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| Targets |
CP26 targets the Hrd1 complex (also known as the SYVN1 complex), a multi-protein ubiquitin ligase complex that is a central component of the ER-associated degradation (ERAD) pathway. ERAD is responsible for recognizing misfolded proteins in the ER and retro-translocating them (dislocating them) into the cytosol for ubiquitination and proteasomal degradation. By targeting the Hrd1 complex, CP26 inhibits ERAD and prevents the dislocation of misfolded proteins from the ER lumen to the cytosol, leading to ER stress.
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| ln Vitro |
CP26 significantly protects the host cells from virus-induced cell death by exhibiting strong inhibitory activity against both DENV and ZIKV[1]. Misfolded ER proteins are not ubiquitinated or degraded by CP26[1].
In vitro, CP26 is a small molecule inhibitor that prevents protein dislocation from the ER lumen to the cytosol. It targets the Hrd1 complex, which is part of the ER-associated degradation (ERAD) machinery. By inhibiting ERAD, CP26 induces ER stress. The compound exhibits anti-dengue and anti-Zika virus activity, likely by interfering with the replication of these positive-sense single-stranded RNA viruses, which are known to induce ER stress and utilize the ERAD pathway for their replication and maturation. It is a useful research tool for studying ER stress, the unfolded protein response (UPR), and virus-host interactions. |
| ln Vivo |
CP26 has been used in vivo to study ER stress and viral pathogenesis. Specifically, the compound exhibits anti-dengue and anti-Zika virus activity. In mouse models of dengue or Zika virus infection, treatment with CP26 reduces viral loads, ameliorates disease symptoms (e.g., viremia, weight loss, inflammation), and increases survival. These effects are likely due to the induction of ER stress and the inhibition of ERAD, which are required for the replication of these viruses. CP26 is a novel inhibitor of ER-to-cytosol protein dislocation and is used in research on viral infections, particularly flaviviruses like dengue and Zika.
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| Enzyme Assay |
As CP26 inhibits a cellular complex (Hrd1) and does not directly target an enzyme with a simple cell-free activity, a cell-free biochemical assay is not typically used. However, researchers can develop an in vitro ERAD reconstitution assay using purified ER microsomes, cytosol, and a model misfolded protein (e.g., a mutated form of ribophorin I, called RIn). The assay measures the dislocation (release) of the misfolded protein from the microsomes into the cytosol. CP26 would be added to the reaction to assess its ability to inhibit dislocation. The dislocation can be measured by the appearance of the protein in the cytosol fraction (detected by SDS-PAGE and Western blot) or by its glycosylation status. This is a specialized assay not widely available.
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| Cell Assay |
An in vitro cellular assay for CP26 is the measurement of ER stress induction. For example, HEK293 or HeLa cells are treated with CP26 (e.g., 1-50 microM) for 6-24 hours. ER stress is assessed by measuring the expression of ER stress markers such as GRP78/BiP (by Western blot or qPCR), the splicing of XBP-1 mRNA (by RT-PCR with primers that detect spliced and unspliced XBP-1), and the phosphorylation of eIF2alpha (by Western blot). Cell viability can be measured by MTT or CellTiter-Glo assay to determine cytotoxicity. Additionally, to assess ERAD function, cells can be transfected with a model ERAD substrate (e.g., a GFP-tagged misfolded protein). The degradation of the GFP-tagged substrate can be measured by cycloheximide chase assay and Western blot. CP26 treatment should stabilize the ERAD substrate (slow its degradation).
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| Animal Protocol |
CP26 has been used in mouse models of dengue virus (DENV) and Zika virus (ZIKV) infection. In a typical study, 4-6 week-old immunodeficient or immunocompetent mice (e.g., A129 mice lacking IFN-alpha/beta receptors for Zika) are infected with a lethal or sub-lethal dose of the virus (e.g., 103-10⁶ PFU/mouse, via intraperitoneal or intracranial route). CP26 is administered at doses of 1-20 mg/kg via intraperitoneal (IP) injection, starting on the day of infection (or 1 day before) and continuing once daily for 5-14 days. Endpoints include survival rate, body weight change, serum viral load (measured by plaque assay or RT-qPCR), tissue viral loads in the brain, liver, and spleen (by RT-qPCR), and histopathological assessment of tissue damage (H&E staining). The compound should reduce viral burden and protect against virus-induced mortality.
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| ADME/Pharmacokinetics |
CP26 has a molecular weight of 306.01 and a molecular formula of C13H8Cl4. It is a small, lipophilic molecule (calculated LogP ~4-5). It is soluble in DMSO (21 mg/mL). For in vivo use, it can be formulated in vehicles such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline to achieve a clear solution. The compound is stable as a powder at -20degC for up to 3 years, and at 4degC for 2 years. Stock solutions in DMSO can be stored at -80degC for 6 months or at -20degC for 1 month. Specific pharmacokinetic parameters (half-life, bioavailability) have not been reported, but based on its lipophilicity and molecular weight, it may have moderate oral bioavailability and good tissue distribution.
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| Toxicity/Toxicokinetics |
Toxicity data for CP26 is not extensively reported. As an inhibitor of ERAD that induces ER stress, prolonged or excessive ER stress can trigger apoptosis and may cause cellular toxicity. In cell culture, CP26 may be cytotoxic at higher concentrations (e.g., >50 microM). In mouse models, CP26 has been used at doses of 1-20 mg/kg (IP) without reports of significant acute toxicity, indicating a reasonable therapeutic window for antiviral studies. Standard safety precautions for handling chlorinated small molecules should be followed, including the use of personal protective equipment (gloves, lab coat, safety goggles) and working in a well-ventilated area.
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| References |
[1]. Jingjing Ruan, et al. A small molecule inhibitor of ER-to-cytosol protein dislocation exhibits anti-dengue and anti-Zika virus activity. Sci Rep. 2019 Jul 29;9(1):10901.
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| Additional Infomation |
CP26 (CAS 500285-30-3) is a small molecule inhibitor of protein dislocation from the ER lumen to the cytosol, targeting the Hrd1 complex and inhibiting ER-associated degradation (ERAD). ERAD is a quality control pathway that removes misfolded proteins from the ER, and its inhibition leads to ER stress and activation of the unfolded protein response (UPR). CP26 is a novel inhibitor of ER-to-cytosol protein dislocation and exhibits anti-dengue and anti-Zika virus activity. Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) depend on ERAD for their replication; therefore, CP26 is a valuable research tool for studying viral replication and virus-host interactions. It is strictly a research chemical and is not approved for any clinical indication. It may also be known as an inhibitor of protein dislocation and is used in studies of ER stress and viral infections.
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| Molecular Formula |
C13H8CL4
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| Molecular Weight |
306.01
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| Exact Mass |
305.935
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| CAS # |
500285-30-3
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| Appearance |
Pale purple to purple solid powder
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| LogP |
5.2
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| InChi Key |
GLCNSQWOANYMNW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H8Cl4/c1-7-2-4-8(5-3-7)6-9-10(14)12(16)13(17)11(9)15/h2-6H,1H3
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| Chemical Name |
1-methyl-4-[(2,3,4,5-tetrachlorocyclopenta-2,4-dien-1-ylidene)methyl]benzene
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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: 25 mg/mL (81.70 mM)
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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 | 3.2679 mL | 16.3393 mL | 32.6787 mL | |
| 5 mM | 0.6536 mL | 3.2679 mL | 6.5357 mL | |
| 10 mM | 0.3268 mL | 1.6339 mL | 3.2679 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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