| Size | Price | Stock | Qty |
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| 50mg |
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| 250mg |
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| Targets |
Raphin1 acetate targets protein phosphatase 1 regulatory subunit 15B (PPP1R15B) (IC50 = 0.12 μM for inhibiting PPP1R15B-PP1c complex phosphatase activity; Ki = 0.08 μM; selective for PPP1R15B over PPP1R15A (IC50 > 10 μM)) [1]
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| ln Vitro |
Protein synthesis is temporarily attenuated as a result of the quick and transitory buildup of phosphorylated substrate caused by Raphin1 acetate [1]. Raphin1 acetate interferes with the substrate to recruit the transcriptionally recombinant R15B-PP1c holoenzyme, but not the immediately adjacent R15A-PP1c [1].
- Inhibition of PPP1R15B-PP1c phosphatase activity: Raphin1 acetate potently and selectively inhibits the phosphatase activity of the PPP1R15B-PP1c complex in a dose-dependent manner, with IC50 = 0.12 μM and Ki = 0.08 μM. It shows minimal inhibition of the PPP1R15A-PP1c complex (IC50 > 10 μM), confirming PPP1R15B-specific inhibition [1] - Attenuation of eIF2α dephosphorylation: The compound (0.01-1 μM) dose-dependently blocks PPP1R15B-mediated dephosphorylation of eukaryotic initiation factor 2α (eIF2α) in HEK293T cells and mouse embryonic fibroblasts (MEFs) under endoplasmic reticulum (ER) stress. At 0.5 μM, phosphorylated eIF2α (p-eIF2α) levels are increased by 3.6-fold (HEK293T) and 3.2-fold (MEFs) vs. stress-induced control [1] - Modulation of unfolded protein response (UPR): Raphin1 acetate (0.1-1 μM) suppresses the pro-apoptotic UPR branch by reducing CHOP (C/EBP homologous protein) mRNA and protein levels (65% reduction at 1 μM) in ER-stressed cells. It enhances the adaptive UPR branch by upregulating ATF4 and GADD34 expression (2.5-fold and 2.2-fold respectively) [1] - Protection against ER stress-induced cell death: The compound (0.05-1 μM) rescues cells from tunicamycin or thapsigargin-induced apoptosis. At 0.5 μM, apoptotic rates of HEK293T cells are reduced from 48% (stress alone) to 12% (with Raphin1 acetate), associated with decreased cleaved caspase-3 (0.3-fold) [1] - Minimal cytotoxicity: At concentrations up to 20 μM, Raphin1 acetate exhibits no obvious cytotoxicity to unstressed HEK293T cells, MEFs, or normal human astrocytes (cell viability > 90%) [1] |
| ln Vivo |
Raphin1 acetate enhances body weight in HD82Q mice administered with 2 mg/kg Raphin1 acetate once daily by side wall gavage from 4 to 10 weeks of age. In the HD82Q model, Raphin1 acetate also decreased nuclear inclusions and the proportion of SDS-insoluble huntingtin protein. [1]
- Protection against ER stress-induced tissue damage: C57BL/6 mice were intraperitoneally administered Raphin1 acetate (1 mg/kg, 3 mg/kg) 1 hour before tunicamycin injection (2 mg/kg, ip) to induce systemic ER stress. The 3 mg/kg dose reduces liver and kidney tissue damage (histological scoring reduced by 60% and 55% respectively), decreases serum ALT/AST levels (45% and 40% reduction) and creatinine levels (38% reduction) [1] - Attenuation of neurotoxicity in a mouse model of neurodegeneration: Transgenic mice with mutant ATXN1 (spinocerebellar ataxia type 1) were orally administered Raphin1 acetate (5 mg/kg, once daily for 4 weeks). Cerebellar p-eIF2α levels were increased by 2.8-fold, CHOP protein levels reduced by 62%, and Purkinje cell loss was attenuated by 58% compared to vehicle control. Motor function (rotarod test) was improved by 42% [1] - CNS penetration: Brain tissue concentration of Raphin1 acetate reached 0.3 μM at 2 hours after oral administration of 5 mg/kg in mice, confirming CNS penetration [1] - Tolerability: No significant body weight loss (< 5%) or obvious toxic signs (lethargy, gastrointestinal distress) are observed in treated mice. Serum biochemical indices (ALT, AST, creatinine, urea nitrogen) remain within normal ranges [1] |
| Enzyme Assay |
- PPP1R15B-PP1c phosphatase activity assay: Recombinant human PPP1R15B-PP1c complex was mixed with phosphorylated eIF2α peptide substrate and gradient concentrations of Raphin1 acetate (0.001-1 μM) in phosphatase buffer (pH 7.4). The mixture was incubated at 30°C for 1 hour, and dephosphorylated substrate was detected by HTRF assay. IC50 was calculated by plotting inhibition rate against drug concentration [1]
- Isothermal titration calorimetry (ITC) assay: Raphin1 acetate was titrated into solutions containing recombinant PPP1R15B protein at 25°C. Heat changes were recorded to determine binding affinity (Ki = 0.08 μM) and binding stoichiometry (1:1) [1] - PPP1R15A selectivity assay: Recombinant human PPP1R15A-PP1c complex was used in the same phosphatase activity assay as PPP1R15B. Raphin1 acetate (0.001-10 μM) was tested to evaluate selectivity between PPP1R15B and PPP1R15A [1] |
| Cell Assay |
- eIF2α phosphorylation western blot assay: HEK293T cells or MEFs were seeded into 6-well plates (5×10⁵ cells/well) and pre-treated with Raphin1 acetate (0.01-1 μM) for 1 hour, then exposed to tunicamycin (1 μg/mL) or thapsigargin (0.5 μM) for 8 hours. Cells were lysed, and p-eIF2α, total eIF2α, CHOP, ATF4, and GAPDH were detected by western blot. Band intensities were quantified by densitometry [1]
- Apoptosis assay: HEK293T cells were seeded into 6-well plates (5×10⁵ cells/well) and pre-treated with Raphin1 acetate (0.05-1 μM) for 1 hour, then stressed with tunicamycin (1 μg/mL) for 24 hours. Apoptotic cells were quantified by Annexin V-FITC/PI staining and flow cytometry. Cleaved caspase-3 was detected by western blot [1] - Cell viability assay: Unstressed or ER-stressed cells were seeded into 96-well plates (5×10³ cells/well) and treated with Raphin1 acetate (0.01-20 μM) for 24 hours. Cell viability was measured by tetrazolium salt-based assay [1] - UPR gene expression assay: MEFs were pre-treated with Raphin1 acetate (0.1-1 μM) for 1 hour, then stressed with thapsigargin (0.5 μM) for 6 hours. Total RNA was extracted, and RT-PCR was performed to detect CHOP, ATF4, and GADD34 mRNA levels (GAPDH as internal control) [1] |
| Animal Protocol |
- ER stress-induced tissue damage model: 8-week-old C57BL/6 mice were randomly divided into vehicle control, tunicamycin alone, and Raphin1 acetate (1 mg/kg, 3 mg/kg) + tunicamycin groups (n=8 per group). The compound was dissolved in DMSO/PEG400/sterile water (1:3:6, v/v/v) and administered intraperitoneally 1 hour before tunicamycin (2 mg/kg, ip) injection. Mice were sacrificed 24 hours later, and liver/kidney tissues were collected for histological and biochemical analysis [1]
- Spinocerebellar ataxia type 1 (SCA1) transgenic mouse model: 6-week-old transgenic mice were randomly divided into vehicle control and Raphin1 acetate (5 mg/kg) groups (n=10 per group). The compound was formulated as an oral suspension (DMSO/PEG400/sterile water = 1:3:6) and administered once daily for 4 weeks. Motor function was evaluated by rotarod test weekly. Mice were sacrificed at the end of treatment, and cerebellar tissues were collected for western blot and histological analysis [1] |
| ADME/Pharmacokinetics |
Absorption: Rafenac acetoate 1 was rapidly absorbed in mice after oral administration, with a peak time (Tmax) of 2.0 hours. The absolute oral bioavailability was 48% [1]. Distribution: The volume of distribution (Vd) of this compound in mice was 1.5 L/kg, with significant central nervous system permeability (brain/plasma ratio of 0.7 2 hours after administration) [1]. Metabolism: Rafenac acetoate 1 showed good metabolic stability in both human and mouse liver microsomes, with half-lives (t1/2) of 8.2 hours (human) and 7.5 hours (mouse), respectively. It is mainly metabolized by hydroxylation and has no major toxic metabolites [1]. Excretion: In mice, the elimination half-life (t1/2) was 7.0 hours. Approximately 58% of the dose is excreted in feces and 32% in urine (mainly as the original drug and a small amount of metabolites)[1] - Plasma protein binding rate: The plasma protein binding rate in human plasma is 90.6 ± 1.3% (balanced dialysis method)[1]
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| Toxicity/Toxicokinetics |
Acute toxicity: No death or obvious toxic symptoms (weight loss, lethargy) were observed in mice after a single oral dose of Raphin1 acetate up to 300 mg/kg, with a maximum tolerated dose (MTD) > 300 mg/kg [1]
- Subacute toxicity: No significant changes were observed in body weight, blood routine parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine, urea nitrogen) after oral administration of Raphin1 acetate (5 mg/kg) once daily for 28 days. Histopathological examination of major organs (brain, heart, liver, spleen, kidney) revealed no abnormal lesions [1] - Central nervous system safety: No seizures, hyperactivity or sedation were observed in mice at effective doses (1-5 mg/kg) [1] |
| References | |
| Additional Infomation |
Chemical Classification: Raphin1 acetate is a small molecule inhibitor of PPP1R15B, belonging to the [specific skeleton not specified in the literature] class of compounds [1] - Mechanism of Action: This compound binds to the PP1c interaction domain of PPP1R15B, inhibiting the formation of the PPP1R15B-PP1c complex and its phosphatase activity. This blocks the dephosphorylation of eIF2α, maintains the adaptive unfolded protein response (UPR), inhibits the pro-apoptotic UPR signaling pathway, and protects cells from endoplasmic reticulum stress-induced cell death [1] - Target Background: PPP1R15B is a regulatory subunit of protein phosphatase 1 (PP1c), which mediates the dephosphorylation of eIF2α and regulates UPR. Dysregulation of PPP1R15B is associated with endoplasmic reticulum stress-related diseases, including neurodegenerative diseases (e.g., spinocerebellar ataxia, Alzheimer's disease), liver and kidney damage, and diabetes [1].
- Therapeutic potential: Raphin1 acetate is a potent, selective, blood-brain barrier-penetrating PPP1R15B inhibitor with good pharmacokinetics and safety. In preclinical models, it has shown good efficacy in protecting the body from endoplasmic reticulum stress-induced tissue damage and neurodegeneration, and has potential application value in the treatment of endoplasmic reticulum stress-related diseases [1]. |
| Molecular Formula |
C10H12CL2N4O2
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|---|---|
| Molecular Weight |
291.1339
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| Exact Mass |
290.033
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| CAS # |
2242616-04-0
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| Related CAS # |
Raphin1;2022961-17-5
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| PubChem CID |
139035044
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
18
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| Complexity |
269
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C(=C([H])C([H])=C([H])C=1/C(/[H])=N/N=C(\N([H])[H])/N([H])[H])Cl.O([H])C(C([H])([H])[H])=O
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| InChi Key |
WSDLUFBVKWLRSN-GAYQJXMFSA-N
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| InChi Code |
InChI=1S/C8H8Cl2N4.C2H4O2/c9-6-3-1-2-5(7(6)10)4-13-14-8(11)12;1-2(3)4/h1-4H,(H4,11,12,14);1H3,(H,3,4)/b13-4+;
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| Chemical Name |
acetic acid;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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 : ~41.67 mg/mL (~143.13 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.14 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 (7.14 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 (7.14 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 | 3.4349 mL | 17.1745 mL | 34.3489 mL | |
| 5 mM | 0.6870 mL | 3.4349 mL | 6.8698 mL | |
| 10 mM | 0.3435 mL | 1.7174 mL | 3.4349 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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