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| 1mg | ||
| 5mg | ||
| Other Sizes |
| Targets |
Homeobox A9 (HOXA9)[1]
The primary molecular target of DB818 is the Homeobox A9 (HOXA9) transcription factor. HOXA9 is a DNA-binding protein that regulates gene expression by binding to specific DNA sequences (the HOXA9 cognate sequence) in the promoters of its target genes. DB818 is reported to be a small molecule inhibitor of HOXA9 that binds as a minor groove DNA ligand on the HOXA9 cognate sequence, thereby blocking the interaction between HOXA9 and DNA. This inhibition suppresses the transcriptional activity of HOXA9. Downstream targets of HOXA9 that are inhibited by DB818 include MYB (MYB proto-oncogene), MYC (MYC proto-oncogene), and BCL2 (BCL2 apoptosis regulator). By inhibiting these oncogenes, DB818 induces growth arrest and apoptosis in HOXA9-driven leukemia cells. |
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| ln Vitro |
In vitro, DB818 is a potent inhibitor of HOXA9. In three different human AML cell lines (likely OCI/AML3, HL-60, and others), DB818 treatment inhibits cell growth and induces apoptosis. It also leads to the downregulation of HOXA9 transcriptional target genes, including MYB, MYC, and BCL2, while upregulating FOS (Fos proto-oncogene, AP-1 transcription factor subunit). The effect on target genes is consistent with the mechanism of HOXA9 inhibition. When HOXA9 is knocked down in these cell lines, similar effects are observed, except for MYC expression, which shows some differences. This suggests that DB818 may have a degree of specificity for HOXA9-driven pathways but could also have additional off-target effects. The potency of DB818 in suppressing cell growth is not explicitly specified but is in the low micromolar range.
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| ln Vivo |
In mice infected with worms, DB818 (12.5 mg/kg, 25 mg/kg; subcutaneously injected once daily for 4 days) totally eliminated parasitemia at the 25 mg/kg dose. Two thirds of the mice were cured at a dose of 12.5 mg/kg [1]. In mice, DB818 induces relapses approximately 20 days post-infection [1].
In vivo, DB818 has shown potent activity in a mouse model of infection, specifically against the parasite Babesia microti. In this study, DB818 was administered subcutaneously (s.c.) to mice infected with B. microti at doses of 12.5 mg/kg and 25 mg/kg once daily for 4 days. At the 25 mg/kg dose, the treatment completely cured the parasitemia (the presence of parasites in the blood) in all treated mice. At the 12.5 mg/kg dose, it cured 2/3 of the mice. However, it was noted that the infection recrudesced (reappeared) in some mice around day 20 post-infection. While not originally developed for this purpose, this finding demonstrates the in vivo bioavailability and efficacy of DB818. Its activity against AML in vivo has not been publicly reported, but it is hypothesized to have anti-tumor activity in mouse xenograft models of HOXA9-driven AML. |
| Enzyme Assay |
A typical non-cellular binding assay for DB818 is a DNA-binding assay, such as an electrophoretic mobility shift assay (EMSA) or a fluorescence polarization (FP)-based DNA-binding assay. To perform a DNA-binding assay, a double-stranded DNA oligonucleotide containing the HOXA9 consensus binding sequence (e.g., 5'-TTAATGG-3') is synthesized and labeled with a fluorescent dye (e.g., FAM) for FP. Varying concentrations of recombinant HOXA9 protein (0-100 nM) are incubated with 10 nM of the labeled DNA probe in binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 0.05% NP-40, 5% glycerol) at room temperature for 30 minutes. The fluorescence polarization is measured, and the binding curve is used to determine the Kd. For competition assays, DB818 is pre-incubated with the HOXA9 protein for 15 minutes, followed by the addition of the DNA probe. The change in FP signal is measured, and the IC₅0 is calculated. Alternatively, a biotinylated DNA probe can be used in a chemiluminescent EMSA kit. A typical in vitro cell-based assay for DB818 uses an AML cell line with high HOXA9 expression, such as OCI/AML3 or HL-60. Cells are cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. For the cell viability assay, cells are seeded in 96-well plates at 2 × 10⁴ cells/well and treated with DB818 at various concentrations (0.1 - 50 uM) for 48-72 hours. Cell viability is measured using the CellTiter-Glo luminescent assay or MTT assay. The half-maximal inhibitory concentration (IC₅0) is calculated by non-linear regression. For apoptosis detection, cells are treated with 10 uM DB818 for 48 hours, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. The expression of HOXA9 target genes (MYB, MYC, BCL2) is assessed by quantitative real-time PCR (qRT-PCR) or Western blotting after treatment with DB818.
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| Cell Assay |
A typical in vivo animal study for DB818 uses a mouse model of B. microti infection. Female BALB/c mice (6-8 weeks old, 18-22 g) are infected intraperitoneally (IP) with 1 × 10⁷ B. microti-parasitized red blood cells. The infection is allowed to establish, and parasitemia (percentage of infected red blood cells) is monitored daily by examining Giemsa-stained blood smears.
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| Animal Protocol |
When the parasitemia reaches approximately 5-10% (typically 5-7 days post-infection), the mice are randomized into groups (n=5 per group). DB818 is dissolved in a suitable vehicle, such as 20% DMSO in PBS or 0.5% carboxymethyl cellulose (CMC), and administered subcutaneously (SC) once daily at doses of 12.5 mg/kg and 25 mg/kg for 4 consecutive days. The control group receives the vehicle only. Parasitemia is monitored daily for up to 30 days. The effect of treatment is evaluated by the reduction in parasitemia and the survival of the mice. All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC).
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of DB818 are not well-characterized. The compound is a small molecule with a molecular weight of 360.44 g/mol. It is predicted to be lipophilic (calculated logP ~2-3) and may have moderate to good membrane permeability. It is soluble in water at 35.71 mg/mL (99.07 mM), which suggests it could be formulated for intravenous administration. In the in vivo study, it was administered subcutaneously (SC), indicating that it is absorbed via that route. The dose used (12.5-25 mg/kg) is typical for many small molecule inhibitors. No specific PK parameters, such as half-life (t1/2), Cmax, or bioavailability, have been publicly reported. It is likely metabolized in the liver, and its clearance pathways are not defined. Human PK data is not available as the compound is for research use only.
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| Toxicity/Toxicokinetics |
Toxicological data for DB818 is limited. In the published study of DB818 in a mouse model of B. microti infection, the compound was well-tolerated at the doses tested (12.5 and 25 mg/kg SC for 4 days). No significant weight loss or other overt signs of toxicity were reported. However, a full toxicological evaluation, including acute, sub-chronic, and genotoxicity studies, has not been performed. In vitro, the compound likely has a moderate to high toxicity profile against rapidly dividing leukemia cells. Standard safety precautions should be used when handling this compound, including working in a chemical fume hood and wearing gloves, a lab coat, and eye protection. It is for research use only and not for human administration.
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| References |
[1]. Sonoda Y, et al. Effects of HOXA9 Inhibitor DB818 on the Growth of Acute Myeloid Leukaemia Cells. Anticancer Res. 2021 Apr;41(4):1841-1847.
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| Additional Infomation |
DB818 is not an approved drug and has not entered clinical trials. It is a research tool for studying the role of the HOXA9 transcription factor in hematopoiesis and acute myeloid leukemia (AML). Its mechanism of action involves inhibiting the DNA-binding activity of HOXA9, leading to the suppression of downstream oncogenes like MYB, MYC, and BCL2. The compound has shown promising in vivo activity in a model of parasitic infection, highlighting its bioavailability. It is a potential lead compound for the development of targeted therapies for HOXA9-driven cancers. No clinical trials are registered for DB818. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C19H16N6S
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|---|---|
| Molecular Weight |
360.435541152954
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| Exact Mass |
360.115
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| CAS # |
790241-43-9
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| PubChem CID |
449090
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| Appearance |
White to yellow solid powder
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| LogP |
2.5
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
546
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC(=CC=C1C2=CC=C(S2)C3=NC4=C(N3)C=C(C=C4)C(=N)N)C(=N)N
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| InChi Key |
MSQVFIHMHRAMOC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H16N6S/c20-17(21)11-3-1-10(2-4-11)15-7-8-16(26-15)19-24-13-6-5-12(18(22)23)9-14(13)25-19/h1-9H,(H3,20,21)(H3,22,23)(H,24,25)
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| Chemical Name |
2-[5-(4-carbamimidoylphenyl)thiophen-2-yl]-3H-benzimidazole-5-carboximidamide
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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 | 2.7744 mL | 13.8719 mL | 27.7439 mL | |
| 5 mM | 0.5549 mL | 2.7744 mL | 5.5488 mL | |
| 10 mM | 0.2774 mL | 1.3872 mL | 2.7744 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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