GPR39, also known as ZnR (zinc sensing receptor), is a member of a large family A of 7-transmembrane (7-TM) containing G protein-coupled receptors (GPCRs).
GPR39, also known as ZnR, is a member of a large family A of 7-transmembrane (7-TM) containing G protein-coupled receptors (GPCRs) [1]. GPR39 is found in all vertebrates and was cloned together with GPR38 as structural homologues to the ghrelin receptor from human fetal brain in 1997 [2] and belongs to the ghrelin receptor subfamily with six of its most closely related receptors [3]. The other members of this family are well-established regulators of metabolism through their ligands: the peptide hormones and neuropeptide regulators ghrelin, motilin, neurotensin and neuromedin U [3]. Conversely, no endogenous peptide ligand has been discovered for GPR39 [4]. Coincidentally, the existence of an ionic zinc (Zn2+) -sensing receptor was indicated in 2001, when extracellular Zn2+ was found to activate intracellular Ca2+ signaling; the receptor then named the Zn2+-sensing receptor (ZnR) and now known to be GPR39 [5]. Indeed, physiological concentrations of Zn2+ were shortly after shown to activate GPR39 [6,7,8].
Zinc is essential for human health and zinc deficiency is recognized as a world-wide malnutrition problem, affecting 17% of the global population [9]. All cell types require Zn2+ for various catalytic and regulatory functions and around 3000 proteins are estimated to have binding sites for Zn2+ [9,10]. Consequently, zinc deficiency affects the functions of multiple tissues and cell signaling pathways; for example, zinc plays a role in immune responses, DNA replication and repair, oxidative stress response, aging, cell cycle progression, homeostasis and apoptosis [9,11,12,13]. Extracellular Zn2+ concentration can increase in many circumstances, such as when Zn2+ is co-released from vesicles with insulin in the pancreatic β-cells or with glutamate in the nerve synapses, or in connection with cell death and injury, which then triggers GPR39-dependent signaling [14,15,16].
Initially, obestatin, a peptide encoded by the ghrelin gene, was suggested to be an endogenous ligand for GPR39, reducing food intake and gastric motility [23]. However, it was later confirmed that obestatin does not activate GPR39 [4,6].
Only a small number of synthetic ligands for GPR39 have been characterized. The best characterized and most widely published agonist to date is TC-G 1008 (originally known as GPR39-C3), but since its discovery, other potent agonists have been characterized. Since the first GPR39 agonist was discovered in 2013 [59] and shortly after in 2014 TC-G 1008, which quickly became commercially available [60], the number of studies focusing on GPR39 agonists has been steadily increasing in recent years. This has permitted the characterization of the physiological functions of GPR39 more specifically, compared to the GPR39-deficient or overexpressing mice, where global systemic compensatory mechanisms may play a role.
The first GPR39 agonist was a piperazine derivative, identified by a screening of GPCR-focused libraries against GPR39 [59]. The agonist was able to activate GPR39 and to induce the intracellular Ca2+ response, but turned out to be only moderately potent and not suitable for in vivo studies [59]. Soon after, in 2014, another research group identified 2-pyridylpyrimidines as potential GPR39 agonists via a reporter gene assay designed for identifying inhibitors of Hedgehog signaling, and then optimized them to improve efficiency and pharmacokinetics, which yielded TC-G 1008 [60]. It is a highly potent and selective GPR39 agonist with EC50 values of <1 nM, and orally bioavailable. When TC-G 1008 treatment was combined with Zn2+, more InsP was generated than with either substance alone, indicating that TC-G 1008 does not compete for binding with Zn2+, but the presence of Zn2+ further potentiates GPR39 activation by TC-G 1008. Originally, TC-G 1008 was also found to robustly induce glucagon-like peptide-1 (GLP-1) levels when orally administered to mice [60]. Later, it has been shown to activate all GPR39 inducible pathways [35]. Since its discovery, TC-G 1008 has proven to be a potent agonist and a very valuable tool in characterizing the physiological functions and therapeutic potential of GPR39 activation, published by a number of studies to date [30,31,32,35,61,62,63,64,65,66,67,68,69,70,71,72]. However, most studies have not verified their findings in GPR39−/− settings, which would be critical in order to confirm that the observed effects are indeed GPR39-dependent.
Additionally, in 2014, a series of cyclohexyl-methyl aminopyrimidine chemotype compounds (CMAPs) activating GPR39 was identified co-incidentally, while similarly screening for compounds that could inhibit Hedgehog signaling [73]. The identified compounds, which inhibited Hedgehog signaling, were also found to increase InsP production, indicating GPCR involvement. GPCR mRNA expression levels were correlated with the compound activity in different cell lines, which indicated the target of the compound to be GPR39, later verified with GPR39-deficient cells. The EC50 of the most potent compound CMAP 7 was 20 nM, thus the compound was not as potent as TC-G 1008. Importantly, this study showed that GPR39 activation interferes with Hedgehog signaling, which plays important roles in tumorigenesis and determines cell fate during development [73]. Shortly after, in 2015, three novel GPR39 agonists were described, AZ7914, AZ4237 and AZ1395 [74]. They were identified by a high throughput screen of the AstraZeneca compound library and were verified by a limited medicinal chemistry program. These compounds were also found to be more potent in the presence of Zn2+ and to interact directly with Zn2+. In a physiological characterization, these agonists were not able to improve glucose tolerance in lean or obese mice or in Zucker fatty rats. All three compounds had higher EC50 values than TC-G 1008, thus being less potent [74].
In 2016, two previously existing kinase inhibitors, the Janus kinase (JAK) inhibitor LY2784544 and the PI3Kβ inhibitor GSK2636771, were identified as novel GPR39 agonists by unbiased small-molecule-based screening of multiple compound libraries using a modified β-arrestin recruitment assay [65]. Their signaling patterns were compared to those of TC-G 1008, and GPR39 activation by all three compounds was shown to be allosterically modulated by physiological concentrations of Zn2+. As with TC-G 1008, the presence of Zn2+ increased the potency of LY2784544 and GSK2636771 to activate GPR39 and downstream signaling. Although LY2784544 and GSK2636771 were suggested to be more potent in activating GPR39 than inhibiting JAK2 and PI3Kβ, respectively, to date, these compounds have not been utilized in other GPR39 studies, likely due to not being exclusively selective for GPR39 [65]. However, one study used LY2784544 as a control for TC-G 1008 and found that both compounds similarly suppressed lipopolysaccharide (LPS) –induced interleukin 6 (IL6) production in macrophages [62]. Furthermore, as both compounds are being tested in clinical trials, their potential to activate GPR39 would be of great interest to study, as GPR39 activation might induce adverse effects or be partly responsible for the positive outcomes. Currently, according to www.clinicaltrials.gov, LY2784544 is being tested in myeloproliferative disorders and GSK2636771 in various cancers, most focusing on cancers with genetic changes of phosphatase and tensin homolog (PTEN).
Another novel set of GPR39 agonists were discovered in 2017 with the help of a homology model-based approach [75]. This model took advantage of a suggested binding site for synthetic ligands in GPR39 and commercially available libraries of synthetic compounds. Interestingly, some of the most potent identified compounds turned out to be inactive alone, but highly potent and selective in the presence of Zn2+ as an allosteric enhancer. Mutational mapping indicated that the synthetic agonists are able to bind to the main ligand binding pocket of GPR39, whereas Zn2+ seems to bind differently when acting as the sole agonist as compared to acting as an allosteric modulator. The selected agonist, TM-N1324 (compound 8 in [75]), had an EC50 of 280 nM and 9 nM in the absence and presence of Zn2+, respectively, thus being potent even in the absence of Zn2+. TM-N1324 could be orally administered and resulted in micromolar plasma concentrations, adequate for maximal activation of GPR39. With the help of this agonist, GPR39 was identified as an important regulator of gastric somatostatin secretion, increasing somatostatin secretion and correspondingly decreasing ghrelin secretion in primary gastric mucosal cells; an effect which was eliminated in GPR39−/− cells [75,76].
The first biased ligand, i.e., a ligand favoring selected signaling pathways over others, for GPR39 was discovered in 2017 [35]. GSB-118 was found through high-throughput screening of a compound library and by utilizing cAMP assays in the presence of Zn2+. It showed functional selectivity by activating the Gαs pathway, resulting in cAMP responses and β-arrestin recruitment, but did not activate the Gαq or Gα12/13 pathways, result in InsP accumulation or Ca2+ response, or desensitize GPR39. GSB-118 did not show any independent agonist activity but acted as a positive allosteric modulator for Zn2+, potentiating its responses [35]. The lack of desensitization observed here could attenuate drug tolerance and therefore, enhance responses to treatment. Development of more biased agonists targeting certain pathways more specifically rather than activating all GPR39-dependent signaling would allow for more targeted therapies and might be a key for future applications of GPR39 agonism for therapeutic purposes. More knowledge about the physiological functions of GPR39 and their molecular mechanisms in each disease is needed, before the development of signaling pathway-targeted therapies is meaningful. However, all identified synthetic ligands have not been fully characterized regarding signal transduction and might include some biased agonists. Despite being highly potent, the majority of these novel agonists presented here are more or less Zn2+-dependent, and thus it remains to be seen if they are sufficiently functional in tissues with lower physiological Zn2+ concentrations, and importantly, if they have therapeutic potential in conditions linked to Zn2+ deficiency. It is also still an open question whether at least some of the synthetic ligands simply function as allosteric activators of Zn2+ and not vice versa, as was suggested for GSB-118 [35]. Additionally, the GPR39-specificity of most ligands in physiological conditions will still need to be determined in GPR39−/− cell and tissue models.
This entry is adapted from the peer-reviewed paper 10.3390/ijms22083872