Trends in GPCR drug discovery: new agents, targets and indications.

All drugs approved in the United States are listed in the Drugs@FDA database. The primary resource for clinical trial registries is the US National Institutes of Health (clinicaltrials.gov), which currently holds information on more than 251,000 clinical trials globally. The current status of agents in clinical trials is available in commercial databases such as CenterWatch’s commercial database, Drugs in Clinical Trials, which covers more than 4,000 such agents in phase I through phase IV trials worldwide or recently discontinued. However, none of these resources is comprehensive with respect to the targets of agents in trials, which can be collected from a combination of public resources, such as Drugbank 22 , Pharos 23 and Open Targets 24 (which also has a disease ontology), literature 25 – 27 and company press releases. By manually curating CenterWatch’s Drugs in Clinical Trials database (data extracted in July 2017) and cross-referencing with public sources, we were able to identify 481 approved drugs that target GPCRs. This accounts for approximately 34% of all FDA-approved drugs, which is similar to previously reported estimates (27-33%) on the number of GPCR-targeting drugs 1 , 25 , 27 . GPCR drug discovery has advanced rapidly. Just in the past five years, 69 new GPCR-targeting drugs have been approved by the FDA (see TABLE 1 for recent new molecular entities (NMEs)). The most recent approval of a GPCR-targeted drug is for abaloparatide 28 , a parathyroid hormone receptor (PTHR1) agonist used to treat postmenopausal women with osteoporosis. The number of agents currently in trials is 320 (67% of the total number of already approved drugs). Interestingly, 114 (36%) of the agents in trials target potentially novel GPCR targets without an approved drug. To study the distribution of established and currently investigated drug targets, we mapped approved drugs and clinical trial candidates onto the GPCR superfamily tree ( FIG. 1a ). The 481 approved drugs mediate their effect via 107 GPCR targets, accounting for 27% of the human non-olfactory GPCRs. Today, all aminergic receptors are established as drug targets, which serve as the targets for 321 of the approved drugs. Currently established GPCR drug targets are utilized by, on average, 10.5 (median = four) distinct approved agents. This indicates a near saturation of the current target space and emphasizes the necessity of expanding to new druggable receptors in order to develop novel medications. Identification and exploitation of new targets is especially warranted for diseases with large unmet medical needs and few current viable targets, such as Alzheimer’s disease. The US Food and Drug Administration recently reported 70%, 33% and 25-30% success rates for phases I, II and III, respectively, for all target families. We estimated the success rates of GPCR-targeted agents in 2013–2017 (up to July 2017), by counting successes as agents that successfully made it to at least one subsequent phase, and failures as agents that were reported as discontinued, terminated or withdrawn; or completed before this period but never progressed. This yielded GPCR agent success rates of 78%, 39% and 29% for phases I, II and III, respectively, which is slightly higher than FDAs average for all investigated agents (see Further information ). This may reflect the high-level experience in targeting GPCRs. Both our and the FDA’s analyses show a clear trend of more failures in later phases. A recent example is the small-molecule drug fasiglifam, an agonist of the free fatty acid 1 (FFA1) receptor (also known as GPR40). Fasiglifam showed efficacy in a phase III trial for diabetes 29 , but further development was terminated due to hepatotoxicity. We identified 64 potentially novel GPCR targets in clinical trials; that is, targets that are not yet modulated by approved drugs. Of these, 35 are peptide/protein-activated GPCRs (a selection of which are shown in Table 2 , including the calcitonin-gene related peptide (CGRP) receptor for the treatment of migraine, the GPR55 receptor for the treatment of epilepsy and the apelin receptor for the treatment of cardiovascular disorders. Furthermore, the chemokine receptors alone have 22 agents in clinical trials for the treatment of cancer, asthma, rheumatoid arthritis, COPD and HIV. The six members of the glucagon receptor family are also a focus of new approaches for the treatment of type 2 diabetes. Notably, while all recognise natural peptides/protein ligands, these targets span a breadth of receptor families and classes. This suggests that general new approaches, such as increased tractability with biologics, have contributed to the overall increased therapeutic targeting.

Interestingly, current clinical trials feature a number of orphan GPCRs, for which endogenous ligands have yet to be discovered. These orphan GPCRs serve as potentially novel targets for treatment of a diverse set of indications, such as GPR119 for treatment of diabetes, leucine-rich repeat-containing G protein-coupled receptors 4 and 5 (LGR4/5) for treatment of gastrointestinal disease, GPR35 for treatment of an allergic inflammatory condition, GPR55 as an antispasmodic target, the proto-oncogene Mas (MAS) for treatment of thrombocytopenia, and GPR84 for treatment of ulcerative colitis. This indicates that the drug discovery process can still take place, and even advance, despite limited knowledge about the endogenous ligand(s) and/or signalling pathway(s). The entry of an agent and target into clinical trials serves as an important qualifier as animal studies, preclinical data and disease relevance look promising enough for involving humans in the following investigations. It can be expected that public information on orphan receptors will increase as candidate drugs progress through clinical trials and/or upon approval.

Our data show that the indications for GPCR-targeted agents are expanding from historically popular areas, such as hypertension, allergy, analgesics, schizophrenia and depression, into novel areas such as Alzheimer’s disease and obesity ( FIG. 3a ). Furthermore, in the past five years GPCRs, have also been targeted for new indications including multiple sclerosis, smoking cessation, short bowel syndrome and hypocalcaemia. Major trends in the indications of GPCR-targeted agents are highlighted below. Grouping the indications of approved GPCR-targeted drugs onto higher-level disease terms utilizing the Open Target 24 ontology shows that central nervous system (CNS) diseases are the most abundant, accounting for 130 (27%) of all of approved GPCR-targeted drugs. Furthermore, 137 GPCR-targeting agents are currently in clinical trials for CNS indications, demonstrating a continued strong interest. An analysis of receptor baseline expression from the human protein atlas 40 , and studies on the mouse brain 41 , show that more than half of all non-olfactory GPCRs are expressed in the cerebral cortex. Malfunctions in GPCR-mediated neurotransmission can lead to multiple neurological and psychiatric disorders, making GPCRs promising targets in such disorders 42 . Multiple sclerosis, Alzheimer’s disease, Huntington’s disease and fragile X sydrome are highlighted here. Multiple sclerosis (MS), the most common chronic autoimmune disorder that affects the CNS, is caused by damage to the insulating myelin covers of axons. Data from clinical studies and animal models have uncovered several GPCRs involved in the pathogenesis of MS 12 , and one GPCR-targeted drug has been approved: fingolimod, a sphingosine 1-phosphate receptor 1 (S1P 1 ) modulator, which reduces relapse rates and the risk of disability progression 43 . Several other S1P 1 receptor modulators — ozanimod, ponesimod and siponimod — are currently in phase II and III trials. These drugs are expected to have advantages over fingolimod, such as higher selectivity for the S1P 1 receptor, faster clearance and improved tissue penetration 44 . Other MS targets include the cannabinoid receptors. A tetrahydrocannabinol and cannabidiol (THC-CBD) oromucosal spray has been tested in phase II and III studies and been shown to reduce spasticity 45 . A GPR55-selective compound in phase II trials may also potentially be better tolerated than comparable antispasmodics. Taken together, the multitude of agents and targets in late-stage clinical trials indicates that additional MS therapies acting through GPCRs are likely to emerge in the near future. GPCRs are also involved in several neurotransmitter systems associated with Alzheimer’s disease (AD), with glutamatergic, serotonergic, adrenergic and peptidergic pathways in particular being deregulated in this neurodegenerative disorder 46 . Targeting these systems might protect against disease progression by modulating the formation of amyloid- β plaques (one of the cardinal disease features) or aberrant signalling following plaque formation 46 . There is a huge unmet medical need for new therapies for AD, particularly those that might modify disease progression, as the small number of existing therapies (most of which act by increasing levels of acetylcholine by inhibiting its breakdown by acetylcholinesterase) only have limited effectiveness at improving disease symptoms. Leuprolide, a gonadotropin-releasing hormone receptor agonist approved to treat prostate cancer, has been tested in a phase III trial for AD, but it failed to meet the primary or secondary endpoints, although there were signs of an effect on disease progression in patients taking an acetylcholinesterase inhibitor 47 . An additional nine GPCR-targeting agents are in clinical trials for treatment of AD ( FIG. 3 ). Serotonin receptor modulators are of particular interest, including 5-HT 6 receptor antagonists to improve disease symptoms (rather than modify the disease course) by promoting the release of acetylcholine 48 . However, Lundbeck recently terminated the development of their 5-HT 6 receptor antagonist idalopirdine owing to insufficient efficacy in phase III trials. Pfizer also terminated a 5-HT 6 receptor antagonist PF-05212377 due to lack of efficacy after a phase II trial in 2016. Phase III trials of another 5-HT 6 receptor antagonist, intepirdine, licensed by Axovant from GlaxoSmithKline, are still ongoing. GPCRs are also potential targets for another neurodegenerative disorder: Huntington’s disease (HD) 49 , for which current therapies can also only improve some symptoms. HD is caused by numerous repetitions of CAG-triplet repeats within the Huntingtin gene (HTT), which lead to the expression of an abnormal pathogenic huntingtin protein. This leads to cell damage; however, the mechanisms are not fully elucidated. Several GPCR pathways are downregulated in HD patients, and two GPCR-targeted agents are currently in clinical trials for HD: the adenosine A 1 receptor antagonist pbf-999 (which is in phase I trials) and the dopamine D 2 receptor antagonist pridopidine (which is in phase III trials). Finally, GPCRs have attracted considerable investment as targets for fragile X syndrome (FXS) — the most common inherited form of intellectual disability and autism —which is caused by alterations in FMR1 , the gene coding for FMRP. Studies in Fmr1 knockout mice, which have been widely used as an animal model for FXS, showed that inhibition of the metabotropic glutamate receptor 5 (mGlu 5 ) improved synaptic function in these animals and highlighted the importance of mGlu 5 in FXS 37 . However, mGlu 5 negative allosteric modulators such as basimglurant, mavoglurant and STX107, failed in phase II trials, as no improvement over placebo could be demonstrated. Agents targeting the GABA B receptor, which improved function in Fmr1 knockout mice, have also been investigated clinically for FXS. However, despite some indications of efficacy in a phase II trial with the GABA B agonist arbaclofen 50 , subsequent phase III trials failed due to lack of efficacy compared with placebo. The growing market share of metabolic disease medications 51 is reflected in the high number of GPCR-targeted agents in clinical trials for diabetes and obesity, with 27 and seven agents targeting GPCRs, respectively (together making ~10% of the total number of agents in clinical trials). 415 million people are estimated to suffer from diabetes mellitus 52 , with 90% of these having been diagnosed with type 2 diabetes. In contrast to type 1 diabetes, which requires regular insulin injections due to failure of the pancreas to produce enough insulin, type 2 diabetes can be treated with medications that stimulate insulin secretion or increase insulin sensitivity 53 . The first GPCR-targeted drug for type 2 diabetes — the GLP1 receptor agonist (or incretin mimetic) exenatide — was approved in 2005. As noted above, there are now a number of other approved peptidic GLP1 receptor agonists, including liraglutide, lixisenatide, dulaglutide and albiglutide. They differ in their durations of action, but are all formulated as injectable drugs. However, semaglutide, a once-weekly GLP1 analogue with significantly improved glycemic control 54 is now also being tested for oral dosing in phase III trials. Advances in small-molecule screening, including structure-based techniques 9 , have identified new non-peptide agonists of the glucagon-like peptide (GLP-1) receptor, including the orally bioavailable TTP273, which is currently in phase II trials. The challenges of treating type 2 diabetes and associated diseases such as diabetic neuropathy and foot ulcers has catalysed investment in further GPCR-targeted agents 32 , 55 .At present, eleven GPCRs mediate the therapeutic effects of approved treatments for these conditions, and agents targeting a further 25 GPCRs are under investigation in clinical trials. Among them is MBX-2982, a small-molecule GPR119 agonist currently in phase II trials. MBX-2982 increases both insulin secretion and GLP1 release 56 , 57 . Another novel target for which agonists stimulate insulin secretion is the FFA1 receptor (also known as GPR40) 34 . Despite the recently discontinued development of the small-molecule agonist fasiglifam (TAK-875) due to hepatotoxicity mentioned already above 29 , the FFA1 receptor remains a viable target, albeit with a need for further characterization of its signalling spectrum, and at least one FFA1 receptor modulator, LY2881835, is in clinical trials. Another novel target for treatment of diabetes is the dopamine D 2 receptor. The first dopaminergic agent, bromocriptine, was recently approved for improved glycemic control and glucose tolerance in type 2 diabetes 58 . Currently, 21 approved drugs with an antineoplastic indication mediate their effect via 15 distinct GPCRs. Among those are degarelix, a gonadotropin-releasing hormone (GnRH) receptor antagonist that is approved for patients with advanced prostate cancer, and vismodegib, a smoothened (SMO) receptor inhibitor for the treatment of basal-cell carcinoma. The most recent FDA approval for a GPCR-targeted agent in oncology was for another SMO receptor inhibitor, sonidegib in 2015, also for the treatment of basal cell carcinoma. An additional 23 GPCR-targeted agents for treating cancer — seven of which have potentially novel targets — are in clinical trials. Chemokine receptors and proteins in the Wnt pathway are among the novel GPCR targets being pursued 13 , often with peptide or mAb therapeutics. For example, CCR2 is the target of the mAb plozalizumab, which is in phase I trials for melanoma, and the small-molecule CCR2 inhibitor CCX872 is in phase I trials for advanced pancreatic cancer. Vantictumab (also known as OMP-18R5) — a mAb specific for the frizzled-7 receptor (FZD7) that has been tested in clinical trials for breast and pancreatic cancer — targets the Wnt signalling pathway, which is dysregulated in many cancers, leading to cancer stem cell activity and tumour growth 59 . Other biologics in clinical development with targets in the Wnt signalling pathway include ipafricept (a fusion protein targeting FZD8), OTSA-101-DTPA- 90 Y (a radiolabelled mAb targeting FZD10) and BNC-101 (a mAb targeting LGR 5 ). Furthermore, several other GPCRs have been suggested as potential cancer targets based on mRNA-expression analyses of tumours 60 . Besides targeting GPCR-mediated cancer pathways directly, over-expressed receptor homo- and heterodimers that are over-expressed in particular cancers might function as selective markers for cancer treatment 61 . Drugs for established GPCR targets in oncology also continue to be investigated, including GnRH antagonists such as relugolix for prostate cancer. GnRH antagonists such as relugolix and elagolix are also being investigated for other hormone-related indications such as endometriosis. Repurposing of existing drugs for new indications can reduce the time and cost to bring a therapeutic to market 62 . Of the approved GPCR-targeted drugs, 160 (33%) have more than one indication and the overall average is 1.5 indications ( FIG. 2d ), demonstrating that many such drugs are already used for several indications. Agents in clinical trials have a similar average number of indications as the approved drugs. Ongoing clinical trials are evaluating the potential to repurpose 51 (11%) of the approved drugs, which account for ~8%, ~7% and ~14% of the agents in Phases I, II and III, respectively ( FIG. 2e ). Repurposed agents are as frequent in the ongoing and discontinued trials indicating that they do not necessarily succeed more often than new agents. This suggests that efficacy is typically the limiting factor, rather than safety.

GPCR drug discovery has gained new momentum, as demonstrated by the high number of new drug targets and the scientific impetus in GPCR structural biology, pharmacology and modelling. Although it is to be expected that many of the GPCR-targeted agents currently in clinical trials will not ultimately gain regulatory approval, the demonstrated druggability of the GPCR protein family and the important role of GPCRs in diseases such as diabetes, obesity, AD and psychiatric disorders, provide a strong driving force for continued drug discovery and development efforts in this field. As the (patho)physiology of GPCRs becomes better characterised, certain groups of receptors may prove intractable, whereas others expand the druggable GPCRome. There is emerging evidence of excreted gut microbiota metabolites that serve as ligands for GPCRs and thereby influence our hormone release 91 . Additionally, many GPCRs have been identified as nutrient sensors, which could serve as potential targets to treat metabolic dysfunction and inflammatory diseases 92 . Furthermore, several orphan receptors may have evolved to recognise pathogens and invoke appropriate immune responses 93 . Characterization of the remaining orphan receptors could reveal new targets for a multitude of indications. Strikingly, the number of orphan receptors could expand significantly, as many of the ~400 olfactory GPCRs are now known to be widely expressed throughout the body and to play important functions beyond the detection of odorants 94 . Olfactory receptors have also been detected as overexpressed in tumour cells 94 . However, some orphan receptors have been shown to not follow the classical paradigm of endogenous activation by an (external) ligand. GPR50 heterodimerizes constitutively and specifically with melatonin receptors MT 1 and MT 2 . The class C orphan receptors GPR156 and GPRC5A-D lack the N-terminus, which contains the orthosteric binding site for the liganded members of this class. Furthermore, the non-orphan protease-activated and adhesion receptors are activated by their own N-terminus upon truncation. These receptors could still be targeted by drugs with a specific mechanism-of-action, such as allosteric modulation, inhibitors interfering with natural protein-protein interactions and antibodies, which are currently used to target e.g. adhesion receptors for antineoplastic treatment 95 . Moving forward in GPCR drug discovery depends on solving several critical issues. Suitable tool compounds are required to establish target biological functions and disease relevance 96 . This could be aided by novel high-throughput ligand identification methods able to probe a larger chemical space, such as DNA-encoded libraries 97 . Furthermore, improved disease models and genetically engineered systems are needed for unambiguous target validation, which could be aided by gene editing technologies such as CRISPR 98 . The huge and heterogeneous published and patented chemical and biological data needs to be made available and structured in long-term committing databases such as ChEMBL, Guide to PHARMACOLOGY, GPCRdb and others. These endeavours are too large for any single entity to pursue, and will have to engage the wider interdisciplinary basic and applied GPCR field, calling for a continued investment in public-private pre-competitive partnerships and consortia to accelerate progress.

More than half of the human genome-encoded non-olfactory GPCRs (n = 227) remain therapeutically unexploited ( FIG. 1a ). Drug targets are often first identified and explored in an academic environment. However, due to the long timespan of drug development, there is usually a lag time of many years or even decades before such discoveries are translated into marketed drugs. For this reason, we wanted to investigate how research results in the form of publication output relate to drug discovery efforts, and if this output could give indications of new trends in GPCR-targeted drug discovery. Gene and protein name searches in PubMed abstracts shows that the chemokine receptor type 4 (CXCR4) and the putative adhesion G protein-coupled receptor E4P (AGRE4) have the most publications (11,123) and fewest publications (0, although this may be in part due to new nomenclature for adhesion receptors), respectively ( FIG. 4a , note logarithmic scale). GPCRs with approved drugs or agents in clinical trials have on average ~400 publications, whereas this figure is only 20 for non-targeted receptors. Receptors with a crystal structure ( FIG. 4b ) also have many publications (this probably actually reflects that crystallization efforts in the past decade or so focused initially on therapeutically important and well-characterized GPCRs). Orphan receptors (purple in FIG. 4a ), with unknown physiological agonists and functions, typically have very few publications. Nonetheless, some orphan receptors, such as GPR143 and GPR15 have over 100 articles. The disproportionate data foundation underlines that the research efforts on many GPCRs are still in their infancy, and further characterisation is needed to assess their role in (patho)physiology. This is in accordance with a general knowledge deficit and lack of funding for many understudied human proteins, as recently brought to attention by an NIH programme “Illuminating the Druggable Genome”. About 63% of the human GPCRs have at least one ligand reported in ChEMBL ( FIG. 4c ), including various promising emerging targets for which agents have not yet been explored in clinical trials. For example, the neuropeptide S system, which was described only a few years ago, has been associated with several mental illnesses 63 , and the relaxin family receptors have recently been examined as potential targets for the treatment of addiction, anxiety, obesity and anorexia 64 . With a range of specific antagonists at hand, modulation of formyl peptide receptors has been suggested to inhibit tumour angiogenesis in glioblastoma and other cancers 65 . Bombesin 3 receptor knockout mice develop metabolic disturbances, obesity and hypertension 66 , 67 . Disruption of galanin receptor signalling in a number of preclinical studies have indicated that this system is involved in a range of pathologies, including Alzheimer’s disease, epilepsy, depression and cancer 68 , 69 . The aforementioned target families are expected to be among the first to enter clinical trials in the coming years due to their promising preclinical findings. To further evaluate potentially active pharmaceutical investigations on promising new targets, we analysed patents filed between 2014 and October 2016 in medical sciences through the World Intellectual Property Organization (WIPO) ( FIG. 4d ). Based on those filings, chemokine receptors, with more than 100 filed patents, are currently the most vigorously pursued target family, followed by calcium-sensing receptors (n = 11), glycoprotein hormone receptors (n = 9) and frizzled receptors (n = 9). In addition, 35 patents towards orphan receptors including GPR84, GPR1, GPR17 and LGR5 have also been filed within this timeframe. The diverse nature of GPCRs is reflected by our analysis of target disease associations from Open Targets ( FIG. 5 ), which shows that GPCRs cover nearly every aspect of human pathophysiology. Strikingly, emerging targets — those that have not yet been targeted and those for which agents are in trials but none have yet been approved, are found in 18 and 19 of the 20 displayed categories, respectively. Of note, most targets in trials have already reached phase III (dark green) indicating that they may soon transition to established targets. The largest shares of non-targeted receptors are found within genetic disorders and immune system diseases (both 27%), and when including targets in trials, more than one third (37% and 36%, respectively) are non-established targets. Furthermore, eye and skin disorders also show a high prevalence (both 27%) of new and unexploited targets. Notably, immunology is a major growth area with a high demand for new targets. Hence, these disease categories may be where we will see the largest proportions of novel targets in the near to intermediate future. The exploitation of the emerging targets will be facilitated by new avenues in GPCR drug discovery, such as the application of structural data, biased signalling, allosteric modulation, de-orphanization, which are described in the following sections. Structure-based drug design has long had a valuable role in drug discovery, particularly for drugs with enzyme targets, such as the HIV protease inhibitor indinavir 70 , the tyrosine kinase inhibitor imatinib 71 and the influenza neuraminidase inhibitor zanamivir 72 . However, until recently, major challenges in applying X-ray crystallography to GPCRs limited the potential for structure-based drug design for such targets. Now, however, thanks to recent breakthroughs in GPCR crystallography 73 , 74 , 44 distinct GPCR structures and 196 ligand–receptor complexes are available across all human GPCR classes A–C and F ( FIG. 6 ) 21 . This has paved the way for novel lead discovery through virtual screening and better off-target rationalisation 75 , 76 . For example, recent docking experiments against the μ opioid receptor structure identified PZM21, a G i protein-biased agonist with potency and efficacy similar to morphine, but with reduced adverse effects in mice 77 . In another exciting example, two chemokine receptor structures —for CCR9 and CCR2 — revealed a previously unknown intracellular binding pocket, which might provide a new strategy for drug design 78 . We investigated if the availability of crystal structures has yet had a measureable impact on the number of agents in clinical trials. FIG. 6a shows that most of the receptors with many approved drugs (red bars) have an early crystal structure published in 2007-2012, with exception of the muscarinic M 1 receptor (ACM1) from 2016. It is likely that these receptors were selected for crystallisation based on their high therapeutic relevance, such as many and early validated disease associations. However, these targets also have a significant proportion of agents in phase I and II trials, where the structural templates could have been available long enough to contribute to the lead discovery and/or optimisation processes. The real impact of structural data may be underestimated due to unpublished proprietary structures. For example, Receptos announced in January 2011 that they were targeting the S1P 1 receptor with a proprietary structure, which was not published until the following year. Receptos’ S1P 1 agonist ozanimod (which is now being developed by Celgene following their acquisition of Receptos) is expected to be submitted for FDA approval in 2017. Furthermore, Heptares’ pipeline (see Further information ) currently lists ten specified, and an even larger number of undisclosed, targets for which agents are in preclinical and clinical trials. Known structure-based candidates that target GPCRs include agents that target the μ opioid receptor for pain 77 , M 1 /M 4 -muscarinic receptors for AD, metabotropic glutamate receptor 5 (mGlu 5 ) for psychiatric disorders, orexin receptor 2 (HCRTR2) for narcolepsy, protease-activated receptor 2 (PAR2) for inflammatory disorders (see Heptares pipeline in Further information ) and the adenosine A 2A receptor for cancer 79 . Activating the appropriate cellular response through one of the four major G α families and their intracellular effectors (such as adenylate cyclase and phospholipase C) or by β-arrestin-dependent activation of kinases and others, is critical for a favourable physiological response 80 – 82 . Recent discoveries of molecules that preferentially trigger one of these pathways — referred to as biased agonists — offer a new mechanism for reducing side-effects 8 , 20 , 83 , 84 . This has led to the notion of distinct receptor conformational states that are stabilized by different ligands, and can lead to activation of specific signalling and regulatory proteins 85 . Several promising agents with bias through β-arrestin, a G protein or allosteric modulation are being investigated in preclinical studies as well as clinical trials 8 . Most notable is the µ-opioid receptor ligand oliceridine (TRV130), currently in phase III trials, which was granted ‘Breakthrough Therapy’ designation by the FDA owing to its improved analgesic profile 86 . Oliceridine, and its current phase I follow-on agent TRV734, do not engage the β-arrestin pathway, which is associated with opioid-induced respiratory depression and constipation 87 . Conversely, no G protein engagement was observed for the β-arrestin-biased ligand TRV027, which targets the angiotensin II type 1 receptor and is a drug candidate for the treatment of acute heart failure 88 . However, in May 2016, Trevena announced that TRV027 failed to meet the primary or secondary endpoints in a phase IIb trial. Therapeutic implications for biased agonists have just started to be elucidated for a number of GPCR systems including adrenergic, angiotensin, opioid, dopamine, serotonin and chemokine receptors 7 . More knowledge on the signalling repertoire of GPCRs will be required to fully exploit the potential of biased signalling and the fine-tuning of intracellular responses to therapy 89 . New ways to validate biased signalling in animal models will help development of the tools needed to transition biased ligands towards preclinical development as drug candidates and beyond 90 .