MLKL-IN-3 (compound 66): Human MLKL (Cysteine86); no inhibition on human RIPK1 and RIPK3 kinases (10 μM concentration shows no kinase activity inhibition) [1]

1. MLKL-IN-3 (compound 66) is a covalent inhibitor of human MLKL, showing potent anti-necroptotic activity in TSZ-induced HT-29 cells with an EC₅₀ of 31 nM [1]
2. MLKL-IN-3 (compound 66) covalently binds to the Cys86 residue of human MLKL, which is confirmed by LC-MS/MS analysis and activity-based protein profiling (ABPP) competition assay; it can efficiently outcompete the MLKL-targeting probe TC13136 for binding to MLKL [1]
3. MLKL-IN-3 (compound 66) has no inhibitory effect on RIPK1 and RIPK3 kinase activity in vitro (10 μM concentration shows no obvious inhibition) and does not affect the phosphorylation status of RIPK1 and MLKL in TSZ-induced HT-29 cells [1]
4. MLKL-IN-3 (compound 66) does not inhibit extrinsic apoptosis (TNFα + CHX, TNFα + Smac mimetic), intrinsic apoptosis (ABT737 + S63845), or ferroptosis (PACMA31 + Regorafenib) in HeLa cells, indicating specific anti-necroptotic activity [1]
5. MLKL-IN-3 (compound 66) partially inhibits MLKL oligomerization in TSZ-induced HT-29 cells but shows a weaker inhibitory effect compared to TC13172; it significantly inhibits MLKL translocation to the cell membrane, which is confirmed by western blot analysis of soluble and membrane fractions and immunofluorescence staining of p-MLKL [1]
6. MLKL-IN-3 (compound 66) has no anti-necroptotic activity in mouse embryonic fibroblast (MEF) cells, as mouse MLKL lacks the Cys86 residue targeted by the compound [1]
7. The anti-necroptotic activity of MLKL-IN-3 (compound 66) is abolished in HT-29 MLKL knockout (KO) cells transfected with MLKL C86S mutant, while it retains activity in cells transfected with MLKL F148A or K157A mutants [1]
8. MLKL-IN-3 (compound 66) has a significantly lower reaction rate with glutathione (GSH) than TC13172 (more than 150-fold lower), indicating better chemical stability and reduced potential off-target effects [1]
9. High concentrations of MLKL-IN-3 (compound 66) do not affect the survival or proliferation of HT-29 cells in the absence of TSZ induction [1]
In HT-29 cells, MLKL-IN-3(compound 66) (1 μM, 24 h) prevents MLKL from translocating to cell membranes[1]. The phosphorylation state of MLKL and RIPK1 is unaffected by MLKL-IN-3[1].

Compound 66, MLKL-IN-3, exhibits T1/2 over a 48-hour period and over 150-fold reduced reaction rates with glutathione (GSH), which may mitigate their cytotoxic effects and off-target consequences[1].

1. RIPK1 kinase activity assay for MLKL-IN-3 (compound 66): The assay is performed in appropriate plates using purified RIPK1 kinase. The reaction system contains ATP and substrate protein, and the kinase activity is evaluated by monitoring the amount of ADP generated via luciferase. The compound is tested at concentrations of 1 μM and 10 μM, with Ponatinib as a positive control. The results show no inhibitory effect on RIPK1 kinase activity [1]
2. RIPK3 kinase activity assay for MLKL-IN-3 (compound 66): Using a similar setup to the RIPK1 kinase assay, purified full-length RIPK3 kinase is used as the target. The compound is tested at 1 μM and 10 μM, and no inhibitory effect on RIPK3 kinase activity is observed after detecting ADP production [1]

1. TSZ-induced necroptosis inhibition assay for MLKL-IN-3 (compound 66): HT-29 cells are seeded in 96-well plates (2×10⁴ cells/well) and cultured overnight at 37℃ with 5% CO₂. The cells are treated with serial concentrations of the compound and TSZ (TNFα 20 ng/mL, Smac mimetic LCL-161 1 μM, ZVAD-FMK 20 μM) for 24 hours. Cell viability is determined by ATP quantification using a luminescent cell viability assay reagent, and luminescence is detected with a microplate reader to calculate the EC₅₀ value [1]
2. MLKL covalent binding verification assay (ABPP) for MLKL-IN-3 (compound 66): HT-29 cells are preincubated with 10 μM of the compound for 2 hours, then 10 μM MLKL probe TC13136 is added and incubated for another 2 hours. Cell lysates are subjected to a click reaction with Biotin-C₂H₄-N₃, TBTA, CuSO₄, and sodium ascorbate for 2 hours. Biotin-modified proteins are enriched and analyzed by western blot with MLKL antibody to verify competitive binding [1]
3. MLKL oligomerization and membrane translocation assay for MLKL-IN-3 (compound 66): HT-29 cells are treated with TSZ in the presence of 1 μM of the compound for 6-8 hours. For oligomerization detection, cell lysates are analyzed by western blot to compare the level of oligomerized MLKL with DMSO control. For membrane translocation detection, cells are fractionated into soluble and membrane fractions, and MLKL levels in each fraction are detected by western blot with GAPDH (soluble fraction control) and COXIV (membrane fraction control); immunofluorescence staining with anti-pMLKL antibody is also performed to observe the cellular localization of p-MLKL [1]
4. MLKL mutant cell assay for MLKL-IN-3 (compound 66): HT-29 MLKL KO cells are transfected with wild-type MLKL or MLKL mutants (C86S, F148A, K157A). The transfected cells are treated with TSZ and the compound for 24 hours, and cell viability is detected by ATP quantification to evaluate the dependence of the compound's activity on specific MLKL residues [1]
5. Cell death specificity assay for MLKL-IN-3 (compound 66): HeLa cells are induced to undergo extrinsic apoptosis (TNFα 50 ng/mL + CHX or Smac mimetic 10 μM), intrinsic apoptosis (ABT737 5 μM + S63845 2 μM), or ferroptosis (PACMA31 1 μM + Regorafenib 10 μM) in the presence of the compound. Cell viability is assessed by ATP levels after 24 hours of incubation to verify the compound's specificity for necroptosis [1]
6. Cross-species anti-necroptosis assay for MLKL-IN-3 (compound 66): Mouse MEF cells are seeded in 96-well plates and treated with TSZ and the compound for 24 hours. Cell viability is measured by ATP levels to evaluate the compound's activity in mouse cells [1]

The chemical stability of MLKL-IN-3 (Compound 66) was assessed by measuring its reaction rate with glutathione (GSH). When incubated with GSH in DPBS buffer, the compound had a half-life (T1/2) of over 48 hours, which is more than 150-fold slower than that of TC13172 (T1/2 = 20 min). This indicates improved stability against endogenous nucleophiles and a lower potential for off-target effects. [1]
Other ADME parameters such as absorption, distribution, metabolism, excretion, and oral bioavailability were not reported. [1]

The potential for off-target effects and cell toxicity was inferred from its low reaction rate with glutathione (GSH). MLKL-IN-3 (Compound 66) showed a reaction half-life (T1/2) of over 48 hours, which is more than 150-fold slower than that of the xanthine inhibitor TC13172. This reduced chemical reactivity suggests a lower potential for covalent binding to undesired proteins, thereby minimizing off-target side effects and cell toxicity. [1]
In a cell toxicity assay, HT-29 cells were treated with MLKL-IN-3 (Compound 66) for up to 4 days. The compound did not affect cell viability or proliferation, indicating low cellular toxicity at the concentrations tested. [1]

[1]. Discovery of a New Class of Uracil Derivatives as Potential Mixed Lineage Kinase Domain-like Protein (MLKL) Inhibitors. J Med Chem. 2022 Oct 13;65(19):12747-12780.

1. MLKL-IN-3 (compound 66) is a uracil-derived covalent MLKL inhibitor, synthesized by conjugating 3-(4-hydroxy-3-((3-methoxy-2-oxopyridin-1(2H)-yl)methyl)phenyl)prop-2-yn-1-yl group to the uracil core, with a molecular formula of C₃₁H₃₀ClN₄O₆ and a molecular weight of 589.18 (m/z [M+H]⁺); its purity is ≥95% as determined by UPLC [1]
2. The warhead of MLKL-IN-3 (compound 66) is the chlorine atom at the 6-position of the uracil ring, which undergoes nucleophilic substitution reaction with the sulfhydryl group of Cys86 on MLKL to form a covalent bond [1]
3. MLKL-IN-3 (compound 66) has a different mode of action from TC13172: TC13172 completely inhibits MLKL oligomerization by forming π-π stacking with Phe148, while MLKL-IN-3 (compound 66) mainly inhibits MLKL membrane translocation with weak effects on oligomerization [1]
4. MLKL-IN-3 (compound 66) is optimized through structure-activity relationship (SAR) studies on uracil derivatives, with 100-fold higher potency than the initial hit compound 9 (EC₅₀=3380 nM) [1]
5. The binding of MLKL-IN-3 (compound 66) to MLKL is independent of the modification state of Cys86 by other inhibitors (e.g., BI-8925) and does not rely on Lys157-mediated π-cation interaction [1]

C31H29CLN4O6

589.04

588.17756

3031406-27-3

168279716

Typically exists as solid at room temperature

3.5

2

6

9

42

1240

0

CC1=CC=C(C=C1)CCC(=O)NC2=C(N(C(=O)N(C2=O)C)CC#CC3=CC(=C(C=C3)O)CN4C=CC=C(C4=O)OC)Cl

MWDOKDNDKHXYHZ-UHFFFAOYSA-N

InChI=1S/C31H29ClN4O6/c1-20-8-10-21(11-9-20)13-15-26(38)33-27-28(32)36(31(41)34(2)30(27)40)17-4-6-22-12-14-24(37)23(18-22)19-35-16-5-7-25(42-3)29(35)39/h5,7-12,14,16,18,37H,13,15,17,19H2,1-3H3,(H,33,38)

N-[6-chloro-1-[3-[4-hydroxy-3-[(3-methoxy-2-oxo-1-pyridinyl)methyl]phenyl]prop-2-ynyl]-3-methyl-2,4-dioxopyrimidin-5-yl]-3-(4-methylphenyl)propanamide

MLKL-IN-3; MLKL-IN 3; CHEMBL5188408; SCHEMBL27384351; MLKL-IN3; 3031406-27-3;

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)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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