Glucagon Receptor; egative allosteric modulator of glucagon receptor

-GCGR inhibitory activity: NNC0640 inhibits glucagon induced cAMP accumulation by binding to the outer surface of the GCGR transmembrane domain, with an IC ₅₀ of 69.2 nM. Its binding mode is confirmed by crystal structure, stabilizing the non activated conformation of the receptor.
-GLP-1R regulatory activity: In vitro experiments, NNC0640 acts as a negative allosteric regulator of GLP-1R, inhibiting GLP-1 mediated cAMP accumulation and partially overlapping with the binding site of PF-06372222. - Literature [1] (Zhang H, et al., 2017) mainly reports the cryo-electron microscopy structure of the full-length human glucagon receptor (GCGR) in complex with glucagon and a G protein. It focuses on analyzing the structural basis of GCGR activation [1]
- Literature [2] (Song G, et al., 2017) describes the X-ray crystal structure of the transmembrane domain of the human GLP-1 receptor (GLP-1R) in complex with two allosteric modulators (PF-06372222 and NNC0640). It explores the binding mode of allosteric modulators to GLP-1R [2]

-GCGR binding experiment: Through surface plasmon resonance (SPR) and crystal structure analysis, the binding affinity (KD) of NNC0640 to the transmembrane domain of GCGR was found to be 0.8 nM. The complex structure showed that the drug stabilized the non activated conformation of the receptor through hydrophobic interactions.
-CAMP accumulation inhibition experiment: In HEK293 cells, NNC0640 inhibited glucagon induced cAMP accumulation in a dose-dependent manner, with an IC ₅₀ of 69.2 nM.

-Receptor activation inhibition experiment: In CHO cells expressing GCGR, NNC0640 (10-1000 nM) significantly inhibited glucagon induced ERK1/2 phosphorylation, with an IC ₅₀ of 75 nM.
-GLP-1R functional experiment: In HEK293-GLP-1R cells, NNC0640 (1-1000 nM) inhibited GLP-1 induced cAMP accumulation, with an IC ₅₀ of 120 nM.

[1]. Structure of the full-length glucagon class B G-protein-coupled receptor. Nature. 2017 Jun 8;546(7657):259-264.

[2]. Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators. Nature. 2017 Jun 8;546(7657):312-315.

Mechanism of Action: NNC0640 stabilizes the inactive conformation of the receptor by binding to the transmembrane domains of GCGR and GLP-1R, thereby blocking ligand-induced signal transduction. Its binding mode provides a template for drug design targeting type B GPCRs. - Significance of Structural Study: The structure of the GCGR-NNC0640 complex (PDB ID: 5W0P) resolved by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, is the first structure of a full-length type B GPCR and its small molecule regulator, revealing the crucial role of the Stark region in receptor activation. Human glucagon receptor GCGR belongs to the class B G protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. This paper reports the 3.0 Å crystal structure of the full-length GCGR in its inactive conformation, which contains an extracellular domain and a transmembrane domain. These two domains are connected by a 12-amino acid residue fragment called the stem structure. The stem structure exhibits a β-sheet conformation, rather than forming an α-helix as previously resolved in the GCGR transmembrane domain. The first extracellular loop is in a β-hairpin conformation and interacts with the stem structure to form a tight β-sheet structure. Hydrogen-deuterium exchange, disulfide crosslinking and molecular dynamics studies have shown that the stem structure and the first extracellular loop play a key role in regulating peptide ligand binding and receptor activation. These in-depth analyses of the full-length GCGR structure have deepened our understanding of the signaling mechanism of class B G protein-coupled receptors. [1]

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Glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) both belong to the secretin-like class B G protein-coupled receptor (GPCR) family and play opposing physiological roles in insulin release and glucose homeostasis. Treatment of type 2 diabetes requires positive regulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner. This paper reports the crystal structures of human GLP-1R transmembrane domain complexes with two different negative allosteric regulators, PF-06372222 and NNC0640, at resolutions of 2.7 Å and 3.0 Å, respectively. Structures show that both GLP-1R and GCGR have a common negative allosteric regulator binding pocket located on the outer side of the V-VII helix in the intracellular half of the receptor. The receptor is in an inactive conformation, and the compound restricts the movement of the intracellular tip of helix VI, which is usually associated with the activation mechanism of class A GPCRs. Molecular modeling and mutagenesis studies have shown that the agonist positive allosteric regulator targets the same general region, but in a different sub-pocket at the junction of helix V and helix VI, which may help to form an intracellular binding site, thereby enhancing G protein coupling. [2]

C29H31N7O4S

573.665944337845

573.22

C, 60.72; H, 5.45; N, 17.09; O, 11.16; S, 5.59

307986-98-7

23549991

White to off-white solid powder

4.7

3

7

8

41

970

0

S(C)(C1=CC=CC(=C1)NC(N(CC1C=CC(C(NC2N=NNN=2)=O)=CC=1)C1C=CC(=CC=1)C1CCCCC1)=O)(=O)=O

PPTKULJUDJWTSA-UHFFFAOYSA-N

InChI=1S/C29H31N7O4S/c1-41(39,40)26-9-5-8-24(18-26)30-29(38)36(25-16-14-22(15-17-25)21-6-3-2-4-7-21)19-20-10-12-23(13-11-20)27(37)31-28-32-34-35-33-28/h5,8-18,21H,2-4,6-7,19H2,1H3,(H,30,38)(H2,31,32,33,34,35,37)

4-[[4-cyclohexyl-N-[(3-methylsulfonylphenyl)carbamoyl]anilino]methyl]-N-(2H-tetrazol-5-yl)benzamide

NNC-0640; NNC0640; 4-(((4-cyclohexylphenyl)-((3-methylsulfonylphenyl)carbamoyl)amino)methyl)-N-(2H-tetrazol-5-yl)benzamide; 4-[[(4-cyclohexylphenyl)-[(3-methylsulfonylphenyl)carbamoyl]amino]methyl]-N-(2H-tetrazol-5-yl)benzamide; RefChem:166214; 307986-98-7; NNC-0640; NNC 0640; 4-[1-(4-Cyclohexylphenyl)-3-(3-methanesulfonylphenyl)ureidomethyl]-N-(2H-tetrazol-5-yl)benzamide; NNC 0640

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: ~250 mg/mL (~435.8 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.63 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 20.8 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: ≥ 2.08 mg/mL (3.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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: ≥ 2.08 mg/mL (3.63 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 20.8 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.)