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| Targets |
7α,25-OHC's primary and most well-characterized target is the G protein-coupled receptor GPR183 (EBI2). The binding of 7α,25-OHC to GPR183 is of high affinity, with a Kd of 450 pM, and it acts as a potent agonist, activating the receptor with an EC50 of 140 pM. This interaction initiates downstream signaling pathways that are critical for immune cell function. GPR183 is a GPCR that couples to Gαi proteins, and its activation leads to a decrease in intracellular cyclic AMP (cAMP) levels. This signaling cascade directs the migration of B cells, T cells, and dendritic cells towards sources of 7α,25-OHC, which is crucial for the proper organization of lymphoid organs, including the positioning of B cells in follicles and germinal centers. The physiological importance of this interaction is highlighted by the fact that 7α,25-OHC is the only oxysterol that fully recapitulates physiological EBI2 signaling. Beyond its role in immune cell trafficking, GPR183 is also expressed in other tissues, and 7α,25-OHC-GPR183 signaling has been implicated in a range of biological processes, including cholesterol metabolism, inflammatory processes, and disease pathophysiology. The specificity of 7α,25-OHC for GPR183, and its inability to induce the thymocyte apoptosis seen with 25-HC, makes it an invaluable tool for specifically studying GPR183-mediated pathways.
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| ln Vitro |
At a half-maximum effective concentration of roughly 500 pM, 7α,25-dihydroxycholesterol (7α,25-OHC) externally increases the migration of B and T cells in EBI2-expressing animals, but has no impact on EBI2-deficient cells [1].
In vitro studies have established 7α,25-OHC as a highly potent and selective agonist for GPR183. Its activity is typically measured using a variety of cell-based functional assays. The most direct measure of its activity is its ability to activate GPR183, which is often assessed by measuring the decrease in intracellular cAMP levels, as GPR183 is a Gαi-coupled receptor. In these assays, 7α,25-OHC demonstrates an EC50 of 140 pM, confirming its picomolar potency. Another common assay is the measurement of receptor internalization or β-arrestin recruitment, which are downstream consequences of GPCR activation. Furthermore, 7α,25-OHC is used in chemotaxis assays to demonstrate its role as a chemoattractant. In these experiments, immune cells (such as B cells) are placed in a chamber, and a gradient of 7α,25-OHC is established. The migration of cells towards the higher concentration of the oxysterol is then quantified, confirming its function in directing immune cell movement. Importantly, in vitro studies also highlight the specificity of 7α,25-OHC. Unlike its precursor 25-HC, which can induce apoptosis in thymocytes, 7α,25-OHC does not exhibit this activity, making it a cleaner tool for studying GPR183-mediated effects without the confounding variable of cell death. These in vitro findings are critical for validating 7α,25-OHC as the primary endogenous ligand for GPR183. |
| ln Vivo |
Restoring 7α,25-dihydroxycholesterol (1 μM; 1.5 hours) makes normal or EBI2-deficient B cells less homing to splenic follicular regions, desensitizing them [1].
In vivo studies have validated the role of 7α,25-OHC as a critical regulator of immune cell migration and lymphoid tissue organization. The compound is an endogenous metabolite, and its physiological functions are studied by modulating its levels or by using GPR183 knockout mice. In vivo, 7α,25-OHC acts as a chemoattractant, directing the migration of B cells, T cells, and dendritic cells that express GPR183. This function is essential for the proper organization of lymphoid tissues, such as the positioning of B cells in follicles and germinal centers, which is crucial for effective humoral immune responses. The in vivo significance of the 7α,25-OHC-GPR183 axis has been demonstrated in various disease models. It has been shown to play a role in metabolic steatosis, neuroinflammation, and pulmonary inflammation, making it a potential therapeutic target for these conditions. Furthermore, because 7α,25-OHC lacks the thymocyte apoptosis activity of its precursor 25-HC, its in vivo effects are more specific to GPR183 signaling, allowing researchers to study the receptor's function without the confounding effects of cell death. These in vivo studies confirm that the 7α,25-OHC-GPR183 axis is a key regulator of the immune system and a promising target for therapeutic intervention. |
| Enzyme Assay |
The in vitro receptor binding and functional assays for 7α,25-OHC are designed to confirm its high-affinity interaction with GPR183 and to characterize its agonist activity. A standard receptor binding assay involves using membranes from cells that overexpress GPR183. Radiolabeled 7α,25-OHC or a labeled competitor is used to determine the binding affinity (Kd). In a typical protocol, increasing concentrations of unlabeled 7α,25-OHC are incubated with the receptor membranes and a fixed concentration of a radiolabeled ligand. After incubation, the bound and free ligand are separated, and the radioactivity is measured. The data is then analyzed using Scatchard or nonlinear regression analysis to determine the Kd, which for 7α,25-OHC is 450 pM. For functional assays, cells expressing GPR183 are used. One common method is to measure the inhibition of forskolin-stimulated cAMP accumulation, as GPR183 is a Gαi-coupled receptor. Cells are pre-incubated with 7α,25-OHC at various concentrations, then stimulated with forskolin to elevate cAMP levels. The amount of cAMP is then quantified using a competitive immunoassay. The EC50 for this response is 140 pM. Another functional assay measures receptor internalization or β-arrestin recruitment using techniques like bioluminescence resonance energy transfer (BRET) or enzyme fragment complementation. These assays provide robust and quantitative measures of 7α,25-OHC's potency and efficacy at GPR183.
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| Cell Assay |
In vitro cell-based assays for 7α,25-OHC are employed to study its role in immune cell function, particularly its chemoattractant properties. A primary assay is the chemotaxis assay, which directly measures the ability of 7α,25-OHC to induce cell migration. In a typical protocol, primary human or murine B cells, T cells, or dendritic cells are isolated and placed in the upper chamber of a transwell plate. The lower chamber contains media with a gradient of 7α,25-OHC at various concentrations. After a defined incubation period (e.g., 2-4 hours), the number of cells that have migrated to the lower chamber is quantified using flow cytometry or a cell counter. This assay demonstrates that 7α,25-OHC is a chemoattractant for immune cells expressing GPR183. Another assay used to study its activity is the measurement of intracellular calcium flux. When GPR183 is activated by 7α,25-OHC, it can lead to a transient increase in intracellular calcium in certain cell types. Cells are loaded with a calcium-sensitive dye (e.g., Fluo-4), and the fluorescence intensity is measured upon the addition of 7α,25-OHC. This provides a real-time readout of receptor activation. Additionally, to confirm the specificity of the response, cells can be pre-treated with a GPR183 antagonist or siRNA to knock down GPR183 expression. These cell-based assays are crucial for understanding the functional consequences of 7α,25-OHC-GPR183 interaction in a physiologically relevant context.
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| Animal Protocol |
In vivo animal experiments for 7α,25-OHC typically involve using mouse models to study its role in immune cell trafficking and disease. A standard protocol involves administering 7α,25-OHC to mice and then assessing the migration of immune cells. For example, labeled B cells can be adoptively transferred into recipient mice, and then 7α,25-OHC or a vehicle control is injected. The distribution of the labeled cells in various lymphoid organs is then analyzed by flow cytometry to determine if they have migrated towards the sites of 7α,25-OHC administration. Alternatively, GPR183 knockout mice are used to study the loss of function. In these models, the role of 7α,25-OHC in organizing lymphoid tissue is assessed by examining the structure of the spleen and lymph nodes and by measuring immune responses to antigens. The compound's role in disease models is also investigated. For example, in models of neuroinflammation or pulmonary inflammation, the levels of 7α,25-OHC are measured, and the effect of modulating its signaling (e.g., by using a GPR183 antagonist) on disease severity is assessed. These in vivo studies are essential for validating the physiological and pathological roles of the 7α,25-OHC-GPR183 axis.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of 7α,25-OHC are typical of an endogenous oxysterol. As a small, lipophilic molecule with a molecular weight of 418.7 g/mol and a molecular formula of C27H46O3, it is expected to be highly bound to plasma proteins and lipoproteins. Its biosynthesis is tightly regulated by the enzymes CH25H and CYP7B1, and its levels are controlled by local production and metabolism rather than by dietary intake. 7α,25-OHC is a potent and selective agonist for GPR183, with an EC50 of 140 pM and a Kd of 450 pM, indicating that it is active at very low concentrations. For research use, it is typically supplied as a solid powder and is soluble in DMSO. It is recommended to store it dry, in the dark, at 0-4°C for short-term use (days to weeks) or at -20°C for long-term storage (months to years). Its half-life in the circulation is expected to be short due to rapid metabolism and clearance, consistent with its role as a signaling molecule. As a research tool, its stability and handling are well-defined to ensure experimental reproducibility.
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| Toxicity/Toxicokinetics |
As an endogenous metabolite, 7α,25-OHC is not considered toxic. It is a naturally occurring molecule that plays a critical role in the immune system. Unlike its precursor 25-HC, which can induce apoptosis in thymocytes, 7α,25-OHC is specifically noted for lacking this activity, making it a more selective tool for studying GPR183 signaling without inducing cell death. This suggests that, at physiological concentrations, it is well-tolerated and does not cause general cytotoxicity. However, the compound's effects are highly context-dependent, as it directs the migration of immune cells. Dysregulation of the 7α,25-OHC-GPR183 axis has been implicated in various diseases, including metabolic steatosis, neuroinflammation, and pulmonary inflammation. This indicates that while the molecule itself is not a conventional toxin, its overproduction or aberrant signaling can contribute to pathology. For research use, it is handled as a standard laboratory chemical, with appropriate precautions to avoid inhalation, ingestion, or skin contact.
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| References | |
| Additional Infomation |
7α,25-Dihydroxycholesterol is a 7α-hydroxysteroid, 25-hydroxysteroid, oxosterol, and 3β-hydroxy-Δ5-steroid. It is a human metabolite and functionally related to cholesterol. There are reports and data regarding the presence of 7α,25-dihydroxycholesterol in the human body.
7α,25-Dihydroxycholesterol is a research tool and is not approved for any clinical or therapeutic use. It is the cognate, high-affinity ligand for the orphan GPCR GPR183 (EBI2) and is recognized as the only oxysterol that fully recapitulates physiological EBI2 signaling. Its primary application is in immunology research to study the migration of B cells, T cells, and dendritic cells. By activating GPR183, 7α,25-OHC directs immune cell trafficking, which is essential for the proper organization of lymphoid organs and the generation of effective humoral immune responses. Beyond immunology, it is also used as a research tool to study its involvement in cholesterol metabolism, inflammatory processes, and disease pathophysiology. Its role in metabolic steatosis, neuroinflammation, and pulmonary inflammation makes it a target of interest for drug discovery. The compound is commercially available as a high-purity reagent (≥98% by HPLC) and is supplied under cold-chain shipping conditions to ensure its stability. Its validated use in these pathways makes it an essential tool for researchers investigating immune function and related diseases. |
| Molecular Formula |
C27H46O3
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| Molecular Weight |
418.652348995209
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| Exact Mass |
418.344
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| CAS # |
64907-22-8
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| PubChem CID |
11954197
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| Appearance |
White to off-white solid powder
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| Density |
1.1±0.1 g/cm3
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| Boiling Point |
548.5±35.0 °C at 760 mmHg
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| Melting Point |
235 - 236 °C
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| Flash Point |
230.5±20.5 °C
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| Vapour Pressure |
0.0±3.3 mmHg at 25°C
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| Index of Refraction |
1.550
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| LogP |
5.75
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
30
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| Complexity |
669
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| Defined Atom Stereocenter Count |
9
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| SMILES |
C[C@H](CCCC(C)(C)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2[C@@H](C=C4[C@@]3(CC[C@@H](C4)O)C)O)C
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| InChi Key |
BQMSKLCEWBSPPY-IKVTXIKFSA-N
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| InChi Code |
InChI=1S/C27H46O3/c1-17(7-6-12-25(2,3)30)20-8-9-21-24-22(11-14-27(20,21)5)26(4)13-10-19(28)15-18(26)16-23(24)29/h16-17,19-24,28-30H,6-15H2,1-5H3/t17-,19+,20-,21+,22+,23-,24+,26+,27-/m1/s1
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| Chemical Name |
(3S,7S,8S,9S,10R,13R,14S,17R)-17-[(2R)-6-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol
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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 |
| 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 : ~4.55 mg/mL (~10.87 mM)
Ethanol :< 1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 5 mg/mL (11.94 mM) in 15% Cremophor EL 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3886 mL | 11.9432 mL | 23.8863 mL | |
| 5 mM | 0.4777 mL | 2.3886 mL | 4.7773 mL | |
| 10 mM | 0.2389 mL | 1.1943 mL | 2.3886 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.
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