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Purity: ≥98%
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.
| Targets |
EBI2 ( IC50 = 53.6 nM )
Epstein-Barr virus-induced seven-transmembrane receptor EBI2 (GPR183) (Ki = 3 nM for [³H]-7α,25-dihydroxycholesterol binding inhibition) [1] |
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| 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].
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| Enzyme Assay |
1. Radioligand binding assay for EBI2:
- Membrane preparations were generated from HEK293 cells stably expressing human EBI2 and resuspended in binding buffer containing MgCl₂ and NaCl to maintain receptor integrity. - Serial concentrations of GSK682753A (0.01–100 nM) were pre-incubated with the membrane preparations for 30 minutes at 25°C to allow potential binding. - [³H]-7α,25-dihydroxycholesterol (1 nM, the radiolabeled endogenous ligand) was added to each reaction, and incubation continued for 60 minutes at 25°C to reach binding equilibrium. - Bound and free ligand were separated by rapid filtration through glass fiber filters pre-soaked in binding buffer. Filters were washed three times with ice-cold buffer to remove unbound ligand. - Radioactivity retained on the filters was measured using a liquid scintillation counter. The percentage of specific binding (relative to vehicle control) was plotted against the log concentration of GSK682753A, and the Ki value was calculated using the Cheng-Prusoff equation based on the IC₅₀ from competition curves [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].
1. Calcium influx assay: - HEK293 cells stably expressing human EBI2 were seeded in 96-well black-walled plates at a density of 2×10⁴ cells/well and cultured overnight at 37°C with 5% CO₂. - Cells were loaded with a calcium-sensitive fluorescent probe (Fluo-4 AM) in Hank's balanced salt solution (HBSS) containing 20 mM HEPES for 60 minutes at 37°C, then washed twice to remove excess probe. - Serial concentrations of GSK682753A (0.01–100 nM) were added to the cells, and pre-incubated for 30 minutes at 37°C. - EBI2 agonist (7α,25-dihydroxycholesterol, 10 nM) was added to trigger calcium release, and fluorescence intensity (excitation 485 nm, emission 525 nm) was measured in real-time using a microplate reader. - The maximum fluorescence response in each well was normalized to the vehicle control (100% response) and blank wells (0% response), and the IC₅₀ was derived from the concentration-response curve [1] 2. Cell chemotaxis assay: - EBI2-transfected HEK293 cells were harvested, resuspended in serum-free RPMI 1640 medium, and adjusted to a density of 5×10⁵ cells/mL. - Serial concentrations of GSK682753A (0.01–100 nM) were mixed with the cell suspension and pre-incubated for 30 minutes at 37°C. - The cell-drug mixture was added to the upper chamber of Transwell inserts (5 μm pore size), and the lower chamber was filled with RPMI 1640 medium containing 10% fetal bovine serum and CCL19 (10 nM, chemotactic agonist). - Plates were incubated for 2 hours at 37°C with 5% CO₂ to allow cell migration. - Non-migrated cells in the upper chamber were removed with a cotton swab, and migrated cells attached to the lower surface of the insert were fixed with 4% paraformaldehyde and stained with crystal violet. - Stained cells were counted under a light microscope (five random fields per insert), and the percentage of migration inhibition was calculated relative to vehicle-treated controls. The IC₅₀ was determined from the concentration-response curve [1] |
| References | |
| Additional Infomation |
1. Drug classification and structure: GSK682753A is a synthetic small molecule belonging to the pyrazolopyrimidine class of compounds, specifically designed as a selective EBI2 inverse agonist/antagonist [1] 2. Mechanism of action: GSK682753A binds to the orthoside of EBI2 and competes with endogenous ligands (7α,25-dihydroxycholesterol, CCL19) for receptor binding sites. This interaction blocks ligand-induced receptor activation and reduces constitutive EBI2 signaling, thereby inhibiting downstream pathways involved in immune cell migration and localization [1] 3. Biological significance: EBI2 (GPR183) is mainly expressed in immune cells (B cells, T cells, dendritic cells) and plays a key role in regulating the migration of immune cells to inflammatory sites. The inhibitory effect of GSK682753A on EBI2 suggests that it may have potential application value in the treatment of autoimmune diseases, allergic diseases and cancer by regulating abnormal immune cell recruitment [1].
4. Research application: GSK682753A can be used as a valuable chemical probe to study the biological characteristics of EBI2, thereby enabling the study of EBI2-mediated signaling pathways and immune cell dynamics in preclinical models [1]. |
| Molecular Formula |
C23H21CL3N2O3
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|---|---|
| Molecular Weight |
479.7834
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| Exact Mass |
478.06
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| CAS # |
1334294-76-6
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| PubChem CID |
126843232
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| Appearance |
White to light yellow solid powder
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| LogP |
5.2
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
31
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| Complexity |
688
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CN(CCC12CN(C(=O)O2)CC3=CC(=C(C=C3)Cl)Cl)C(=O)/C=C/C4=CC=C(C=C4)Cl
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| InChi Key |
RDDLWMWIRXGSJM-XBXARRHUSA-N
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| 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
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| Synonyms |
GSK-682753A; GSK682753A; GSK 682753A
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO: ≥ 100 mg/mL (~208.4 mM)
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| 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. View More
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. |
| 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 |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 Structure of the EBI2-selective inverse agonist GSK682753A.J Biol Chem.2011 Aug 19;286(33):29292-302. |
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 GSK682753A does not affect cell surface expression (A) or constitutive internalization of EBI2 (B–D).J Biol Chem.2011 Aug 19;286(33):29292-302. |
 A, EBI2 is constitutively active via ERK.
Overexpression of EBI2 increases basal B cell migration and potentiates antibody-induced B cell proliferation. |
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