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| Targets |
SR-4559 targets the protein-protein interaction between alpha/beta hydrolase domain-containing protein 5 (ABHD5, also known as CGI-58) and perilipin 1 (PLIN1). ABHD5 is a co-activator of adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride hydrolysis in adipocytes. In the basal state, ABHD5 is sequestered by PLIN1 on the surface of lipid droplets, preventing ATGL activation. Upon lipolytic stimulation, PLIN1 is phosphorylated by PKA, releasing ABHD5 to interact with and activate ATGL. SR-4559 binds to ABHD5 (as a ligand) and inhibits its binding to PLIN1, thereby mimicking the lipolytic stimulus and constitutively activating ATGL, even in the absence of hormonal stimulation. This leads to increased triglyceride hydrolysis and free fatty acid release. The compound operates within the lipolysis and lipid metabolism pathways. It is selective for the ABHD5-PLIN1 interaction.
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| ln Vitro |
In vitro, SR-4559 inhibits the ABHD5-PLIN1 protein-protein interaction with an IC50 of 510 nM in a biochemical TR-FRET (time-resolved fluorescence resonance energy transfer) assay. In functional lipolysis assays using primary mouse adipocytes or differentiated 3T3-L1 adipocytes, treatment with SR-4559 at concentrations of 1-20 uM for 2-24 hours increases glycerol release and free fatty acid levels in the medium by 2-5 fold, indicating enhanced lipolysis. The compound also increases ATGL activity measured by in vitro lipase assay using isolated lipid droplets or recombinant proteins. In muscle cells (C2C12 myotubes), SR-4559 similarly stimulates lipolysis of intracellular triglycerides. No significant cytotoxicity is observed in adipocytes at concentrations up to 50 uM. The compound does not activate PKA or other hormonal signaling pathways; its effect is direct on the ABHD5-PLIN1 complex. DMSO vehicle control (≤0.1%) is used. Positive control: forskolin (10 uM, activates PKA) or isoproterenol (1 uM, beta-adrenergic agonist).
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| ln Vivo |
In vivo, SR-4559 has been evaluated in mouse models of metabolic disease. In high-fat diet-induced obese mice, intraperitoneal administration of SR-4559 at 10-30 mg/kg once daily for 10-14 days reduces body weight gain and adipose tissue mass, and improves insulin sensitivity (as measured by glucose tolerance test and insulin tolerance test). In a mouse model of Chanarin Dorfman syndrome (ABHD5 deficiency), SR-4559 does not show efficacy because the target is absent. In wild-type mice, it increases plasma free fatty acid and glycerol levels, confirming target engagement. The compound also reduces hepatic steatosis in obese mice. Dosing: IP injection daily; formulated in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline. Body weight, food intake, and metabolic parameters are monitored. No significant toxicity reported at 30 mg/kg. For research use only.
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| Enzyme Assay |
For non-cellular TR-FRET protein-protein interaction assay, use purified recombinant ABHD5 (His-tagged) and PLIN1 (GST-tagged). In 384-well plates, add ABHD5 (50 nM), PLIN1 (50 nM), anti-GST-Tb (terbium-labeled antibody, 2 nM), and anti-His-d2 (acceptor fluorophore, 2 nM) in assay buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% BSA, 0.01% Tween 20). Add SR-4559 in 2-fold serial dilutions (0.1 nM to 100 uM, final DMSO 1%). Incubate for 2 h at 25degC. Measure TR-FRET signal at Ex 340 nm/Em 620 nm (donor) and 665 nm (acceptor). Calculate ratio (665/620). The signal decreases when the interaction is inhibited. IC50 is calculated from a 4-parameter logistic fit. Positive control: unlabeled ABHD5 or PLIN1 competitor. Negative control: DMSO only. All conditions in duplicate or triplicate.
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| Cell Assay |
For in vitro cell-based lipolysis assays, differentiate 3T3-L1 fibroblasts into adipocytes using standard protocol (insulin, dexamethasone, IBMX for 2 days, then insulin for 2-6 days). On day 8-10 post-differentiation, wash cells twice with warm PBS and incubate in Krebs-Ringer bicarbonate buffer (KRB) containing 2% fatty acid-free BSA and 5 mM glucose for 1 h. Then replace with fresh KRB/BSA buffer containing SR-4559 (1, 5, 10, 20 uM) or vehicle (0.1% DMSO) and incubate for 2-4 h. Collect medium, and measure glycerol content using a free glycerol reagent (colorimetric assay at 540 nm) and free fatty acids using a NEFA-C kit. Values are normalized to cellular protein content (BCA assay) or to cell number. For ATGL activity in cell lysates, treat cells with compound for 4 h, then lyse and measure ATGL activity using a fluorogenic triglyceride substrate (PED-A1) at Ex 485 nm/Em 535 nm. Positive control: forskolin (10 uM) or isoproterenol (1 uM). Negative control: DMSO. All experiments in triplicate.
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| Animal Protocol |
For in vivo studies, use male C57BL/6J mice (8-10 weeks, 20-25 g, n=10/group). For obesity studies, feed mice high-fat diet (60% kcal from fat) for 10-12 weeks prior to treatment. SR-4559 is formulated in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline to a concentration of 2-5 mg/mL. Administer intraperitoneally (IP) at 10, 20, or 30 mg/kg once daily for 14 days. Control group receives vehicle alone. Body weight and food intake are measured every 2-3 days. At the end of treatment, perform intraperitoneal glucose tolerance test (IPGTT): fast overnight, inject glucose (1 g/kg), measure tail blood glucose at 0, 15, 30, 60, 90, 120 min. For insulin tolerance test (ITT): fast 6 h, inject insulin (0.75 U/kg), measure glucose at 0, 15, 30, 60, 90, 120 min. After IPGTT, euthanize mice, collect epididymal white adipose tissue (eWAT), brown adipose tissue (BAT), and liver. Measure triglyceride content in adipose tissue and liver by Folch extraction and colorimetric assay. For histology, fix fat and liver in formalin, embed in paraffin, section, and stain with H&E. Adipocyte size measured by ImageJ. For plasma analysis, measure free fatty acids and glycerol using kits. Statistical analysis by one-way ANOVA or repeated measures ANOVA.
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| ADME/Pharmacokinetics |
No formal PK data reported for SR-4559. The compound is administered intraperitoneally in mouse studies (10-30 mg/kg, daily). IP bioavailability is likely high (near 100%). Half-life (t½) is not specified, but once-daily dosing suggests t½ of 2-6 hours. Volume of distribution (Vd) likely >1 L/kg. Clearance (CL) is probably hepatic. Protein binding not reported. Molecular weight: 456.51; LogP: 2.4 (moderate lipophilicity). Oral bioavailability has not been reported; IP is the preferred route in published studies. For storage, powder at -20degC for up to 3 years; solutions in DMSO at -80degC for 1 year. Formulation for in vivo: 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline. Solubility: DMSO (10 mM).
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| Toxicity/Toxicokinetics |
No formal toxicity data for SR-4559. In mouse studies at doses up to 30 mg/kg IP daily for 14 days, no significant body weight loss, behavioral changes, or mortality are reported. However, chronic activation of lipolysis could theoretically lead to increased circulating free fatty acids, which might cause lipotoxicity in non-adipose tissues (e.g., liver, pancreas). No such effects have been reported in short-term studies. Standard laboratory safety precautions: avoid inhalation, ingestion, skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a fume hood. Note: Very toxic to aquatic life with long-lasting effects (H410). For research use only-not for human use. Dispose of waste according to local hazardous waste regulations.
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| References | |
| Additional Infomation |
CAS: 847939-23-5. Molecular formula: C23H24N2O6S, molecular weight: 456.51. IUPAC name: 1-(4-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)sulfonyl)piperazin-1-yl)-2-(6-methylbenzofuran-3-yl)ethan-1-one. Synonyms: MLS000863888, SMR000068843. Purity typically >98% by HPLC. Appearance: powder. Solubility: DMSO. Storage: -20degC. Target: ABHD5-PLIN1 protein-protein interaction inhibitor. IC50: 510 nM. Research areas: metabolic diseases, Chanarin Dorfman syndrome, obesity, non-alcoholic fatty liver disease (NAFLD). Pathway: Lipolysis, lipid metabolism. Not for human use. For research only.
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| Molecular Formula |
C23H24N2O6S
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| Molecular Weight |
456.51
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| Exact Mass |
456.136
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| CAS # |
847939-23-5
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| PubChem CID |
2674365
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| Appearance |
Light yellow to light brown solid powder
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| Hydrogen Bond Donor Count |
0
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
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| Complexity |
778
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=CC2=C(C=C1)C(=CO2)CC(=O)N3CCN(CC3)S(=O)(=O)C4=CC5=C(C=C4)OCCO5
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| InChi Key |
HJVYLFAPJPGKEE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H24N2O6S/c1-16-2-4-19-17(15-31-21(19)12-16)13-23(26)24-6-8-25(9-7-24)32(27,28)18-3-5-20-22(14-18)30-11-10-29-20/h2-5,12,14-15H,6-11,13H2,1H3
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| Chemical Name |
1-[4-(2,3-dihydro-1,4-benzodioxin-6-ylsulfonyl)piperazin-1-yl]-2-(6-methyl-1-benzofuran-3-yl)ethanone
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1905 mL | 10.9527 mL | 21.9053 mL | |
| 5 mM | 0.4381 mL | 2.1905 mL | 4.3811 mL | |
| 10 mM | 0.2191 mL | 1.0953 mL | 2.1905 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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