RIP3 (IC50 = 20.8 nM)

HS-1371 shows an inhibitory effect on S227 auto-phosphorylation of RIP3 at the basal level. It completely inhibits TNF-induced necroptosis signaling, as evidenced by the absence of RIP3 and MLKL phosphorylation in HT-29 cells. By preventing the formation of necrosome complexes, HS-1371's inhibition of RIP3 kinase activity demonstrates disruption of MLKL recruitment. HS-1371 prevents the necroptosis that TNF causes in cells. It prevents necroptotic cell death caused by RIP3 but does not prevent apoptotic cell death[1].

Reaction Biology Corp used radiometric kinase assays ([γ-32P]ATP) to determine all compounds' inhibitory activities toward RIP3. Myelin basic protein (MBP) in a freshly made reaction buffer (20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% BRIJ-35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO) was used to measure the enzymatic activity of RIP3. Each putative RIP3 inhibitor was dissolved in 100% DMSO at particular concentrations, serially diluted with epMotion 5070 in DMSO, and then tested. The reaction buffer received additions of human RIP3 and 20 μM peptide substrate (MBP). The kinase reaction mixture was administered the candidate inhibitor dissolved in DMSO using Acoustic technology (Echo550; nanoliter range), and the reaction mixture was then incubated for 20 min at room temperature. A final ATP concentration of 10 μM and 33P-ATP with a specific activity of 10 μCi/μL were added to the reaction mixture to start the enzymatic reaction. After allowing the reaction mixture to sit at room temperature for two hours, radioactivity was then measured using the filter binding technique. The percentage of remaining kinase activity in relation to the vehicle (dimethyl sulfoxide) reaction was used to calculate the biochemical potency of an inhibitor at a given concentration. The PRISM program was then used to obtain IC50 values and curve fits. Due to its high biochemical potency against numerous kinases, including RIP3, the ATP-competitive inhibitor staurosporine (STSP) was used in this study as a positive control. [1]

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Enzymatic assays[1]
The inhibitory activities of all compounds toward RIP3 were measured by Reaction Biology Corp by means of radiometric kinase assays ([γ-32P]ATP). The enzymatic activity of RIP3 was monitored using 20 μM of myelin basic protein (MBP) dissolved in freshly prepared reaction buffer (20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% BRIJ-35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). Each putative RIP3 inhibitor was dissolved in 100% DMSO at specific concentrations and serially diluted with epMotion 5070 in DMSO. Human RIP3 and 20 μM of peptide substrate (MBP) were added to the reaction buffer. After delivering the candidate inhibitor dissolved in DMSO to the kinase reaction mixture using Acoustic technology (Echo550; nanoliter range), the reaction mixture was incubated for 20 min at room temperature. To initiate the enzymatic reaction, 33P-ATP with specific activity of 10 μCi/μL was added to the reaction mixture to reach a final ATP concentration of 10 μM. Radioactivity was then monitored using the filter binding method after incubation of the reaction mixture for 2 h at room temperature. At given concentrations of inhibitor, biochemical potency was measured by the percent remaining kinase activity with respect to the vehicle (dimethyl sulfoxide) reaction. Curve fits and IC50 values were then obtained using the PRISM program (GraphPad Software). The ATP-competitive inhibitor staurosporine (STSP) was employed as a positive control in this study because of its high biochemical potency against various kinases including RIP3.

Nec-1 (40 μM), DAB (5 μM), or HS-1371 were pretreated in HT-29 cells for 2 hours before being given TSZ for 4 hours. Anti-RIP3 antibody was used to immunoprecipitate cell lysates.

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Immunoblot analysis and immunoprecipitation [1]
Cells were rinsed in cold phosphate-buffered saline (PBS) and lysed in M2 buffer containing 20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, and 1 μg/mL leupeptin. The cell extracts were subjected to western blot analysis. For immunoprecipitation, lysates were mixed and precipitated with antibody and protein A-agarose beads overnight or for 4 h at 4 °C. Bound proteins were removed by boiling in SDS and resolved by SDS-PAGE, and immunoblotting was visualized by enhanced chemiluminescence.

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Cytotoxicity assays [1]
Cell viability was determined using tetrazolium dye colorimetric tests (the MTT assay) read at 570 nm. PI staining was quantified using propidium iodide. Lactate dehydrogenase (LDH) leakage was quantified using the CellTiter-Glo Luminescent Cell Viability Assay kit according to the manufacturer’s instructions. The LDH absorbance was read at 490 nm, and the mean ± STDEV of duplicates is presented.

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Immunofluorescence staining [1]
HT-29 cells were fixed in 4% paraformaldehyde for 10 min. To stain phospho-MLKL, cells were permeabilized with 0.25% Triton X-100 for 10 min. After incubation in a blocking buffer (10% fetal bovine serum in DPBS) for 30 min, the primary antibody to phospho-MLKL was incubated overnight at 4 °C, and FITC-conjugated secondary antibody (goat anti-rabbit IgG, 1:250, dilution) was incubated for 1 h at room temperature. A mounting medium containing DAPI was used for counterstaining. Representative images were taken by confocal microscope.

[1]. HS-1371, a novel kinase inhibitor of RIP3-mediated necroptosis. Exp Mol Med. 2018 Sep 20;50(9):125.

Necroptosis is a type of programmed cell death that usually occurs under apoptosis-deficient conditions. Receptor-interacting protein kinase-3 (RIP3, or RIPK3) is a central player in necroptosis, and its kinase activity is essential for downstream necroptotic signaling events. Since RIP3 kinase activity has been associated with various diseases, the development of specific RIP3 inhibitors is an attractive strategy for therapeutic application. In this study, we identified a potent RIP3 inhibitor, HS-1371, by the extensive screening of chemical libraries focused on kinases. HS-1371 directly binds to RIP3 in an ATP-competitive and time-independent manner, providing a mechanism of action. Moreover, the compound inhibited TNF-induced necroptosis but did not inhibit TNF-induced apoptosis, indicating that this novel inhibitor has a specific inhibitory effect on RIP3-mediated necroptosis via the suppression of RIP3 kinase activity. Our results suggest that HS-1371 could serve as a potential preventive or therapeutic agent for diseases involving RIP3 hyperactivation.[1]

C24H24N4O

384.473565101624

384.195

C, 74.97; H, 6.29; N, 14.57; O, 4.16

2158197-70-5

134817449

White to off-white solid powder

4

1

4

4

29

517

0

VPVLPCIBKVWFDT-UHFFFAOYSA-N

InChI=1S/C24H24N4O/c1-17-2-5-21(6-3-17)29-24-10-13-26-23-14-18(4-7-22(23)24)19-15-27-28(16-19)20-8-11-25-12-9-20/h2-7,10,13-16,20,25H,8-9,11-12H2,1H3

4-(4-methylphenoxy)-7-(1-piperidin-4-ylpyrazol-4-yl)quinoline

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)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.34 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 16.7 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: ≥ 1.67 mg/mL (4.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (4.34 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 16.7 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.)