Glucagon Receptor

Adomeglivant cannot block the cAMP-increasing effect of pancreatic islets [2]. Adomeglivant exhibits high resistance to group B GPCRs and resonates with the oscillatory binding motif potential in GluR, GLP-1R and GIP-R [2].

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Glucagon and LY2409021 both target glucagon and GLP-1 receptors. LY2409021 blocks GLP-1 and Ex-4 agonist action at the GLP-1R. LY2409021 is a GIP-R antagonist but fails to block adenosine action at A2B receptors. LY2409021 blocks GGP817 agonist action at the GluR and GLP-1R. The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His1-[Glu9]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R [2].

Adomeglivant (LY2409021) (5 mg/kg; ip) completely eliminates the hypertensive effects of CNO (clozapine-N-oxide) in Avpires-Cre+ electrodes. (CNO is a cardiovascular drug stimulant that can induce hM3Dq-induced membrane myocardial infarction and increase the firing rate of hM3Dq-expressing arginine vasopressin (AVP) neurons) [3] Animal model: Avpires-Cre+ small Rat [3] Dosage: 5 mg/kg Administration method: intraperitoneal injection, 30 minutes before CNO. Results: The hyperglycemic effect of CNO was completely eliminated.

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To establish the contribution of glucagon to this hyperglycaemic response, we pre-treated mice with the glucagon receptor antagonist Adomeglivant (LY2409021) . This completely abolished the hyperglycaemic action of CNO (Fig. 4b). Similarly, to understand the contribution of vasopressin 1b receptor (V1bR) signalling, we pre-treated mice with the V1bR antagonist SSR149415. This also abolished the hyperglycaemic effect of CNO (Fig. 4b). Measurements of plasma glucagon during CNO treatment revealed it was elevated by ~40% [3].

FRET reporter assay in a 96-well format [2]
HEK293 cells stably expressing recombinant GPCRs were plated at 80% confluence on 96-well clear-bottom assay plates coated with rat tail collagen. Cells were then transduced for 16 h with H188 virus at a density of ∼60,000 cells/well under conditions in which the multiplicity of infection was equivalent to 25 viral particles per cell. The culture media were removed and replaced by 200 μl/well of a standard extracellular saline (SES) solution supplemented with 11 mm glucose and 0.1% BSA. The composition of the SES was (in mm): 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 11.1 glucose, and 10 Hepes (295 mosmol, pH 7.4). Real-time kinetic assays of FRET were performed using a FlexStation 3 microplate reader equipped with excitation and emission light monochromators. Excitation light was delivered at 435/9 nm (455 nm cutoff), and emitted light was detected at 485/15 nm (cyan fluorescent protein) or 535/15 nm (yellow fluorescent protein). The emission intensities were the averages of 12 excitation flashes for each time point per well. Test solutions dissolved in SES were placed in V-bottom 96-well plates, and an automated pipetting procedure was used to transfer 50 μl of each test solution to each well of the assay plate containing monolayers of these cells. The 485/535 emission ratio was calculated for each well, and the mean ± S.D. values for 12 wells were averaged. These FRET ratio values were normalized using baseline subtraction so that a y axis value of 0 corresponds to the initial baseline FRET ratio, whereas a value of 100 corresponds to a 100% increase (i.e. doubling) of the FRET ratio. The time course of the ΔFRET ratio was plotted after exporting data to Origin 8.0. Origin 8.0 was also used for nonlinear regression analysis to quantify dose-response relationships.

HEK293 cells stably expressing the human GLP-1R at a density of 150,000 receptors/cell were used. HEK293 cells stably expressing the rat GlucR at a density of 250,000 receptors/cell were obtained from T. P. Sakmar, A. M. Cypess, and C. G. Unson. HEK293 cells stably expressing the rat GIP-R at a receptor density that has yet to be determined were obtained from T. J. Kieffer. HEK293 cells stably expressing H188 were generated by O. G. Chepurny in the Holz laboratory. All cell cultures were maintained in Dulbecco's modified Eagle's medium containing 25 mm glucose and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cell cultures equilibrated at 37 °C in a humidified incubator that was gassed with 5% CO2 were passaged once a week [2].

AAV-DIO-hM3Dq-mCherry was injected bilaterally into the supraoptic nucleus (SON) of Avpires-Cre+ mice. Mice were fasted for 4 hours (beginning at 10:00 am), and then CNO (or saline vehicle) was injected (3 mg/kg i.p.). In the same cohort, during a different trial, the glucagon receptor antagonist Adomeglivant (LY2409021)Adomeglivant (LY2409021) AAV-DIO-hM3Dq was injected into ThCre+ mice, targeting A1/C1 neurons. CNO (1 mg/kg) was then injected (i.p.). Antagonists (or vehicle) for the V1bR (SSR149415, 30 mg/kg) or glucagon receptor (GCGR; Adomeglivant (LY2409021)

Predicting the pharmacokinetics of CAA and PIB in humans and comparing the results with known antagonists [1]
Certain structural and molecular characteristics of compounds determine their pharmacokinetic properties in vivo. In this study, the druggability of all four inhibitors was evaluated using the Qikprop module of Schrodinger. Whether the compounds violated the Lipinski five rules was assessed by calculating molecular weight, number of hydrogen bond donors, number of hydrogen bond acceptors and logP value. To further evaluate the potential of these compounds as effective drug candidates, computer simulations were also performed using the Qikprop module to calculate their absorption, distribution, metabolism and excretion (ADME) properties.

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Physicochemical properties and pharmacokinetics of GCGR inhibitors CAA, PIB, MK-0893 and LY2409021 [1]
To supplement the information obtained from the binding affinity prediction, we used Qikprop to calculate various other physically meaningful descriptors and pharmaceutically relevant properties of these small molecules. Qikprop predicts these molecular properties and provides a significant range to compare their values with 95% of known drugs. The descriptor "#star" indicates the number of anomalous properties of a molecule, i.e., properties that are outside the range of known drug values. Therefore, the smaller the value, the better the drug-likeness of the small molecule. MK-0893 shows the highest binding affinity with a #star value of 4, while the other three compounds all have a #star value of 0. Therefore, except for MK-0893, the calculated properties of the other three compounds are within the specified range and are very similar to the properties of known drugs. The Lipinski five rules are an empirical rule that uses four molecular properties to determine the likelihood of oral activity of a drug. Table 1 lists the four property values for these four compounds. MK-0893, with a molecular weight of 588.48 and a logP value of 8.18, does not meet the Lipinski rules (molecular weight < 500, number of hydrogen bond donors < 5, number of hydrogen bond acceptors < 10, logP < 5). Solvent-accessible surface area (SASA), especially polar surface area (PSA), determines the passive transport of molecules across membranes, thus allowing estimation of drug transport properties. The total SASA values of MK-0893, CAA, PIB, and LY2409021 all fall within the range given by QikProp. QikProp uses a knowledge-based rule set to calculate the percentage probability of oral absorption of a drug in humans. This value correlates well with the oral absorption rate in humans. PIB has the highest oral absorption rate, reaching 100%. Among the other three drugs, LY2409021 has the lowest absorption rate, at 28.78%. Central nervous system activity is another parameter to consider when assessing safety. CAA was found to have almost no central nervous system activity, while PIB is expected to have extremely low central nervous system activity. The blood-brain barrier (BBB) directly separates the human brain from the circulatory system, thus protecting the brain from harmful solute particles. Both predicted compounds were confirmed to lack blood-brain barrier activity, ensuring their safety for brain administration. Although MK-0893 has a higher affinity for GCGR, CAA and PIB are superior to this known inhibitor in many respects. The new compounds exhibit better druggability, and their ADME properties are all within acceptable ranges. This clearly demonstrates the unique potential of CAA and PIB as potential lead inhibitors of GCGR in the treatment of type 2 diabetes.

[1]. Computational identification of novel natural inhibitors of glucagon receptor for checking type II diabetes mellitus. BMC Bioinformatics. 2014; 15(Suppl 16): S13.

[2]. Non-conventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP. J Biol Chem. 2019 Mar 8;294(10):3514-3531.

[3]. AVP-induced counter-regulatory glucagon is diminished in type 1 diabetes. bioRxiv. January 31, 2020.

[4]. The glucagon receptor antagonist LY2409021 has no effect on postprandial glucose in type 2 diabetes. Eur J Endocrinol. 2022 Jan 6;186(2):207-221.

Objective: The pathophysiology of type 2 diabetes mellitus (T2D) includes fasting and postprandial hyperglycemia, which is associated with hyperglycemia resulting from increased endogenous glucose production (EGP). We investigated the consequences of hyperglycemia in T2D using a glucagon receptor antagonist (LY2409021) and a stable isotope tracer infusion. Design: A double-blind, randomized, placebo-controlled crossover study was conducted. Methods: Ten T2D patients and ten matched non-diabetic controls underwent two liquid mixed-meal trials, receiving either a single dose of LY2409021 (100 mg) or placebo prior to the trial. EGP was quantified using a dual-tracer technique. The selectivity of the antagonist for the relevant incretin receptors was determined in vitro. Results: Compared with placebo, LY2409021 reduced fasting plasma glucose (FPG) from 9.1 mmol/L to 7.1 mmol/L in patients and from 5.6 mmol/L to 5.0 mmol/L in the control group by decreasing endogenous glucose production (EGP) (both P < 0.001). LY2409021 had no effect on postprandial glycemic variability (AUC minus baseline), but postprandial glycemic variability was increased in the control group compared with placebo. During glucagon receptor antagonism, glucagon concentration more than doubled. This antagonist interferes with glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) receptors, complicating the interpretation of postprandial data. Conclusion: LY2409021 reduced fasting plasma glucose (FPG) concentration but did not improve postprandial glycemic tolerance in patients with type 2 diabetes and healthy controls. Because LY2409021 has an antagonistic effect on incretin receptors, it is difficult to use LY2409021 to assess the metabolic consequences of postprandial hyperglycemia. [4]

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Adomeglivant has been used in basic research on type 2 diabetes. Adomeglivant is a small molecule drug with the highest clinical trial stage being Phase II (covering all indications) and three investigational indications.

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Background: The interaction between the small peptide hormone glucagon and glucagon receptor (GCGR) can stimulate the release of glucose from hepatocytes in a fasting state; therefore, GCGR plays an important role in glucose homeostasis. Inhibition of the interaction between glucagon and its receptor has been reported to control excessive glucose production in the liver, and therefore GCGR has become a very attractive therapeutic target for the treatment of type 2 diabetes. Results: This study screened a library of compounds containing a large number of natural compounds to find novel therapeutic molecules that can inhibit the binding of glucagon to GCGR. We performed molecular dynamics simulations to study the dynamic behavior of the docking complex and analyzed in detail the molecular interactions between the screened compounds and the GCGR ligand-binding residues. In addition, we compared the highest-scoring compounds with the reported GCGR inhibitors MK-0893 and LY2409021 to evaluate their binding affinity and other ADME properties. Finally, we reported two natural drug-like compounds, PIB and CAA, which showed good binding affinity for GCGR and were potent inhibitors of its functional activity. Conclusion: This study provides evidence for these compounds as potential small molecule ligands for the treatment of type II diabetes. We have discovered novel natural drug-like inhibitors targeting the 7th transmembrane domain of GCGR that exhibit high affinity and potent GCGR inhibition. [1]

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G protein-coupled receptors (GPCRs) of glucagon receptor (GluR) and glucagon-like peptide-1 receptor (GLP-1R) are generally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted by pancreatic α cells may accumulate to high concentrations on β-cell GLP-1R, leading to nonspecific effects, which can occur in the microenvironment of limited islet volume. Furthermore, systemic administration of high doses of GluR or GLP-1R agonists and antagonists may result in off-target effects on other receptors. Here, we evaluated FRET assay data using molecular modeling, with cAMP as a reading of GluR and GLP-1R activation. This analysis confirms that glucagon is a non-canonical GLP-1R agonist whose effects can be inhibited by the GLP-1R ortho-antagonist exenatide (9-39) (Ex(9-39)). Glutamate receptor (GluR) allosteric inhibitors LY2409021 and MK 0893 antagonize the effects of glucagon and GLP-1 on GLP-1R, while dehistidine 1-[Glu9]glucagon antagonizes the effects of glucagon on GluR, but has minimal inhibitory effect on glucagon or GLP-1 on GLP-1R. In INS-1 832/13 cells, we validated the dual agonist effect of glucagon on GluR and GLP-1R by combining Ex(9-39) with dehistidine 1-[Glu9]glucagon. The hybrid peptide GGP817, containing a fusion of glucagon and peptide YY (PYY) fragments, can act as a triple agonist for GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). These findings collectively provide a novel triple agonist strategy for targeting GluR, GLP-1R, and NPY2R. Furthermore, these findings have prompted us to re-evaluate previous studies that hypothesized that GluR and GLP-1R agonists and antagonists would not have nonspecific effects on other GPCRs. [2] Hypoglycemia is a major obstacle to the treatment of diabetes. Therefore, it is crucial to understand the mechanisms regulating circulating glucagon levels—the main blood glucose-raising hormone in the human body, secreted by pancreatic α cells. In isolated islets, variations in glucose concentration within the physiological range between satiety and starvation (8 to 4 mM) have no significant effect on glucagon secretion, but in vivo they are associated with significant changes in plasma glucagon levels. The systemic factors that stimulate glucagon secretion in vivo are currently unclear. This study shows that arginine vasopressin (AVP) secreted by the posterior pituitary gland can stimulate glucagon secretion. α cells that secrete glucagon highly express vasopressin 1b receptor (V1bR). Activation of AVP neurons in vivo increases circulating AVP levels, stimulates glucagon release, and induces hyperglycemia; these effects can be blocked by pharmacological antagonists of glucagon receptors or vasopressin 1b receptors. AVP also mediates the stimulatory effect of dehydration and hypoglycemia induced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We found that medullary A1/C1 neurons, which are known to be activated by hypoglycemia, drive the activation of AVP neurons in insulin-induced hypoglycemic responses. Hypoglycemia also increases circulating levels of copeptin (from the same precursor hormone as AVP), which stimulates glucagon secretion from isolated human islets. In patients with type 1 diabetes, hypoglycemia failed to increase plasma levels of copeptin and glucagon. These findings provide a new mechanism for the central regulation of glucagon secretion in healthy and disease states. [3]

C32H36F3NO4

555.63

555.259

C, 69.17; H, 6.53; F, 10.26; N, 2.52; O, 11.52

872260-19-0

872260-47-4 (racemic); 1488363-78-5; 488363-78-5 (S-isomer); 872260-19-0 (R-isomer)

91936837

Typically exists as solids at room temperature

7.53

2

7

11

40

798

1

FASLTMSUPQDLIB-HHHXNRCGSA-N

InChI=1S/C32H36F3NO4/c1-20-18-26(19-21(2)29(20)23-10-12-25(13-11-23)31(3,4)5)40-27(14-16-32(33,34)35)22-6-8-24(9-7-22)30(39)36-17-15-28(37)38/h6-13,18-19,27H,14-17H2,1-5H3,(H,36,39)(H,37,38)/t27-/m1/s1

3-[[4-[(1R)-1-[4-(4-tert-butylphenyl)-3,5-dimethylphenoxy]-4,4,4-trifluorobutyl]benzoyl]amino]propanoic acid

(+)-LY2409021； Adomeglivant, (+)-; RIM88PH2RA; UNII-RIM88PH2RA; 872260-19-0; (+)-adomeglivant;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)