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| Targets |
Obtustatin triacetate specifically targets the integrin alpha1beta1 (VLA-1, very late antigen-1), which is a cell surface receptor for collagen and laminin. Integrin alpha1beta1 is expressed on various cell types, including endothelial cells, fibroblasts, smooth muscle cells, and some immune cells (activated T cells). It plays a critical role in cell adhesion, migration, proliferation, and survival, particularly on type IV collagen (a major component of basement membranes). Obtustatin binds to the alpha1 subunit of the alpha1beta1 integrin with high affinity and selectivity, blocking the interaction between alpha1beta1 and its primary ligand, type IV collagen. By inhibiting alpha1beta1-mediated adhesion, Obtustatin disrupts endothelial cell migration and tube formation (angiogenesis). It is a potent and selective inhibitor of integrin alpha1beta1 adhesion to type IV collagen.
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| ln Vitro |
In vitro studies have characterized the inhibitory activity of Obtustatin. Using a solid-phase integrin binding assay, Obtustatin inhibits the binding of purified integrin alpha1beta1 to type IV collagen with an IC50 in the low nanomolar range (typically IC50 ∼ 20-100 nM). It also inhibits alpha1beta1 binding to laminin but is less potent. In cell adhesion assays using human umbilical vein endothelial cells (HUVECs), Obtustatin (0.1-10 microM) inhibits cell adhesion to type IV collagen by >80% but does not inhibit adhesion to fibronectin or vitronectin, confirming selectivity for alpha1beta1-mediated adhesion. The compound also inhibits HUVEC migration on type IV collagen in a wound healing assay and tube formation on Matrigel, demonstrating its anti-angiogenic properties. It shows no activity against alpha2beta1, alpha5beta1, alphavbeta3, or alphaIIbbeta3 integrins. It is a potent and selective inhibitor.
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| ln Vivo |
The in vivo activity of Obtustatin triacetate has been demonstrated in models of angiogenesis and cancer. In the chick chorioallantoic membrane (CAM) assay, Obtustatin (10-50 microg/embryo) inhibits angiogenesis, reducing the number and length of blood vessels. In a murine Matrigel plug assay, subcutaneous injection of Matrigel containing bFGF (or VEGF) and Obtustatin (10-100 microg/plug) results in reduced hemoglobin content and endothelial cell infiltration compared to control plugs. In a mouse model of tumor angiogenesis (e.g., subcutaneous B16 melanoma or Lewis lung carcinoma), systemic administration of Obtustatin (1-10 mg/kg, IV or IP) reduces tumor growth and microvessel density. These effects are attributed to inhibition of alpha1beta1 integrin-mediated endothelial cell functions. It is a specific inhibitor of alpha1beta1 integrin adhesion to type IV collagen.
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| Enzyme Assay |
A cell-free integrin binding assay is used to determine the affinity and selectivity of Obtustatin triacetate. Purified human integrin alpha1beta1 (5 nM) is immobilized on a 96-well plate by overnight incubation at 4degC. The plate is blocked with 3% BSA. Varying concentrations of Obtustatin triacetate (0.1 nM-10 microM) are added and incubated for 1 hour. Then, biotinylated type IV collagen (0.5-1 microg/mL) is added and incubated for an additional 2 hours. After washing, HRP-conjugated streptavidin is added, followed by TMB substrate. The absorbance is read at 450 nm. The IC50 is calculated from the dose-response curve. For specificity screening, the same protocol is performed with purified alpha2beta1, alpha5beta1, alphavbeta3, and alphaIIbbeta3 integrins, using their respective ligands (e.g., type I collagen for alpha2beta1, fibronectin for alpha5beta1 and alphavbeta3). Obtustatin does not inhibit these integrins at concentrations up to 10 microM, demonstrating >100-fold selectivity for alpha1beta1.
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| Cell Assay |
HUVECs are used for cell adhesion and migration assays. For adhesion assays, 96-well plates are coated with type IV collagen (10 microg/mL) or other extracellular matrix proteins (type I collagen, fibronectin, vitronectin, 10 microg/mL) overnight at 4degC, then blocked with 1% BSA. HUVECs are detached with trypsin, washed, and resuspended in serum-free medium. Cells (2×10⁵ cells/well) are pre-incubated with Obtustatin triacetate (0.01-10 microM) for 15 minutes at 37degC, then added to the coated wells and allowed to adhere for 30-60 minutes at 37degC. Non-adherent cells are removed by gentle washing. Adherent cells are fixed, stained with crystal violet, lysed, and quantified by absorbance at 590 nm. For migration assays, confluent HUVEC monolayers in 6-well plates are wounded with a pipette tip. Obtustatin (0.1-10 microM) is added, and wound closure is monitored by microscopy at 0, 6, 12, 24, and 48 hours. The percentage of wound closure is calculated. For tube formation assays, HUVECs are seeded on Matrigel-coated 96-well plates (1×10⁴ cells/well) in the presence of Obtustatin (0.1-10 microM) and incubated for 6-18 hours at 37degC. Tube formation (number of nodes, branch points, total tube length) is quantified using ImageJ. Obtustatin inhibits tube formation.
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| Animal Protocol |
The chick chorioallantoic membrane (CAM) assay is a standard in vivo model for studying angiogenesis. Fertilized chicken eggs are incubated at 37degC for 8-10 days. A small window is cut in the shell, and a sterile filter disk or silicone ring is placed on the CAM. Obtustatin triacetate (10-50 microg in 20-50 microL sterile saline) is applied to the disk. Control eggs receive saline alone. Eggs are returned to the incubator for 48-72 hours. The CAM is then fixed with 4% paraformaldehyde, and the disk area is excised. Blood vessels are visualized under a dissecting microscope, and the number of blood vessel branch points, vessel density, or hemoglobin content in the CAM tissue is quantified. A reduction in angiogenesis (e.g., >50% reduction in vessel number) compared to control indicates anti-angiogenic activity. For tumor models, female C57BL/6 mice (6-8 weeks) are injected subcutaneously with 2×10⁵ B16-F10 melanoma cells in the right flank. When tumors reach 50-100 mm3, mice are treated with Obtustatin (1-10 mg/kg) by intraperitoneal (IP) or intravenous (IV) injection daily for 10-14 days. Tumor volume is measured every 2-3 days. At study termination, tumors are excised and weighed. Tumor microvessel density is assessed by CD31 immunohistochemistry.
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| ADME/Pharmacokinetics |
Obtustatin triacetate has a molecular weight of approximately 4,500-4,800 Da (41 amino acids). The lyophilized powder should be stored at -20degC in a sealed container, protected from moisture, where it is stable for up to 3 years. For solution storage, it should be kept at -80degC for up to 6 months. The triacetate salt form enhances water solubility. The peptide is soluble in water (1-5 mg/mL) and PBS (pH 7.4). For in vivo studies, the compound can be dissolved in sterile saline or PBS. The non-RGD disintegrin has 8 disulfide bonds. It is isolated from snake venom. The product is for research use only.
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| Toxicity/Toxicokinetics |
This product is for research use only and is not for human therapeutic use. No specific toxicity data is available for Obtustatin. As a peptide derived from snake venom, it may be immunogenic, and repeated administration may elicit an antibody response. At research doses (1-10 mg/kg), no significant acute toxicity was observed in mice. Standard laboratory safety practices (gloves, lab coat, safety glasses) should be followed. Avoid inhalation of powder and contact with skin and eyes. The compound is not an FDA-approved drug. It is a research tool for integrin alpha1beta1 studies.
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| References | |
| Additional Infomation |
Integrins are heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix (ECM) interactions. The alpha1beta1 integrin is a receptor for collagens (types I, IV, VI, IX, XIII) and laminins. It is involved in angiogenesis, fibrosis, and immune responses. Obtustatin is one of the few selective alpha1beta1 inhibitors and is widely used as a chemical probe to study the role of alpha1beta1 in these processes. Unlike RGD disintegrins that target alphaIIbbeta3 (platelet aggregation) and alphavbeta3 (angiogenesis), Obtustatin has a distinct binding mechanism. It is a 41-residue peptide and is a non-RGD disintegrin. This product is not a drug. Supplier information must not be included.
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| Molecular Formula |
C184H284N52O57S8.3C2H4O2
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| Molecular Weight |
4573.21
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| Related CAS # |
Obtustatin
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| Appearance |
White to off-white solid powder
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 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.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.2187 mL | 1.0933 mL | 2.1866 mL | |
| 5 mM | 0.0437 mL | 0.2187 mL | 0.4373 mL | |
| 10 mM | 0.0219 mL | 0.1093 mL | 0.2187 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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