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.

Adomeglivant has been validated in basic scientific research on type 2 diabetes. Adomeglivant is a small molecule drug that has completed the most Phase II clinical trials (covering all indications) and has three investigational indications. Background: The interaction between the small peptide hormone glucagon and its receptor (GCGR) stimulates hepatocytes to release glucose during fasting; therefore, GCGR plays an important role in glucose homeostasis. It has been reported that inhibiting the interaction between glucagon and its receptor can control excessive hepatic glucose production, thus GCGR has become an attractive therapeutic target for type 2 diabetes. Results: In this study, we screened a large library 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. Furthermore, we compared the highest-scoring compounds with 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 exhibited 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 against type II diabetes. We identified novel natural drug-like inhibitors targeting the seventh transmembrane domain of GCGR that exhibited high binding affinity and potent inhibitory activity against GCGR. [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 in the islets, resulting in nonspecific effects on GLP-1R on β cells, which may occur in the microenvironment of limited islet volume. In addition, systemic administration of high doses of GluR or GLP-1R agonists and antagonists may lead to off-target effects on other receptors. Here, we utilize molecular modeling to evaluate data obtained from a FRET assay that uses cAMP as a readout for 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 either glucagon or GLP-1 on GLP-1R. In INS-1 832/13 cells, we validated the dual agonist effect of glucagon on both GluR and GLP-1R when Ex(9-39) was used in combination with dehistidine 1-[Glu9]glucagon. The hybrid peptide GGP817, which contains a fusion of glucagon and peptide YY (PYY), can act as a triple agonist of GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). These findings together provide a novel triple agonist strategy for targeting GluR, GLP-1R, and NPY2R. In addition, these findings also prompt 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 that regulate 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) do not significantly affect glucagon secretion, but in vivo they are associated with significant changes in plasma glucagon levels. It is currently unclear what the systemic factors that stimulate glucagon secretion in vivo are. This study demonstrates that arginine vasopressin (AVP), secreted by the posterior pituitary gland, stimulates glucagon secretion. Alpha cells that secrete glucagon highly express vasopressin 1b receptors (V1bR). In vivo activation of AVP neurons 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, known to be activated by hypoglycemia, drive AVP neuron activation in insulin-induced hypoglycemic responses. Hypoglycemia also increases circulating levels of copeptin (derived 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 both healthy and disease states. [3]

C32H36F3NO4

555.6278

555.259

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

1488363-78-5

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

91933867

White to off-white solid powder

1.2±0.1 g/cm3

656.7±55.0 °C at 760 mmHg

350.9±31.5 °C

0.0±2.1 mmHg at 25°C

1.542

7.53

2

7

11

40

798

1

FC(C([H])([H])C([H])([H])[C@@]([H])(C1C([H])=C([H])C(C(N([H])C([H])([H])C([H])([H])C(=O)O[H])=O)=C([H])C=1[H])OC1C([H])=C(C([H])([H])[H])C(=C(C([H])([H])[H])C=1[H])C1C([H])=C([H])C(=C([H])C=1[H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])(F)F

FASLTMSUPQDLIB-MHZLTWQESA-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-/m0/s1

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

LY-2409021; LY2409021; LY 2409021

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)

DMSO: ~100 mg/mL (~180 mM)
Ethanol: ~100 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.50 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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