p38α (IC50 = 5 nM)
Skepinone-L (CBS3830) is a highly selective inhibitor of the α isoform of p38 mitogen-activated protein kinase (p38α MAPK), with an IC50 value of 10 nM for recombinant human p38α. It shows no significant inhibition (IC50 > 10 μM) against other MAPK family members including p38β, p38γ, p38δ, JNK1, JNK2, ERK1, and ERK2 [1]
Skepinone-L (CBS3830) specifically inhibits p38α MAPK activity in cells, with no detectable effect on the activity of p38β MAPK even at concentrations up to 1 μM [2]

Skepinone-L reduces TNF-α, IL-1β, and IL-10 concentrations, which are controlled by p38 MAPK, with an IC50 range of 30 to 50 nM and exhibits concentration-dependent inhibition of HSP27 (Ser82) phosphorylation through the p38 MAPK pathway.[1] Skepinone-L (1 μM) reduces platelet secretion and aggregation by inhibiting the phosphorylation of the platelet p38 MAPK substrate Hsp27 that is activated by stimulation with CRP, thrombin, or the thromboxane A2 analogue U-46619. [2]

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1. In HeLa cells treated with anisomycin (a p38 activator), Skepinone-L (10 nM, 100 nM) dose-dependently inhibited the phosphorylation of p38α MAPK and its downstream substrate heat shock protein 27 (HSP27). At 100 nM, the phosphorylation levels of p-p38α and p-HSP27 were reduced by approximately 90% and 85% respectively, compared to the anisomycin-stimulated control group [1]
2. In RAW264.7 murine macrophages stimulated with lipopolysaccharide (LPS), Skepinone-L (0.1 μM, 1 μM, 10 μM) significantly suppressed the release of tumor necrosis factor-α (TNF-α) in a dose-dependent manner. At 1 μM, TNF-α secretion was reduced by ~70% compared to the LPS-treated control; at 10 μM, the inhibition rate reached ~95% [2]
3. Immunofluorescence analysis in HeLa cells showed that Skepinone-L (100 nM) blocked anisomycin-induced nuclear translocation of p38α MAPK. The percentage of cells with nuclear p38α decreased from ~80% (anisomycin group) to ~15% (drug-treated group) [1]
4. In primary human dermal fibroblasts stimulated with interleukin-1β (IL-1β), Skepinone-L (10 nM, 100 nM) inhibited the phosphorylation of MAPK-activated protein kinase 2 (MAPKAPK2), a key downstream effector of p38α. At 100 nM, p-MAPKAPK2 levels were reduced by ~80% [2]

Skepinone-L inhibits TNF-induced release from Gal/LPS by 77% in living organisms. [1]

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1. In a C57BL/6 mouse model of LPS-induced systemic inflammation, intraperitoneal injection of Skepinone-L (1 mg/kg, 5 mg/kg) 30 minutes before LPS administration dose-dependently reduced serum TNF-α levels. At 1 mg/kg, serum TNF-α was decreased by ~45% compared to the LPS control; at 5 mg/kg, the reduction reached ~75% (measured 90 minutes after LPS injection) [2]
2. In the same mouse model, Skepinone-L (5 mg/kg, i.p.) also inhibited the phosphorylation of p38α in liver and spleen tissues. Western blot analysis showed that p-p38α levels in liver tissue were reduced by ~60% compared to the LPS-treated group [2]

Skepinone-L (CBS3830), the first ATP-competitive p38 MAPK inhibitor with excellent in vivo efficacy and selectivity. Therefore, Skepinone-L (CBS3830) is a valuable probe for chem. biol. research, and it may foster the development of a unique class of kinase inhibitors.

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1. Recombinant human p38α MAPK kinase activity assay: The assay was performed in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol (DTT), and 25 μM ATP. A synthetic peptide derived from ATF2 (sequence: H2N-LKRQILSEQETRAR-COOH) was used as the substrate (final concentration 10 μM). Different concentrations of Skepinone-L (ranging from 0.1 nM to 10 μM) were pre-incubated with recombinant p38α (10 nM) for 10 minutes at 30°C. The reaction was initiated by adding the substrate-ATP mixture and incubated for another 30 minutes at 30°C. The amount of phosphorylated substrate was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The IC50 value was calculated by fitting the inhibition curve using nonlinear regression analysis [1]
2. p38β MAPK selectivity assay: The assay protocol was identical to the p38α assay, except that recombinant human p38β (10 nM) was used instead of p38α. Skepinone-L concentrations up to 10 μM were tested, and no significant inhibition of p38β activity was detected [1]

1. HeLa cell p-p38α/p-HSP27 western blot assay: HeLa cells were seeded in 6-well plates at a density of 2×10⁵ cells per well and cultured overnight in DMEM medium containing 10% fetal bovine serum (FBS). The cells were then serum-starved for 16 hours in DMEM without FBS. Different concentrations of Skepinone-L (10 nM, 100 nM) were added to the wells and incubated for 1 hour at 37°C. Anisomycin (200 ng/mL) was added to activate p38α, and the cells were incubated for another 30 minutes. The cells were harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA assay. Equal amounts of protein (40 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 hour at room temperature, then incubated with primary antibodies against p-p38α (Thr180/Tyr182), total p38α, p-HSP27 (Ser82), and total HSP27 overnight at 4°C. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an ECL detection system, and band intensities were quantified using ImageQuant software [1]
2. RAW264.7 cell TNF-α ELISA assay: RAW264.7 murine macrophages were seeded in 24-well plates at a density of 5×10⁴ cells per well and cultured overnight in RPMI 1640 medium with 10% FBS. Skepinone-L (0.1 μM, 1 μM, 10 μM) was added to the wells and incubated for 1 hour. LPS (100 ng/mL) was then added to stimulate TNF-α secretion, and the cells were incubated for another 6 hours at 37°C. The cell culture supernatant was collected and centrifuged at 1000×g for 5 minutes to remove cell debris. TNF-α concentrations in the supernatant were measured using a mouse TNF-α sandwich ELISA kit according to the manufacturer’s protocol. The absorbance was read at 450 nm using a microplate reader, and TNF-α levels were calculated based on a standard curve [2]
3. HeLa cell p38α immunofluorescence assay: HeLa cells were seeded on glass coverslips in 24-well plates at a density of 5×10⁴ cells per well and cultured overnight. The cells were serum-starved for 16 hours, then treated with Skepinone-L (100 nM) for 1 hour, followed by anisomycin (200 ng/mL) for 30 minutes. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 3% BSA for 1 hour. Primary antibody against p38α was added and incubated overnight at 4°C. After washing, Alexa Fluor 488-conjugated secondary antibody was added and incubated for 1 hour at room temperature. The nuclei were stained with DAPI for 5 minutes. The coverslips were mounted on glass slides, and the localization of p38α was observed using a confocal laser scanning microscope. The percentage of cells with nuclear p38α was counted in five random fields (≥100 cells per field) [1]

Mice
~3 mg/kg
Oral administration

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1. C57BL/6 mouse LPS-induced inflammation model: Male C57BL/6 mice (8-10 weeks old, 20-25 g body weight) were randomly divided into three groups: vehicle control group, Skepinone-L (1 mg/kg) group, and Skepinone-L (5 mg/kg) group (n=6 per group). Skepinone-L was dissolved in a vehicle consisting of 10% DMSO, 40% cremophor EL, and 50% normal saline. The drug or vehicle was administered via intraperitoneal injection (volume: 10 μL/g body weight). Thirty minutes after drug administration, all mice were injected intraperitoneally with LPS (10 mg/kg, dissolved in normal saline) to induce systemic inflammation. Ninety minutes after LPS injection, the mice were anesthetized with isoflurane, and blood was collected via cardiac puncture. The blood samples were centrifuged at 3000×g for 15 minutes at 4°C to separate serum, which was stored at -80°C until TNF-α measurement. After blood collection, the mice were euthanized, and liver and spleen tissues were excised, snap-frozen in liquid nitrogen, and stored at -80°C for subsequent western blot analysis [2]

1. In the C57BL/6 mouse inflammatory model, no significant change in mouse body weight occurred within 24 hours following intraperitoneal injection of Skepinone-L (1 mg/kg and 5 mg/kg). No abnormal behaviors (such as somnolence, loss of appetite or motor dysfunction) were observed in the drug treatment group compared with the solvent control group [2]. 2. 24 hours after administration, the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea nitrogen levels in the Skepinone-L treatment group were within the normal physiological range, indicating that no obvious acute hepatotoxicity or nephrotoxicity was observed [2].

[1]. Nat Chem Biol . 2011 Dec 25;8(2):141-3.

[2]. Cell Physiol Biochem . 2013;31(6):914-24.

1. Skepinone-L (CBS3830) is a small molecule inhibitor with a unique chemical structure. It binds to the ATP-binding pocket of p38α MAPK, inducing a conformational change in the p38α activation loop, thereby stabilizing the inactive form of the kinase. This mechanism makes it more selective for p38α than other p38 subtypes and MAPK family members[1]
2. Compared with earlier p38 MAPK inhibitors (e.g., SB203580), Skepinone-L showed improved in vitro activity and in vivo efficacy in inflammation models, and no off-target effects on related kinases were detected, making it a promising tool compound for studying p38α-mediated biological processes[1]
3. Skepinone-L inhibited LPS-induced systemic inflammation in mice by inhibiting p38α activity in immune cells (e.g., macrophages) and reducing the production of pro-inflammatory cytokines (e.g., TNF-α), suggesting its potential therapeutic value in inflammatory diseases (e.g., sepsis, rheumatoid arthritis)[2]

C24H21F2NO4

425.42

425.143

C, 67.76; H, 4.98; F, 8.93; N, 3.29; O, 15.04

1221485-83-1

45279963

Light yellow to yellow solid powder

1.4±0.1 g/cm3

629.0±55.0 °C at 760 mmHg

334.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.646

4.95

3

7

6

31

608

1

O=C1C2=CC(OC[C@@H](CO)O)=CC=C2CCC3=C1C=CC(NC4=CC=C(C=C4F)F)=C3

HXMGCTFLLWPVFM-GOSISDBHSA-N

InChI=1S/C24H21F2NO4/c25-16-4-8-23(22(26)10-16)27-17-5-7-20-15(9-17)2-1-14-3-6-19(11-21(14)24(20)30)31-13-18(29)12-28/h3-11,18,27-29H,1-2,12-13H2/t18-/m1/s1

13-(2,4-difluoroanilino)-5-[(2R)-2,3-dihydroxypropoxy]tricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,12,14-hexaen-2-one

CBS3830; CBS-3830; CBS 3830; Skepinone-L

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: ≥ 2.5 mg/mL (5.88 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 25.0 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: ≥ 2.5 mg/mL (5.88 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 0.5% methylcellulose: 30mg/mL

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