NF-κB; TBK1 (IKKβ = 3.598 μM); IKKα (IC50 = 8.975 μM)
Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) with an IC50 of 8.97 µM. [1]
Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) with an IC50 of 3.59 µM. [1]

INH14 inhibited TLR2-mediated NF-κB activation in a dose-dependent manner in HEK293-TLR2 cells stimulated with Pam3CSK4 (P3), with an IC50 of 4.127 µM in a luciferase reporter assay. [1]
INH14 (10 µM) also inhibited NF-κB activation induced by stimulation of IL-1 receptor (with IL-1β) or TNF receptor (with TNFα) in HEK293 cells. [1]
INH14 (15 µM) reduced TNFα production in RAW264.7 mouse macrophages stimulated with TLR2 ligand P3 or TLR4 ligand LPS, as measured by ELISA. A similar reduction was observed in human primary monocytes stimulated with P3, LPS, or IL-1β. [1]
INH14 decreased NF-κB transcriptional activity induced by overexpression of various proteins in the TLR2 signaling pathway (Mal, MyD88, IRAK1, TRAF6, TAK1/TAB1, IKKα, IKKβ) in HEK293 cells. [1]
INH14 inhibited NF-κB activity (using a kb3.luc reporter) but not AP-1 activity (using an AP1.luc reporter) in HEK293-TLR2 cells stimulated with TLR2 ligands. [1]
Immunoblotting analysis showed that INH14 treatment significantly reduced lipopeptide (P3)-induced IκBα degradation in HEK293-TLR2 cells, but did not affect the phosphorylation of p38 or JNK. [1]
INH14 decreased NF-κB transcriptional activity induced by TLR3 stimulation (with PolyI:C) or by overexpression of the TRIF adaptor protein in HEK293 cells, but did not affect IRF3-dependent promoter (INFβ.luc) activity. [1]
INH14 (20 µM) decreased the constitutive NF-κB activity in ovarian cancer SKOV3 cells by approximately 50% in a luciferase reporter assay. Immunoblotting confirmed reduced IκBα degradation in SKOV3 cells treated with INH14. [1]
In a wound-healing assay, INH14 (20 µM) inhibited the migration of SKOV3 ovarian cancer cells after 48 hours of treatment. [1]

INH14 (5 µg/g, i.p. for 2 hours) decreases inflammation brought on by lipopeptide in mice[1].

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In a mouse model of lipopeptide-induced inflammation, intraperitoneal pretreatment with INH14 (5 mg/kg) 1 hour before injection of Pam2CSK4 (P2, 2.5 mg/kg) significantly reduced the serum level of TNFα measured 2 hours post-injection, compared to the vehicle control group. No pathological effects (e.g., weight loss, abnormal movements, dyspnea) were observed in the treated animals. [1]

A kinase assay was performed using recombinant IKKα enzyme. The enzyme (15 ng per reaction) was incubated with ATP (50 µM) and a substrate peptide (0.2 mg/mL) in the presence of vehicle or increasing concentrations of INH14 at room temperature for 1 hour. The production of ADP was quantified using a chemiluminescence-based detection method (ADP-Glo assay). The IC50 value for IKKα inhibition was determined to be 8.97 µM. [1]
A similar kinase assay was performed using recombinant IKKβ enzyme (20 ng per reaction) with ATP (25 µM) and substrate peptide. The assay was conducted in the presence of vehicle or increasing concentrations of INH14. The IC50 value for IKKβ inhibition was determined to be 3.59 µM. [1]

Luciferase Reporter Assay: HEK293 or HEK293-TLR2 cells were transfected with an NF-κB-dependent luciferase reporter plasmid (Elam.luc or kb3.luc) and a constitutively active Renilla plasmid for normalization. After transfection, cells were treated with INH14 or vehicle for 1 hour, followed by stimulation with specific ligands (e.g., P3, IL-1β, TNFα, PolyI:C) or by transfection with plasmids expressing signaling proteins. After incubation (typically 5 hours post-stimulation or as specified), cells were lysed, and luciferase activity was measured. The signal was normalized to Renilla activity to quantify NF-κB activation. [1]
Enzyme-Linked Immunosorbent Assay (ELISA): Cells (e.g., RAW264.7 macrophages, human primary monocytes) were pretreated with INH14 or vehicle, then stimulated with inflammatory ligands (e.g., P3, LPS, IL-1β). Cell culture supernatants were collected after a specified incubation period (e.g., 9 hours or overnight). The concentration of secreted TNFα in the supernatant was quantified using commercial ELISA kits according to the manufacturer's instructions. [1]
Immunoblotting (Western Blot): Cells (e.g., HEK293-TLR2, SKOV3) were treated with INH14 and stimulated as indicated. Cells were then washed, lysed in a buffer containing protease and phosphatase inhibitors, and centrifuged. Protein concentrations were determined, and equal amounts of lysate were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked. Membranes were incubated overnight at 4°C with primary antibodies (e.g., anti-IκBα, anti-phospho-p38, anti-phospho-JNK, anti-β-actin), followed by incubation with HRP-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence detection reagents and imaged. Band intensity was quantified using appropriate software. [1]
Cell Viability Assay (CCK-8): Human primary monocytes were seeded in 96-well plates and incubated overnight with INH14 (20 µM), vehicle (DMSO), or a positive control (SDS). A tetrazolium salt (WST-8) was then added to each well, and cells were incubated for an additional hour at 37°C. The formation of formazan dye by dehydrogenase activity in viable cells was measured by the increase in absorbance at 450 nm using a plate reader. [1]
Wound-Healing Assay: SKOV3 ovarian cancer cells were seeded in 6-well plates to form a confluent monolayer. A scratch was made in the monolayer using a pipette tip. The medium was replaced with low-serum medium containing INH14 (20 µM), vehicle, or a control inhibitor (5Z-7-oxozeaenol, 20 µM). Photographs of the scratch were taken immediately after wounding and after 48 hours of incubation using a light microscope. The width of the wound gap was measured at multiple points using image analysis software to quantify cell migration. [1]

8-week old, male, pathogen-free C57BL/6J mice[1]
5 µg/g, one hour before Pam2CSK4 injection
I.P. for 2 hours

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Lipopeptide-Induced Inflammation Model: Eight-week-old male C57BL/6J mice were used. INH14 was dissolved in a vehicle of DMSO/NaCl. Mice were pretreated with a single intraperitoneal injection of INH14 at a dose of 5 mg/kg body weight. One hour later, mice received an intraperitoneal injection of the TLR2 ligand Pam2CSK4 (P2) at a dose of 2.5 mg/kg body weight. Blood samples (25 µL) were collected from the tail vein immediately before (0 hour) and 2 hours after the P2 injection. Serum was separated by centrifugation and stored at -20°C until analysis. Serum TNFα levels were quantified by ELISA. [1]

INH14 at a concentration of 20 µM was non-toxic to human primary monocytes after overnight incubation, as confirmed by CCK-8 cell viability assays. [1] In mouse studies, intraperitoneal injection of INH14 (5 mg/kg) did not result in pathological effects such as weight loss, abnormal movement, or dyspnea. [1]

[1]. INH14, a Small-Molecule Urea Derivative, Inhibits the IKKα/β-Dependent TLR Inflammatory Response. Chembiochem. 2019 Mar 1;20(5):710-717.

INH14 is a fragmented small molecule (approximately 240 Da) with a biarylurea backbone. It was initially identified as a TLR2-mediated inhibitor of NF-κB activation. [1]
Its mechanism of action involves the inhibition of IKKα and IKKβ kinases, which are key regulators of both classical and non-classical NF-κB signaling pathways. By inhibiting IKK, INH14 prevents the phosphorylation and subsequent degradation of IκBα, thereby blocking the nuclear translocation of NF-κB and its pro-inflammatory transcriptional activity. [1]
INH14 exhibits broad anti-inflammatory activity by inhibiting downstream signaling pathways of a variety of receptors, including TLR2, TLR4, IL-1R, and TNF-R, which ultimately converge on the IKK complex. [1] Molecular docking studies have shown that INH14 may bind to the hinge region of IKKβ, with its urea moiety forming hydrogen bonds with the protein backbone. [1] This compound can reduce the constitutive activity and cell migration ability of NF-κB in ovarian cancer cells, suggesting that it may have potential applications in tumor treatment in addition to simple anti-inflammatory therapy. [1]

C15H16N2O

240.3

240.13

C, 74.97; H, 6.71; N, 11.66; O, 6.66

200134-22-1

200134-22-1

295048

White to off-white solid powder

3.8

2

1

3

18

253

0

CPCZNJSFVOOZOG-UHFFFAOYSA-N

InChI=1S/C15H16N2O/c1-2-12-8-10-14(11-9-12)17-15(18)16-13-6-4-3-5-7-13/h3-11H,2H2,1H3,(H2,16,17,18)

1-(4-ethylphenyl)-3-phenylurea

INH14; INH-14; INH 14

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: ~48 mg/mL (~199.9 mM)
Ethanol: ~18 mg/mL (~74.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.66 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.66 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 20.8 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.)