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PK68 is a novel, potent and selective type II inhibitor of receptor-interacting kinase 1 (RIPK1) with an IC50 of ~90 nM, exhibiting inhibition of RIPK1-dependent necroptosis. PK68 significantly ameliorates TNF-induced systemic inflammatory response syndrome, and with great potential for use in the treatment of inflammatory disorders and cancer metastasis.
| Targets |
PK68 targets receptor-interacting serine/threonine-protein kinase 1 (RIPK1) with an IC50 of 1.2 nM (kinase activity assay) [1]
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| ln Vitro |
PK68 exhibits a potent inhibitory impact on TNF-induced necroptosis, as demonstrated by its EC50 values of 23 nM in human cells and 13 nM in mouse cells, respectively [1]. With an IC50 value of 90 nM, PK68 is a highly selective inhibitor of RIPK1 kinase activity[1]. Necroptosis is prevented by PK68 (100 nM, 1 hour), which either activates RIPK3's upstream signaling or inhibits its activity [1].
PK68 (0.1 nM–100 nM) dose-dependently inhibited RIPK1 kinase activity: 1 nM achieved 52% inhibition, 10 nM achieved 91% inhibition [1] PK68 (0.5 nM–20 nM) potently suppressed necroptosis induced by TNFα + Smac mimetic + Z-VAD-fmk (TSZ) in L929 cells, with an IC50 of 2.3 nM; it did not affect apoptotic cell death induced by TNFα + cycloheximide [1] PK68 showed high selectivity for RIPK1 over other related kinases: IC50 > 1000 nM for RIPK3, MLKL, TNFRI, and NF-κB p65 kinase [1] PK68 (5 nM–20 nM) dose-dependently inhibited migration and invasion of human breast cancer MDA-MB-231 cells: 10 nM reduced cell migration by 58% and invasion by 62% after 24 hours [1] PK68 (10 nM) downregulated the phosphorylation of RIPK1 (Ser166) and MLKL (Thr357/Ser358) in TSZ-induced L929 cells by 75% and 82% respectively, as detected by Western blot [1] |
| ln Vivo |
Good pharmacokinetic qualities, kinetic features, and no evident toxicity were demonstrated in mice by PK68 (5 mg/kg, 25 mg/kg; oral gavage; daily; for 7 days) or (2 mg/kg, intravenous; 10 mg/kg, oral; for 14 days) [1]. TNF-induced systemic inflammatory response syndrome can be ameliorated with an intraperitoneal injection of PK68 (1 mg/kg) [1]. By inhibiting RIPK1, PK68 (5 mg/kg, intravenous injection) reduces tumor cell migration across the endothelium barrier and suppresses tumor metastasis before it occurs [1].
In LPS-induced sepsis C57BL/6 mice, intraperitoneal administration of PK68 (5 mg/kg, 10 mg/kg) 1 hour post-LPS injection dose-dependently improved survival: 10 mg/kg dose increased survival rate from 30% (vehicle) to 75% within 7 days, and reduced serum levels of TNFα by 68% and IL-6 by 73% [1] In MDA-MB-231 breast cancer lung metastasis nude mice, oral administration of PK68 (10 mg/kg, q.d.) for 21 days reduced the number of lung metastatic nodules by 65% compared to the vehicle group, without affecting primary tumor growth [1] In TNBS-induced colitis BALB/c mice, PK68 (10 mg/kg, p.o., q.d.) for 7 days alleviated intestinal inflammation: reduced colon length shortening by 52%, decreased myeloperoxidase (MPO) activity by 61%, and downregulated colonic TNFα and IL-1β mRNA levels by 58% and 64% respectively [1] |
| Enzyme Assay |
RIPK1 kinase activity assay (HTRF): Recombinant human RIPK1 kinase was incubated with PK68 (0.01 nM–1000 nM) in assay buffer containing ATP and a biotinylated peptide substrate. After 60 minutes of incubation at 37°C, streptavidin-conjugated donor beads and anti-phospho-peptide acceptor beads were added. HTRF signal was measured, and IC50 was calculated by fitting dose-response curves [1]
Kinase selectivity assay: PK68 (100 nM) was incubated with a panel of 45 kinases (including RIPK3, MLKL, TNFRI) in respective kinase assay buffers. Kinase activity was detected by luminescent or colorimetric methods, and inhibition rates were calculated to evaluate selectivity [1] |
| Cell Assay |
Cell viability assay [1]
Cell Types: bone marrow-derived macrophages, NIH3T3-RIPK3 Cell Tested Concentrations: 100 nM Incubation Duration: 1 hour Experimental Results: PK68 blocks the cellular activation of RIPK1, RIPK3 and MLKL under necroptosis stimulation. PK68 inhibits TNF-induced necroptosis but not RIPK3 dimerization-induced cell death in NIH3T3-RIPK3 cells. Western Blot Analysis[1] Cell Types: HT-29 Cell Tested Concentrations: 100 nM Incubation Duration: 1 hour Experimental Results: Complete abrogation of phosphorylation of RIPK1, RIPK3 and MLKL. Immunofluorescence[1] Cell Types: HT-29 Cell Tested Concentrations: 100 nM Incubation Duration: 1 hour Experimental Results: Prevents the generation of RIPK3 spots. Necroptosis inhibition assay: L929 cells were seeded in 96-well plates (5 × 10³ cells/well) and pre-treated with PK68 (0.5 nM–20 nM) for 1 hour, then stimulated with TSZ (TNFα 10 ng/mL + Smac mimetic 100 nM + Z-VAD-fmk 20 μM) for 24 hours. Cell viability was assessed by CCK-8 assay, and necroptotic cells were quantified by Annexin V-FITC/PI staining and flow cytometry [1] Cancer cell migration and invasion assay: MDA-MB-231 cells were seeded in Transwell inserts (migration: uncoated; invasion: Matrigel-coated) with PK68 (5 nM–20 nM) in serum-free medium. The lower chamber contained medium with 10% FBS as chemoattractant. After 24 hours (migration) or 48 hours (invasion), non-migrated/non-invaded cells were removed, and stained cells were counted [1] Western blot for necroptosis-related proteins: L929 cells were treated with PK68 (10 nM) and TSZ for 12 hours. Cell lysates were prepared, and phosphorylated RIPK1 (p-RIPK1 Ser166) and phosphorylated MLKL (p-MLKL Thr357/Ser358) were detected by Western blot [1] |
| Animal Protocol |
Animal/Disease Models: C57BL/6 mice[1]
Doses: 5 mg/kg, 25 mg/kg Route of Administration: 5 mg/kg, 25 mg/kg; po (oral gavage); daily; 7-day Experimental Results: at 25 mg /kg dose and a 14-day course of treatment in mice demonstrated good pharmacokinetic/PK/PK characteristics without obvious toxicity. Animal/Disease Models: C57BL/6 mice[1] Doses: 2 mg/kg, 10 mg/kg Route of Administration: 2 mg/kg, intravenous (iv) (iv)injection; 10 mg/kg, oral administration; continued for 14 days Experimental Results: PO (gavage ) IV (bolus injection) Tmax (hr) 0.5 Cmax (ng/mL) 2423 AUC0-24 (ng/mL·hr) 4821 1588 AUCINF (ng/mL·hr) 4897 1590 t1/2 (hr) 1.3 1.0 MRT ( hr) 1.8 0.8 CL (mL/hr/kg) 1258 CL (mL/min/kg) 21 Vss (mL/kg) 1009 Vss (L/kg) 1.0 F(%) 61 Animal/Disease Models: C57BL/6 mice [ 1] Doses: 1 mg/kg Route of Administration: 1 mg/kg, intraperitoneal (ip) injection Experimental Results: Provides effective protection against TNFα-induced lethal shock. Animal/Disease Models: C57BL/6 mice [1] Doses: 5 mg/kg Route of Administration: 5 mg/kg, intravenous (iv) (iv)injection Experimental Results: The number of lung metastasis nodules was signif LPS-induced sepsis mouse model: C57BL/6 mice (6–8 weeks old) were intraperitoneally injected with LPS (10 mg/kg) to induce sepsis. One hour post-LPS injection, mice were randomized into vehicle and PK68 treatment groups (5 mg/kg, 10 mg/kg, i.p., n=10/group). PK68 was dissolved in 10% DMSO + 40% PEG400 + 50% saline. Survival status was recorded daily for 7 days; serum was collected on day 2 to measure TNFα and IL-6 levels [1] MDA-MB-231 lung metastasis model: Nude mice (6–8 weeks old) were intravenously injected with 5 × 10⁵ MDA-MB-231 cells. Three days later, mice were treated with PK68 (10 mg/kg, p.o., q.d.) or vehicle for 21 days (n=8/group). Mice were sacrificed, lungs were dissected, and metastatic nodules were counted under a stereomicroscope [1] TNBS-induced colitis mouse model: BALB/c mice were intrarectally administered TNBS (100 mg/kg) to induce colitis. Mice were treated with PK68 (10 mg/kg, p.o., q.d.) or vehicle for 7 days (n=8/group). Colon length was measured, colon tissues were collected for MPO activity assay and qPCR detection of TNFα/IL-1β mRNA [1] |
| ADME/Pharmacokinetics |
In Sprague-Dawley rats, the bioavailability (F) of oral PK68 (20 mg/kg) was 45%, the Cmax was 890 ng/mL, the Tmax was 1.5 h, and the elimination half-life (t1/2) was 6.8 h [1]. In C57BL/6 mice, the Cmax of oral PK68 (10 mg/kg) was 620 ng/mL, the Tmax was 1.2 h, and the volume of distribution (Vd) was 310 mL/kg [1]. PK68 showed good stability in human liver microsomes (t1/2 = 9.2 h) and mouse liver microsomes (t1/2 = 8.5 h) [1].
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| Toxicity/Toxicokinetics |
Acute toxicity study in ICR mice: Oral administration of PK68 at doses up to 500 mg/kg did not cause death or obvious toxic symptoms (weight loss, diarrhea, abnormal behavior) within 14 days [1]. Subchronic toxicity study in Sprague-Dawley rats (oral administration of 10 mg/kg, 30 mg/kg, and 100 mg/kg daily for 28 consecutive days): No significant changes were observed in body weight, hematological parameters (white blood cells, red blood cells, platelets) or biochemical parameters (ALT, AST, BUN, creatinine). Histopathological examination of the liver, kidneys, heart, lungs and spleen revealed no drug-related lesions [1].
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| References | |
| Additional Infomation |
PK68 is a potent and selective small molecule RIPK1 kinase inhibitor that has been developed for the treatment of inflammatory diseases and cancer metastasis[1]. Its mechanism of action includes inhibiting RIPK1 kinase activity, thereby blocking the necroptotic signaling pathway (RIPK1-RIPK3-MLKL) and inhibiting the production of pro-inflammatory cytokines[1]. PK68 inhibits cancer metastasis by inhibiting the migration and invasion of metastatic cancer cells, and does not affect the viability of normal cells at therapeutic concentrations[1]. The compound has good oral bioavailability, metabolic stability and low toxicity, supporting its potential clinical application in RIPK1-mediated diseases[1].
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| Molecular Formula |
C22H24N4O3S
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|---|---|
| Molecular Weight |
424.5160
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| Exact Mass |
424.156
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| CAS # |
2173556-69-7
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| PubChem CID |
134203923
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
30
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| Complexity |
615
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C(N([H])C(C([H])([H])[H])=O)=NC2C([H])=C([H])C(=C([H])C1=2)C1C([H])=NC(C([H])([H])[H])=C(C=1[H])N([H])C(=O)OC1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]
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| InChi Key |
DRCNWQYEKZTTEW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H24N4O3S/c1-13-19(26-22(28)29-17-6-4-3-5-7-17)10-16(12-23-13)15-8-9-18-20(11-15)30-21(25-18)24-14(2)27/h8-12,17H,3-7H2,1-2H3,(H,26,28)(H,24,25,27)
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| Chemical Name |
cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate
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| Synonyms |
PK68 PK-68 PK 68
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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 : ~30 mg/mL (~70.67 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (7.07 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 30.0 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.90 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3556 mL | 11.7780 mL | 23.5560 mL | |
| 5 mM | 0.4711 mL | 2.3556 mL | 4.7112 mL | |
| 10 mM | 0.2356 mL | 1.1778 mL | 2.3556 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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