| Size | Price | Stock | Qty |
|---|---|---|---|
| 50mg |
|
| Targets |
PPP1R15A (stress-induced regulatory subunit of protein phosphatase 1)
|
|---|---|
| ln Vitro |
Sephin1 (50 μM) selectively disrupted the PPP1R15A-PP1c complex in HeLa cells treated with 2.5 μg/ml tunicamycin, while sparing the PPP1R15B-PP1c complex, as shown by immunoprecipitation and immunoblotting. [1]
Sephin1 (50 μM) prolonged eIF2α phosphorylation on Ser51 after tunicamycin stress, delayed translation recovery (measured by 35S methionine incorporation), and reduced expression of the pro-apoptotic protein CHOP while prolonging ATF4 expression. [1] Sephin1 (50 μM) did not affect eIF2α signaling in the absence of stress. [1] Sephin1 did not inhibit the catalytic subunit PP1c (Figure S3). [1] Sephin1 protected wild-type cells from cytotoxic ER stress induced by tunicamycin in a dose-dependent manner (protection abolished in Ppp1r15a mutant cells, Figure 1F). [1] Sephin1 lacked measurable α2-adrenergic agonist activity in a cell-based assay using cells expressing recombinant human α2A adrenergic receptor, in contrast to guanabenz (GBZ). [1] |
| ln Vivo |
In MPZmutant mice (Charcot-Marie-Tooth 1B model), oral Sephin1 (1 mg/kg twice daily) for 3 months completely prevented motor defects detected by rotarod at 4 months of age. [1]
Sephin1 (1 mg/kg twice daily for 5 months) rescued myelin thickness around axons in sciatic nerves (measured by g-ratio) and reduced levels of ER stress markers (e.g., CHOP, BiP) in MPZmutant mice. [1] In SOD1G93A (SOD1mutant) ALS mouse model, oral Sephin1 (5 mg/kg once daily from 4 to 11 weeks of age) almost completely prevented progressive weight loss and motor deficits (rotarod analysis at 110 days of age). [1] Sephin1 (5 mg/kg once daily) prevented motor neuron loss (NeuN staining) in lumbar spinal cord of SOD1mutant mice. [1] Sephin1 prevented accumulation of insoluble SOD1mutant and decreased ER stress markers (CHOP, BiP) in transgenic spinal cords. [1] |
| Enzyme Assay |
Biotinylated Sephin1 selectively captured a recombinant fragment of PPP1R15A (amino acids 325-636) on neutravidin beads, but did not capture the related PPP1R15B fragment (amino acids 340-698), as revealed by Coomassie-stained gel analysis of input and bound fractions. [1]
Immunoprecipitation of PPP1R15 complexes from cell lysates treated with vehicle, 50 μM GBZ, or 50 μM Sephin1 for 6 hours was analyzed by immunoblotting to assess disruption of the PPP1R15A-PP1c interaction. Sephin1 selectively disrupted the stress-induced PPP1R15A-PP1c complex while sparing the constitutive PPP1R15B-PP1c complex. [1] |
| Cell Assay |
HeLa cells were treated with 2.5 μg/ml tunicamycin in the presence or absence of 50 μM Sephin1 for indicated times, then lysed and analyzed by immunoblotting for phosphorylated eIF2α (Ser51), CHOP, and ATF4. [1]
Newly synthesized proteins were labeled with 35S methionine in HeLa cells treated with tunicamycin with or without 50 μM GBZ or Sephin1; translation recovery was monitored by autoradiography and Coomassie-stained gel. [1] Cell viability protection assay: Wild-type or Ppp1r15a mutant (mut/mut) cells were treated with increasing doses of Sephin1 in the presence of 2.5 μg/ml tunicamycin; cell survival was measured to determine dose-dependent protection. Sephin1 protected wild-type but not Ppp1r15a mutant cells. [1] α2-adrenergic activity assay: Cells expressing recombinant human α2A adrenergic receptor were treated with GBZ or Sephin1 to measure agonist activity; Sephin1 showed no measurable activity. [1] |
| Animal Protocol |
Mice: MPZmutant mice (CMT1B model) and SOD1G93A (SOD1mutant) ALS model mice, as well as wild-type controls. [1]
For MPZmutant mice: Oral administration of Sephin1 at 1 mg/kg twice daily starting from postnatal day 28 to 61 for body weight monitoring, or for 3 months (rotarod) or 5 months (histology). [1] For SOD1mutant mice: Oral administration of Sephin1 at 5 mg/kg once daily from 4 to 11 weeks of age (body weight) or until 110 days of age (rotarod, histology). [1] Rotarod test: Mice were placed on an accelerating rotarod; performance was measured before and after treatment (1-5 mg/kg Sephin1). Sephin1 did not decrease rotarod performance. [1] Morris water maze: Mice treated with 1 mg/kg Sephin1 twice daily for 4 weeks were trained to locate a hidden platform over 5 consecutive days (5 trials/day); quadrant occupancy was measured after platform removal. [1] Fear conditioning: Mice treated with 1 mg/kg Sephin1 twice daily for 4 weeks underwent conditioning with light/tone (conditioned stimulus) and foot shock (unconditioned stimulus); freezing responses were measured during context and auditory cue testing. [1] |
| ADME/Pharmacokinetics |
After oral administration of Sephin1 at 1 or 10 mg/kg, the molecule rapidly disappeared from plasma. [1]
Sephin1 concentrated in the nervous system, reaching concentrations 7 to 44 times higher in the brain and sciatic nerve (up to ~1 μM) than in the plasma, similar to guanabenz. [1] Pharmacokinetic data were shown in Figure S5 (plasma disappearance and tissue distribution). [1] |
| Toxicity/Toxicokinetics |
Sephin1 (1 to 5 mg/kg) did not cause any decrease in rotarod performance in mice, in contrast to guanabenz which produced dose-dependent reductions. [1]
Chronic treatment with Sephin1 (1 mg/kg twice daily for 1 month) had no measurable adverse effects on body weight gain in mice. [1] Sephin1 did not impair spatial learning or memory in the Morris water maze; Sephin1-treated mice showed normal learning and memory. [1] Sephin1 did not affect fear conditioning responses (contextual and auditory cue testing). [1] Sephin1 lacked α2-adrenergic agonist activity, thus avoiding the central hypotensive and sedative side effects of guanabenz. [1] No adverse effects on general health or memory were observed in multiple experimental paradigms. [1] |
| References | |
| Additional Infomation |
Sephin1 (selective inhibitor of a holophosphatase) is a small molecule derived from guanabenz (GBZ) that safely and selectively inhibits the stress-induced PPP1R15A regulatory subunit of protein phosphatase 1 in vivo, sparing the constitutive PPP1R15B. [1]
By prolonging eIF2α phosphorylation, Sephin1 enhances the adaptive integrated stress response, increases availability of chaperones, and protects cells from otherwise lethal protein misfolding stress. [1] Sephin1 prevented two unrelated protein misfolding diseases in mice: Charcot-Marie-Tooth 1B (CMT1B) and amyotrophic lateral sclerosis (ALS). [1] This demonstrates that regulatory subunits of phosphatases can serve as drug targets to safely manipulate cellular proteostasis for therapeutic benefit. [1] |
| Molecular Formula |
C8H9N4CL
|
|---|---|
| Molecular Weight |
196.63
|
| Exact Mass |
196.052
|
| CAS # |
13098-73-2
|
| Related CAS # |
Icerguastat;951441-04-6
|
| PubChem CID |
9561611
|
| Appearance |
White to off-white solid powder
|
| Density |
1.35g/cm3
|
| Boiling Point |
374.2ºC at 760 mmHg
|
| Flash Point |
180.1ºC
|
| Vapour Pressure |
8.5E-06mmHg at 25°C
|
| Index of Refraction |
1.626
|
| LogP |
2.347
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
2
|
| Rotatable Bond Count |
2
|
| Heavy Atom Count |
13
|
| Complexity |
210
|
| Defined Atom Stereocenter Count |
0
|
| Chemical Name |
(2E)-2-[(2-Chlorophenyl)methylene]-hydrazinecarboximidamide
|
| Synonyms |
Sephin-1 Sephin 1 Sephin1
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| 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
|
|---|---|
| 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 | 5.0857 mL | 25.4285 mL | 50.8569 mL | |
| 5 mM | 1.0171 mL | 5.0857 mL | 10.1714 mL | |
| 10 mM | 0.5086 mL | 2.5428 mL | 5.0857 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
