GSK682753A is a novel, potent, and selective inverse agonist of the epstein-barr virus-induced receptor 2 (EBI2) with an IC50 of 53.6 nM. It has recently been demonstrated that the constitutively active seven-transmembrane Epstein-Barr virus-induced receptor 2 (EBI2) coordinates the location of B cells within the follicle. No endogenous or artificial ligand has been found to date that modifies EBI2 activity. Here, we report on the high potency and effectiveness of an inverse agonist, GSK682753A, which specifically inhibited the constitutive activity of EBI2. This compound demonstrated a potency of 2.6-53.6 nm and an inhibitory efficacy of 75% in guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding as well as cAMP-response element-binding protein-based reporter assays. Furthermore, we demonstrate the insensitive activation of extracellular signal-regulated kinase (ERK) by EBI2. The inhibition of ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation by GSK682753A was intriguing because it was equally potent. The proliferation of murine B cells stimulated by antibodies in vivo was significantly enhanced when EBI2 was overexpressed in comparison to WT cells; however, this was likewise diminished in B cells lacking EBI2. The proliferation was always inhibited by blocking EBI2 constitutive activity. Interestingly, compared to EBI2-deficient B cells, WT or EBI2-overexpressing B cells showed a much stronger (32-fold) suppression. To find potential molecular binding determinants in EBI2, GSK682753A was lastly screened against a library of EBI2 mutants. Since Ala substitution caused IC(50) to decrease by more than 500 times, Phe(111) at position III:08/3.32 was found to be essential for GSK682753A inverse agonism. We have now presented the first ligand that targets EBI2. This molecule therefore functions as a powerful lead compound and a helpful tool for additional EBI2 characterization.
Physicochemical Properties
| Molecular Formula | C23H21CL3N2O3 |
| Molecular Weight | 479.7834 |
| Exact Mass | 478.06 |
| CAS # | 1334294-76-6 |
| PubChem CID | 126843232 |
| Appearance | White to light yellow solid powder |
| LogP | 5.2 |
| Hydrogen Bond Donor Count | 0 |
| Hydrogen Bond Acceptor Count | 3 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 31 |
| Complexity | 688 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | C1CN(CCC12CN(C(=O)O2)CC3=CC(=C(C=C3)Cl)Cl)C(=O)/C=C/C4=CC=C(C=C4)Cl |
| InChi Key | RDDLWMWIRXGSJM-XBXARRHUSA-N |
| InChi Code | InChI=1S/C23H21Cl3N2O3/c24-18-5-1-16(2-6-18)4-8-21(29)27-11-9-23(10-12-27)15-28(22(30)31-23)14-17-3-7-19(25)20(26)13-17/h1-8,13H,9-12,14-15H2/b8-4+ |
| Chemical Name | 8-[(E)-3-(4-chlorophenyl)prop-2-enoyl]-3-[(3,4-dichlorophenyl)methyl]-1-oxa-3,8-diazaspiro[4.5]decan-2-one |
| Synonyms | GSK-682753A; GSK682753A; GSK 682753A |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | EBI2 ( IC50 = 53.6 nM ) |
| ln Vitro | GSK682753 is a highly potent and selective inverse agonist that inhibits both G protein-dependent and likely G protein-independent signals for both human and murine EBI2. This compound has a potency of 2.6-53.6 nM and an inhibitory efficacy of 75% in guanosine5'-3-O-(thio)-triphosphate (GTPγS) binding assays and cAMP-response element-binding protein-based reporter assays. GSK682753A inhibits EBI2 with an IC50 of 53.6 nM in a dose-dependent manner. Similar potency is shown by GSK682753A's inhibition of ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation[1]. |
| Cell Assay | GSK682753A's impact on cAMP-induced CREB activation is quantified. When the transfection is stopped with a DMSO concentration following compound addition of 0.1%, GSK682753A is added at different concentrations. The LucLite substrate is used to measure the CREB activity 24 hours after transfection[1]. |
| References |
[1]. Ligand modulation of the Epstein-Barr virus-induced seven-transmembrane receptor EBI2: identification of a potent and efficacious inverse agonist. J Biol Chem. 2011 Aug 19;286(33):29292-302. |
Solubility Data
| Solubility (In Vitro) | DMSO: ≥ 100 mg/mL (~208.4 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.21 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 sonication. 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. Solubility in Formulation 2: 2.08 mg/mL (4.34 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.34 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0843 mL | 10.4214 mL | 20.8429 mL | |
| 5 mM | 0.4169 mL | 2.0843 mL | 4.1686 mL | |
| 10 mM | 0.2084 mL | 1.0421 mL | 2.0843 mL |