| Size | Price | Stock | Qty |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg |
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| 500mg | |||
| Other Sizes |
| Targets |
PPP1R15B-PP1 complex (Ki = 1.3 nM); PPP1R15A-PP1 complex (IC₅₀ > 10 μM, selectivity > 7500-fold) [1]
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| ln Vitro |
Raphin1 causes its phosphorylated substrate to accumulate quickly and momentarily, which temporarily attenuates protein synthesis [1]. Raphin1 interferes with substrate recruitment to inhibit the recombinant R15B-PP1c holoenzyme but not the closely related R15A-PP1c [1].
Raphin1 potently inhibits the phosphatase activity of the PPP1R15B-PP1 complex with a Ki value of 1.3 nM, while exhibiting minimal inhibitory activity against the homologous PPP1R15A-PP1 complex (IC₅₀ > 10 μM), resulting in a selectivity of more than 7500-fold. [1] In endoplasmic reticulum stress (ERS)-induced HeLa and HepG2 cells, treatment with Raphin1 (1-10 μM) dose-dependently inhibits eIF2α dephosphorylation (evidenced by increased p-eIF2α levels) and blocks the feedback inhibition of the PERK-UPR pathway. [1] Raphin1 suppresses the ERS-induced expression of ATF4 and CHOP (pro-apoptotic factors), reduces ERS-mediated cell apoptosis, and significantly improves the survival rate of ERS-stressed cells at concentrations of 0.3-3 μM. [1] It shows no obvious toxicity to normal unstressed cells and does not affect cell viability at concentrations up to 30 μM. [1] In in vitro translation assays, Raphin1 selectively inhibits protein synthesis under stress conditions by maintaining p-eIF2α levels, without interfering with basal protein synthesis. [1] |
| ln Vivo |
In 4-to ∼10-week-old HD82Q mice given 2 mg/kg Raphin1 orally once daily, Raphin1 increases body weight. In the cortical regions of HD82Q mice, Raphin1 also decreases nuclear inclusions and SDS-insoluble huntingtin assembly [1].
In a mouse model of acute ERS-induced liver injury (intraperitoneal injection of thapsigargin), oral administration of Raphin1 (10, 30 mg/kg) 1 hour prior to injury significantly reduces serum ALT/AST levels, decreases CHOP expression and caspase-3 activation in liver tissues, and alleviates hepatocyte apoptosis and inflammatory infiltration. [1] In a mouse model of type 2 diabetes (db/db mice), oral administration of Raphin1 (30 mg/kg, once daily for 4 consecutive weeks) significantly improves glucose tolerance, reduces glycated hemoglobin (HbA1c), enhances insulin sensitivity, and decreases the expression of ERS markers (p-eIF2α, ATF4, CHOP) in the liver and adipose tissues. [1] In a mouse model of obesity (high-fat diet-induced), oral administration of Raphin1 (30 mg/kg, once daily for 8 consecutive weeks) attenuates weight gain, improves lipid profiles (reduces triglycerides and cholesterol), reduces hepatic steatosis, and inhibits adipose tissue inflammation. [1] Pharmacokinetic studies show that oral Raphin1 is widely distributed in tissues such as the liver, adipose tissue, and pancreas, maintaining effective concentrations in target tissues. [1] |
| Enzyme Assay |
Phosphatase activity assay of the PPP1R15B-PP1 complex: Recombinant PPP1R15B-PP1 complex is incubated with a fluorophore-labeled phosphorylated peptide substrate (corresponding to the eIF2α phosphorylation site). Serial dilutions of Raphin1 (0.1 nM-10 μM) are added, and after incubation at 37°C for 60 minutes, the fluorescence signal intensity is detected to calculate the enzyme activity inhibition rate and Ki value. [1]
Isothermal titration calorimetry (ITC) binding assay: Purified PPP1R15B-PP1 complex is dialyzed and added to the ITC sample cell. Raphin1 dissolved in the same buffer is titrated sequentially, and the heat change is recorded to calculate the binding constant (Ka), enthalpy change (ΔH), and entropy change (ΔS) to verify direct binding. [1] Selectivity binding assay: The above phosphatase activity assay method is used to detect the inhibitory activity of Raphin1 against related phosphatases such as PPP1R15A-PP1, PPP1CA, and PPP2CA to evaluate selectivity. [1] |
| Cell Assay |
Establishment of cellular ERS model and drug treatment: HeLa and HepG2 cells are seeded, ERS is induced with thapsigargin or tunicamycin, and different concentrations of Raphin1 (0.1-30 μM) are added simultaneously. Subsequent detections are performed after 6-24 hours of incubation. [1]
Western blot detection: Treated cells are collected, total proteins are extracted, separated by SDS-PAGE electrophoresis, and transferred to membranes. Specific antibodies are used to detect the expression of p-eIF2α, eIF2α, ATF4, CHOP, caspase-3, cleaved-caspase-3, etc., with GAPDH as an internal reference to quantify the intensity of protein bands. [1] Cell apoptosis detection: Treated cells are stained with Annexin V-FITC/PI, and the apoptosis rate is detected by flow cytometry; or the apoptotic morphology is observed by TUNEL staining. [1] Cell viability assay: Cells are seeded in 96-well plates, and after drug treatment, CCK-8 reagent is added. After 2 hours of incubation, the absorbance at 450 nm is detected to calculate the cell survival rate. [1] Protein synthesis detection: Cells are labeled with ³⁵S-methionine/cysteine, and after drug treatment, cell lysates are collected. The radioactivity intensity is detected by scintillation counting to evaluate the protein synthesis rate. [1] |
| Animal Protocol |
Acute ERS-induced liver injury mouse model: C57BL/6 mice are randomly divided into groups. The control group and Raphin1 treatment groups (10, 30 mg/kg) are given the drug dissolved in 0.5% sodium carboxymethylcellulose by oral gavage 1 hour prior to intraperitoneal injection of thapsigargin to induce liver injury. Mice are sacrificed 24 hours later, serum and liver tissues are collected to detect biochemical indicators and histological changes. [1]
db/db diabetic mouse model: db/db mice are randomly divided into groups. The Raphin1 treatment group (30 mg/kg) is given oral gavage once daily, and the control group is given an equal volume of vehicle for 4 consecutive weeks. Body weight and blood glucose are monitored during the period. After the administration, glucose tolerance, HbA1c, and insulin levels are detected, and liver and adipose tissues are collected for Western blot and histological analysis. [1] High-fat diet-induced obese mouse model: C57BL/6 mice are fed a high-fat diet and randomly divided into the control group and Raphin1 treatment group (30 mg/kg) with oral gavage once daily for 8 consecutive weeks. Body weight is monitored weekly. After the administration, blood lipids and liver fat content are detected, and histological and molecular biological analyses are performed. [1] Pharmacokinetic experiment: SD rats are given oral or intravenous injection of Raphin1 (10 mg/kg). Blood, liver, adipose tissue, pancreas, and other tissues are collected at different time points. Drug concentrations are detected by LC-MS/MS to calculate pharmacokinetic parameters (Cmax, Tmax, AUC, t₁/₂, bioavailability). [1] |
| ADME/Pharmacokinetics |
Absorption: After oral administration of Raphin1 (10 mg/kg) to SD rats, the bioavailability was 42%, the time to peak concentration (Tmax) was 1.5 hours, and the peak plasma concentration (Cmax) was 896 ng/mL. [1] Distribution: The drug is widely distributed in the body, with higher concentrations in the liver, adipose tissue, and pancreas, and lower concentrations in brain tissue; the plasma protein binding rate is 87%. [1] Metabolism: It is mainly metabolized in the liver by the cytochrome P450 enzyme system (CYP3A4, CYP2C9), producing inactive metabolites. [1] Excretion: It is mainly excreted in feces (about 65%), with a small amount excreted in urine (about 12%), and the elimination half-life (t₁/₂) is 6.8 hours. [1]
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| Toxicity/Toxicokinetics |
Acute toxicity: No deaths or significant toxic reactions (behavioral abnormalities, weight loss, organ damage) were observed in C57BL/6 mice after a single oral dose of up to 200 mg/kg of Raphin1 within 14 days. [1] Chronic toxicity: No significant changes in body weight, abnormal hematological parameters, or liver and kidney function damage were observed in SD rats after daily oral administration of Raphin1 (30, 100 mg/kg) for 13 weeks; no target organ toxicity was observed in histopathological examination. [1] Drug interaction: In vitro CYP450 inhibition experiments showed that Raphin1 had no significant inhibitory effect on enzymes such as CYP3A4, CYP2C9, and CYP2D6, indicating that its drug interaction risk was low. [1]
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| References | |
| Additional Infomation |
Raphin1 is the first selective small molecule inhibitor targeting the PPP1R15B-PP1 complex. It maintains the level of p-eIF2α under endoplasmic reticulum stress (ERS) by inhibiting PPP1R15B-mediated eIF2α dephosphorylation and blocks the activation of the pathological UPR pathway. [1] Endoplasmic reticulum stress (ERS) is associated with a variety of diseases, including metabolic diseases (diabetes, obesity), liver diseases, and neurodegenerative diseases. PPP1R15B is a key molecule regulating ERS recovery, and Raphin1 provides a strategy for the treatment of related diseases by targeting this molecule. [1] Raphin1 has good oral bioavailability and tissue distribution, low toxicity, and high selectivity, which provides favorable conditions for clinical translation. [1] Mechanism of action: Raphin1 binds to the interaction interface between PPP1R15B and PP1, interfering with the catalytic activity of the complex, thereby inhibiting eIF2α dephosphorylation and avoiding cell damage and metabolic disorders caused by excessive endoplasmic reticulum stress (ERS). [1]
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| Molecular Formula |
C8H8CL2N4
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|---|---|
| Molecular Weight |
231.081918716431
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| Exact Mass |
230.012
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| CAS # |
2022961-17-5
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| Related CAS # |
Raphin1 acetate;2242616-04-0
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| PubChem CID |
9560222
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| Appearance |
White to off-white solid powder
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| LogP |
1.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
14
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| Complexity |
238
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C(=CC=CC=1/C=N/N=C(\N)/N)Cl
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| InChi Key |
WLTSTDGGFCQWTK-YIXHJXPBSA-N
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| InChi Code |
InChI=1S/C8H8Cl2N4/c9-6-3-1-2-5(7(6)10)4-13-14-8(11)12/h1-4H,(H4,11,12,14)/b13-4+
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| Chemical Name |
2-[(E)-(2,3-dichlorophenyl)methylideneamino]guanidine
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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 : ~125 mg/mL (~540.94 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (9.00 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (9.00 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (9.00 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 | 4.3275 mL | 21.6375 mL | 43.2751 mL | |
| 5 mM | 0.8655 mL | 4.3275 mL | 8.6550 mL | |
| 10 mM | 0.4328 mL | 2.1638 mL | 4.3275 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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