After TNF-α activation, tikinib (10–10,000 nM; 24 hours) causes MDA-MB-231 cells to undergo apoptosis [1]. Takinib (10 μM; 0–1 hour) decreases p65 and IKK phosphorylation [1]. The chemical foundation for PfPK9 (KD(app) 0.46 μM) malaria inhibitor development is provided by tikinib [3]. TAK1Thr184/187, STAT3Tyr705, and STAT3Ser727 phosphorylation are induced in RASF treated with IL-1β (10 ng/mL; 30 minutes) by tikinib (2 hours; 0.1-20 μM; human RASF) [4].

After TNF-α activation, tikinib (10–10,000 nM; 24 hours) causes MDA-MB-231 cells to undergo apoptosis [1]. Takinib (10 μM; 0–1 hour) decreases p65 and IKK phosphorylation [1]. The chemical foundation for PfPK9 (KD(app) 0.46 μM) malaria inhibitor development is provided by tikinib [3]. TAK1Thr184/187, STAT3Tyr705, and STAT3Ser727 phosphorylation are induced in RASF treated with IL-1β (10 ng/mL; 30 minutes) by tikinib (2 hours; 0.1-20 μM; human RASF) [4].

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In human rheumatoid arthritis synovial fibroblasts (RASFs) stimulated with IL-1β (10 ng/mL), pretreatment with Takinib (0.1–20 µM) for 24 hours led to a dose-dependent and statistically significant reduction in the secretion of pro-inflammatory mediators ENA-78/CXCL5, IL-6, and MCP-1/CCL2. IL-8 secretion showed a decreasing trend and reached significance at 10 µM Takinib. However, the inhibitory effects of Takinib were generally less potent compared to the TAK1 inhibitors 5Z-7-oxozeaenol (5Z/O, 1 µM) and NG-25 (1 µM). [4]
In lipopolysaccharide (LPS, 10 µg/mL)-stimulated THP-1 monocyte-derived macrophages, pretreatment with 1 µM Takinib for 24 hours reduced the secretion of IL-1β by approximately 26% and TNFα by approximately 54% compared to LPS-stimulated controls. Again, the inhibition by Takinib was less effective than that of 5Z/O and NG-25. [4]
Western blot analysis of IL-1β-stimulated human RASFs showed that pretreatment with Takinib (10 µM) increased the phosphorylation of TAK1 at Thr184/187, while simultaneously inhibiting the phosphorylation of STAT3 at both Tyr705 and Ser727 residues. It also showed a modest inhibition of JNK phosphorylation. [4]
In contrast, Takinib (10 µM) pretreatment did not inhibit STAT3 phosphorylation in human RASFs stimulated via canonical IL-6 trans-signaling (using IL-6 and soluble IL-6 receptor). [4]
In IL-1β-stimulated human RASFs, pretreatment with Takinib (10 µM) significantly reduced the nuclear translocation of phosphorylated STAT3 (Tyr705), as shown by Western blot analysis of purified nuclear extracts. However, it did not inhibit the nuclear translocation of NF-κB p65. [4]
A STAT3 DNA-binding activity assay using nuclear extracts from IL-1β-stimulated human RASFs demonstrated that pretreatment with Takinib (10 µM) significantly inhibited the DNA-binding activity of STAT3, to a level comparable to the JAK inhibitor tofacitinib (5 µM). [4]
In LPS-stimulated THP-1 macrophages, pretreatment with Takinib increased LPS-induced TAK1 phosphorylation at Thr184/187. However, it did not lead to significant inhibition of downstream signaling effectors such as phosphorylated JNK, phosphorylated c-Jun, phosphorylated NF-κB p65, or phosphorylated IκBα. STAT3 signaling was also unaffected by Takinib in this cell type and stimulation context. [4]

In a mouse model of type II collagen-induced arthritis (CIA) of rheumatoid arthritis, tikinib (50 mg/kg; i.p.; once daily on days 18–36) lowers clinical scores [4]. ?In Hodgkin's lymphoma xenografted NSG mice, takinib (50 mg/kg; oral gavage; daily till day 17) inhibits the formation of tumors [5].

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Takinib (50 mg/kg, daily intraperitoneal injection) significantly reduced clinical arthritis scores in collagen-induced arthritis (CIA) mice from day 30 to 36 compared to vehicle control.
Takinib attenuated body weight loss in CIA mice.
Takinib reduced overall joint pathology by ~30%, including inflammation, cartilage damage, pannus formation, bone resorption, and periosteal bone formation.
The most pronounced effects were observed in knee joints, with ~40% reduction in inflammation and cartilage damage.
Similar but less pronounced trends were seen in forepaws and hind paws. [2]

Western Blot Analysis[1]
Cell Types: Breast cancer cell line MDA-MB-231
Tested Concentrations: 10 μM
Incubation Duration: 5, 15, 30, 60 minutes
Experimental Results: IKK and p65 were maximally phosphorylated at 15 minutes, indicating activation of NF -κB pathway, whereas p38 phosphorylation peaked at 30 min.

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Western Blot Analysis[4]
Cell Types: IL-1β treatment (10 ng/mL; 30 minutes) RASF
Tested Concentrations: 0.1-20 µM
Incubation Duration: 2 hrs (hours)
Experimental Results: Induction of TAK1Thr184/187, STAT3Tyr705 and STAT3Ser727 phosphorylation.

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Cytokine Production ELISA: Human RASFs or differentiated THP-1 macrophages were seeded in culture plates. After reaching appropriate confluence, cells were serum-starved overnight. Cells were pre-treated with Takinib or other inhibitors at indicated concentrations for 2 hours, followed by stimulation with IL-1β (10 ng/mL for RASFs) or LPS (10 µg/mL for THP-1) for 24 hours. After incubation, conditioned media was collected, centrifuged to remove particulates, and the supernatants were analyzed for specific cytokines (e.g., IL-6, IL-8, CXCL5, MCP-1, IL-1β, TNFα) using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. [4]
Western Blot Analysis: Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined. Equal amounts of protein (25 µg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with Tris-buffered saline containing Tween 20 (TBST) and 5% non-fat dry milk, then incubated overnight with specific primary antibodies. Protein bands were visualized using chemiluminescence, and band intensities were analyzed using image analysis software. β-actin was used as a loading control for total cell lysates. [4]
Cell Fractionation for Nuclear Extract: Human RASFs grown in plates were pretreated, stimulated, washed with cold phosphate-buffered saline (PBS), and lysed with a cytoplasmic lysis buffer. The cell suspension was incubated on ice, and nuclei were pelleted by centrifugation. The supernatant (cytoplasmic extract) was saved. The nuclear pellet was washed, then lysed with RIPA buffer. After incubation on ice and centrifugation, the supernatant (nuclear extract) was collected for subsequent Western blot analysis or DNA-binding assays. [4]
STAT3 DNA-Binding Assay: Nuclear extracts (5 µg) isolated from treated human RASFs were used. The DNA-binding activity of STAT3 was measured using a commercial transcription factor assay kit according to the manufacturer's protocol. Briefly, nuclear extracts were incubated in wells coated with a specific DNA sequence bound by STAT3. After binding, a primary antibody against STAT3 was added, followed by a secondary antibody conjugated to horseradish peroxidase (HRP). A colorimetric substrate was added, and absorbance was measured to quantify STAT3 DNA-binding activity. [4]
Molecular Docking Simulation: To investigate potential direct interaction, computational docking studies were performed. The three-dimensional structure of human STAT3 (from Protein Data Bank) was prepared. The structure of Takinib was also prepared. Docking calculations were performed using specialized software. A grid was generated around the binding site of a known STAT3 inhibitor. Takinib was docked into this site, first with standard precision and then with extra precision settings. The flexibility of both the ligand and key receptor residues was considered in some analyses (induced fit docking). The resulting binding poses, interaction energies, and specific interactions (e.g., hydrogen bonds, hydrophobic contacts, π-π stacking) with STAT3 residues were analyzed. [4]

Animal/Disease Models: Male DBA/1 mouse (CIA arthritis model) [4]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; one time/day starting on days 18-36
Experimental Results: compared to vehicle control , clinical arthritis scores diminished.

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Animal/Disease Models: Female NSG mice (8 weeks old) [5]
Doses: 50 mg/kg
Route of Administration: po (oral gavage); one time/day until 17 days
Experimental Results: Tumor growth slowed down and tumor size/weight diminished.

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Male DBA/1 mice were immunized with type II collagen in Freund’s complete adjuvant on day 0 and day 21 to induce arthritis.
Mice were randomized into treatment groups on day 18 and treated daily with takinib (50 mg/kg) or vehicle via intraperitoneal injection until day 36.
Clinical scores for each paw were assessed daily from day 18 to 36 by a blinded experimenter.
On day 36, mice were euthanized for histopathological analysis of joints. [2]

Takinib was rapidly cleared from plasma after intraperitoneal injection (50 mg/kg) in mice, with a half-life (t_1/2) of 21 minutes. The half-lives in different tissues were approximately 0.25 hours in the heart, approximately 4.6 hours in the spleen, approximately 0.37 hours in the tumor, and approximately 0.15 hours in the muscle. [2]

MTT assays showed that treatment with 10 µM Takinib for 24 hours resulted in cytotoxicity in human RASF cells. The reduction in pro-inflammatory mediators observed at this higher concentration may be partly attributed to the loss of cell viability. [4]

[1]. Takinib, a Selective TAK1 Inhibitor, Broadens the Therapeutic Efficacy of TNF-α Inhibition for Cancer and Autoimmune Disease. Cell Chem Biol. 2017 Aug 17;24(8):1029-1039.

[2]. Scarneo SA, et.al. Pharmacological inhibition of TAK1, with the selective inhibitor takinib, alleviates clinical manifestation of arthritis in CIA mice. Arthritis Res Ther. 2019 Dec 17;21(1):292.

[3]. Plasmodium PK9 Inhibitors Promote Growth of Liver-Stage Parasites. Cell Chem Biol. 2019 Mar 21;26(3):411-419.e7.

[4]. Panipinto PM, et.al. Takinib Inhibits Inflammation in Human Rheumatoid Arthritis Synovial Fibroblasts by Targeting the Janus Kinase-Signal Transducer and Activator of Transcription 3 (JAK/STAT3) Pathway. Int J Mol Sci. 2021;22(22):12580. Published 2021 Nov 22.

[5]. Song Z,et.al. Essential role of the linear ubiquitin chain assembly complex and TAK1 kinase in A20 mutant Hodgkin lymphoma. Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):28980-28991.

This study challenges the view that Takinib is a specific TAK1 inhibitor. It provides evidence that in human RASF cells, the anti-inflammatory effect of Takinib is mainly achieved by inhibiting the JAK/STAT3 pathway, including reducing the phosphorylation level of STAT3 (Tyr705 and Ser727), inhibiting its nuclear translocation, and inhibiting its DNA binding activity. [4] This study suggests that the mechanism of action of Takinib may be cell type and stimulation-dependent. Takinib inhibits STAT3 in IL-1β-stimulated RASF cells, but has no inhibitory effect in IL-6-stimulated RASF cells or LPS-stimulated THP-1 macrophages. [4] Molecular docking simulations predict that Takinib can directly bind to the SH2 domain of the STAT3 protein and interact with key residues (e.g., E638, Q644, Y657, and W623) through hydrogen bonding and hydrophobic interactions, similar to known STAT3 inhibitors. This provides a structural hypothesis for its observed inhibitory effect on STAT3 activity. [4] The results showed that Takinib induced phosphorylation of TAK1 at Thr184/187 (an activation marker) in IL-1β-stimulated RASF cells and LPS-stimulated macrophages, contrary to the expected effect of direct TAK1 kinase inhibitors. This raises questions about its selectivity for TAK1 in certain inflammatory settings. [4]

C18H18N4O2

322.3611

322.142

1111556-37-6

37750349

White to off-white solid powder

2.3

2

3

5

24

470

0

O=C(C1=CC=CC(C(N)=O)=C1)NC1=NC2=CC=CC=C2N1CCC

UOZVVPXKJGOFIG-UHFFFAOYSA-N

InChI=1S/C18H18N4O2/c1-2-10-22-15-9-4-3-8-14(15)20-18(22)21-17(24)13-7-5-6-12(11-13)16(19)23/h3-9,11H,2,10H2,1H3,(H2,19,23)(H,20,21,24)

3-N-(1-Propylbenzimidazol-2-yl)benzene-1,3-dicarboxamide

EDHS-206; EDHS 206; EDHS206

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 : ~50 mg/mL (~155.11 mM)

Solubility in Formulation 1: 2.5 mg/mL (7.76 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (7.76 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)