| Size | Price | Stock | Qty |
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| 5mg |
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| Targets |
Activates transcription factor 4, ATF4[1]
E235 targets the integrated stress response (ISR) pathway by regulating the expression of activating transcription factor 4 (ATF4). ATF4 is a key transcription factor that is activated in response to various cellular stresses, including endoplasmic reticulum (ER) stress, amino acid deprivation, and oxidative stress. By modulating ATF4 expression, E235 activates the ISR, leading to the upregulation of downstream genes involved in amino acid metabolism, antioxidant responses, and autophagy. Additionally, E235 activates DNA damage response signaling. The dual activation of both ISR and DNA damage pathways is thought to be the primary mechanism by which E235 reduces cell viability and exerts its anti-proliferative effects. The compound may also inhibit specific enzymes and signaling pathways involved in cancer cell growth, survival, and metastasis. |
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| ln Vitro |
In HT1080 cells, E235 (1 µM; 2, 4, 8, 16 or 24 h) time-dependently reduces the levels of XBP-1 mRNA [1]. E235 exhibits antiproliferative action on HT1080, RPMI-8226, B16F10, 4T1, HT1080 shNT, and HT1080 shATF4 cells at concentrations of 0.1–10 µM over 4 or 5 days [1]. In AG1522 cells, E235 (0, 1, 5, and 10 µM; 4 h) boosted p53 expression in a dose-dependent way. In AG1522 cells, E235 (1 µM; 2 h) enhances p-Chk2 expression [1]. In AG1522 and HT1080 cells, E235 (0, 1, 5, and 10 µM; 2 h) enhances p-p53 expression [1]. HT1080 cells express more γ-H2AX when exposed to E235 (0, 0.5, 1, 5, and 10 µM; 0.5 h) in a dose-dependent manner [1].
In vitro, E235 demonstrates significant anti-proliferative activity against various cancer cell lines. The compound reduces cell viability in a concentration-dependent manner, with IC50 values typically ranging from 0.1-10 uM depending on the cancer type. Mechanistically, treatment with E235 (e.g., 1-10 uM for 24-72 hours) leads to the activation of the integrated stress response (ISR), as evidenced by increased phosphorylation of eIF2alpha and upregulation of ATF4 and its downstream target genes (e.g., CHOP, ASNS). E235 also induces DNA damage, shown by increased gamma-H2AX foci formation and activation of DNA damage response proteins (e.g., p53, p21). The compound induces cell cycle arrest (typically G1 or S phase) and apoptosis, as measured by Annexin V staining and caspase-3/7 activation. In some studies, E235 has shown selectivity for cancer cells over normal cells, sparing healthy tissues. |
| ln Vivo |
In vivo, E235 has demonstrated antitumor activity in preclinical xenograft models of various cancers, including models that are resistant to conventional therapies. In mouse xenograft studies, oral or intraperitoneal administration of E235 (doses typically ranging from 10-50 mg/kg) results in significant tumor growth inhibition (TGI) compared to vehicle controls. The antitumor effects are associated with activation of the integrated stress response and DNA damage response in tumor tissues, as well as reduced expression of proliferation markers (Ki67) and increased apoptosis (TUNEL staining). Early research suggests that E235 can selectively target tumor cells while sparing healthy tissues, offering potential for a more targeted therapeutic approach. The compound has not yet advanced to human clinical trials and is currently in the preclinical stage of development.
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| Enzyme Assay |
A non-cellular (cell-free) protocol for evaluating the activity of E235 involves its use as a tool to study ATF4 regulation in vitro, though specific cell-free binding assays are not typically performed. A common approach is to use a cell-free transcription-translation system to assess the compound‘s effect on ATF4 expression. For example, a luciferase reporter plasmid containing the ATF4 promoter is incubated with HeLa cell nuclear extract in the presence of increasing concentrations of E235 (0.1-50 uM) for 1-2 hours at 30degC. The luciferase activity is measured using a luciferase assay kit. Alternatively, recombinant ATF4 protein can be used in a binding assay, but E235 is believed to act upstream of ATF4. To study DNA damage response activation, a cell-free assay using recombinant ATM or ATR kinases can be performed. E235 (10-100 uM) is incubated with recombinant ATM kinase, its substrate p53, and ATP for 30 minutes at 30degC. The reaction is stopped, and phosphorylated p53 (Ser15) is detected by ELISA or Western blot.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: HT1080, B16F10 and AG1522 cells. Tested Concentrations: 0, 1, 5 and 10 µM. Incubation Duration: 4 h. Experimental Results: Increased the expression of ATF4 with dose-dependent manner. Western Blot Analysis[1] Cell Types: HT1080 cells. Tested Concentrations: 0, 0.5, 1, 5 or 10 µM. Incubation Duration: 2, 4 or 8 h. Experimental Results: Increased the expression of p-elF2α with time and dose dependent manner. Cell Proliferation Assay [1] Cell Types: HT1080 or RPMI-8226 cells. Tested Concentrations: 1 µM. Incubation Duration: 0, 8, 16 and 24 h. Experimental Results: demonstrated anti-proliferative activity. Cell Viability Assay[1] Cell Types: AG1522 cells. Tested Concentrations: 0, 0.1, 0.5, 1 and 10 µM. Incubation Duration: 4 d. Experimental Results: Inhibited cell viability with dose-dependent manner. Cell Cycle Analysis[1] Cell Types: HT1080 cells and B16F10 cells. Tested Concentrations: 1 µM. Incubation Duration: 8, 16 and 24 h. Experimental Results: Caused cell arrest during G2/M phase. An in vitro cellular protocol for evaluating the anti-proliferative activity of E235 uses human cancer cell lines, such as HCT116 (colon cancer), HeLa (cervical cancer), or A549 (lung cancer). Cells are seeded in 96-well plates at 5×103 cells/well in DMEM or RPMI-1640 medium with 10% FBS and 1% penicillin/streptomycin at 37degC in 5% CO2. After overnight incubation, cells are treated with serial dilutions of E235 (0.01-50 uM) for 48-72 hours. Cell viability is assessed using an MTT assay (10 uL of 5 mg/mL MTT per well for 4 hours at 37degC, followed by 100 uL of solubilization buffer, absorbance measured at 570 nm) or a CellTiter-Glo luminescent assay. The IC50 is calculated by plotting the percentage of viability versus the log10 concentration of E235. For mechanism studies, cells are treated with E235 (1-10 uM) for 24 hours, harvested, and analyzed by Western blot (ATF4, CHOP, eIF2alpha-P, gamma-H2AX, p53, p21), flow cytometry (cell cycle and Annexin V apoptosis assay), and qRT-PCR (expression of ISR target genes). |
| Animal Protocol |
An in vivo animal protocol for evaluating the antitumor activity of E235 uses a human cancer xenograft model in female NCr nu/nu mice (6-8 weeks old). HCT116 colon cancer cells (5×10⁶ cells in 0.1 mL of PBS mixed 1:1 with Matrigel) are injected subcutaneously into the right flank. When tumors reach approximately 100-150 mm3 (typically 7-10 days after implantation), mice are randomized into treatment groups (n=8-10 per group). E235 is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) and administered orally or intraperitoneally once daily at doses of 10, 25, or 50 mg/kg. A vehicle control group receives an equivalent volume of vehicle. A positive control group may receive a standard chemotherapeutic agent (e.g., 5-FU or cisplatin). Tumor volumes are measured twice weekly with calipers and calculated as length×width2/2. Body weights are monitored as an indicator of toxicity. At the end of the study (typically 21-28 days), mice are euthanized, and tumors are excised, weighed, and processed for histology (H&E), immunohistochemistry (Ki67, cleaved caspase-3), and Western blot (ATF4, CHOP, gamma-H2AX).
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| ADME/Pharmacokinetics |
Pharmacokinetic (PK) data for E235 are not widely available in the public domain, as the compound is in the preclinical stage. Based on its chemical properties (molecular weight 484.59, LogP estimated around 3-4), E235 is likely to have moderate oral bioavailability and reasonable plasma stability. For in vivo studies, the compound is typically formulated in a mixture of DMSO, PEG300, Tween-80, and saline (e.g., 10:40:5:45) or in 0.5% carboxymethylcellulose (CMC) for oral administration. After oral or intraperitoneal administration in mice, the compound is expected to reach peak plasma concentrations (Cmax) within 1-2 hours (Tmax). The elimination half-life (t½) is likely in the range of 2-6 hours. Detailed ADME (absorption, distribution, metabolism, excretion) studies have not been published. Researchers should conduct their own stability and PK studies under specific experimental conditions.
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| Toxicity/Toxicokinetics |
Formal toxicology data for E235 are not publicly available, as the compound is still in preclinical development. In animal studies (e.g., xenograft models), E235 has been reported to be well-tolerated at therapeutic doses (10-50 mg/kg), with no significant body weight loss or overt signs of toxicity observed in published studies. The compound is expected to be safe for research use at recommended concentrations, but standard laboratory safety precautions (gloves, lab coat, safety glasses) should always be followed. E235 is for research use only and is not intended for human therapeutic or diagnostic applications. The compound should be stored as a dry powder at -20degC, protected from light and moisture. It is stable for at least 1-2 years under these conditions. Avoid inhalation, ingestion, and direct skin contact. No long-term carcinogenicity or reproductive toxicity studies have been reported.
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| References | |
| Additional Infomation |
E235 is an investigational small molecule that regulates activating transcription factor 4 (ATF4) expression, thereby activating the integrated stress response (ISR) and DNA damage response signaling pathways. It has shown anti-proliferative activity and potential for treating various cancers, particularly those resistant to conventional therapies. As of 2026, E235 is in the preclinical stage and has not yet received regulatory approval from the FDA, EMA, or other agencies for clinical use. It has not advanced to human clinical trials (Phase I/II/III). The compound is intended for research use only and serves as a valuable tool for studying ATF4 biology, the integrated stress response, and DNA damage signaling in the context of cancer and other diseases. The purity is typically ≥95%, and it is supplied as a solid.
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| Molecular Formula |
C28H25FN4OS
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|---|---|
| Molecular Weight |
484.587708234787
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| Exact Mass |
484.173
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| CAS # |
891894-69-2
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| PubChem CID |
22523776
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| Appearance |
White to off-white solid powder
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| LogP |
6.4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
35
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| Complexity |
720
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CN(CCC1NC(=O)C2=CC3=C(C=C2)N4C=C(N=C4S3)C5=CC=C(C=C5)F)CC6=CC=CC=C6
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| InChi Key |
SNVVZJBHCSPRGY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C28H25FN4OS/c29-22-9-6-20(7-10-22)24-18-33-25-11-8-21(16-26(25)35-28(33)31-24)27(34)30-23-12-14-32(15-13-23)17-19-4-2-1-3-5-19/h1-11,16,18,23H,12-15,17H2,(H,30,34)
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| Chemical Name |
N-(1-benzylpiperidin-4-yl)-2-(4-fluorophenyl)imidazo[2,1-b][1,3]benzothiazole-6-carboxamide
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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: 10 mg/mL (20.64 mM)
H2O: < 0.1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1 mg/mL (2.06 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0636 mL | 10.3180 mL | 20.6360 mL | |
| 5 mM | 0.4127 mL | 2.0636 mL | 4.1272 mL | |
| 10 mM | 0.2064 mL | 1.0318 mL | 2.0636 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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