functional selectivity, and protean agonism, among other terms [19]. It is helpful to discriminate between the many forms of differential pharmacology on a mechanistic basis. Thus, functional selectivity refers to differential pharmacological effects across any number of assays in any number of systems, and is therefore a very broad category that can include mechanistic differentiation at the level of pharmacokinetics, molecular target(s), intrinsic efficacy, and target receptor conformation. There are numerous examples of each type of functional selectivity driving important differences in in vivo pharmacology (Table 1). Ligand bias, however, is solely related to target receptor conformation and is intrinsic to the ligand–receptor complex when a compound stabilizes different receptor conformations compared to a reference ligand. One of the important features of intrinsic ligand bias is that it is more likely to be system-independent. Unlike functional selectivity, which is driven by pharmacokinetics, engagement of multiple targets, or differential amplification of signals downstream of a partial agonists, each of which largely depends on tissue-specific factors, ligand bias reflects different thermodynamic contributions of ligand binding energy to stabilize distinct conformations of the ligand-bound receptor, and thus the unmasking of different receptor-coupling mechanisms. Although membrane composition and expression/regulation of receptorcoupling proteins such as G proteins, b-arrestins, and receptor trafficking machinery can influence the pharmacology downstream of a biased ligand, the core differentiation may be preserved across experimental systems, as noted in several examples of biased ligands described below. Thus, in vitro assays, measured appropriately (see below), can identify intrinsic bias associated with specific ligand-receptor states that are somewhat insulated from cellular or tissue factors; this may explain the successes to date in translating data from in vitro overexpressed receptor systems to in vivo differentiation of biased and unbiased ligands. The different GPCR conformations stabilized by biased and unbiased ligands are most often inferred from differences in receptor-selective signals measured in vitro. This has been done using comprehensive approaches such as proteomics and with more focused assays [20–22]. These approaches represent both integrative downstream signals, which capture the influences of multiple receptorcoupling mechanisms such as proteome pathway clustering, cellular impedance, and MAPK activation, and discriminating proximal readouts, which more directly measure receptor-coupling mechanisms such as second messenger signals, receptor–effector coupling (e.g., barrestin recruitment), and receptor trafficking [23,24]. Either approach can effectively identify biased ligands, but it is important to recognize the shortfalls of each.


Review


Table 1. Types of functional selectivity. GPCR functional selectivity, a differentiated pharmacological profile triggered by a single receptor family, can be achieved through distinct mechanisms. The select examples shown here highlight these different mechanisms, and illustrate the unique features of functional selectivity engendered by intrinsic ligand bias, which engages differential pharmacology at the level of the receptor, not upstream (as in pharmacokinetic or subfamily target selectivity) or downstream (as in differential amplification of partial agonist signals) Mechanism Example Pharmacokinetics Peripheral restriction

Target selectivity

Histamine receptors b-Adrenoceptors

Binding affinity Efficacy

Target (compound) m-opioid receptor agonist (loperamide) m-opioid receptor antagonist (MeNTX, alvimopan) k-Opioid receptor agonist (CR665) H2 histamine receptor blocker(cimetidine, ranitidine) b1 adrenoceptor blocker (metoprolol, bisoprolol) S1P1 modulator (fingolimod)

Receptor degradation vs recycling to cell surface Partial agonist m-Opioid receptor (buprenorphine) Dopamine receptor (aripiprazole) Inverse agonist

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b2 adrenergic receptor (bisoprolol, nadolol) TRV027

G protein bias

TRV130

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Calcium-sensing receptor (cinacalcet) CXCR4 (ATI-2341)

Focused screening of proximal signals can be precise, highthroughput, and carefully quantified, but may miss important signal transduction pathways. Distal integrative signals capture a fuller set of receptor functions but can be more difficult to quantify, to prove target specificity, and to ascribe to specific biased signaling pathways. Measurement In some cases, ligand bias is evident from qualitative comparisons of data from different assays systems, as when the relative potencies or efficacies of two compounds are reversed in different systems [25]. However, in many cases the varying sensitivities of assay formats necessitate quantitative measures of ligand bias to discriminate intrinsic bias from functional selectivity arising from differential amplification of signals downstream of a partial agonist. Several methods have been developed and used to characterize biased ligands [26–28], and have enhanced the rigor of biased ligand detection. However, it is important to note the assumptions inherent in these models, which can lead investigators astray. These include assumptions that the assays are reporting effects from only one molecular target, that assay conditions influence ligand concentration or ligand–receptor interactions equally across assays, and that assays are equivalently sensitive to receptor binding kinetics. For these reasons, biased ligand identification is most robust when using (i) assays of receptor-proximal signals to reduce the chances of assay signal contamination by off-target pharmacology; (ii) controls for receptor specificity; (iii) matching assay conditions (e.g., cell type and assay medium) to reduce the

Pharmacology Slows GI motility, no CNS effects Blocks opioid-induced constipation without reducing analgesia Analgesia without CNS effects Inhibit stomach acid without CNS and antiinflammatory effects Block cardiac inotropy and chronotropy without blocking broader B2AR function Higher affinity than S1P maintains interaction after internalization to promote downregulation of receptor Ceiling effect for opioid pharmacology may improve safety Mood stabilizer; blocks hyperdopaminergia without adverse effects of full dopamine antagonism Reduces constitutive activity of b-adrenergic receptors Blocks effects of RAS activation on vasculature and kidney while stimulating contractility and prosurvival signals in heart Stimulates increased analgesia, reduced constipation, and reduced respiratory depression vs morphine Preferential effect on membrane ruffling vs calcium or MAPK Stimulates Gai coupling but not Ga13 or b-arrestin coupling

chances of non-receptor influences on assay readouts; and (iv) matching assay duration to prevent receptor binding kinetics or compound degradation from artificially reduced potency or efficacy in one assay. Most importantly, followup confirmatory studies, preferably with orthogonal assay systems, greatly increase confidence in the discrimination of biased and unbiased ligands [22,29]. Translation of mechanistic insights to novel pharmacology: compounds in the clinic Since the recognition of ligand bias, numerous groups have speculated that biased ligands could deliver novel pharmacology distinct from classical agonists or antagonists [25,30–32]. Broadly, ligand bias may differentiate in several ways. Biased ligands may eliminate on-target adverse events by avoiding undesirable signaling pathways, increase the efficacy of agonists and antagonists by avoiding or stimulating specific positive or negative feedback loops in signaling pathways, and reveal previously unappreciated pharmacology to endow agonists or antagonists with new benefits. Numerous examples of each have been proffered; here we highlight several targets and novel ligands that illustrate the promise of biased ligands as novel GPCR drugs. b-Arrestin-biased ligands at the angiotensin II type 1 receptor Signaling at the angiotensin II (AngII) type 1 receptor (AT1R) is perhaps the best-studied example of ligand bias. Studies using signal transduction inhibitors, RNA interference, and knockout mice showed that AT1R supports 3


Review robust signaling through both G protein- and b-arrestindependent pathways [6]. These included evidence that barrestin-dependent AT1R ligands are anti-apoptotic and cardioprotective [33,34]. The discovery that Sar1 Ile4 Ile4angiotensin (SII) induces AT1R-dependent mitogen-activated protein kinase (MAPK) activation but not phosphatidylinositol turnover [35] and that this discrepancy is driven by recruitment of b-arrestins in the absence of G protein signaling [36] first established the capability of AT1R to support ligand bias. Later work showed that SII stimulated the contractility of isolated mouse cardiomyocytes via AT1R and b-arrestin-2, again without stimulating G protein coupling [37]. The absence of G protein coupling suggested that b-arrestin-biased ligands would decrease blood pressure akin to angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme (ACE) inhibitors. This led to the hypothesis that optimization of compounds such as SII could deliver a novel therapeutic approach to cardiovascular disease by exerting the renal and vascular effects of an ARB with the additional benefits of enhanced myocardial contractility and survival provided by b-arrestin-2 engagement [30]. On the basis of this hypothesis, a peptide optimization campaign identified TRV027 (also known as TRV120027) as a potent, selective, b-arrestin-biased ligand [22]. TRV027 binds competitively at AT1R with respect to AngII and ARBs, and stimulates a subset of the biochemical signals that AngII does in vitro. This is consistent with a mechanism of stabilization of discrete subsets of the full set of active AT1R conformations. In rodents, TRV027 and a related peptide, TRV120023, decrease blood pressure and increase cardiac contractility via b-arrestin-2 [22,38], as predicted by earlier in vitro studies. The absence of detectable G protein coupling from these ligands in multiple assays suggests that enhanced cardiac contractility is not driven by direct calcium mobilization as occurs with classic inotropes and with AngII. Chronic infusion of TRV120023 in normal rats and rats co-infused with AngII showed that the b-arrestin-biased ligand increased calcium sensitivity, likely through post-translational modification of cardiac myofilaments [39]. TRV027 was progressed to translational preclinical studies in the tachypaced canine heart failure model, where it reduced pulmonary capillary wedge pressure (a clinical correlate of dyspnea), reduced systemic and renal vascular resistance, and increased cardiac output [40]. These findings are all consistent with the in vitro and rodent studies of SII, TRV027, and TRV120023. The pharmacology of TRV027 was preserved in tachypaced canines co-administered furosemide, suggesting that b-arrestinbiased AT1R ligands may have benefits when used in conjunction with loop diuretics [41]. These data suggest that TRV027 may provide important benefit in acute heart failure (AHF), for which current use of loop diuretics to offload fluid, nitrates to reduce blood pressure, and inotropes to support cardiac output has failed to alleviate the tremendous burden of symptoms, mortality, and rehospitalization associated with AHF [42– 44]. TRV027 may be able to quickly and safely improve hemodynamics and preserve renal function to allow loop diuretics to more effectively offload fluid, while supporting 4


β-arrestin-biased ligand Angiotensin II type 1 receptor Cell membrane

Gαq β-arresn

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↑ Cardiac contraclity ↓ Cardiac cell apoptosis TRENDS in Pharmacological Sciences

Figure 1. Clinical hypothesis for b-arrestin-biased ligands at the angiotensin II type 1 receptor (AT1R). Preclinical data suggest that Gaq coupling at AT1R is linked to vasoconstriction and sodium and fluid retention, whereas b-arrestin-2 recruitment is associated with increased cardiomyocyte contractility and protection against cardiac apoptosis. The renin–angiotensin system (RAS) is activated in acute heart failure, elevating angiotensin II concentrations to exert both the deleterious and beneficial AT1R pathways. b-Arrestin-biased ligands such as TRV027 competitively bind AT1R, stabilizing a receptor conformation that facilitates phosphorylation by GPCR kinases (GRKs) and recruitment of b-arrestin to bind the phosphorylated receptor, all without promoting G protein coupling. This novel class of ligand may thus block harmful AT1R effects while preserving or enhancing beneficial effects.

end-organ perfusion with the combined vascular and cardiac benefits unique to the biased ligand mechanism [45]. Despite their well-established benefit in hypertension and chronic heart failure, ACE inhibitors and ARBs are not used in the treatment of AHF, in part because they can cause prolonged hypotension and reduce cardiac output [45,46]. In this regard, TRV027 is positioned to unlock the AT1R for use in a new indication: as a rapidly acting and rapidly reversible peptide can afford control of vasoactive effects to prevent prolonged hypotension, and as a barrestin-biased ligand it can directly support cardiac contractility and preserve cardiac function (Figure 1). Early clinical trials of TRV027 have demonstrated a rapidly acting and rapidly reversible decrease in blood pressure in healthy volunteers [47] and stabilization of chronic heart failure [48]. Importantly, TRV027 is only active when the renin–angiotensin system – a pathophysiological cornerstone of AHF that is not directly targeted by current AHF therapies – is activated. This supports the mechanistic basis of TRV027 function and provides an intrinsic safety feature: TRV027 pharmacology should be substantially blunted if dosed to patients who do not in fact suffer from the intended target pathophysiology. The discovery of biased ligands at the AT1R uncovered novel receptor pharmacology that could be stimulated in the context of otherwise well-described receptor antagonism. This feature of b-arrestin-biased ligands such as TRV027 also mitigates adverse on-target pharmacology associated with full receptor antagonism in the context of AHF. A Phase IIb trial of TRV027 in AHF is currently under way (clinicaltrials.gov identifier NCT01966601) and will further test if biased ligands can deliver superior therapeutic profiles compared to traditional GPCR-targeted drugs.


Review G protein-biased ligands at the m opioid receptor The m opioid receptor (MOR) is a striking example of pathway validation dissecting on-target benefits from adverse effects. Mu opioid agonists such as morphine, fentanyl, and oxycodone provide powerful analgesia through the MOR, but their clinical utility is greatly complicated by nausea, vomiting, constipation, postoperative ileus, respiratory depression, sedation, dependence, and abuse, all also mediated by the MOR [49]. Several approaches have been taken to mitigate these on-target adverse effects. Peripherally restricted antagonists such as methylnaltrexone and alvimopan significantly reduce constipation when co-administered with opioid analgesics [50,51]. Partial agonists such as buprenorphine display ceiling effects for analgesia and respiratory depression but are functionally selective in some systems [52]. Multimodal agonists such as tapentadol engage additional analgesic mechanisms [53]. Each of these has been described as functionally selective, which is accurate at the organismal level as discussed above, but these approaches do not intrinsically dissociate analgesia from ontarget adverse effects at the level of the receptor. For example, peripheral mu opioid antagonists reduce constipation but do not reduce adverse CNS effects and do not directly increase analgesia. Likewise, multimodal mu opioid agonist/norepinephrine transporter inhibitor molecules offer additive or synergistic analgesic mechanisms in some but not all pain models [54]. Thus, 50% of patients report moderate-to-severe pain following surgery despite opioid use, in large part because the on-target adverse effects of opioids prevent physicians from successfully escalating the dosage to adequately relive pain [55]. Mu opioid agonists were used therapeutically long before the molecular target was identified and shown to mediate all classic opioid pharmacology [56]. The loss of both opioid analgesia and adverse opioid pharmacology in MOR knockout mice suggested that on-target adverse effects were inextricable from analgesic efficacy for mu opioid agonists. A series of experiments in b-arrestin-2 knockout mice served as pathway validation to show that opioid pharmacology can in fact be dissociated into discrete signals: compared to wild type mice, morphine is more analgesic [57], less prone to receptor desensitization and tolerance [58], and causes less respiratory depression and constipation [59] in b-arrestin-2 knockout mice. The effects of b-arrestin-2 ablation on analgesia were reproduced by RNA interference in the periaqueductal grey [60] and spinal cord [61]. The influence of barrestins on subjective opioid effects such as liking, dependence, and withdrawal is less clear: morphine in b-arrestin2 knockout animals leads to increased liking [62] but some reduced signs of withdrawal [63]. The interactions of liking, dependence, and withdrawal in opioid abuse are complicated, so it is difficult to predict what effect if any b-arrestins might have on human opioid misuse and abuse. Opioid analgesia is mediated by G protein-coupled ion channel inhibition to hyperpolarize nociceptive fibers [64], so the inhibitory role of b-arrestin-2 is consistent with its ability to reduce G protein coupling; however, the mechanisms by which b-arrestin-2 regulate gastrointestinal, respiratory function, and subjective effects are unknown. The improved profile of morphine in b-arrestin-2 knockout mice suggested that a G protein-biased ligand devoid of


G protein-biased ligand μ-opioid receptor Cell membrane

Gαi

β-arresn

Increased analgesia

Respiratory depression nausea & conspaon TRENDS in Pharmacological Sciences

Figure 2. Clinical hypothesis for G-protein-biased ligands at the m-opioid receptor (MOR). According to preclinical data, Gai coupling at the MOR is linked to powerful opioid analgesia, whereas b-arrestin-2 recruitment is associated with respiratory and gastrointestinal dysfunction, as well as inhibition of the analgesic effect. Opioids such as morphine offer powerful analgesia but the threat of dose-limiting adverse events often prevents adequate pain relief. G-protein-biased ligands such as TRV130 bind the MOR and stabilize receptor conformations that promote G protein coupling but do not permit appreciable GRK-mediated receptor phosphorylation or b-arrestin-2 recruitment. By evading the on-target adverse events and inhibition of analgesic signals mediated by b-arrestin-2, this novel class of opioid may offer increased analgesia with improved safety and tolerability compared to currently prescribed opioids.

b-arrestin recruitment might offer increased analgesia with improved safety and tolerability (Figure 2). TRV130 was identified from a lead optimization campaign based on a hit screened from a diversity library [39,65] and is a novel small-molecule chemotype targeting MORs. It is potent and highly selective, and is a strong agonist of G protein coupling at the MOR with intrinsic efficacy comparable to that of morphine and greater than that of the partial agonist buprenorphine. By contrast, TRV130 stimulates nearly undetectable levels of b-arrestin recruitment compared to morphine and other strong opioid agonists. In rodents, TRV130 is a potent analgesic in several pain models, but exerts reduced gastrointestinal dysfunction and respiratory depression at equianalgesic doses compared to morphine (Figure 3). Thus, genetic manipulation of morphine responses successfully predicted biased ligand pharmacology, supporting the use of pathway validation in addition to traditional target validation to exploit the potential of biased ligands to deliver differentiated pharmacology. TRV130 has progressed to early clinical trials, and a study for a single ascending dose revealed dose-linear exposure and dose- and exposure-related pupil constriction as a marker of CNS mu opioid activity [66]. The molecule was well tolerated across a range of doses and exposures associated with marked pupil constriction, supporting further investigation of TRV130 and its differentiation from unbiased ligands. Future studies will carefully compare TRV130 to morphine to test the clinical translation of preclinical differentiation of biased and unbiased ligands. TRV130 appears to be the first biased ligand poised for head-to-head 5


Review


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Figure 3. TRV130 causes less constipation and respiratory depression than morphine does at equianalgesic doses. Antinociceptive efficacy was measured in (A) a mouse hot-plate test at 568C and (D) and a rat hot-plate test at 528C. To illustrate the therapeutic margin, these data are shown in panels B and E for morphine equivalents, with TRV130 doses normalized by the mouse or rat hot-plate ED50 ratio for morphine and TRV130. On this equianalgesic basis, (C) gastrointestinal dysfunction measured in a glass bead test of mouse colonic motility and (F) respiratory depression measured as rat blood carbon dioxide are displayed as therapeutic indices. The data illustrate an improvement in on-target adverse pharmacology by a biased ligand. Figures adapted from previously published data [29].

testing against an unbiased ligand in humans, and thus will be an important benchmark for the ligand bias concept. If successful, the molecule could solve the great challenge of opioid analgesia by allowing caregivers to safely and tolerably escalate analgesia to provide pain relief to the large proportion of patients who do not receive tolerable pain relief with currently available options. TRV130 is positioned first for intravenous use in place of current opioids, for which postoperative nausea and vomiting can be serious and costly, the threat of respiratory depression requires vigilance and limits dosing especially in at-risk patients, and reduced gastrointestinal motility prolongs hospitalization in cases of postoperative ileus. However, the same on-target adverse effects are problematic in chronic opioid therapy as well, particularly for gastrointestinal motility, which is especially problematic in chronic opioid use and is only partially alleviated by expensive adjunct therapy with peripheral MOR antagonists [51,67]. For these reasons, TRV130 and G-protein-biased MOR ligands like it may provide valuable new options for a range of pain indications. The successful translation of findings in b-arrestin knockout mice to differential pharmacology of biased MOR ligands exemplifies a rational basis for using pathway validation to dissect pluripotent receptor function, and then identifying 6

new ligands that selectively target the clinically beneficial signaling pathway to deliver an improved therapeutic profile. TRV130 is now entering mid-stage clinical trials. Evidence of differential pharmacology at other GPCRs: paths to future drug discovery? Evidence has accumulated for a number of GPCRs that biased ligands may offer safer, better-tolerated, or more efficacious drugs, in some cases suggesting a path to successful drug development for targets that have been abandoned because of on-target adverse pharmacology. Some of these hypotheses relate to selective engagement of G protein and b-arrestin signals as discussed above, but others relate to additional receptor signaling and regulatory mechanisms. Several receptors discussed below highlight the range of potential mechanisms for biased ligands to deliver value in drug discovery. Biasing allosteric modulators at the calcium-sensing receptor Cinacalcet, used to treat chronic kidney disease with secondary hyperparathyroidism, is a positive allosteric modulator of the calcium-sensing receptor (CaSR) and thus potentiates the effect of calcium on CaSR function. Recent


Review work demonstrated that cinacalcet and two other allosteric modulators preferentially modulate some CaSR signals over others in vitro, classifying these ligands as biasing allosteric modulators [68]. Both the positive allosteric modulators cinacalcet and NPS-R568 and the negative allosteric modulator NPS-2143 modulate membrane ruffling more potently than pERK or calcium signals, indicating that the allosteric interaction favors a receptor state linked to membrane ruffling. Thus, biasing allosteric modulators stabilize receptor conformations that alter both affinity for orthosterically binding ligands (e.g., the endogenous ligand) and the relative strength of different receptor signals. Other work demonstrated orthosteric biased ligands at the CaSR [69], further suggesting that the CaSR can adopt signal-selective conformations. Together these findings suggest that biased ligands may offer more refined and therapy-targeted pharmacology at the CaSR [70]. This example also highlights the utility of allosteric modulators to engender bias; a drug need not displace an agonist to stabilize receptor conformations that restrict some signalcoupling mechanisms while stimulating others. Ligand-selective trafficking of the sphingosine-1phosphate type 1 receptor Lymphocytes migrate from lymphoid organs to the bloodstream in a chemotactic response to sphingosine-1-phospate (S1P), activating the S1P1 receptor. This is rapidly followed by migration from blood to target tissues against the S1P gradient in a carefully coordinated cycle of receptor activation and desensitization involving GRK2 [71]. Fingolimod (also known as FTY720) was recently approved as a disease-modifying therapy for multiple sclerosis. Fingolimod is a prodrug; its activity is mediated by a phosphorylated active form that binds S1P1 and disrupts homing of lymphocytes to the CNS, thereby reducing autoimmune neuroinflammation [72]. However, phospho-fingolimod is not an antagonist but rather an agonist causing transient S1P1 activation. This activation promotes receptor internalization and degradation, unlike internalization and recycling stimulated by endogenous S1P [73]. This ligand-selective receptor trafficking may result from increased affinity of phospho-fingolimod compared to the endogenous ligand, and not from intrinsic bias at the receptor. This form of functional selectivity may impact S1P1 pharmacology more broadly because it was shown that receptor degradation is required for S1P1-mediated vascular leakage but not lymphopenia [74]. This suggests that future generations of S1P1 modulators may differentiate at the level of receptor trafficking, and may be an opportunity for biased ligands or other forms of functional selectivity to provide therapeutic differentiation. Other receptors It has been shown that numerous other receptors signal through parallel pathways or support ligand bias. A comprehensive description of these receptors is beyond the scope of this review, but several exemplars illustrate the range of receptors and biological systems for which biased ligands may be valuable. For example, some dopaminergic behavioral effects are b-arrestin-2-dependent [75], suggesting that the well-known benefits and drawbacks of


dopamine receptor modulation in CNS disease could be improved by biased ligands; examples of biased ligands targeting the D2 dopamine receptor suggest the feasibility of this approach [76]. Similarly, a GRK–b-arrestin–MAPK pathway mediates stress- and aversion-associated effects of kappa opioid receptor agonists, suggesting that biased kappa opioid ligands may provide analgesia without the dysphoric effects associated with classic kappa opioid agonists [77]. The importance of GRKs and b-arrestins in physiological function are highlighted by the chemokine receptor CXCR4: mutations deleting the receptor carboxyterminus, required for phosphorylation and ensuing barrestin coupling, generate a G-protein-biased receptor variant that causes WHIM syndrome [78,79]. It was discovered that a CXCR4 allosteric modulator peptide is biased towards Gai coupling and away from Ga13 and barrestin coupling [80], suggesting that CXCR4 could be targeted by compounds with novel pharmacological profiles. The free fatty acid receptor GPR120 stimulates antiinflammatory signals via b-arrestin-2 and insulin sensitivity via Gaq/11, suggesting that dietary omega-3 fatty acids exert benefit through parallel signaling pathways [81]. Endogenous biased ligands have also been described: the chemokine receptor CCR7 is activated by both CCL19 and CCL21, which are equivalent for G protein coupling but differ in their GRK and b-arrestin engagement [11,12]. Other examples of the breadth of biased ligand pharmacology include the nicotinic acid receptor HCA2 (GPR109a), which mediates the antilipolytic effects of niacin through G protein coupling and of niacin-mediated flushing through a b-arrestin-mediated prostaglandin pathway [82]. This suggested an avenue for increasing high-density lipoprotein (HDL) levels without the tolerability limitations of niacin; however, other work showed that the antilipolytic effect of HCA2 does not translate to increased HDL [83]. HCA2 continues to be studied for its potential in regulating atherosclerosis and vascular inflammation [84]. These and other examples indicate that ligand bias may be achievable for a wide range of receptors. Translation of concept to new medicines The identification of a biased ligand suitable for clinical development from a medicinal chemistry perspective can be challenging. Optimization for bias in addition to potency at the receptor, selectivity, and pharmaceutical properties adds a degree of complexity to such an effort. Highthroughput screens utilizing assays of G protein coupling, b-arrestin recruitment, and other receptor functions have identified hits with varying degrees of bias, but bias can also be engendered by iterative optimization of unbiased hits. Similar to other drug discovery paradigms, the key is to develop a solid understanding of the structure–activity relationship (SAR) of the chemical series. Biased ligand optimization, however, further necessitates understanding the SAR related to bias. Subtle modifications to the chemical structure can result in profound changes to the level of bias, and the SAR for bias does not always translate across related chemotypes. However, it is possible to identify key substituents or specific substitution locations that lead to robust bias that can be maintained even when significant changes are made elsewhere in the molecule. Once the SAR 7


Review of a chemical series in a biased ligand program is understood, optimization of the drug-like properties of compounds without eroding bias requires careful medicinal chemistry. Although the entire process of lead optimization is more complex for these programs, it is feasible, as demonstrated with TRV130 [65]. Once discovered and optimized, biased ligands offer promise for the development of differentiated new medicines for treating significant patient needs. Although the development path for each new drug is specific to the characteristics of the molecule and the therapy area, the underpinning feature of any development program should be to show how the new medicine enhances clinical care and meets patient needs. In cases in which the drug target has a precedent and unbiased drugs are currently used, this requires demonstration of clinical benefits, for example reduced side effects and/or enhanced efficacy, and translation of those benefits into clinically meaningful measures early in the development program. To achieve this, experimental methods may be used to great effect in early development to differentiate new biased compounds from unbiased drugs. If the biased compound unlocks a new indication for a receptor already targeted, as with TRV027 for example, experimental methods may also be used to demonstrate pharmacology and help identify the active dose range in humans, but in these cases inclusion of an active comparator is not necessary or indeed feasible. The crucial first step in a development program is to identify the right molecule with the optimal pharmacology to bring forward into the clinic. Concluding remarks In summary, the concept of biased ligands targeting GPCRs has gained significant traction in recent years. The number of biased ligands reported in the literature continues to grow. With several biased ligands in clinical proof-of-concept studies, and a growing number of receptor targets that may offer improved therapeutic profiles with biased ligands, GPCRs may deliver a new generation of important medicines. References 1 Lefkowitz, R.J. (2004) Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol. Sci. 25, 413–422 2 Pitcher, J.A. et al. (1998) G protein-coupled receptor kinases. Annu. Rev. Biochem. 67, 653–692 3 DeWire, S.M. et al. (2007) b-Arrestins and cell signaling. Annu. Rev. Physiol. 69, 483–510 4 Shukla, A.K. et al. (2011) Emerging paradigms of b-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem. Sci. 36, 457–469 5 Hunyady, L. and Catt, K.J. (2006) Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol. Endocrinol. 20, 953–970 6 DeWire, S.M. and Violin, J.D. (2011) Biased ligands for better cardiovascular drugs: dissecting G-protein-coupled receptor pharmacology. Circ Res. 109, 205–216 7 Schmid, C.L. and Bohn, L.M. (2009) Physiological and pharmacological implications of b-arrestin regulation. Pharmacol. Ther. 121, 285–293 8 Samama, P. et al. (1993) A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636 9 Spengler, D. et al. (1993) Differential signal transduction by five splice variants of the PACAP receptor. Nature 365, 170–175 8


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9

Signal transduction mechanism of biased ligands at histamine H2 receptors.

Biased agonism at G protein-coupled receptors: the promise and the challenges--a medicinal chemistry perspective.

Biased signaling at chemokine receptors.

Evidence for biased agonists and antagonists at the endothelin receptors.

Preventing ARDS: progress, promise, and pitfalls.

Evaluating Youth Development Programs: Progress and Promise.

CG Receptors.

Robotic surgery: the promise and finally the progress.

Integrated pest management in the U.S.: progress and promise.

Biased agonism at kappa opioid receptors: Implication in pain and mood disorders.

The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders.

Corrigendum: biased signaling of protease-activated receptors.

Biased signaling of protease-activated receptors.

Biased ligands: pathway validation for novel GPCR therapeutics.

Diabetes care progress report: shows promise, could do better.

Urinary stone disease: Great progress with promise for the future.

WHO FCTC article 5.3: promise but little progress.

Fcγ receptors and ligands and cardiovascular disease.

α-Conotoxin MrIC is a biased agonist at α7 nicotinic acetylcholine receptors.

Identification of N-arachidonoyl dopamine as a highly biased ligand at cannabinoid CB1 receptors.

A Practical Guide to Approaching Biased Agonism at G Protein Coupled Receptors.

UTP is not a biased agonist at human P2Y(11) receptors.

Endogenous ligands of opiate receptors.

Patents related to EPH receptors and ligands.