Discovery of a novel potent GPR40 full agonist

Compound 12 is a GPR40 agonist that realizes the full magnitude of efficacy possible via GPR40 receptor agonism. In vitro and in vivo studies demonstrated superior glucose lowering by 12 compared to fasiglifam (TAK-875), in a glucose dependent manner. The enhanced efficacy observed with the full agonist 12 was associated with both direct and indirect stimulation of insulin secretion.     

Pharmacophore-guided repurposing of fibrates and retinoids as GPR40 allosteric ligands with activity on insulin release

A classical drug repurposing approach was applied to find new putative GPR40 allosteric binders. A two-step computational protocol was set up, based on an initial pharmacophoric-based virtual screening of the DrugBank database of known drugs, followed by docking simulations to confirm the interactions between the prioritised compounds and GPR40. The best-ranked entries showed binding poses comparable to that of TAK-875, a known allosteric agonist of GPR40. Three of them (tazarotenic acid, bezafibrate, and efaproxiral) affect insulin secretion in pancreatic INS-1 832/13 β-cells with EC₅₀ in the nanomolar concentration (5.73, 14.2, and 13.5 nM, respectively). Given the involvement of GPR40 in type 2 diabetes, the new GPR40 modulators represent a promising tool for therapeutic intervention towards this disease. The ability to affect GPR40 was further assessed in human breast cancer MCF-7 cells in which this receptor positively regulates growth activities (EC₅₀ values were 5.6, 21, and 14 nM, respectively).     

Role of GPR40 in fatty acid action on the beta cell line INS-1E

GPR40 is a G protein-coupled receptor expressed preferentially in beta cells, that has been implicated in mediating free fatty acid-stimulated insulin release. GPR40 RNAi impaired the ability of palmitic acid (PA) to increase both insulin secretion and intracellular calcium ([Ca2+]i). The PA-dependent [Ca2+]i increase was attenuated by inhibitors of Galphaq, PLC, and SERCA. Thus GPR40 activates the Galphaq pathway, leading to release of Ca2+ from the ER. Yet the GPR40-dependent [Ca2+]i rise was dependent on extracellular Ca2+ and elevated glucose, and was blocked by inhibition of L-type calcium channels (LTCC) or opening of the K(ATP) channel; this suggests that GPR40 promotes Ca2+ influx through up-regulation of LTCC pre-activated by glucose and membrane depolarization. Taken together, the data indicate that GPR40 mediates the increase in [Ca2+]i and insulin secretion through the Galphaq-PLC pathway, resulting in release of Ca2+ from the ER and leading to up-regulation of Ca2+ influx via LTCC.     

Discovery of 3-aryl-3-ethoxypropanoic acids as orally active GPR40 agonists

The G protein-coupled receptor 40 (GPR40) mediates enhancement of glucose-stimulated insulin secretion in pancreatic β cells. The GPR40 agonist has been attracting attention as a novel insulin secretagogue with glucose dependency for the treatment of type 2 diabetes. The optimization study of compound 1 led to a potent and bioavailable GPR40 agonist 24, which showed insulin secretion and glucose lowering effects in rat OGTT. Compound 24 is a potential lead compound for a novel insulin secretagogue with a low risk of hypoglycemia.     

GPR40 is expressed in glucagon producing cells and affects glucagon secretion

The free fatty acid receptor, GPR40, has been coupled with insulin secretion via its expression in pancreatic beta-cells. However, the role of GPR40 in the release of glucagon has not been studied and previous attempts to identify the receptor in alpha-cells have been unfruitful. Using double-staining for glucagon and GPR40 expression, we demonstrate that the two are expressed in the same cells in the periphery of mouse islets. In-R1-G9 hamster glucagonoma cells respond dose-dependently to linoleic acid stimulation by elevated phosphatidyl inositol hydrolysis and glucagon release and the cells become increasingly responsive to fatty acid stimulation when overexpressing GPR40. Isolated mouse islets also secrete glucagon in response to linoleic acid, a response that was abolished by antisense treatment against GPR40. This study demonstrates that GPR40 is present and active in pancreatic alpha-cells and puts further emphasis on the importance of this nutrient sensing receptor in islet function.     

GPR40 gene expression in human pancreas and insulinoma

To assess gene expression of a membrane-bound G-protein-coupled fatty acid receptor, GPR40, in the human pancreas and islet cell tumors obtained at surgery were analyzed. The mRNA level of the GPR40 gene in isolated pancreatic islets was approximately 20-fold higher than that in the pancreas, and the level was comparable to or rather higher than that of the sulfonylurea receptor 1 gene, which is known to be expressed abundantly in human pancreatic beta cells. A large amount of GPR40 mRNA was detected in tissue extracts from two cases of insulinoma, whereas the expression was undetectable in glucagonoma or gastrinoma. The present study demonstrates that GPR40 mRNA is expressed predominantly in pancreatic islets in humans and that GPR40 mRNA is expressed solely in human insulinoma among islet cell tumors. These results indicate that GPR40 is probably expressed in pancreatic beta cells in the human pancreas.     

Quercetin-3-oleoyl derivatives as new GPR40 agonists: Molecular docking studies and functional evaluation

The G-protein-coupled receptor 40 (GPR40) is an attractive molecular target for the treatment of type 2 diabetes mellitus. Previously, based on the natural oleic acid substrate, an exogenous ligand for this receptor, named AV1, was synthesized. In this context, here we validated the activity of AV1 as a full agonist, while the corresponding catechol analogue, named AV2, was investigated for the first time. The ligand-protein interaction between this new molecule and the receptor was highlighted in the lower portion of the GPR40 groove that generally accommodates DC260126. The functional assays performed have demonstrated that AV2 is a suitable GPR40 partial agonist, showing a therapeutic potential and representing a useful tool in the management of type 2 diabetes.     

Discovery of HWL-088: A highly potent FFA1/GPR40 agonist bearing a phenoxyacetic acid scaffold

Based on a previously reported phenoxyacetic acid scaffold, compound 7 (HWL-088) has been identified as a superior free fatty acid receptor 1 (FFA1) agonist by comprehensive structure-activity relationship study. Our results indicated that the introduction of ortho-fluoro greatly increased the activity of phenoxyacetic acid series, and the unique structure-activity relationship in biphenyl moiety is different from previously reported FFA1 agonists. Moreover, the modeling study was also performed to better understand the binding mode of present series. Compound 7 significantly improved glucose tolerance both in normal and diabetic models, and even exerted greater potential on glucose control than that of TAK-875. These findings provided a novel candidate HWL-088, which is currently in preclinical study to evaluate its potential for the treatment of diabetes.     

Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice

G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic β-cells and enhances the effect of glucose-stimulated insulin secretion (GSIS) on activation. The development of an oral GPR119 agonist that specifically targets the first phase of GSIS represents a promising strategy for the treatment of type 2 diabetes. In the present study, we evaluated the therapeutic potential of a novel small molecule GPR119 agonist, AS1535907, which was modified from the previously identified 2,4,6-tri-substituted pyrimidine core agonist AS1269574. AS1535907 displayed an EC₅₀ value of 4.8 μM in HEK293 cells stably expressing human GPR119 and stimulated insulin secretion in rat islets only under high-glucose (16.8 mM) conditions. In isolated perfused pancreata from normal rats, AS1535907 enhanced the first phase of insulin secretion at 16.8 mM glucose, but had no effect at 2.8 mM glucose. In contrast, the sulfonylurea glibenclamide predominantly induced insulin release in the second phase at 16.8 mM glucose and also markedly stimulated insulin secretion at 2.8 mM glucose. In in vivo studies, a single 10 μM administration of AS1535907 to diabetic db/db mice reduced blood glucose levels due to the rapid secretion of insulin secretion following oral glucose loading. These results demonstrate that GPR119 agonist AS1535907 has the ability to stimulate the first phase of GSIS, which is important for preventing the development of postprandial hypoglycemia. In conclusion, the GPR119 agonist AS1535907 induces a more rapid and physiological pattern of insulin release than glibenclamide, and represents a novel strategy for the treatment of type 2 diabetes.     

Design,synthesis and biological evaluation of indane derived GPR40 agoPAMs

GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet β-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet β-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.     

Glucagon increases insulin levels by stimulating insulin secretion without effect on insulin clearance in mice

Circulating insulin is dependent on a balance between insulin appearance through secretion and insulin clearance. However, to what extent changes in insulin clearance contribute to the increased insulin levels after glucagon administration is not known. This study therefore assessed and quantified any potential effect of glucagon on insulin kinetics in mice. Prehepatic insulin secretion in mice was first estimated following glucose (0.35 g/kg i.v.) and following glucose plus glucagon (10 μg/kg i.v.) using deconvolution of plasma C-peptide concentrations. Plasma concentrations of glucose, insulin, and glucagon were then measured simultaneously in individual mice following glucose alone or glucose plus glucagon (pre dose and at 1, 5, 10, 20 min post). Using the previously determined insulin secretion profiles and the insulin concentration-time measurements, a population modeling analysis was applied to estimate the one-compartment kinetics of insulin disposition with and without glucagon. Glucagon with glucose significantly enhanced prehepatic insulin secretion (Cmax and AUC_0-20) compared to that with glucose alone (p < 0.0001). From the modeling analysis, the population mean and between-animal SD of insulin clearance was 6.4 ± 0.34 mL/min for glucose alone and 5.8 ± 1.5 mL/min for glucagon plus glucose, with no significant effect of glucagon on mean insulin clearance. Therefore, we conclude that the enhancement of circulating insulin after glucagon administration is solely due to stimulated insulin secretion.     

Free fatty acid receptor 1 (FFA<Subscript>1</Subscript>R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion

Free fatty acids (FFA) have generally been proposed to regulate pancreatic insulin release by an intracellular mechanism involving inhibition of CPT-1. The recently de-orphanized G-protein coupled receptor, FFA₁R/GPR40, has been shown to be essential for fatty-acid-stimulated insulin release in MIN6 mouse insulinoma cells. The CPT-1 inhibitor, 2-bromo palmitate (2BrP), was investigated for its ability to interact with mouse FFA₁R/GPR40. It was found to inhibit phosphatidyl inositol hydrolysis induced by linoleic acid (LA) (100 μM in all experiments) in HEK293 cells transfected with FFA₁R/GPR40 and in the MIN6 subclone, MIN6c4. 2BrP also inhibited LA-stimulated insulin release from mouse pancreatic islets. Mouse islets were subjected to antisense intervention by treatment with a FFA₁R/GPR40-specific morpholino oligonucleotide for 48 h. Antisense treatment of islets suppressed LA-stimulated insulin release by 50% and by almost 100% when islets were pretreated with LA for 30 min before applying the antisense. Antisense treatment had no effect on tolbutamide-stimulated insulin release. Confocal microscopy using an FFA₁R/GPR40-specific antibody revealed receptor expression largely localized to the plasma membrane of insulin-producing cells. Pretreating the islets with LA for 30 min followed by antisense oligonucleotide treatment for 48 h reduced the FFA₁R/GPR40 immunoreactivity to background levels. The results demonstrate that FFA₁R/GPR40 is inhibited by the CPT-1 inhibitor, 2BrP, and confirm that FFA₁R/GPR40 is indeed necessary, at least in part, for fatty-acid-stimulated insulin release. A. Salehi and E. Flodgren contributed equally to this work     

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.     

Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations

Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually beta-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of beta-cell mitochondrial proteins obtained by SELDI-TOF-MS.     

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve β-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve β-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC₅₀ value of 2.5 μM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic β-cell line MIN-6 only under high-glucose (16.8 mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8 mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.     

A low-protein diet during pregnancy alters glucose metabolism and insulin secretion

In pancreatic islets, glucose metabolism is a key process for insulin secretion, and pregnancy requires an increase in insulin secretion to compensate for the typical insulin resistance at the end of this period. Because a low-protein diet decreases insulin secretion, this type of diet could impair glucose homeostasis, leading to gestational diabetes. In pancreatic islets, we investigated GLUT2, glucokinase and hexokinase expression patterns as well as glucose uptake, utilization and oxidation rates. Adult control non-pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) from days 1 to 15 of pregnancy. The insulin secretion in 2.8 mmol l(-1) of glucose was higher in islets from LPP rats than that in islets from CP, CNP and LPNP rats. Maximal insulin release was obtained at 8.3 and 16.7 mmol l(-1) of glucose in LPP and CP groups, respectively. The glucose dose-response curve from LPNP group was shifted to the right in relation to the CNP group. In the CP group, the concentration-response curve to glucose was shifted to the left compared with the CNP group. The LPP groups exhibited an "inverted U-shape" dose-response curve. The alterations in the GLUT2, glucokinase and hexokinase expression patterns neither impaired glucose metabolism nor correlated with glucose islet sensitivity, suggesting that β-cell sensitivity to glucose requires secondary events other than the observed metabolic/molecular events.     

AMG 837: a novel GPR40/FFA1 agonist that enhances insulin secretion and lowers glucose levels in rodents

Agonists of GPR40 (FFA1) have been proposed as a means to treat type 2 diabetes. Through lead optimization of a high throughput screening hit, we have identified a novel GPR40 agonist called AMG 837. The objective of these studies was to understand the preclinical pharmacological properties of AMG 837. The activity of AMG 837 on GPR40 was characterized through GTPγS binding, inositol phosphate accumulation and Ca(2+) flux assays. Activity of AMG 837 on insulin release was assessed on isolated primary mouse islets. To determine the anti-diabetic activity of AMG 837 in vivo, we tested AMG 837 using a glucose tolerance test in normal Sprague-Dawley rats and obese Zucker fatty rats. AMG 837 was a potent partial agonist in the calcium flux assay on the GPR40 receptor and potentiated glucose stimulated insulin secretion in vitro and in vivo. Acute administration of AMG 837 lowered glucose excursions and increased glucose stimulated insulin secretion during glucose tolerance tests in both normal and Zucker fatty rats. The improvement in glucose excursions persisted following daily dosing of AMG 837 for 21-days in Zucker fatty rats. Preclinical studies demonstrated that AMG 837 was a potent GPR40 partial agonist which lowered post-prandial glucose levels. These studies support the potential utility of AMG 837 for the treatment of type 2 diabetes.     

Time and amplitude regulation of pulsatile insulin secretion by triggering and amplifying pathways in mouse islets

Glucose-induced insulin secretion is pulsatile. We investigated how the triggering pathway (rise in β-cell [Ca²⁺]_i) and amplifying pathway (greater Ca²⁺ efficacy on exocytosis) influence this pulsatility. Repetitive [Ca²⁺]_i pulses were imposed by high K⁺+ diazoxide in single mouse islets. Insulin secretion (measured simultaneously) tightly followed [Ca²⁺]_i changes. Lengthening [Ca²⁺]_i pulses increased the duration but not the amplitude of insulin pulses. Increasing glucose (5–20 mmol/l) augmented the amplitude of insulin pulses without changing that of [Ca²⁺]_i pulses. Larger [Ca²⁺]_i pulses augmented the amplitude of insulin pulses at high, but not low glucose. In conclusion, the amplification pathway ensures amplitude modulation of insulin pulses whose time modulation is achieved by the triggering pathway.     

Synergistic effect on insulin secretion from INS-1 cells of a sulfonylurea and a phosphodiesterase 3 inhibitor

The effects on insulin secretion from INS-1 cells of varying concentrations of the sulfonylurea glyburide and the PDE3 inhibitor milrinone, separately and in combination were measured. Over a range of concentrations the effects of the two drugs in combination were more than additive. A response surface model was fit to the data and was found to describe the data well. From this model, it was apparent that a significant synergistic effect upon insulin secretion existed over a wide range of combinations of the two drugs.