P28 targets Mixed Lineage Kinase Domain-like Protein (MLKL).
It covalently modifies the cysteine residue in the FSNRSNICRFLTASQDK peptide at the N-terminus of human MLKL. The EC50 of MLKL binding to P28 is 10.3 μM, as determined by spectral shift dose-response curves. [1]

n FADD-knockout Jurkat T cells, P28 potently inhibits TNF-α-induced necroptosis with an IC50 < 1 μM, as measured by cell viability assay. [1]
At 0.1 μM, P28 demonstrates greater potency than necrosulfonamide (NSA) in preventing cell death after necroptotic stimulation, as assessed by LDH release. [1]
Flow cytometry analysis using Annexin V/PI staining shows that P28 treatment (at 1, 3, and 10 μM) reduces late apoptosis and cell death, with the proportion of Annexin V⁺PI⁺ cells being 23.25%, 2.52%, and 1.1%, respectively, compared to 39.04% in necroptosis-induced control cells. [1]
In HT-29 cells, P28 treatment (3 μM) decreases the phosphorylation of MLKL located in the plasma membrane after TSZ-induced necroptosis, as shown by immunofluorescence and plasma membrane fraction analysis. [1]
Transmission electron microscopy analysis reveals that P28 (3 μM) reverses TSZ-induced plasma membrane rupture and permeability in HT-29 cells. [1]
Under nonreducing SDS-PAGE conditions, P28 (3 μM) considerably decreases TSZ-induced MLKL oligomerization in HT-29 cells. [1]
In hepatic stellate cells (LX-2), P28 treatment (3 μM) inhibits the mRNA expression of fibrosis markers (α-SMA, collagen-1), chemokines (CXCL1, CXCL2), MLKL, and adhesion molecule (ICAM1) induced by necroptosis stimulation, as measured by quantitative RT-PCR. [1]
Immunofluorescence staining shows that P28 (3 μM) decreases α-SMA expression in LX-2 cells under necroptosis conditions. [1]
In U937 monocytes, P28 treatment (1, 3, and 10 μM) inhibits TSZ-induced expression of cytokines (TNF-α, CCL2), chemokines (CXCL1, CXCL2), and adhesion molecule (ICAM1) in a dose-dependent manner, as measured by quantitative RT-PCR. [1]
Flow cytometry analysis reveals that P28 treatment (1, 3, and 10 μM) dose-dependently reduces CCR2 expression in TSZ-treated U937 cells. [1]
LC-MS/MS analysis confirms that P28 covalently modifies the cysteine residue in the FSNRSNICRFLTASQDK peptide at the N-terminus of human MLKL. [1]
Compared to NSA, P28 shows lower cytotoxicity in FADD-knockout Jurkat T cells at 10 μM. [1]

The binding interaction between P28 and recombinant MLKL was assessed using spectral shift analysis. The concentration of fluorescently labeled MLKL was kept constant, while the concentration of P28 was varied from 24 nM to 100 μM. The dose-response curve was generated, and the EC50 value was calculated. The half-maximal effective concentration (EC50) of MLKL binding to P28 was determined to be 10.3 μM. [1]
The covalent binding site of P28 on MLKL was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Recombinant MLKL protein was treated with P28, followed by enzymatic digestion. The resulting peptide fragments were analyzed using an Orbitrap Exploris 480 mass spectrometer equipped with a nanoelectrospray source. The samples were loaded onto a C18 trap column, desalted, and then separated on a C18 analytical column. The mobile phases were water and acetonitrile, each containing 0.1% formic acid. The LC gradient was run with increasing acetonitrile concentration over time. Mass spectrometry was operated in data-dependent mode, switching between MS1 and MS2 scans. Full-scan MS1 spectra were acquired in the Orbitrap, and MS2 spectra were acquired using higher-energy collisional dissociation (HCD). The resulting fragment ions (b8 to b16 and y10 to y16) from the peptide FSNRSNICRFLTASQDK were analyzed. The m/z values of the unmodified and P28-modified peptides differed by the mass of P28 (394.1288 for +1 charge or 197.0644 for +2 charge), confirming covalent modification of the cysteine residue. [1]

For cell proliferation and cytotoxicity assays, cells were seeded in 96-well plates and incubated for 24 hours. After drug treatment, proliferation was assessed using the Cell Counting Kit-8, with absorbance measured at 450 nm. Cell ATP content was assessed using the CellTiter-Glo 2.0 assay, and luminescence was measured. For LDH release assays, cells were seeded in 96-well plates and incubated for 24 hours, then cell toxicity was evaluated using an LDH cytotoxicity detection kit. [1]
For flow cytometry-based cell death analysis, cells (1 × 10⁶ cells/well) were plated in 100 mm dishes and treated for 24 hours. Cells were harvested and incubated with Annexin V (2 μg/mL) and propidium iodide (PI, 10 μg/mL). Necroptosis was evaluated based on cell morphology (swelling, detachment, membrane rupture) and Annexin V/PI staining. [1]
For immunofluorescence staining, cells were seeded on eight-well chamber slides, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 10% normal goat serum and 1% BSA. Cells were incubated with primary antibody overnight at 4°C, followed by fluorescent secondary antibody for 1 hour at room temperature, and mounted with DAPI mounting solution. [1]
For RNA isolation and quantitative RT-PCR, total RNA was extracted using TRIzol reagent and converted to complementary DNA using a PrimeScript RT reagent kit. Polymerase chain reactions were performed using a LightCycler 480 system with SYBRGreen I Mastermix. [1]
For nonreducing SDS-PAGE, samples were prepared in buffer lacking 2-mercaptoethanol or dithiothreitol to preserve disulfide bonds. [1]
For cytosol and nuclear fraction extraction, cells were processed using a cell fractionation kit via buffer extraction. [1]
For plasma membrane protein extraction, cells were lysed in buffer mixture, homogenized using a syringe, and centrifuged at 700g for 10 minutes. The supernatant was further centrifuged at 10,000g for 30 minutes, and the resulting supernatant was collected as the cytoplasmic fraction. [1]
For transmission electron microscopy, cells were fixed in 2.5% glutaraldehyde buffer with 0.1 M PBS for 8 hours, postfixed in 1% osmium tetrazide, dehydrated in graded ethanol, and embedded in resin. Sections were cut using an ultramicrotome, stained with uranyl acetate and lead citrate, and imaged. [1]

In FADD-knockout Jurkat T cells, P28 at 10 μM shows less cytotoxicity compared to necrosulfonamide (NSA). NSA treatment at 10 μM resulted in 97.26% cell death, while P28 showed significantly reduced toxicity at the same concentration. [1]
In contrast to NSA and TC13172, which exhibit cytotoxicity at concentrations ≥ 10 μM with lower therapeutic ranges, P28 was developed using a xanthine scaffold to avoid cytotoxicity. The methyl sulfone functional group of TC13172 is activated via hydrogen bonding with cysteine 86 and generates a byproduct (CH₃SO₂H) with strong toxicity. [1]

[1]. Novel Inhibitor of Mixed-Lineage Kinase Domain-Like Protein: The Antifibrotic Effects of a Necroptosis Antagonist. ACS Pharmacology & Translational Science, 2023 Sep 11;6(10):1471-1479.

P28 is a novel MLKL inhibitor developed based on the structures of NSA and TC13172, selected after screening 100 compounds. It demonstrates potent necroptosis inhibition and antifibrotic effects. [1]
P28 directly inhibits MLKL phosphorylation and oligomerization after necroptosis induction, inhibits immune cell death, and reduces the expression of adhesion molecules. Additionally, P28 reduces the activation of hepatic stellate cells and the expression of hepatic fibrosis markers induced by necroptosis stimulation. [1]
Unlike NSA and TC13172, which show cell toxicity and species specificity, P28 treatment does not induce cytotoxicity. [1]
P28 has several advantages over previously developed necroptosis inhibitors: (1) it avoids the cytotoxicity associated with the methyl sulfone functional group of TC13172 by using a xanthine scaffold; (2) it shows higher inhibitory efficacy than previously developed drugs as measured by LDH release from necroptotic cells; (3) it efficiently reduces necroptosis-induced chemokine and adhesion molecule expression in immune cells and hepatic stellate cells. [1]
Limitations include that P28 does not show a significantly lower concentration than previously developed MLKL inhibitors, and further improvement in its efficacy is required. [1]
The findings suggest that targeting MLKL with P28 is a promising strategy for treating inflammatory and liver diseases, including nonalcoholic fatty liver disease (NAFLD), in which necroptosis has been implicated in disease progression. [1]

C20H18N4O5

394.38

394.1277196

White to off-white solid powder

1.4

1

6

5

29

769

0

CN1C(=NC2=C1C(=O)N(C(=O)N2CC#CC3=CC(=CC=C3)O)C)/C=C/C(=O)OC

PURCTTCEAJLVTC-MDZDMXLPSA-N

InChI=1S/C20H18N4O5/c1-22-15(9-10-16(26)29-3)21-18-17(22)19(27)23(2)20(28)24(18)11-5-7-13-6-4-8-14(25)12-13/h4,6,8-10,12,25H,11H2,1-3H3/b10-9+

methyl (E)-3-[3-[3-(3-hydroxyphenyl)prop-2-ynyl]-1,7-dimethyl-2,6-dioxopurin-8-yl]prop-2-enoate

MLKL-IN-6; MLKL-IN 6; MLKL-IN6; MLKL inhibitor P28; TP prodrug CX-23; Triptolide prodrug CX-23; MLKL-IN-P28;

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: 100 mg/mL (253.6 mM)

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.)