TRAF6-Ubc13; NF-κB

The interaction between TRAF6 and Ubc13 is inhibited by C25-140 in a dose-dependent manner[1].
Effectively inhibiting TRAF6-mediated ubiquitin chain formation is C25-140 (10–30 μM; 2 hours)[1].
C25-140 affects TNFα-induced phosphorylation of IκBα as well as NF-κB-induced target gene expression[1].
In the context of cytokine activation, C25-140 effectively inhibits IL-1β- and TNFα mediated receptor signaling[1].

In a mouse model of autoimmune psoriasis caused by R 837, C25-140 (1.5 mg/kg; applied topically to the right ear and the shaved back; twice daily for 6 days) reduces the symptoms of the condition[1].
In the collagen-induced arthritis (CIA) model, C25-140 (6–14 mg/kg; given i.p.; twice daily for 14 days) shows a dose-dependent improvement of RA disease outcome[1].
The Cmax, AUC, t1/2, and Vd are 9.7 g/mL, 274083 ng min/mL, 80.62 min, and 4.13 L/kg, respectively, after treatment with C25-140 (10 mg/kg; i.v.)[1].
Treatment with C25-140 (10 mg/kg; p.o.) reveals that the Cmax, AUC, t1/2, and Vd are, respectively, 3.4 g/mL, 124034 ng min/mL, 127.33 min, and 13.3 L/kg.
Treatment with C25-140 (10 mg/kg; i.p.) reveals that the Cmax, AUC, t1/2, and Vd are, respectively, 4.2 g/mL, 100 000 ng min/mL, 184 min, and 25.6 L/kg.

Effects of C25-140 on TNFα-induced NF-κB activation and P/I-induced MAPK activation. A, MEF cells were treated with C25-140 and TNFα-induced IκBα phosphorylation was analyzed. C25-140 reduced IκBα phosphorylation. pIκBα levels were densitometrically quantified in relation to β-Actin. Error bars indicate mean +/- S.D.; n = 3 biological replicates were quantified; unpaired t-test (two-tailed); ***P<0.001, ****P<0.0001. B, Target gene (ICAM-1 and A20) expression is also diminished after C25-140 treatment and TNFα stimulation. Error bars indicate mean +/- S.D.; n = 3 biological replicates; unpaired t-test (two-tailed). **P<0.01, ***P<0.001, ****P<0.0001. C, Jurkat T-cells were treated with C25-140 and P/I-induced JNK phosphorylation was analyzed. C25-140 reduced JNK phosphorylation indicating inhibition of MAPK signaling; D = DMSO.

C25-140 is not toxic and does not affect cell cycle phases. A, Viability of MEF cells and Jurkat T-cells was evaluated after dose-dependent treatment of cells with C25-140 for 24h using the CellTiter-Glo assay that measures levels of cellular ATP (n = 3). No substantial signs of toxicity was evident. B, The effect of C25-140 on cell cycle was investigated after propidium iodide staining of Jurkat T-cells and subsequent analysis of the cell cycle phases by flow cytometry. 24 and 72 hours treatment had no effect on the cell cycle (n=3)[1].

R 837-induced psoriasis mouse model (male BALB/c mice)[1]
~1.5 mg/kg
Topically to the shaved back and the right ear; twice daily for 6 days

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Psoriasis mouse study (in vivo)[1]
To evaluate the anti-inflammatory activity of the compound C25-140, an Imiquimod (IMQ)-induced psoriasis mouse model was carried out. Per group (vehicle– and C25-140–treated), eight male BALB/c mice were shaved in the back (1.5 × 2 cm), and IMQ and C25-140 were applied topically to the shaved back and the right ear every day for 6 days. Here, C25-140 was dissolved in acetone and applied at a total dosage of 500 μg/day (2 × 250 μg), representing a total of ∼1.5 mg/kg per application. Mice were monitored, and daily scores were independently collected on a scale from 0 to 4: 0 = none; 1 = slight; 2 = moderate; 3 = marked; and 4 = very marked. Ear thickness was measured by electronic calipers as an indicator of edema. IL-17 cytokine secretion of the right ear tissue was measured by ELISA. Bioethical permission for the IMQ-induced psoriasis study was granted under IACUC approval number 17-063 (to W. B.).[1]

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Collagen-induced arthritis model (in vivo)[1]
To test the potential of C25-140 to ameliorate RA symptoms, a CIA model in DBA1/J mice was performed. Each group of 10 mice was subjected to a single subcutaneous injection of collagen/complete Freund's adjuvant emulsion (0.05 ml/mouse; 100 μg of collagen/CFA). After 20 days (day 21), mice were booster-injected with collagen (0.05 ml/mouse; 100 μg/mouse of collagen in incomplete Freund's adjuvant). On day 28, mice were scored for their arthritic index and for a subsequent 14 days intraperitoneally dosed twice daily with vehicle: 6 mg/kg C25-140, 10 mg/kg C25-140, and 14 mg/kg C25-140. 10 mg/kg prednisolone was applied once daily. Mice were daily scored for macroscopic signs of arthritis. Thereby, the following scoring index was applied to each individual paw: 0 = no visible effects of arthritis; 1 = edema and/or erythema of one digit; 2 = edema and/or erythema of two digits; 3 = edema and/or erythema of more than two digits; 4 = severe arthritis of entire paw and digits. Single paw scores were added and recorded. On day 42, animals were euthanized, limbs were collected, and histopathology was performed using toluidine blue staining with scoring (from 0 to 5) of several parameters (summed score, inflammation, pannus, bone resorption, cartilage damage, periosteal bone formation, and bone width). [1]

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ADME and pharmacokinetic studies [1]
All ADME (plasma stability, plasma protein binding, microsomal stability, log D, Caco-2 assay, CYP450 inhibition, and hERG predictor assay) as well as pharmacokinetic studies (Tmax, Cmax, AUC0–240 min, mean residence time, elimination t½, elimination rate constant, volume of distribution, and clearance) of C25-140 were conducted by using their standard protocols. Pharmacokinetic measurements were done by intraperitoneal, intravenous, or peroral application of 10 mg/kg C25-140.

[1].Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity. J Biol Chem. 2018 Aug 24;293(34):13191-13203.

Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys63-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.[1]

C26H31N7O

457.570644617081

457.259

C, 68.25; H, 6.83; N, 21.43; O, 3.50

1358099-18-9

1358099-18-9

53093165

White to off-white solid powder

3.6

0

5

6

34

678

0

HFKVRXVHZGBRKF-UHFFFAOYSA-N

InChI=1S/C26H31N7O/c1-18-23(19(2)32(29-18)25-11-10-24-28-27-20(3)33(24)30-25)9-12-26(34)31-15-13-22(14-16-31)17-21-7-5-4-6-8-21/h4-8,10-11,22H,9,12-17H2,1-3H3

1-(4-benzylpiperidin-1-yl)-3-[3,5-dimethyl-1-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrazol-4-yl]propan-1-one

C25140; C 25140; C-25140; C25-140; C25-140; 1358099-18-9; 1-(4-Benzylpiperidin-1-yl)-3-(3,5-dimethyl-1-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-1H-pyrazol-4-yl)propan-1-one; 6-{4-[3-(4-benzylpiperidin-1-yl)-3-oxopropyl]-3,5-dimethyl-1H-pyrazol-1-yl}-3-methyl[1,2,4]triazolo[4,3-b]pyridazine; 1-(4-BENZYLPIPERIDIN-1-YL)-3-(3,5-DIMETHYL-1-{3-METHYL-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL}-1H-PYRAZOL-4-YL)PROPAN-1-ONE; 1-(4-benzylpiperidin-1-yl)-3-[3,5-dimethyl-1-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrazol-4-yl]propan-1-one; 1-(4-benzylpiperidin-1-yl)-3-(3,5-dimethyl-1-{3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl}pyrazol-4-yl)propan-1-one; SCHEMBL19974504; C-25-140; C25 140

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: 41.7~92 mg/mL (91.1~201.1 mM)
Ethanol 92 mg/mL (~201.1 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.55 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 (4.55 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 20.8 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: ≥ 2.08 mg/mL (4.55 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.)