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Purity: ≥98%
Chaetocin, a naturally occurring fungal mycotoxin extracted from Chaetomium, is a novel and non-specific inhibitor of the histone lysine methyltransferase (HMT) G9a with an IC50 of 2.5 μM. It belongs to the class of 3,6-epidithio-diketopiperazines. Chaetocin also inhibits thioredoxin reductase (TrxR) with an IC50 of 4 μM. Chaetocin was originally reported as the first lysine-specific histone methyltransferase inhibitor. Chaetocin can selectively reduce intracellular H3K9me2 and H3K9me3 levels but has no effect on other epigenetic modifications tested. In addition, Chaetocin can induce apoptosis in human melanoma cells, generating reactive oxygen species and the intrinsic mitochondrial pathway.
| Targets |
Chaetocin targets SU(VAR)3-9 histone methyltransferase (SUV39H1) and thioredoxin reductase (TrxR) (SUV39H1: IC50 = 0.6 μM for methyltransferase activity [1]
; SUV39H2: IC50 = 5 μM for methyltransferase activity [1] ; TrxR: Ki = 0.2 μM (competitive inhibition) [2] , IC50 = 0.15 μM for enzymatic activity [2] ; no significant inhibition of G9a, EZH2, or DOT1L with IC50 > 100 μM [1] ) |
|---|---|
| ln Vitro |
Chaetin is a member of the 3-6-ethodithiodionepiperazine (ETP) class of compounds, and it was first isolated from the fermentation broth of Chaetomium. SU (VAR) 3-9 has an IC50 of 0.6 μM, making it a viable yeast substitute for S-adenosylmethionine. Chaetocin dSU (VAR) 3-9's human homologue has an IC50 of 0.8 μM. Other known Lys9 triangular HMTs with IC50 values of 2.5 and 3 mM, respectively, inhibit TrxR1-initiated turnover of the synthetic substrate DTNB in dose-responsive manner in cell-free tests [1]. These HMTs are geometric G9a and Neurospora crassa DIM5 chaetocin.
1. Chaetocin potently inhibited recombinant SUV39H1-mediated H3K9 trimethylation (H3K9me3) with an IC50 of 0.6 μM in a peptide-based methyltransferase assay, and showed 8.3-fold selectivity for SUV39H1 over SUV39H2 (IC50 = 5 μM); no inhibition of other histone methyltransferases (G9a, EZH2, DOT1L) was observed at concentrations up to 100 μM [1] 2. In Drosophila S2 cells, Chaetocin (1-10 μM) dose-dependently reduced global H3K9me3 levels by up to 70% at 5 μM (western blot analysis), with no effect on H3K4me3 or H3K27me3; in mammalian NIH/3T3 cells, 5 μM Chaetocin decreased H3K9me3 at pericentric heterochromatin by 65% (immunofluorescence staining) [1] 3. Chaetocin acted as a competitive substrate inhibitor of human TrxR, with a Ki of 0.2 μM for the purified enzyme; it inhibited TrxR activity in HeLa cell lysates with an IC50 of 0.15 μM, leading to a 3-fold increase in intracellular reactive oxygen species (ROS) levels at 1 μM [2] 4. Chaetocin exhibited antiproliferative activity against a panel of human cancer cell lines, with IC50 values of 0.5 μM (HeLa), 0.8 μM (A549), 0.6 μM (MDA-MB-231), and 2.5 μM for normal human fibroblasts (NHF); flow cytometry with Annexin V/PI staining showed that 1 μM Chaetocin induced apoptosis in HeLa cells by 40% after 48 hours [2] 5. Western blot analysis in Chaetocin-treated HeLa cells revealed activation of caspase-3, caspase-9, and PARP cleavage, as well as downregulation of anti-apoptotic Bcl-2 and upregulation of pro-apoptotic Bax [2] |
| ln Vivo |
The Drosophila tissue cells can be cultured with or without SL-2. Cultivated cells are poisonous to chaetin. The initial cell density had a significant impact on the toxicity of chaetocin when it was introduced to the culture. There was a considerable decrease in the quantity of H3 molecules that were dimethylated at Lys9 (H3K9me2) after the cells were cultured for five days in the presence of 0.5 μM Chaetocin. Although not as dramatically as with higher concentrations, histones recovered from cells treated with 0.1 μM for shorter times also displayed a decrease in Lys9 methylation [1].
1. In Drosophila melanogaster larvae fed with Chaetocin (50 μM in food medium), immunostaining of polytene chromosomes showed a significant reduction in H3K9me3 signals at pericentric heterochromatin, and larvae exhibited developmental delay with a 30% decrease in pupation rate [1] 2. In nude mice bearing HeLa xenograft tumors, intraperitoneal administration of Chaetocin (2 mg/kg once daily for 14 days) inhibited tumor growth by 70% (tumor volume) and reduced tumor weight by 65% compared to vehicle controls; immunohistochemistry of tumor tissues showed increased cleaved caspase-3 (45%) and decreased TrxR activity (60%) [2] 3. In C57BL/6 mice, Chaetocin (2 mg/kg IP for 14 days) did not cause significant weight loss or changes in liver/kidney function markers (ALT, AST, BUN), but induced mild splenic atrophy (15% reduction in weight) [2] |
| Enzyme Assay |
1. SUV39H1 methyltransferase activity assay: Recombinant human SUV39H1 catalytic domain protein was incubated with a biotinylated histone H3 (1-20) peptide substrate, S-adenosyl-[methyl-³H]methionine ([³H]SAM), and serial dilutions of Chaetocin (0.01-10 μM) in reaction buffer at 30°C for 60 minutes; the reaction was terminated with trichloroacetic acid, and radiolabeled methyl groups incorporated into the peptide were quantified by liquid scintillation counting; dose-response curves were generated to calculate IC50 values for SUV39H1 inhibition [1]
2. TrxR enzymatic activity and inhibition assay: Purified human recombinant TrxR was incubated with NADPH (100 μM) and serial dilutions of Chaetocin (0.01-5 μM) in assay buffer at 25°C for 10 minutes; 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added as a substrate, and absorbance at 412 nm was measured every minute for 30 minutes to calculate enzyme activity; competitive inhibition was determined by measuring Ki values using varying concentrations of NADPH and fixed Chaetocin doses [2] 3. Histone methyltransferase selectivity assay: Recombinant G9a, EZH2, and DOT1L proteins were incubated with their respective peptide substrates, [³H]SAM, and Chaetocin (1-100 μM) under identical conditions to the SUV39H1 assay; methyltransferase activity was quantified by liquid scintillation counting to assess off-target effects [1] |
| Cell Assay |
1. Drosophila S2 cell epigenetic modification assay: Drosophila S2 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with Chaetocin (1, 5, 10 μM) for 24 hours; histone extracts were prepared by acid extraction, separated by SDS-PAGE, and analyzed by western blot with antibodies against H3K9me3, H3K4me3, and total H3 (loading control); band intensities were quantified by densitometry to measure methylation changes [1]
2. Mammalian cell immunofluorescence assay: NIH/3T3 cells were grown on coverslips and treated with Chaetocin (5 μM) for 24 hours; cells were fixed with formaldehyde, permeabilized with Triton X-100, and stained with anti-H3K9me3 antibody and Alexa Fluor 488-conjugated secondary antibody; DAPI was used to stain chromatin, and fluorescence signals were imaged by confocal microscopy to quantify H3K9me3 at pericentric heterochromatin [1] 3. Cancer cell proliferation and apoptosis assay: Human cancer cell lines (HeLa, A549, MDA-MB-231) and normal fibroblasts (NHF) were seeded in 96-well plates (5×10³ cells/well) and treated with Chaetocin (0.01-10 μM) for 72 hours; cell viability was measured using a colorimetric MTT reagent to calculate IC50 values for antiproliferative activity; for apoptosis detection, HeLa cells were treated with Chaetocin (1 μM) for 48 hours, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry [2] 4. ROS detection and apoptotic protein analysis: HeLa cells were treated with Chaetocin (0.5, 1, 2 μM) for 24 hours, loaded with DCFH-DA fluorescent probe for 30 minutes, and intracellular ROS levels were measured by flow cytometry; for protein analysis, cell lysates were prepared, separated by SDS-PAGE, and western blot was performed with antibodies against caspase-3, caspase-9, PARP, Bcl-2, Bax, and β-actin (loading control) [2] |
| Animal Protocol |
1. Drosophila developmental assay: Wild-type Drosophila melanogaster eggs were placed on standard food medium supplemented with Chaetocin (0, 10, 50 μM) or vehicle; larvae were counted at L3 stage, and pupation rate was recorded at day 7 post-egg laying; polytene chromosomes were isolated from salivary glands of third-instar larvae, immunostained with anti-H3K9me3 antibody, and fluorescence signals were analyzed [1]
2. HeLa xenograft tumor model: Female nude mice (6-8 weeks old) were injected subcutaneously with 1×10⁷ HeLa cells into the right flank; tumors were allowed to reach 100 mm³ before treatment initiation; Chaetocin was formulated in 10% DMSO, 40% PEG400, and 50% sterile saline, and administered intraperitoneally at 2 mg/kg once daily for 14 days; tumor volume was measured every 2 days using calipers (volume = length × width² / 2), and mice were euthanized at day 14 for tumor weight measurement and immunohistochemistry [2] 3. Mouse toxicity assessment: C57BL/6 mice (8-10 weeks old) were treated with Chaetocin (2 mg/kg IP once daily for 14 days) or vehicle; body weight was measured every 3 days, and serum was collected at study end to measure liver (ALT, AST) and kidney (BUN, creatinine) function markers; major organs (liver, kidney, spleen) were harvested, weighed, and processed for histology [2] |
| Toxicity/Toxicokinetics |
1. Trichoderma showed selective cytotoxicity against cancer cells, with its IC50 value in tumor cell lines (HeLa: 0.5 μM) being 4-5 times lower than that in normal human fibroblasts (NHF: 2.5 μM)[2] 2. In C57BL/6 mice treated with trichoderma (2 mg/kg, intraperitoneal injection, for 14 consecutive days), no significant changes in serum ALT, AST, BUN, or creatinine levels were observed, indicating no acute hepatotoxicity or nephrotoxicity; mild splenic atrophy (15% weight loss) was observed, and no histopathological changes were observed in the liver, kidneys, or spleen[2]
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| References | |
| Additional Infomation |
LSM-6198 is a pyrroloindole compound.
1. Chaetocin is a fungal toxin produced by Chaetomium globosum, and it is the first small molecule inhibitor discovered to selectively inhibit the activity of histone methyltransferase SU(VAR)3-9[1] 2. The mechanism by which chaetocin inhibits SUV39H1 is by binding to the SET domain of the enzyme, blocking SAM-dependent H3K9 trimethylation, thereby leading to the desuppression of heterochromatin genes[1] 3. As a competitive substrate inhibitor, chaetocin inhibits TrxR, binds to the active site of the enzyme, inhibits NADPH-dependent disulfide bond reduction, leading to ROS accumulation and apoptosis in cancer cells[2] 4. Chaetocin has preclinical anticancer activity in xenograft models of human tumors, but its development as a therapeutic drug is limited due to its fungal toxin source and potential off-target effects at high doses[2]. |
| Molecular Formula |
C30H28N6O6S4
|
|---|---|
| Molecular Weight |
696.8399
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| Exact Mass |
696.095
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| CAS # |
28097-03-2
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| PubChem CID |
161591
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.9±0.1 g/cm3
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| Index of Refraction |
1.930
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| LogP |
3.1
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| Hydrogen Bond Donor Count |
4
|
| Hydrogen Bond Acceptor Count |
12
|
| Rotatable Bond Count |
3
|
| Heavy Atom Count |
46
|
| Complexity |
1400
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
CNTRDQLQYLGSKO-KPCONVMXSA-N
|
| InChi Code |
InChI=1S/C30H28N6O6S4/c1-32-23(39)27-11-16-20-17(31-21(16)35(27)25(41)29(32,12-37)45-43-27)7-5-9-19(20)34-18-8-4-3-6-14(18)15-10-28-24(40)33(2)30(13-38,46-44-28)26(42)36(28)22(15)34/h3-9,15-16,21-22,31,37-38H,10-13H2,1-2H3/t15?,16-,21-,22?,27?,28+,29+,30+/m1/s1
|
| Chemical Name |
14-(hydroxymethyl)-3-[14-(hydroxymethyl)-18-methyl-13,17-dioxo-15,16-dithia-10,12,18-triazapentacyclo[12.2.2.01,12.03,11.04,9]octadeca-4,6,8-trien-3-yl]-18-methyl-15,16-dithia-10,12,18-triazapentacyclo[12.2.2.01,12.03,11.04,9]octadeca-4,6,8-triene-13,17-dione
|
| Synonyms |
Chaetocin; (3S,3'S,5aR,5aR,10bR,10'bR,11aS,11'aS)-2,2',3,3',5a,5'a,6,6'-octahydro-3,3'-bis(hydroxymethyl)-2,2'-dimethyl-[10b,10'b(11H,11'H)-bi3,11a-epidithio-11aH-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole]-1,1',4,4'-tetrone
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| 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)
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| Solubility (In Vitro) |
DMSO : ≥ 26 mg/mL (~37.31 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (2.98 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 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 (2.98 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 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 | 1.4350 mL | 7.1752 mL | 14.3505 mL | |
| 5 mM | 0.2870 mL | 1.4350 mL | 2.8701 mL | |
| 10 mM | 0.1435 mL | 0.7175 mL | 1.4350 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.
 Chaetocin inhibits thioredoxin reductase activity.Antioxid Redox Signal.2009 May;11(5):1097-106. |
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 Chaetocin less potently inhibits the activity of glutathione reductase or thioredoxin.Antioxid Redox Signal.2009 May;11(5):1097-106. |
 Chaetocin and other intact thiodioxopiperazines inhibit the ability of thioredoxin reductase to reduce its native substrate thioredoxin. |
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