| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Colibactin 742 targets DNA, forming interstrand crosslinks (ICLs) through cyclopropane ring-opening, likely alkylating adenine-N3 residues on opposing strands. This DNA damage activates the Fanconi anemia (FA) DNA repair pathway, leading to FANCD2 monoubiquitination and recruitment, as well as γH2AX focus formation. The compound also induces replication fork stalling and fork fusion events. The gem-dimethyl derivative 22 (lacking cyclopropanes) and the left/right fragments 23 and 24 do not form DNA ICLs, confirming that both cyclopropane warheads are required for crosslinking. The inactive analog colibactin 746 (with methyl groups replacing cyclopropanes) does not induce DNA damage. [1][2]
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| ln Vitro |
Colibactin 742 induces DNA interstrand crosslinks (ICLs) in linearized pUC19 DNA at pH 5.0 and pH 7.0, with activity at concentrations as low as 10-100 nM. The linear precursor 9 shows similar crosslinking activity. In contrast, the gem-dimethyl derivative 22, left fragment 23, and right fragment 24 fail to form ICLs. The ICLs derived from colibactin 742 are stable under denaturing conditions. [1]
In IEC-6 rat intestinal epithelial cells, treatment with 3-100 μM colibactin 742 for 24 hours induces γH2AX foci formation in a concentration-dependent manner, while the inactive analog 746 does not. In human colonic organoids, 48-hour treatment with 10-100 μM 742 induces γH2AX foci and reduces organoid diameter and viability. [3] In human colonic epithelial FHC cells, 4-12 hour treatment with 10-100 μM 742 induces transcriptional activation of p53 signaling, cell cycle arrest/senescence pathways, and ER stress pathways. Upstream regulator analysis identifies p53 and CDKN2A as predicted activated regulators. Genes involved in anti-proliferation (CDKN1A, BTG2) and apoptosis (FAS, DAPK1, BBC3) are upregulated. [3] In RWPE-1 prostate epithelial cells, colibactin 742 treatment (10-40 μM for 1 hour) significantly increases replication fork stalling. At 20 μM, it induces a 2.2-fold increase in replication fork fusion events (asymmetrically sized and labeled forks on the same fiber), a source of chromosomal translocations. [2] In HCT 116 (mismatch repair-deficient) colon cancer cells, chronic exposure to 20 μM colibactin 742 for ten 48-hour cycles increases the total number of single nucleotide variants (SNVs) and indels compared to control (746-treated). The mutational signature includes contributions from pks-associated SBS88, MMRd-associated SBS44, and SBS17. Significant increases in T>N and [T>N]T substitutions within AT-rich motifs are observed. [3] Cytotoxicity: In HeLa cells, colibactin 742 shows an IC50 of 5.2 ± 2.1 μM (72-hour treatment), while the linear precursor 9 shows an IC50 of 7.2 ± 1.7 μM. The inactive analog 746 shows no significant toxicity. [1] |
| ln Vivo |
In Galleria mellonella larvae, oral gavage of 7.42 μg colibactin 742 (approximately 30 mg/kg assuming 250 mg larval weight) induces significant DNA damage in the intestinal epithelium after 24 hours, as measured by alkaline comet assay (increased tail moment and tail intensity). The inactive analog 746 does not induce significant DNA damage. Intrahemocelie injection of 7.42 μg 742 causes increased cuticular melanization compared to 746. [3]
In a germ-free Apcᴹⁱⁿ/⁺ DSS colitis-associated cancer mouse model, colonization with pks⁺ E. coli (NC101) increases the total number of somatic SNPs in colonic tumors and enhances the proportion of mutations attributed to the mismatch repair deficiency-associated signature SBS15 compared to colonization with colibactin-deficient ΔclbP mutant. However, colibactin 742 itself was not administered to mice due to synthetic quantity limitations. [3] |
| Cell Assay |
γH2AX immunofluorescence in IEC-6 cells: Cells were seeded in 8-well chamber slides, serum-starved overnight, then treated with DMSO, 3 μM MMC (positive control), or colibactin 742 or 746 at indicated concentrations (3-100 μM) for 24 hours. Cells were fixed, permeabilized, blocked, and incubated with anti-phospho-Histone H2AX (Ser139) rabbit monoclonal antibody (1:800) overnight at 4°C, followed by Alexa Fluor 488 secondary antibody. Nuclei were counterstained with DAPI. Cells with >5 foci/nucleus were scored as positive. [3]
Human colonoid culture and treatment: Human colonoids were cultured in Matrigel domes in human colonoid media. After passage, colonoids were treated with 742 or 746 at 10-100 μM for 48 hours. For γH2AX detection, colonoids were fixed, permeabilized, and stained with anti-γH2AX antibody (1:400) and DAPI. Z-stacks were merged by maximum intensity projection. For diameter and viability quantification, 30 organoids per condition were measured; viability was defined as organoids >30 μm diameter without luminal debris. [3] Cytotoxicity assay (CellTiter-GLO) in HeLa cells: HeLa cells were treated with serial dilutions of colibactin 742 or linear precursor 9 (24 mM to 100 μM) for 72 hours. Cell viability was measured using CellTiter-GLO luminescent assay, normalized to tamoxifen (60 μM, 100% effect) and DMSO (0% effect). IC50 values were calculated as 5.2 ± 2.1 μM (742) and 7.2 ± 1.7 μM (9). [1] DNA fiber assay in RWPE-1 cells: Cells were pulse-labeled with 100 μM IdU for 20 min, then treated with colibactin 742 (10, 20, or 40 μM) for 1 hour, followed by 300 μM CldU for 20 min. DNA fibers were combed using a molecular combing system, stained with anti-IdU and anti-CldU antibodies, and imaged by confocal microscopy. Fork stalling and fork fusion events were quantified. [2] Chronic exposure in HCT 116 cells: A clonal HCT 116 cell population was treated with 20 μM 742 or 746 for 48 hours (approximate LC50), washed, and allowed to recover for 48-96 hours. This cycle was repeated 10 times. Cells were then subcloned by sparse seeding and expanded for 30-32 days before whole-genome sequencing and RNA-seq analysis. [3] |
| Animal Protocol |
Galleria mellonella oral gavage: Colibactin 742 or 746 was dissolved in DMSO and administered by oral gavage at 7.42 μg per larva (approximately 30 mg/kg assuming average larval weight of 250 mg) in a final volume of 10 μL. Blue food coloring (1:10,000) was added to visualize successful gavage. After 24 hours, the midgut from 5 larvae per treatment was dissected, pooled, and epithelial cells were dissociated for alkaline comet assay. [3]
Galleria mellonella intrahemocelie injection: Compounds (7.42 μg in 10 μL) were injected into the last proleg of G. mellonella larvae. After 24 hours, health index and melanization scores were quantified using a standardized rubric. [3] |
| Toxicity/Toxicokinetics |
In HeLa cells, colibactin 742 has an IC50 of 5.2 ± 2.1 μM after 72-hour treatment, indicating cytotoxicity at low micromolar concentrations. The linear precursor 9 has a similar IC50 (7.2 ± 1.7 μM). The inactive analog 746 shows no significant cytotoxicity. [1]
In human colonic organoids, treatment with 100 μM colibactin 742 for 48 hours significantly reduces organoid viability and diameter, while 746 has minimal effect. [3] In RWPE-1 cells, colibactin 742 at 10-40 μM induces replication fork stalling and fork fusion events, indicating genotoxic stress. [2] In Galleria mellonella larvae, oral administration of 742 induces significant DNA damage in intestinal epithelial cells (increased comet tail moment and tail intensity), while 746 does not. [3] |
| References |
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| Additional Infomation |
Synthesis and stability: Colibactin 742 is prepared by a convergent synthesis (Scheme 1 in reference 1). The linear precursor 9 undergoes rapid cyclodehydration (5-10 min at 22°C in protic solvents) to form the colibactin warheads. The ring isomer 4b is the major product (4b:4a ~8:1), arising from addition of C44 to the C41 ketone. This cyclization is irreversible. [1]
Comparison to natural colibactin: Colibactin 742 lacks the labile C36-C37 1,2-diketone present in natural colibactin 770, which is the locus of instability. This modification confers enhanced stability while retaining the ability to form DNA ICLs, confirming that the diketone is not essential for crosslinking. [1] Comparison to inactive analog 746: Colibactin 746 has the two cyclopropane warheads replaced by unlinked methyl groups. It does not induce DNA damage, γH2AX foci, or transcriptional activation of DNA damage response pathways. [1][3] Mechanism of action: Colibactin binds DNA in the minor groove and alkylates adenine-N3 residues via cyclopropane ring-opening, forming interstrand crosslinks. This activates the Fanconi anemia repair pathway, leading to replication fork stalling, fork fusion, and chromosomal translocations. The combination of colibactin and dihydrotestosterone (DHT) in prostate cells induces synergistic increases in genomic instability, structural variants, somatic SNVs, and kataegis. [1][2][3] |
| Molecular Formula |
C37H42N8O5S2
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|---|---|
| Molecular Weight |
742.90998506546
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| Exact Mass |
742.27
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| CAS # |
2916559-62-9
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| PubChem CID |
162413463
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| Appearance |
Typically exists as solid at room temperature
|
| LogP |
-1.3
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| Hydrogen Bond Donor Count |
5
|
| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
11
|
| Heavy Atom Count |
52
|
| Complexity |
1730
|
| Defined Atom Stereocenter Count |
2
|
| SMILES |
S1C(CCC2=CSC(CNC(CC3=C(C4CC[C@H](C)N=4)C(NC43CC4)=O)=O)=N2)=NC(=C1)C1(CNC(C1C1=C(C2CC[C@H](C)N=2)C(NC21CC2)=O)=O)O
|
| InChi Key |
MUFRDBRHJIYYPD-AYLSQICOSA-N
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| InChi Code |
InChI=1S/C37H42N8O5S2/c1-18-3-6-22(40-18)28-21(35(9-10-35)44-32(28)47)13-25(46)38-14-27-42-20(15-51-27)5-8-26-43-24(16-52-26)37(50)17-39-34(49)31(37)30-29(23-7-4-19(2)41-23)33(48)45-36(30)11-12-36/h15-16,18-19,31,50H,3-14,17H2,1-2H3,(H,38,46)(H,39,49)(H,44,47)(H,45,48)/t18-,19-,31?,37?/m0/s1
|
| Chemical Name |
N-[[4-[2-[4-[3-hydroxy-4-[6-[(2S)-2-methyl-3,4-dihydro-2H-pyrrol-5-yl]-5-oxo-4-azaspiro[2.4]hept-6-en-7-yl]-5-oxopyrrolidin-3-yl]-1,3-thiazol-2-yl]ethyl]-1,3-thiazol-2-yl]methyl]-2-[6-[(2S)-2-methyl-3,4-dihydro-2H-pyrrol-5-yl]-5-oxo-4-azaspiro[2.4]hept-6-en-7-yl]acetamide
|
| Synonyms |
Colibactin 742; Colibactin-742; 2916559-62-9; orb1744645;
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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) |
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
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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 | 1.3461 mL | 6.7303 mL | 13.4606 mL | |
| 5 mM | 0.2692 mL | 1.3461 mL | 2.6921 mL | |
| 10 mM | 0.1346 mL | 0.6730 mL | 1.3461 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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