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]
TC13172 targets mixed lineage kinase domain-like protein (MLKL), a key regulator of programmed necrosis (necroptosis). MLKL is the terminal effector of the necroptosis pathway downstream of RIPK1 and RIPK3. By covalently inhibiting MLKL, TC13172 blocks MLKL oligomerization and plasma membrane translocation, preventing the execution of necroptotic cell death.

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].
In vitro, TC13172 effectively inhibits TSZ combination (TNF-α, Smac mimetic, and Z-VAD-FMK)-induced necroptosis in HT-29 cells with an EC50 of 2 nM. At 100 nM, it blocks TSZ-induced MLKL oligomerization and plasma membrane translocation. The compound shows significant selectivity for MLKL over RIPK1 and RIPK3 at 10 µM.

In vivo, TC13172 has been studied in models of necroptosis-related diseases. By inhibiting MLKL, the compound blocks necroptotic cell death, which may have therapeutic potential in inflammatory diseases, ischemia-reperfusion injury, and neurodegenerative conditions where necroptosis plays a pathogenic role. Detailed in vivo efficacy data are available in research publications.

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]
In vitro enzyme assays for TC13172 measure its inhibition of MLKL activity. The compound's covalent binding to MLKL is assessed using activity-based protein profiling or mass spectrometry. Selectivity is confirmed by testing against RIPK1 and RIPK3 at 10 µM. The compound's potency is evaluated in cell-free systems measuring MLKL phosphorylation or oligomerization.

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]
In vitro cell-based assays for TC13172 use HT-29 cells treated with the TSZ combination (TNF-α, Smac mimetic, and Z-VAD-FMK) to induce necroptosis. Cells are treated with serial dilutions of the compound, and cell viability is measured to determine the EC50 for inhibition of necroptosis (2 nM). MLKL oligomerization and plasma membrane translocation are assessed by Western blotting and immunofluorescence.

In vivo animal models for TC13172 include mouse models of necroptosis-related diseases such as inflammatory bowel disease, ischemia-reperfusion injury, or systemic inflammatory response syndrome. The compound is administered orally or intraperitoneally, and its effects on tissue damage, inflammatory markers, and survival are evaluated. Pharmacodynamic studies measure MLKL inhibition in target tissues.

TC13172 has a molecular weight of 388.4 g/mol and a molecular formula of C17H16N4O5S. It is soluble in DMSO at 20 mg/mL (51.49 mM). For in vivo administration, it can be formulated in 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline at 2 mg/mL. The compound is stored as a powder at -20°C for up to 3 years.

The toxicity profile of TC13172 is characterized in preclinical safety studies. As an MLKL inhibitor, the compound may affect necroptosis-dependent physiological processes. The compound is for research use only and is not approved for clinical use. Appropriate safety precautions should be taken during handling.

[1]. Discovery of a new class of highly potent necroptosis inhibitors targeting the mixed lineage kinase domain-like protein.ChemCommun (Camb). 2017 Mar 28;53(26):3637-3640.

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]
TC13172 is a mixed lineage kinase domain-like protein (MLKL) inhibitor with single nanomolar potency. It is a covalent inhibitor that selectively targets MLKL over RIPK1 and RIPK3 at 10 µM. TC13172 effectively inhibits necroptosis in HT-29 cells with an EC50 of 2 nM. The compound is also known as TC-13172. It is intended for laboratory research use only.

C17H16N4O5S

388.397742271423

390.1

C, 52.30; H, 4.65; N, 14.35; O, 20.49; S, 8.21

2093393-05-4

2093393-05-4

134691750

Off-white to light yellow solid powder

0.7

1

6

3

27

795

0

S(C)(C1=NC2=C(C(N(C)C(N2CC#CC2C=CC=C(C=2)O)=O)=O)N1C)(=O)=O

ZTQLCNOQWGSELY-UHFFFAOYSA-N

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

3-[3-(3-hydroxyphenyl)prop-2-ynyl]-1,7-dimethyl-8-methylsulfonylpurine-2,6-dione

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;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~25 mg/mL (~64.4 mM)

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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)