Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi,GRK2, and Arrestin3

Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G_i-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH₄Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca²⁺ mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.     

Differential tissue and ligand-dependent signaling of GPR109A receptor: Implications for anti-atherosclerotic therapeutic potential

Until recently, the anti-atherosclerotic effects of niacin were attributed primarily to its lipid modification properties mediated by adipocyte G-protein coupled receptor GPR109A, though recent studies have raised significant doubts about this mechanism. In fact, in rodents it has recently been demonstrated that niacin inhibits progression of atherosclerosis through actions on immune cells, particularly via macrophage-expressed GPR109A, independent of lipid-modifying properties. Here, we studied GPR109A signal transduction in human Langerhans cells, macrophages and adipocytes. We find that the consequences of receptor activation are profoundly influenced by cellular context and that ligand-biased signaling significantly impacts functionally relevant signaling. In Langerhans cells, niacin initiates GPR109A-mediated signaling pathways (Erk1/2 and Ca^2 +) responsible for the release of vasodilatory prostanoids, while the synthetic GPR109A agonist MK-0354 fails to elicit any signaling, providing a mechanistic basis for the latter compound's inability to cause flushing. While GPR109A mediates inhibition of cAMP in adipocytes, in macrophages GPR109A signaling via G_βγ subunits results in paradoxical augmentation of intracellular cAMP levels. Also, in macrophages niacin and GPR109A full agonists induce Erk1/2 and Ca^2 + signaling, release of prostanoids, upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulation of reverse cholesterol transport in GPR109A dependent manner. A mechanism is presented in which signals from the autocrine action of released prostanoids and G_i protein mediated cAMP augmentation are integrated leading to modulation of reverse cholesterol transport regulatory components. These studies provide key insights into mechanisms by which GPR109A may influence cholesterol efflux in macrophages; a process that may be at least partially responsible for niacin's anti-atherosclerotic activity. MK-0354 does not induce niacin-like GPR109A signaling in macrophages, suggesting that biased agonists devoid of the flushing side-effect may also lack properties required for macrophage-mediated anti-atherosclerotic effects.     

GPR109A alleviate mastitis and enhances the blood milk barrier by activating AMPK/Nrf2 and autophagy

Mastitis causes great psychological and physical pain among women. Our previous studies found that niacin has anti-inflammatory effect, and the realization of this function depends on GPR109A. However, there are no previous reports about the anti-inflammatory function of GPR109A in mastitis. In our study, we observed the effect of niacin on the WT and GPR109A^-/- mice mastitis model. The results showed that administration of niacin to WT mice reduced the damage, proinflammatory mediators and protected the integrity of the blood milk barrier in mammary gland. While in GPR109A^-/- mice, there was no effect on the above indexes. In mammary epithelial cells, GPR109A was able to promote autophagy and Nrf2 nuclear import through AMPK. In LPS-induced mammary epithelial cells, niacin inhibited the LPS-induced inflammatory response and downregulation of tight junction proteins, and these effects were eliminated by knocking down GPR109A, blocking autophagy or inhibiting Nrf2 nuclear import. These results indicate that in mastitis, GPR109A promotes autophagy and Nrf2 nuclear import through AMPK, thereby inhibiting inflammatory damage to the mammary gland and repairing the blood milk barrier. Our results suggested that GPR109A may be a potential target for the treatment of mastitis.     

Upregulation of GPR109A in Parkinson’s Disease

Background

Anecdotal animal and human studies have implicated the symptomatic and neuroprotective roles of niacin in Parkinson’s disease (PD). Niacin has a high affinity for GPR109A, an anti-inflammatory receptor. Niacin is also thought to be involved in the regulation of circadian rhythm. Here we evaluated the relationships among the receptor, niacin levels and EEG night-sleep in individuals with PD.

Methods and Findings

GPR109A expression (blood and brain), niacin index (NAD-NADP ratio) and cytokine markers (blood) were analyzed. Measures of night-sleep function (EEG) and perceived sleep quality (questionnaire) were assessed. We observed significant up-regulation of GPR109A expression in the blood as well as in the substantia nigra (SN) in the PD group compared to age-matched controls. Confocal microscopy demonstrated co-localization of GPR109A staining with microglia in PD SN. Pro and anti-inflammatory cytokines did not show significant differences between the groups; however IL1-β, IL-4 and IL-7 showed an upward trend in PD. Time to sleep (sleep latency), EEG REM and sleep efficiency were different between PD and age-matched controls. Niacin levels were lower in PD and were associated with increased frequency of experiencing body pain and decreased duration of deep sleep.

Conclusions

The findings of associations among the GPR109A receptor, niacin levels and night-sleep function in individuals with PD are novel. Further studies are needed to understand the pathophysiological mechanisms of action of niacin, GPR109A expression and their associations with night-sleep function. It would be also crucial to study GPR109A expression in neurons, astrocytes, and microglia in PD. A clinical trial to determine the symptomatic and/or neuroprotective effect of niacin supplementation is warranted.     

Fluorinated pyrazole acids are agonists of the high affinity niacin receptor GPR109a

A series of 5-alkyl pyrazole-3-carboxylic acids were prepared and found to act as potent and selective agonists of the human GPCR, GPR109a, the high affinity nicotinic acid receptor. No activity was observed at the highly homologous low affinity niacin receptor, GPR109b. A further series of 4-fluoro-5-alkyl pyrazole-3-carboxylic acids were shown to display similar potency. One example from the series was shown to have improved properties in vivo compared to niacin.     

Discovery of orally bioavailable and novel urea agonists of the high affinity niacin receptor GPR109A

A urea class of high affinity niacin receptor agonists was discovered. Compound 1a displayed good PK, better in vivo efficacy in reducing FFA in mouse than niacin, and no vasodilation in a mouse model. Compound 1q demonstrated equal affinity to GPR109A as niacin.     

Distinct Kinetic and Spatial Patterns of Protein Kinase C (PKC)- and Epidermal Growth Factor Receptor (EGFR)-dependent Activation of Extracellular Signal-regulated Kinases 1 and 2 by Human Nicotinic Acid Receptor GPR109A

Nicotinic acid (niacin) has been widely used as a lipid-lowering drug for several decades, and recently, orphan G protein-coupled receptor GPR109A has been identified as a receptor for niacin. Mechanistic investigations have shown that, upon niacin activation, GPR109A couples to a G_i protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for GPR109A signaling remain largely unknown. Using CHO-K1 cells stably expressing GPR109A and A431 cells, which are a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by niacin was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2 activation via the matrix metalloproteinase/epidermal growth factor receptor transactivation pathway at both early and later time points (2–5 min); this pathway was distinct from the PKC pathway-mediated ERK1/2 phosphorylation that occurs at early time points (≤2 min) in response to niacin. Overexpression of Gβγ subunit scavengers βARK1-CT and the Gα subunit of transducin led to a significant reduction of ERK1/2 phosphorylation, suggesting a critical role for βγ subunits in GPR109A-activated ERK1/2 phosphorylation. Using arrestin-2/3-specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2 and arrestin-3 were not involved in GPR109A-mediated ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to niacin GPR109A receptors initially activate G_i, leading to dissociation of the Gβγ subunit from activated G_i, and subsequently induce ERK1/2 activation via two distinct pathways, one PKC-dependent pathway occurring at a peak time of ≤2 min and the other matrix metalloproteinase-dependent growth factor receptor transactivation occurring at both early and later time points (2–5 min).     

3-Nitro-4-amino benzoic acids and 6-amino nicotinic acids are highly selective agonists of GPR109b

A series of 3-nitro-4-substituted-aminobenzoic acids were prepared and found to act as potent and highly selective agonists of the orphan human GPCR GPR109b, a low affinity receptor for niacin. No activity was observed at the closely homologous high affinity niacin receptor, GPR109a. A second series, comprising 6-amino-substituted nicotinic acids was, also prepared and several analogues showed comparable activity to the nitroaryl series.     

5-N,N-Disubstituted 5-aminopyrazole-3-carboxylic acids are highly potent agonists of GPR109b

A series of 5-N,N-disubstituted-5-aminopyrazole-3-carboxylic acids were prepared and found to act as highly potent and selective agonists of the G-Protein Coupled Receptor (GPCR) GPR109b, a low affinity receptor for niacin and some aromatic d-amino acids. Little activity was observed at the highly homologous higher affinity niacin receptor, GPR109a.     

Discovery of structurally novel,potent and orally efficacious GPR119 agonists

Screening hit 5 was identified in a biochemical screen for GPR119 agonists. Compound 5 was structurally novel, displayed modest biochemical activity and no oral exposure, but was structurally distinct from typical GPR119 agonist scaffolds. Systematic optimization led to compound 36 with significantly improved in vitro activity and oral exposure, to elevate GLP1 acutely in an in vivo mouse model at a dose of 10 mg/kg.     

Synthesis and structure–activity relationship of fused-pyrimidine derivatives as a series of novel GPR119 agonists

A series of fused-pyrimidine derivatives have been discovered as potent and orally active GPR119 agonists. A combination of the fused-pyrimidine structure and 4-chloro-2,5-difluorophenyl group provided the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-dioxide derivative 14a as a highly potent GPR119 agonist. Further optimization of the amino group at the 4-position in the pyrimidine ring led to the identification of 2-{1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}acetamide (16b) as an advanced analog. Compound 16b was found to have extremely potent agonistic activity and improved glucose tolerance at 0.1 mg/kg po in mice. We consider compound 16b and its analogs to have clear utility in exploring the practicality of GPR119 agonists as potential therapeutic agents for the treatment of type 2 diabetes mellitus.     

Design and biological evaluation of tetrahydropyridine derivatives as novel human GPR119 agonists

A series of novel tetrahydropyridine derivatives were prepared and evaluated using cell-based measurements. Systematic optimization of general structure G-1 led to the identification of compound 35 (EC₅₀ = 4.9 nM) and 37 (EC₅₀ = 8.8 nM) with high GPR119 agonism activity and moderate clog P. Through single and long-term pharmacodynamic experiments, we found that compound 35 showed a hypoglycemic effect and may have an effect on improving basal metabolic rate in DIO mice. Both in vitro and in vivo tests indicated that compound 35 was a potential potent GPR119 agonist in allusion to T2DM treatment.     

Evidence for constitutive dimerization of niacin receptor subtypes

The recently deorphanized niacin receptor subtypes NIACR1 (GPR109A) and NIACR2 (GPR109B) play an essential role in the regulation of metabolic processes and immune reactions. Both receptors belong to the G-protein-coupled receptor (GPCR) family, whose members have traditionally been treated as monomeric entities, but now appear to exist and function as both homodimers and heterodimers. In this study, a close physical interaction is shown between the highly homologous niacin receptor subtypes, NIACR1 and NIACR2, using bioluminescence resonance energy transfer (BRET²) in living cells. The extent of homo- and hetero-dimerization of the niacin receptors did not vary after activation of the receptors with selective agonists, indicating that the dimerization state of NIACR1 and NIACR2 is not regulated by ligand binding. Moreover, detection of niacin receptor dimers in both plasma membrane- and endoplasmic reticulum-enriched fractions suggests that they are formed early in the biosynthetic pathway. Taken together, these results demonstrate that niacin receptor dimerization is a constitutive process occurring early during biosynthesis.     

Design and optimization of 2,3-dihydrobenzo[b][1,4]dioxine propanoic acids as novel GPR40 agonists with improved pharmacokinetic and safety profiles

GPR40 has become a new potential therapeutic target for the treatment of diabetes due to its role in mediating the enhancement of glucose-stimulated insulin secretion in pancreatic β cells with a low risk of hypoglycemia. As an effort to extend the chemical space and identify structurally distinct GPR40 agonists with improved liver safety, a novel series of fused-ring phenyl propanoic acid analogues were designed. Comprehensive structure-activity relationship studies around novel scaffolds were conducted and led to several analogues exhibited potent GPR40 agonistic activities and high selectivity against other fatty acid receptors. Further evaluation of pharmacokinetic (PK) profiles and in vivo efficacy identified compound 40a with excellent PK properties and significant glucose-lowering efficacy during an oral glucose tolerance test. In addition, compound 40a displayed lower hepatobiliary transporter inhibition and favorable druggability. All results indicate that compound 40a is a promising candidate for further development.     

Design and synthesis of 1-(1-benzothiophen-7-yl)-1H-pyrazole,a novel series of G protein-coupled receptor 52 (GPR52) agonists

G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-d-aspartate receptors via intracellular cAMP accumulation. Therefore, GPR52 agonists are expected to alleviate symptoms of psychotic disorders. A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as GPR52 agonists was designed and synthesized based on compound 1b. Compound 1b has been reported by our group as the first orally active GPR52 agonist, but high lipophilicity and poor aqueous solubility still remained as issues for candidate selection. To resolve these issues, replacement of the benzene ring at the 7-positon of compound 1b with heterocylic rings, such as pyrazole and pyridine, was greatly expected to reduce lipophilicity to levels for which calculated logD values were lower than that of compound 1b. While evaluating the pyrazole derivatives, introduction of a methyl substituent at the 3-position of the pyrazole ring led to increased GPR52 agonistic activity. Moreover, additional methyl substituent at the 5-position of the pyrazole and further introduction of hydroxy group to lower logD led to significant improvement of solubility while maintaining the activity. As a result, we identified 3-methyl-5-hydroxymethyl-1H-pyrazole derivative 17 (GPR52 EC₅₀?=?21?nM, E_max?=?103%, logD?=?2.21, Solubility at pH 6.8?=?21?μg/mL) with potent GPR52 agonistic activity and good solubility compared to compound 1b. Furthermore, this compound 17 dose-dependently suppressed methamphetamine-induced hyperlocomotion in mice.     

Design,synthesis, and biological evaluation of aryl N-methoxyamide derivatives as GPR119 agonists

A series of N-methoxyamide derivatives was identified and evaluated as GPR119 agonists. Several N-methoxyamides with thienopyrimidine and pyridine scaffolds showed potent GPR119 agonistic activities. Among them, compound 9c displayed good in vitro activity and potency. Moreover, compound 9c lowered glucose excursion in mice in an oral glucose tolerance test and increased GLP-1 secretion in intestinal cells.     

Discovery of a nortropanol derivative as a potent and orally active GPR119 agonist for type 2 diabetes

The lead optimization studies of a series of GPR119 agonists incorporating a nortropanol scaffold are described. Extensive structure-activity relationship (SAR) studies of the lead compound 20f led to the identification of compound 36j as a potent, single digit nanomolar GPR119 agonist with high agonist activity. Compound 36j was orally active in lowering blood glucose levels in a mouse oral glucose tolerance test and increased plasma insulin levels in a rat hyperglycemic model. It showed good to excellent pharmacokinetic properties in rats and monkeys and no untoward activities in counter-screen assays. Compound 36j demonstrated an attractive in vitro and in vivo profile for further development.     

Potent tricyclic pyrazole tetrazole agonists of the nicotinic acid receptor (GPR109a)

Tricyclic pyrazole tetrazoles which are potent partial agonists of the high affinity niacin receptor, GPR109a, have been discovered and optimized. One of these compounds has proven to be effective at lowering free fatty acids in vitro and in vivo.     

Pyrido pyrimidinones as selective agonists of the high affinity niacin receptor GPR109A: Optimization of in vitro activity

Pyrido pyrimidinones are selective agonists of the human high affinity niacin receptor GPR109A (HM74A). They show no activity on the highly homologous low affinity receptor GPR109B (HM74). Starting from a high throughput screening hit the in vitro activity of the pyrido pyrimidinones was significantly improved providing lead compounds suitable for further optimization.     

The psoriasis drug monomethylfumarate is a potent nicotinic acid receptor agonist

Nicotinic acid has been used for several decades to treat dyslipidemia. In mice, the lipid-lowing effect of nicotinic acid is mediated by the Gi coupled receptor PUMA-G. In humans, high (GPR109A) and low (GPR109B) affinity nicotinic acid receptors have been characterized. Here we identify monomethylfumarate as a GPR109A agonist. Monomethylfumarate is the active metabolite of the psoriasis drug Fumaderm. We show that monomethylfumarate activates GPR109A in a calcium based aequorin assay, cAMP assay and demonstrate competitive binding with nicotinic acid. We show that GPR109A is highly expressed in neutrophils and epidermal keratinocytes, and that its expression is increased in human psoriatic lesions. Our findings provide evidence that GPR109A is a target for the drug Fumaderm and suggest that niacin should be investigated to treat psoriasis in addition to its role in treating lipid disorders.