| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
DOT1L 0.17 nM (IC50)
Dot1L-IN-5 specifically targets and inhibits the enzyme activity of Disruptor of Telomeric Silencing 1-like (DOT1L). DOT1L is a histone methyltransferase that catalyzes the methylation of histone H3 at lysine 79 (H3K79). The compound binds to the active site of DOT1L with high affinity, blocking its ability to transfer methyl groups from its cofactor, S-adenosyl-L-methionine (SAM), to the H3K79 residue. |
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| ln Vitro |
Dot1L-IN-5 (Compound 11) is investigated in cellular experiments to examine the capacity to block the dimethylation of H3K79 in HeLa cells (ED50 H3K79me2 Elisa=2.9 nM) and HOXA9 gene expression in Molm-13 cells (ED50 HOXA9 RGA=30 nM)[1].
In vitro, Dot1L-IN-5 potently inhibits DOT1L enzymatic activity with an IC50 of 0.17 nM in a biochemical assay. In cellular experiments, it blocks the dimethylation of H3K79 (H3K79me2) in HeLa cells (ED50 = 2.9 nM). It also inhibits the expression of the downstream target gene HOXA9 in Molm-13 cells (ED50 = 30 nM), demonstrating its ability to suppress the oncogenic transcriptional program driven by DOT1L in leukemia cells. |
| ln Vivo |
In NOD-SCID mice, subcutaneous injection of Dot1L-IN-5 (Compound 11) at a dose of 75 mg/kg once a day for 20 days does not suppress the formation of tumors. 73% growth inhibition is achieved with Dot1L-IN-5 (subcutaneous injection; 75 mg/kg twice day for 20 days)[1]. Although the global H3K79 dimethylation level in the tumor is strongly inhibited by both treatment regimens, the effective regimen is better at lowering the target genes MEIS1 and HOXA9's mRNA expression[1].
In vivo efficacy of Dot1L-IN-5 was assessed in NOD-SCID mice bearing MV4-11 tumor xenografts. A dosing regimen of 75 mg/kg administered subcutaneously twice daily for 20 days resulted in 73% tumor growth inhibition. While once-daily dosing did not cause tumor regression, it did inhibit H3K79 dimethylation in the tumors. The more frequent regimen was required to effectively reduce the mRNA expression of the target genes HOXA9 and MEIS1. |
| Enzyme Assay |
For a cell-free system, purified recombinant DOT1L enzyme, a biotinylated histone H3 peptide (residues 1-40), and 3H-S-adenosyl-L-methionine (3H-SAM) are incubated in a reaction buffer. After the reaction, the peptide is captured on streptavidin-coated plates, and the amount of incorporated 3H is measured by scintillation counting. The compound is added to the reaction to determine its IC50 by assessing the reduction in methylation.
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| Cell Assay |
In cellular assays, cells (e.g., HeLa for mechanistic studies or MV4-11 for cell viability) are seeded and treated with varying concentrations of Dot1L-IN-5. To assess target engagement, cells are lysed and H3K79me2 levels are measured by a H3K79me2-specific ELISA (ED50 = 2.9 nM). For functional activity, gene expression changes (e.g., HOXA9) are quantified by quantitative RT-PCR (ED50 = 30 nM). Cell viability is measured by a standard assay like CellTiter-Glo.
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| Animal Protocol |
Animal/Disease Models: NOD-SCID (severe combined immunodeficient) mouse bearing subcutaneous (sc) MV4-11 tumor xenografts[1]
Doses: 75 mg/kg Route of Administration: subcutaneous (sc) injection; at 75 mg/kg once or twice (two times) daily for 20 days Experimental Results: one time/day administration did not Lead to tumor growth inhibition, 73% growth inhibition was measured in the twice (two times) daily treated group. For in vivo studies, MV4-11 human leukemia cells are injected subcutaneously into NOD-SCID mice. Once tumors reach a suitable volume, mice are randomized into treatment groups. Dot1L-IN-5 is dissolved in a suitable vehicle (e.g., a DMSO-based formulation) and administered by subcutaneous injection. Two dosing regimens are compared: once daily (75 mg/kg) and twice daily (75 mg/kg) for 20 days. Tumor volume is measured with calipers to calculate TGI, and tissues are harvested for biomarker analysis (e.g., H3K79me2, HOXA9 mRNA). |
| ADME/Pharmacokinetics |
Specific ADME (Absorption, Distribution, Metabolism, and Excretion) data for Dot1L-IN-5 is not presented. The compound is described as having activity via subcutaneous injection, as used in efficacy models. It is soluble in DMSO at 250 mg/mL, facilitating the preparation of stock solutions for in vitro and in vivo dosing. The in vivo dosing frequency (twice daily) suggests that its half-life may be relatively short.
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| Toxicity/Toxicokinetics |
No specific preclinical toxicology reports for Dot1L-IN-5 are provided. The primary findings from in vivo studies relate to its efficacy. At the efficacious dose (75 mg/kg twice daily), the compound was tolerated in mice for the 20-day study duration, as indicated by the ability to continue dosing and measure endpoints. Specific adverse effects or detailed toxicological assessments are not described in the available literature.
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| References | |
| Additional Infomation |
DOT1L is an essential oncogenic driver in MLL-rearranged leukemias, making it a validated therapeutic target. Dot1L-IN-5 is a highly potent tool compound used to study this dependency. Its ability to effectively block H3K79 methylation and inhibit the expression of critical downstream genes like HOXA9 and MEIS1 demonstrates its utility as a chemical probe. It is in preclinical research and is not approved for clinical use.
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| Molecular Formula |
C23H19CLF2N8O5S
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|---|---|
| Molecular Weight |
592.96
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| Exact Mass |
592.085
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| CAS # |
2565705-03-3
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| PubChem CID |
145704696
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| Appearance |
White to off-white solid powder
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| LogP |
3.7
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
15
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
40
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| Complexity |
970
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1=CC2OC(F)(F)OC=2C([C@H](NC2C=CC(=CC=2NC2N=C(OC)N=C(N=2)N)S(=O)(N)=O)C2N=CC=CC=2Cl)=C1
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| InChi Key |
BAQOPDGRUNKWLM-KRWDZBQOSA-N
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| InChi Code |
InChI=1S/C23H19ClF2N8O5S/c1-37-22-33-20(27)32-21(34-22)31-15-10-11(40(28,35)36)7-8-14(15)30-17(18-13(24)5-3-9-29-18)12-4-2-6-16-19(12)39-23(25,26)38-16/h2-10,17,30H,1H3,(H2,28,35,36)(H3,27,31,32,33,34)/t17-/m0/s1
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| Chemical Name |
3-[(4-amino-6-methoxy-1,3,5-triazin-2-yl)amino]-4-[[(S)-(3-chloropyridin-2-yl)-(2,2-difluoro-1,3-benzodioxol-4-yl)methyl]amino]benzenesulfonamide
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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: 250 mg/mL (421.61 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.51 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (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 DMSO stock solution (20.8 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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 (3.51 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 DMSO stock solution (20.8 mg/mL) to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6865 mL | 8.4323 mL | 16.8645 mL | |
| 5 mM | 0.3373 mL | 1.6865 mL | 3.3729 mL | |
| 10 mM | 0.1686 mL | 0.8432 mL | 1.6865 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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