| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Mixed lineage kinase domain-like protein (MLKL) [1]
|
|---|---|
| ln Vitro |
MLKL-IN-7 (compound 9) exhibited anti-necroptosis activity in a human HT-29 cell necroptosis model (induced by TNF-α, Smac mimetic, and Z-VAD, TSZ) with an EC50 of 148.4 nM. [1]
In a wash/no-wash assay, HT-29 cells pretreated with MLKL-IN-7 for 3 hours, 1 hour, or 5 minutes, followed by washing and TSZ treatment for 8 hours, showed that the compound remained active (inhibited necroptosis) when preincubated for 3 hours or 1 hour, but wash-off activity decreased significantly when preincubated for only 5 minutes. This indicates that MLKL-IN-7 covalently binds to MLKL in a time-dependent manner. [1] Western blot analysis of TSZ-treated HT-29 cells incubated with 2 μM MLKL-IN-7 for 6 hours showed that, similar to the known MLKL inhibitor NSA, MLKL-IN-7 did not inhibit the phosphorylation of RIPK1, RIPK3, or MLKL, suggesting it functions by blocking oligomerization and membrane translocation of phosphorylated MLKL rather than upstream phosphorylation events. [1] In mouse L929 cells induced with TNF-α and Z-VAD-FMK (TZ), MLKL-IN-7 showed no apparent activity at 20 μM. [1] |
| ln Vivo |
Due to the inactivity of MLKL-IN-7 in mouse L929 cells (attributed to the deficiency of the Cys86 residue in mouse MLKL), in vivo experiments were not performed. [1]
|
| Cell Assay |
Necroptosis induction and cell viability assay: HT-29 cells seeded in 96-well plates were pre-treated with Z-VAD-FMK (20 mM) and Smac mimetic (10 μM) for 30 minutes. Cells were then treated with TNF-α (1 ng/μL) for 8-10 hours. Cell viability was measured using a luminescent cell viability assay kit. Reagents were added to the wells, incubated at 37°C for 5 minutes, and luminescence was detected. [1]
Western blot analysis: HT-29 cells were seeded in 60×15 mm dishes, incubated for 48 hours, then transferred to 6-well plates and pretreated with TNF-α, Smac mimetic, and Z-VAD-FMK. After compound intervention (2 μM for 6 hours), cells were collected, lysed with Nonidet P40 lysis buffer containing protease inhibitor, PMSF, and phosphatase inhibitor cocktail. Protein lysates were centrifuged at 12,000 rpm, 4°C for 10 minutes. Protein concentration was determined by BCA method. Samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, incubated with primary antibodies (1:1000) overnight at 4°C, then with secondary antibodies (1:10000) for 1 hour at room temperature. Blots were analyzed using a bio-rad imaging system and Image J software. Antibodies used included those against phosphorylated RIPK1 (p-RIPK1), RIPK1, phosphorylated RIPK3 (p-RIPK3), RIPK3, phosphorylated MLKL (p-MLKL), MLKL, and GAPDH. [1] Compound wash/no-wash assay: HT-29 cells were seeded in 96-well plates at 20,000 cells per well. After 8 hours, cells were treated with MLKL-IN-7 for 5 minutes, 1 hour, or 3 hours. The medium was then washed away, cells were washed three times with PBS, and treated with TSZ for 8 hours. Cells treated with compound without washing served as controls. Cell viability was measured using a luminescent cell viability assay kit. [1] Mouse L929 cell assay: L929 cells were induced with TNF-α and Z-VAD-FMK (TZ) and incubated with MLKL-IN-7 at 20 μM; no apparent activity was observed. [1] |
| References | |
| Additional Infomation |
MLKL-IN-7 (compound 9) is a quinazoline-containing fused heterocyclic MLKL inhibitor designed based on the structure of Necrosulfonamide (NSA). It was synthesized by replacing the pyrazine component of NSA with a quinazoline ring (specifically 2-aminoquinazoline). [1]
Molecular docking of MLKL-IN-7 with the hMLKL executioner domain (PDB ID: 6ZZ1) predicted that the compound covalently binds to Cys86 of MLKL and forms three π-π stacking interactions with the protein, including one additional π-π stacking interaction with Phe148 compared to NSA, which is suggested to be essential for improved anti-necroptosis activity. [1] MLKL-IN-7 acts by covalently binding to MLKL and blocking the oligomerization and membrane translocation of phosphorylated MLKL, without affecting the upstream phosphorylation of RIPK1, RIPK3, or MLKL. [1] |
| Molecular Formula |
C21H15N5O5S2
|
|---|---|
| Molecular Weight |
481.50
|
| Exact Mass |
481.051460
|
| PubChem CID |
171038133
|
| Appearance |
Typically exists as solid at room temperature
|
| LogP |
3.5
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
9
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
33
|
| Complexity |
835
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
C1=CC=C2C(=C1)C=NC(=N2)NS(=O)(=O)C3=CC=C(C=C3)NC(=O)/C=C/C4=CC=C(S4)[N+](=O)[O-]
|
| InChi Key |
URTZWFXYHGBPQY-YRNVUSSQSA-N
|
| InChi Code |
InChI=1S/C21H15N5O5S2/c27-19(11-7-16-8-12-20(32-16)26(28)29)23-15-5-9-17(10-6-15)33(30,31)25-21-22-13-14-3-1-2-4-18(14)24-21/h1-13H,(H,23,27)(H,22,24,25)/b11-7+
|
| Chemical Name |
(E)-3-(5-nitrothiophen-2-yl)-N-[4-(quinazolin-2-ylsulfamoyl)phenyl]prop-2-enamide
|
| Synonyms |
MLKL-IN-7; CHEMBL5566501; MLKL-IN7; CHEMBL-5566501;
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| 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
|
|---|---|
| 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 | 2.0768 mL | 10.3842 mL | 20.7684 mL | |
| 5 mM | 0.4154 mL | 2.0768 mL | 4.1537 mL | |
| 10 mM | 0.2077 mL | 1.0384 mL | 2.0768 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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