| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg | |||
| Other Sizes |
Purity: ≥98%
TC13172 (TC-13172) is a mixed Lineage Kinase Domain-Like protein (MLKL) inhibitor with single nanomolar potency. As evidence that MLKL should be considered a druggable target, TC13172 may offer useful tools for studying the biological function of MLKL.
| Targets |
MLKL (EC50 = 2±0.6 nM); TC13172 (compound 15): Human MLKL (Cysteine86), no inhibition on human RIP1 and RIP3 kinases (10 μM concentration shows no kinase activity inhibition) [1]
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|---|---|
| ln Vitro |
1. TC13172 (compound 15) acts as a covalent necroptosis inhibitor; in HT-29 cells with no wash treatment, its EC50 for anti-necroptosis is 2.03 nM, and with wash treatment to remove unbound compound, the EC50 is 98.57 nM [1]
2. TC13172 (compound 15) targets the C86 site of human MLKL: in RIP3-HeLa cells with MLKL knockout (KO), the cells resist TSZ-induced necroptosis; after transfecting RIP3-HeLa-MLKL KO cells with wild-type human MLKL, 200 nM of this compound can protect cells from TSZ-induced necroptosis, while it has no protective effect on cells transfected with C86S mutant MLKL [1] 3. TC13172 (compound 15) shows no anti-necroptosis activity in mouse MEF, mouse L929 and rat L6 cells at 5 μM concentration [1] 4. In the TSZ-induced HT29 cell death assay, TC13172 (compound 15) has an EC50 of 2 nM for anti-necroptosis, which is the most potent MLKL inhibitor reported in the study [1] 5. 10 μM of TC13172 (compound 15) exhibits no inhibitory effect on the kinase activity of human RIP1 and RIP3 [1] The HT-29 cell line is used to assess the anti-necroptosis potency of TC13172; the EC50 value is 20.6 nM. The inhibition potency of TC13172 against cell necroptosis is 2 nM. By firmly attaching to Cys-86, TC13172 prevents MLKL from functioning. The fact that TC13172 decreases MLKL levels in the membrane phase without affecting MLKL's phosphorylation shows that these MLKL inhibitors prevent MLKL from moving to the cell membrane and preventing necroptosis[1]. |
| Enzyme Assay |
1. RIP1 kinase activity assay for TC13172 (compound 15): The assay is conducted in white 384-well plates, with an assay buffer containing 25 mM HEPES (pH7.2), 20 mM MgCl₂, 12.5 mM MnCl₂, 5 mM EGTA, 2 mM EDTA, 12.5 mM β-glycerol phosphate and 2 mM DTT. Human RIPK1 is first incubated with the compound or DMSO control for 15 minutes, then an ATP/MBP substrate mixture is added to initiate the kinase reaction. After a 90-minute reaction at room temperature, ADP-Glo reagent and detection solution are added following the kit protocol, and luminescence is measured to evaluate kinase activity [1]
2. RIP3 kinase activity assay for TC13172 (compound 15): The assay is performed in white 384-well plates with almost the same conditions as the RIP1 kinase assay, the only difference being that the assay buffer contains 5 mM MgCl₂ instead of 20 mM MgCl₂ and 12.5 mM MnCl₂. Human RIP3 is incubated with the compound or DMSO control for 15 minutes, followed by the addition of ATP/MBP substrate mixture to start the reaction. The reaction proceeds for 90 minutes at room temperature, then ADP-Glo reagent and detection solution are added, and luminescence detection is carried out to assess kinase activity [1] |
| Cell Assay |
1. Covalent binding verification cell assay for TC13172 (compound 15): HT-29 cells are incubated with the compound for 2 hours, then unbound compound is washed away using PBS buffer and fresh medium is replaced. TNF-ɑ/Smac mimetic/z-VAD (TSZ) is added to the medium to induce necroptosis. Cell viability is detected using a cell viability assay kit by measuring ATP levels, with NSA as a positive control and Nec-1 as a negative control [1]
2. MLKL C86 targeting verification cell assay for TC13172 (compound 15): MLKL is knocked out in RIP3-HeLa cells to obtain RIP3-HeLa-MLKL KO cells; the KO cells are then transfected with wild-type human MLKL or C86S mutant MLKL expression constructs. The transfected cells are treated with 200 nM of the compound and TSZ to induce necroptosis, and cell viability is detected to verify the targeting site, with NSA (5 μM) as a positive control [1] 3. Cross-species anti-necroptosis activity cell assay for TC13172 (compound 15): Mouse MEF, mouse L929 and rat L6 cells are incubated with 5 μM of the compound for 2 hours, then TSZ or TNF-ɑ/zVAD (TZ) is added to induce necroptosis. Cell viability is detected using a cell viability assay kit to evaluate the anti-necroptosis activity of the compound in different species cells [1] 4. TSZ-induced HT29 cell death inhibition assay for TC13172 (compound 15): 3,000 HT-29 cells are plated in each well of a 96-well plate and cultured overnight at 37℃. The cells are co-treated with TSZ and serial concentrations of the compound for 24 hours, then cell viability is detected using a luminescent cell viability assay kit, and luminescence is recorded with a plate reader to calculate the EC50 value of anti-necroptosis [1] 5. High-throughput screening cell assay for TC13172 (compound 15): 2,000 HT-29 cells are seeded in each well of a 384-well assay plate, 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 mM z-VAD are added to induce necroptosis, and the compound is added at the corresponding concentration. After 24 hours, cell viability is determined by measuring ATP levels with a cell viability assay [1] |
| References | |
| Additional Infomation |
1. TC13172 (compound 15) is a novel covalent MLKL inhibitor with a hydroxylphenyl propynyl group on its molecular structure, and its synthesis is based on the structural modification of compound 1 with the introduction of sulfone groups that contribute to covalent binding [1]
2. The anti-necroptosis potency of TC13172 (compound 15) is significantly higher than that of other reported inhibitors (NSA: EC50=447 nM; GW806742X: EC50=589 nM; compound 12: EC50=180 nM; compound 14: EC50=19 nM; compound 16: EC50=27 nM) in the TSZ-induced HT29 cell death assay [1] 3. TC13172 (compound 15) exerts its anti-necroptosis effect by covalently binding to the Cysteine86 site of human MLKL, which is a key amino acid residue for the biological function of MLKL; the mutation of C86 to S86 abrogates the protective effect of the compound on TSZ-induced necroptosis [1] 4. TC13172 (compound 15) has species selectivity, showing potent anti-necroptosis activity in human HT-29 and RIP3-HeLa cells but no activity in mouse and rat cells, indicating its MLKL targeting is specific to human MLKL [1] |
| Molecular Formula |
C17H16N4O5S
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|---|---|
| Molecular Weight |
388.397742271423
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| Exact Mass |
390.1
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| Elemental Analysis |
C, 52.30; H, 4.65; N, 14.35; O, 20.49; S, 8.21
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| CAS # |
2093393-05-4
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| Related CAS # |
2093393-05-4
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| PubChem CID |
134691750
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
0.7
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
27
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| Complexity |
795
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C)(C1=NC2=C(C(N(C)C(N2CC#CC2C=CC=C(C=2)O)=O)=O)N1C)(=O)=O
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| InChi Key |
ZTQLCNOQWGSELY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H16N4O5S/c1-19-13-14(18-16(19)27(3,25)26)21(17(24)20(2)15(13)23)9-5-7-11-6-4-8-12(22)10-11/h4,6,8,10,22H,9H2,1-3H3
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| Chemical Name |
3-[3-(3-hydroxyphenyl)prop-2-ynyl]-1,7-dimethyl-8-methylsulfonylpurine-2,6-dione
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| Synonyms |
2093393-05-4; TC13172; TC 13172; TC-13172; 3-[3-(3-hydroxyphenyl)prop-2-yn-1-yl]-8-methanesulfonyl-1,7-dimethyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione; 3-(3-(3-hydroxyphenyl)prop-2-yn-1-yl)-8-methanesulfonyl-1,7-dimethyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione; RefChem:491592; 827-170-6;
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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: ~25 mg/mL (~64.4 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.25 mg/mL (5.79 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 22.5 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: ≥ 2.25 mg/mL (5.79 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 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5747 mL | 12.8733 mL | 25.7467 mL | |
| 5 mM | 0.5149 mL | 2.5747 mL | 5.1493 mL | |
| 10 mM | 0.2575 mL | 1.2873 mL | 2.5747 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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