Molecular pharmacology of GPCRs

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This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc

This themed section of the British Journal of Pharmacology stems from the ninth in the series of MPGPCR meetings, held as a joint initiative with the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists in Melbourne, Australia, from 28 to 30 November 2016. The articles provide insights into how GPCR environment can influence function, the identification of new drug targets based on allostery, biased agonism and oligomerization and the importance of taking into account sexual dimorphism in developing animal models for the evaluation of potential treatments for cardiovascular disease. In addition, novel approaches are presented for the treatment of metabolic disease, disturbances of calcium homeostasis and migraine.

GPCRs are amongst the most commonly examined targets for drug development. As outlined in the article by Desai and Miller (2018), the early stages of drug development often involve the use of recombinant systems that may assume that the cellular environment of the receptor has little influence on the cellular response. While much work has studied the influence of agonists, antagonists, G proteins and other receptors present in the plasma membrane on receptor conformation, it is now becoming increasingly clear that other membrane proteins and lipids also influence receptor function (Prieto et al., 1990). Phospholipids, sphingolipids, glycolipids and sterols, in particular cholesterol, influence membrane fluidity and GPCR function. In addition, it is clear that lipid rafts rich in these components form membrane microdomains that concentrate signalling and regulatory proteins (Pike, 2003; Ray et al., 2016). There are now many examples demonstrating the influence of cholesterol on GPCR function and regulation (see Desai and Miller, 2018). The lipid composition of the cell membrane also influences the distribution and function of the primary GPCR effectors, G proteins. The molecular and cellular studies that establish the importance of plasma membrane organization on GPCR function and regulation inform clinical studies of factors such as diet and disease pathology on drug sensitivity. The authors conclude with a specific example of the importance of membrane environment in the determination of GPCR function. The http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=76 has an important role in appetite control and is sensitive to membrane cholesterol content (Desai and Miller 2012) as, in patients with a high incidence of obesity and metabolic disease, reduced CCK responsiveness was observed and correlated with high cholesterol levels. Although other factors are also involved, these observations may contribute to the poor outcomes of clinical trials involving CCK₁ receptor agonists.

The ‘textbook’ view of GPCR signalling describes the interaction of a hormone/neurotransmitter with a receptor located at the cell surface followed by signal transduction and amplification to produce second messengers that alter cellular function. However, as reviewed by Jong et al. (2018), it is apparent that many GPCRs are localized to intracellular cell membranes where they may be associated with different signalling systems and regulation. GPCRs are found not only in the endoplasmic reticulum (ER) where they are synthesized, folded, modified and assembled but also in nuclear membranes, vesicles, mitochondria and nucleoplasm (Jong et al., 2018). Receptors can achieve nuclear localization by a number of mechanisms, and some GPCRs possess nuclear localization signals to facilitate this. GPCRs such as the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=348 are synthesized conventionally in the ER, translocate to the cell surface to be activated, internalized but then are transported to the nucleus. Others, such as the http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=4, are localized at the nucleus and are activated by ligands transported through the plasma membrane by the http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=196#1021 (Wu and O'Connell, 2015). Alternatively, nuclear receptors can be accessed by ligands that are cell permeable or produced intracellularly. In addition, the nucleus or nuclear membranes contain a repertoire of G proteins and second messenger synthesizing and inactivating proteins (Campden et al., 2015), although the physiological effects brought about by activation of the same receptor in different locations may differ. For instance, blockade of nuclear http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=293 inhibits pain, whereas blockade of cell surface receptors has no effect. This stresses the importance of development of ligands that can target receptors in specific locations, another approach to the development of biased ligands.

There is now great interest in exploiting allostery, oligomerization and biased agonism as paradigms for novel drug discovery. The interest stems from the potential to activate signal transduction pathways necessary for mediating the therapeutic effect while exerting minimal on‐target adverse effects. The article on new paradigms in adenosine receptor pharmacology by Vecchio et al. (2018) examines how these concepts necessitate modifications to the classical ternary complex model of signal transduction. The formation of oligomers between receptors offers opportunities for drug discovery (Pin et al., 2007). While homomers and heteromers have been described for adenosine receptors, the physiological importance of these interactions has yet to be demonstrated in most cases. On the other hand, considerable progress has been made with allosteric ligands that recognize a site that is topographically distinct yet conformationally linked to the orthosteric binding site (May et al., 2007). There are allosteric ligands that act at all four adenosine receptors with most information available for the adenosine http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=18 including molecular modelling, mutagenesis, pharmacological and crystal structure studies (Glukhova et al., 2017). Drugs acting at allosteric sites offer the potential of greater selectivity, limiting maximal effect and probe dependence. A particularly interesting example is the development of bitopic ligands such as VCP746, which combines an A₁ receptor positive allosteric modulator with adenosine via an aromatic linker and six carbon alkyl linker (Valant et al., 2014). This compound also displays biased agonism and produces a comparatively weak Ca²⁺ mobilization response compared with other G‐protein‐linked pathways. This enables VCP746 to display cardioprotective actions without the typical A₁ receptor ‐mediated bradycardia (Valant et al., 2014). The studies provide interesting examples of how the new paradigms of drug action can influence the safety profile, specificity and translational success of ligands acting at adenosine receptors.

The review by Mouat et al. (2018) focuses on sexual dimorphism and its influence on cardiovascular pathology and physiology. They focus on the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=221, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=219 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=220 endothelin receptors and eicosanoid GPCRs as examples of systems that display sexual dimorphic responses. GPER is clearly important for the vasodilatory effects of oestrogens in the vasculature (Lindsey et al., 2011) with the receptor being located on both endothelial cells and smooth muscle cells. Vasodilation is mediated both by release of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2509 from the endothelial cells and by generation of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2352 in vascular smooth muscle cells. Somewhat surprisingly, although GPER expression seems to be up‐regulated by oestrogens, GPER protein levels are similar in the heart of both sexes, and there is little evidence suggesting that there are sex differences in the effects of the receptor. GPER also seems to be involved in down‐regulation of angiotensin http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=348 that may contribute to their cardioprotective effects. GPER also inhibits vasoconstrictor responses to http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=989, and ET‐1 levels are generally lower in women. In addition, the G_q/11‐coupled ET_A receptor that is largely responsible for the vasoconstrictor actions of ET‐1 is down‐regulated by female sex hormones, although the differential expression of ET_A/ET_B receptors is retained in postmenopausal women, suggesting that oestrogen is not the only player. GPER also influence the biosynthesis of prostanoids (Meyer et al., 2015), which in turn affects responses of prostanoid receptors. http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4482 is a potent activator of platelet aggregation, vasoconstriction and vascular smooth muscle cell proliferation acting through http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=346 that are up‐regulated by male sex hormones. These effects are antagonized by http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1915 acting on http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=345, which are also up‐regulated by sex hormones. The authors make a strong case for animal studies being conducted in both sexes to take account of sex‐dependent differences in cardiovascular physiology.

Although GPCRs are generally regarded as important targets for drug discovery, only recently have successful therapeutic agents been developed for the treatment of diabetes and obesity (Sloop et al., 2018). Some of these attempts, such as the development of agonists at the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=30, were frustrated by the differences in physiology between animal models and humans (Larsen et al., 2002). More recently, clinical trials involving the lysophospholipid receptor http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=126 and the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=225 have failed due to lack of efficacy or toxicity (Mancini and Poitout, 2015). However, the successful introduction of agonists active at the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=249 that improve glycaemic control and reduce body weight is a major advance. Much of this stems from a good understanding of the physiology of the incretins, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3542 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5194 (Drucker, 2006) and GLP‐1 receptor agonists, including http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1135 and GLP‐1 analogues. Other approaches have included bariatric surgery that increases gastrointestinal release of GLP‐1 as well as targeting receptors on intestinal L cells that control release of the peptide hormone. The article concludes by posing a number of key questions that need to be considered by groups wishing to pursue drug discovery targeting GPCRs for the treatment of metabolic disease.

The sometimes long journey of a GPCR, which appears on the basis of biology to be an attractive target, through to an effective therapeutic agent in the clinic is exemplified by the review by Dehvari et al. (2018). Selective β₃‐adrenoceptor agonists such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=567 were shown to be very effective at increasing adipose tissue lipolysis while having little effect on atrial contraction or tracheal/uterine relaxation in rodents (Arch et al., 1984). However, the first generation compounds proved disappointing in humans and had low efficacy. Subsequently, compounds were developed with high efficacy at the human β₃‐adrenoceptor, and these have found a niche for the treatment of overactive bladder (Yamaguchi and Chapple, 2007). This reintroduction of β₃‐adrenoceptor agonists into the clinic has re‐awakened interest in a potential treatment for metabolic disorders particularly since the identification of active brown adipose tissue in humans (Nedergaard et al., 2007). A recent study showed that the β₃‐adrenoceptor agonist http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7445 increased metabolic rate and adipose tissue glucose uptake in human subjects, although the high doses used caused adverse cardiac side effects (Cypess et al., 2015). Further clinical trials are in progress. The β₃‐adrenoceptor may also be a target for the treatment of cardiac failure although mirabegron failed to reach the primary endpoint in this indication. However, in a subset of patients with a left ventricular ejection fraction <40%, there was improvement, encouraging another clinical trial over a longer period. Interpretation of studies with mirabegron is complicated by actions on other receptors, transporters and liver enzymes even though it seems effective in treating lower urinary tract symptoms. Old target it may be, but the β₃‐adrenoceptor would appear to have clinical potential.

Several reviews reflect the interest in the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=54 as a target for the treatment of defects of calcium homeostasis. In the first by Hannan et al. (2018), the authors begin by highlighting the disorders associated with mutations of the CaS receptor and partner proteins and discuss treatment options. In addition to naturally occurring ligands, there are now positive and negative allosteric modulators of CaS receptor function. Allosteric calcimimetics mimic or enhance the effect of Ca²⁺ at the CaS receptor, and examples such as http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3308 have found a role in the treatment of types of hyperparathyroidism (Hannan et al., 2018). A number of negative allosteric modulators have also been developed, but clinical trials of their potential use for the treatment of osteoporosis have been disappointing (Fitzpatrick et al., 2011). Studies of the allosteric binding sites identified by calcimimetics and calcilytics demonstrate similar but not identical sites that may underpin biased signalling profiles observed with allosteric modulators of different chemical classes (Leach et al., 2016). The review goes on to describe key studies that demonstrate the effectiveness of calcimimetics and calcilytics in animal models and in the clinic. The studies bring together the evidence that supports the concept that calcimimetics and calcilytics acting allosterically provide a targeted approach to the treatment of diseases involving mutations of the CaS receptor, Gα₁₁ and AP2σ proteins. In diseases involving hypercalcaemia, calcimimetics lower serum Ca²⁺ and parathyroid concentrations in patients, whereas calcilytics improve hypocalcaemia and hypercalciuria in animal models.

The second article on Ca²⁺ metabolism contrasts the profile of strontium (Sr²⁺) ranelate, an approved treatment for osteoporosis, with three structurally distinct positive CaS receptor allosteric modulators (Diepenhorst et al., 2018). Sr²⁺ is effective in osteoporosis but is associated with an increased incidence of cardiac disorders and thromboembolic events that have led to its being withdrawn from the market. This approach has been compared with the use of calcimimetics in combination with Ca²⁺ to determine whether the biased signalling profile displayed by the different chemical classes of modulators could be utilized for the treatment of osteoporosis. All three classes of calcimimetics potentiated Sr²⁺‐mediated activation of the CaS receptor in a similar manner to Ca²⁺, but the effects did not translate for two of the compounds into inhibition of human osteoclast maturation and function. In contrast, cinacalcet did inhibit osteoclast function, perhaps by allosteric modulation of the CaS receptor or possibly by hitherto unappreciated off‐target actions.

Finally, the role of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2258 in migraine has been examined by Sundrum and Walker (2018). They stress that the heterogeneity of migraine pathophysiology makes it unlikely that a single approach will benefit all patients. PACAP appears to be one of the players in migraine pathogenesis and is located in several sites in the trigeminovascular system and has overlapping biology with the vasodilator http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=681. PACAP pharmacology is complex with the peptide activating three receptors, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=370, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=371 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=372, with additional complexity added by numerous PAC₁ splice variants and with the ability of VPAC₁ and VPAC₂ receptors to interact with RAMPS, resulting in altered ligand binding, regulation and trafficking (Christopoulos et al., 2003). The three PACAP receptors however display similar signalling profiles, and all couple to Gα_s and Gα_q proteins, although there is some evidence that different ligands may promote biased signalling (Walker et al., 2014). The evidence supports the view that PAC₁ receptors have a role in the pathogenesis of migraine, whereas activation of VPAC₁ and VPAC₂ receptors (while causing headache) does not precipitate migraine. Development of molecules that specifically target PAC₁ receptors will be essential in determining whether PACAP has an important role in migraine.

The editors hope that this themed section on GPCRs provides insights into the ways in which new approaches and paradigms are influencing the development of new treatments for a variety of diseases.

Conflict of interest

The authors wish to acknowledge a conflict of interest and highlight that they are co‐authors of the article by Diepenhorst et al. (2018), in this issue.

Langmead, C. J. , and Summers, R. J. (2018) Molecular pharmacology of GPCRs. British Journal of Pharmacology, 175: 4005–4008. 10.1111/bph.14474.