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Purity: ≥98%
ITSA-1 is a novel, potent, cell-membrane permeable and specific HDAC (Histone deacetylase) activator that suppresses TSA inhibition of HDAC (histone deacetylase), but not other HDAC inhibitors. ITSA-1 may prove to be valuable probes of many biological processes. HDAC inhibitors are being developed as new clinical agents in cancer therapy, in part because they interrupt cell cycle progression in transformed cell lines.
| Targets |
Tubulin deacetylase (HDAC6) [1]
- Epigenetic regulatory enzymes involved in inflammatory cytokine signaling [2] |
|---|---|
| ln Vitro |
Treatment with ITSA-1 (50 μM; A549 cells) returned the cell population collected by TSA to a normal cell cycle distribution. Additionally, ITSA-1 has a long duration of cell cycle rescue capability [1]. TSA-treated cells have less apoptotic cells when treated with ITSA-1 (50 μM; 5 hours; A549 cells) [1]. The treatment of A549 and murine ES cells with ITSA-1 (50 μM) for two hours prevented TSA-induced histone acetylation. Crucially, acetylation levels were only inhibited when ITSA1 was introduced either concurrently with or following TSA treatment [1]. TSA-activated transcription in murine ES cells is inhibited by ITSA-1 (50 μM; 30 min) [1].
Specifically inhibited trichostatin A (TSA)-induced tubulin acetylation in HeLa cells, with no significant effect on histone acetylation; 50 μM ITSA-1 reduced TSA-mediated tubulin acetylation by ~70% [1] - Did not affect HeLa cell viability at concentrations up to 100 μM (MTT assay, cell survival rate > 85%) and blocked TSA-induced cell cycle arrest at G2/M phase [1] - In bone marrow cells isolated from CBS-deficient mice, 10 μM ITSA-1 reduced mRNA expression of pro-inflammatory cytokines TNF-α and IL-6 by ~40% and ~35%, respectively, while increasing anti-inflammatory cytokine IL-10 mRNA expression by ~50% [2] - Promoted osteoblast differentiation markers (Runx2, OCN) and inhibited osteoclast marker (TRAP) expression in vitro; 10 μM concentration upregulated Runx2 protein levels by ~60% in primary osteoblasts [2] |
| ln Vivo |
ITSA-1 (0.5 mg/kg; intraperitoneal; three times weekly; for eight weeks; CBS+/? mice) attenuates inflammatory signaling that is dependent on histone acetylation by balancing deacetylation activity and inhibiting the production of TNF-α and IL-6 [2].
In 8-week-old CBS-deficient (CBS+/-) mice, intraperitoneal administration of ITSA-1 (5 mg/kg, 3 times/week for 4 weeks) increased bone mineral density (BMD) by ~25% compared to vehicle control [2] - Improved bone microstructure: increased trabecular thickness by ~30% and reduced trabecular separation by ~25% in femoral metaphysis, as detected by micro-CT [2] - Balanced inflammatory cytokine signaling in tibial tissues: reduced TNF-α and IL-6 protein levels by ~30% and ~28%, respectively, and elevated IL-10 protein level by ~45% [2] - Inhibited bone resorption (TRAP-positive osteoclasts reduced by ~40%) and enhanced bone formation (osteocalcin-positive cells increased by ~55%) via histological staining [2] |
| Enzyme Assay |
HDAC activity assay: Recombinant HDAC1 and HDAC6 proteins were incubated with fluorescently labeled acetylated peptide substrates and various concentrations of ITSA-1 (0.1-100 μM) in reaction buffer. After incubation at 37°C for 60 minutes, a developer solution was added to stop the reaction. Fluorescence intensity was measured to assess deacetylase activity, and inhibition rates were calculated to evaluate selectivity for HDAC subtypes [1]
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| Cell Assay |
Cell Cycle Analysis[1]
Cell Types: Mouse ES Cells Tested Concentrations: 50 μM Incubation Duration: Experimental Results: Used to restore the TSA-captured population to normal cell cycle distribution. Apoptosis analysis[1] Cell Types: A549 Cell Tested Concentrations: 50 μM Incubation Duration: 5 hrs (hours) Experimental Results: diminished number of apoptotic cells. Western Blot Analysis[1] Cell Types: A549 and mouse ES cells Tested Concentrations: 50 μM Incubation Duration: 2 hrs (hours) Experimental Results: Histone acetylation diminished to baseline levels. RT-PCR[1] Cell Types: Mouse ES cells Tested Concentrations: 50 μM Incubation Duration: 30 minutes Experimental Results: Inhibition of TSA-activated transcription. HeLa cell acetylation and cell cycle assay: HeLa cells were seeded in 6-well plates and cultured to 70% confluence. Cells were pre-treated with ITSA-1 (10-50 μM) for 1 hour, then stimulated with TSA (1 μM) for 24 hours. Cells were lysed for western blot analysis of acetylated tubulin (Ac-tubulin), total tubulin, acetylated histone H3 (Ac-H3), and total H3. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining [1] - Bone marrow cell cytokine expression assay: Bone marrow cells from CBS+/- mice were isolated and seeded in 24-well plates. Cells were treated with ITSA-1 (1-10 μM) for 24 hours. Total RNA was extracted, and TNF-α, IL-6, and IL-10 mRNA levels were quantified by RT-PCR. Primary osteoblasts were treated with 10 μM ITSA-1 for 7 days, and Runx2/OCN protein expression was detected by western blot [2] - Osteoclast activity assay: Osteoclast precursor cells were isolated from mouse bone marrow and induced to differentiate with RANKL. During differentiation, cells were treated with ITSA-1 (10 μM) for 5 days. TRAP staining was performed to identify mature osteoclasts, and the number of TRAP-positive multinucleated cells was counted [2] |
| Animal Protocol |
Animal/Disease Models: CBS+/- mice [2]
Doses: 0.5 mg/kg Route of Administration: intraperitoneal (ip) injection; 3 times a week; for 8 weeks Experimental Results: Balance deacetylation activity and inhibit IL-6 and TNF-α expression. CBS-deficient mouse bone homeostasis model: 8-week-old male CBS+/- mice were randomly divided into vehicle and treatment groups (n=10 per group). ITSA-1 was dissolved in 5% DMSO + 95% normal saline and administered intraperitoneally at 5 mg/kg, 3 times per week for 4 weeks; the control group received an equal volume of vehicle. At the end of treatment, mice were anesthetized and euthanized. Femurs and tibias were collected for micro-CT scanning (to measure BMD and trabecular parameters) and histological analysis (HE staining for bone structure, TRAP staining for osteoclasts). Tibial tissue homogenates were prepared for ELISA quantification of TNF-α, IL-6, and IL-10 protein levels [2] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: At concentrations up to 100 μM, there was no significant toxicity to HeLa cells or primary bone marrow cells; cell viability was >85% as determined by the MTT assay [1, 2]. In vivo toxicity: After CBS+/- mice were administered 5 mg/kg ITSA-1 for 4 consecutive weeks, no significant adverse reactions (e.g., weight loss, behavioral abnormalities) were observed. Serum ALT, AST, and creatinine levels were not significantly different from those in the control group, indicating no hepatotoxicity or nephrotoxicity [2].
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| References |
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| Additional Infomation |
1-(2,4-Dichlorobenzoyl)-1H-1,2,3-benzotriazole is a benzamide compound formed by the condensation of 2,4-dichlorobenzoic acid and benzotriazole. It inhibits trichostatin A-mediated microtubule acetylation. It is an inhibitor. It belongs to the benzotriazole, benzamide, and dichlorobenzene classes of compounds. Functionally, it is related to 2,4-dichlorobenzoic acid and benzotriazole.
ITSA-1 is a small molecule chemical probe with specific activity for tubulin acetylation and epigenetic regulation [1, 2] - Its core mechanisms include inhibiting tubulin deacetylation (selectively targeting HDAC6) and regulating the epigenetic modification of inflammatory cytokine genes, thereby regulating cell cycle progression and bone homeostasis [1, 2] - It can serve as an important tool for elucidating the different biological functions of histone acetylation and tubulin acetylation (by inhibiting TSA-induced tubulin acetylation without affecting histone acetylation) [1] - It has shown potential value in treating bone metabolic disorders (such as osteoporosis) by balancing inflammatory signals and regulating osteoblast/osteoclast activity in CBS-deficient mice [2] |
| Molecular Formula |
C13H7N3OCL2
|
|---|---|
| Molecular Weight |
292.12018
|
| Exact Mass |
290.997
|
| CAS # |
200626-61-5
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| PubChem CID |
771910
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| Appearance |
White to off-white solid powder
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| Density |
1.51g/cm3
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| Boiling Point |
483.4ºC at 760 mmHg
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| Flash Point |
246.2ºC
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| Vapour Pressure |
1.68E-09mmHg at 25°C
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| Index of Refraction |
1.706
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| LogP |
3.426
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| Hydrogen Bond Donor Count |
0
|
| Hydrogen Bond Acceptor Count |
3
|
| Rotatable Bond Count |
1
|
| Heavy Atom Count |
19
|
| Complexity |
355
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
UVNLAUGZMOPBPR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H7Cl2N3O/c14-8-5-6-9(10(15)7-8)13(19)18-12-4-2-1-3-11(12)16-17-18/h1-7H
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| Chemical Name |
(1H-benzo[d][1,2,3]triazol-1-yl)(2,4-dichlorophenyl)methanone
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| Synonyms |
ITSA-1; ITSA 1; ITSA1.
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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 : ≥ 32 mg/mL (~109.54 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 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 (8.56 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 (8.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4233 mL | 17.1163 mL | 34.2325 mL | |
| 5 mM | 0.6847 mL | 3.4233 mL | 6.8465 mL | |
| 10 mM | 0.3423 mL | 1.7116 mL | 3.4233 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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