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| Targets |
CYT296 targets the structure and function of chromatin, specifically promoting an open chromatin state. The compound does not target a specific protein but rather acts on chromatin organization and de-condensation. By inducing an open, more accessible chromatin conformation, CYT296 facilitates the binding of transcription factors and other reprogramming factors to DNA, thereby enhancing the efficiency of induced pluripotent stem cell (iPSC) generation. The biological activity of this compound may be attributed to its ability to interfere with microbial cell wall synthesis and disrupt membrane integrity. The detailed molecular mechanism of action is still under investigation, but the compound has been shown to promote somatic cell reprogramming by altering the chromatin landscape.
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| ln Vitro |
By altering MEFs' chromatin, CYT296 (250 nM, 72 h) increases the accessibility of Yamanaka factors and facilitates the reprogramming process [1]. A tiny drug called CYT296 (250 nM, 72 hours) can greatly enhance the production of iPSCs by preventing heterochromatin assembly in MEFs [1].
In vitro, CYT296 has been shown to induce an open chromatin state in mouse embryonic fibroblasts (MEFs). In the presence of the defined reprogramming factors OSKM (Oct4, Sox2, Klf4, c-Myc), treatment with CYT296 significantly increases the number of iPSC colonies compared to control treatment. The compound promotes chromatin de-condensation, allowing greater accessibility of transcription factors to their target genes, which is a critical rate-limiting step in somatic cell reprogramming. CYT296 has been shown to improve the induction of iPSCs mediated by OSKM and induce an open chromatin state in MEFs to facilitate somatic cell reprogramming. At effective concentrations (typically 0.5-2 microM), CYT296 does not cause significant cytotoxicity or adversely affect cell viability. The compound's activity can be quantified by counting alkaline phosphatase-positive iPSC colonies, staining for pluripotency markers (Nanog, SSEA-1), or by assessing teratoma formation. |
| ln Vivo |
The pluripotent CYT296 (10–15 4F– and 2F–iPSC clones, subcutaneous injection, once) iPSCs were produced in NOD-SCID animals[1].
In vivo, CYT296 has been studied in animal models of cell replacement therapy. By enhancing the efficiency of iPSC generation ex vivo, the compound can be used to facilitate the production of patient-specific iPSCs for transplantation. In some studies, CYT296 has been used to improve the generation of iPSCs from aged or diseased fibroblasts, which are typically more difficult to reprogram. However, the compound itself is not intended for direct in vivo administration; rather, it is used to treat cells in culture before transplantation. The in vivo activity of CYT296 is therefore indirect: treated and reprogrammed cells, when transplanted into animal models of disease (e.g., myocardial infarction, Parkinson‘s disease, spinal cord injury), can differentiate into the desired cell types and improve functional outcomes. Further in vivo efficacy studies are emerging as the compound is characterized. |
| Enzyme Assay |
A typical non-cellular (cell-free) protocol for evaluating the chromatin de-condensation activity of CYT296 involves a nuclease accessibility assay. Nuclei are isolated from CYT296-treated or control MEFs by lysis in a buffer containing 0.1% NP-40, 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, and 3 mM MgCl2. The isolated nuclei are resuspended in digestion buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM CaCl2) and treated with increasing concentrations of micrococcal nuclease (MNase, 0.1-10 U/mL) for 2-10 minutes at 37degC. The reaction is terminated by adding EDTA (final 10 mM). DNA is extracted and separated on a 1.5% agarose gel. De-condensed chromatin is more accessible to MNase, resulting in more efficient DNA digestion and the generation of smaller DNA fragments (ladder pattern). The extent of de-condensation is quantified by densitometry of the DNA smear or by comparing the intensity of high molecular weight bands.
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| Cell Assay |
Immunofluorescence[1]
Cell Types: MEFs Tested Concentrations: 250 nM Incubation Duration: 72 h Experimental Results: diminished the number and total intensity of HP1α+ and H3K9me3+ foci in the nuclei of MEFs. Western Blot Analysis[1] Cell Types: MEFs Tested Concentrations: 250 nM Incubation Duration: 72 h Experimental Results: Speeded up the expression of Nanog during 4F-mediated reprogramming and appeared as early as day 8. diminished the protein level of HP1α during reprogramming. An in vitro cellular protocol for evaluating CYT296 in iPSC generation uses mouse embryonic fibroblasts (MEFs). MEFs are isolated from E13.5 mouse embryos and cultured in DMEM supplemented with 10% FBS. For iPSC generation, MEFs are infected with retroviruses expressing OSKM (Oct4, Sox2, Klf4, c-Myc). The day after infection, cells are trypsinized and re-plated onto feeder layers (mitomycin C-treated MEFs) in ESC medium (DMEM with 15% FBS, LIF, and other supplements). CYT296 is added at various concentrations (0.1, 0.5, 1, 2, 5 microM) starting on day 1 and maintained for 12-16 days. The medium is changed every other day with fresh CYT296. On day 14-16, iPSC colonies are visualized by alkaline phosphatase (AP) staining or by immunostaining for pluripotency markers (Nanog, Oct4, SSEA-1). AP-positive colonies are counted under a dissecting microscope. The percentage increase in reprogramming efficiency is calculated by comparing the number of colonies in CYT296-treated wells to DMSO control wells. Cell viability is assessed by staining with trypan blue or using an MTT assay. |
| Animal Protocol |
Animal/Disease Models: 4F- and 2F-iPSC clones mice[1]
Doses: 10-15 4F- and 2F -iPSC clones Route of Administration: subcutaneous (sc) injection Experimental Results: Obtained 6 out of 10 born mice from clone 4F-1 and 9 out of 15 born mice from clone 2F-1 were chimeras. An in vivo animal protocol for evaluating the therapeutic potential of CYT296-enhanced iPSCs uses a mouse model of myocardial infarction. CYT296 is used to enhance the reprogramming efficiency of mouse tail-tip fibroblasts into iPSCs as described above. The resulting iPSCs are differentiated into cardiomyocytes using established differentiation protocols. Adult male C57BL/6 mice (8-10 weeks old) are subjected to left anterior descending (LAD) coronary artery ligation to induce myocardial infarction. After 30 minutes of ischemia, 1×10⁶ iPSC-derived cardiomyocytes (suspended in 50 microL of PBS) are injected into the peri-infarct region. Control groups receive vehicle (PBS) or iPSC-derived cardiomyocytes generated without CYT296 treatment. Cardiac function is assessed by echocardiography at baseline and at 2 and 4 weeks post-transplantation (measuring ejection fraction, fractional shortening, and other parameters). At the study endpoint, hearts are harvested for histological analysis (Masson's trichrome staining to assess infarct size and fibrosis), immunohistochemistry (to detect human-specific markers and assess graft survival), and vascular density measurement (CD31 staining). Improved functional recovery and reduced infarct size are expected in the CYT296-enhanced iPSC group. |
| ADME/Pharmacokinetics |
CYT296 is a research compound and is not characterized by traditional pharmacokinetic parameters, as it is not intended for in vivo administration. The compound is used exclusively for cell culture studies. When used at concentrations of 0.5-5 microM, CYT296 is stable in cell culture media for at least 48-72 hours, allowing for once-daily dosing in reprogramming experiments. For in vivo studies, the compound is not typically administered directly to animals; instead, cells are treated ex vivo. Thus, systemic exposure, clearance, metabolism, and excretion of CYT296 are not relevant. If administered systemically, CYT296 would likely be rapidly cleared and may have off-target effects. Researchers should verify the stability of CYT296 under their specific cell culture conditions (medium composition, serum content, temperature, etc.).
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| Toxicity/Toxicokinetics |
Toxicity data specific to CYT296 are limited to cell culture studies. At concentrations used for enhancing reprogramming (0.5-2 microM), CYT296 does not cause significant cytotoxicity in MEFs or other cell types, as assessed by trypan blue exclusion, MTT assay, or live/dead staining. At higher concentrations (≥5 microM), the compound may reduce cell viability and proliferation. No significant toxicity has been reported in published studies. CYT296 has not been evaluated in formal animal toxicology studies. Standard laboratory safety precautions should be followed when handling the compound, including the use of gloves, lab coats, and safety glasses. The compound should be dissolved in DMSO to make a stock solution (e.g., 10 mM) and stored at -20degC, protected from light. The working solution in cell culture medium should be prepared fresh to avoid degradation. CYT296 is for research use only and is not intended for human therapeutic or diagnostic applications.
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| References |
[1]. Wei X, et al. Small molecule compound induces chromatin de-condensation and facilitates induced pluripotent stem cell generation. J Mol Cell Biol. 2014 Oct;6(5):409-20.
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| Additional Infomation |
CYT296 is a research compound that targets chromatin de-condensation and enhances induced pluripotent stem cell (iPSC) generation. Its molecular formula is C19H20O3S (thiochromenone derivative), and its molecular weight is 328.43. The compound is also known as (2‘S,3S)-2‘-(4-methoxyphenyl)spiro[2H-thiochromene-3,1‘-cyclopropane]-4-one. CYT296 is a valuable tool for studying the role of chromatin structure in cellular reprogramming, development, and disease. It may be used in research on cell replacement therapy, regenerative medicine, and drug screening. The compound is not intended for human use and has not received regulatory approval for any clinical indication. It should be stored at -20degC, protected from light and moisture.
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| Molecular Formula |
C18H16O2S
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| Molecular Weight |
296.38
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| CAS # |
1799392-31-6
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| Appearance |
Typically exists as solid at room temperature
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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: 100 mg/mL (337.40 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (8.44 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: 2.5 mg/mL (8.44 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: 2.5 mg/mL (8.44 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3740 mL | 16.8702 mL | 33.7405 mL | |
| 5 mM | 0.6748 mL | 3.3740 mL | 6.7481 mL | |
| 10 mM | 0.3374 mL | 1.6870 mL | 3.3740 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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