STAT3 ; NF-κB; IKK2 (IC50 = 17.9 nM)

TPCA-1 has an IC50 of 17.9 nM in a time-resolved fluorescence resonance energy transfer assay to inhibit the activity of human IKK-2. Additionally, it has been shown that TPCA-1 competes with ATP. Additionally, TPCA-1 has IC50 values against IKK-1 and JNK3 of 400 nM and 3600 nM, respectively. TPCA-1 exhibits concentration-dependent inhibition of TNF-α, IL-6, and IL-8 production, with IC50 values of 170, 290, and 320 nM, respectively. [1] NFκB-dependent IL8 gene expression, TNF-induced RelA (p65) nuclear translocation, and glioma cell proliferation are all inhibited by TPCA-1. Importantly, TPCA-1 completely blocks IFN-induced gene expression of MX1 and GBP1, while only slightly affecting the expression of ISG15. [2]

Murine collagen-induced arthritis (CIA) is less severe when TPCA-1 is administered prophylactically at doses of 3, 10, or 20 mg/kg, intravenously, every day. The effects of the anti-rheumatic drug etanercept when given prophylactically at 4 mg/kg, i.p., every other day are comparable to the effects of the significantly reduced disease severity and delayed disease onset caused by the administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. In the paw tissue of TPCA-1 and etanercept-treated mice, nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, are markedly decreased. In addition, administration of TPCA-1 in vivo significantly reduces collagen-induced T cell proliferation ex vivo.

A time-resolved fluorescence resonance energy transfer assay is used to determine the activity of recombinant human IKK-2 (residues 1-756) expressed in baculovirus as an N-terminally GST-tagged fusion protein. In a nutshell, IKK-2 (5 nM final) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM CHAPS, pH 7.4, with 1 mM DTT and 0.01% w/v BSA) is added to wells containing various concentrations of the substance or dimethyl sulfoxide (DMSO) vehicle (3% final). In a total volume of 30 L, GST-IB substrate (25 nM final) and ATP (1 μM final) are added to start the reaction. After 30 minutes of incubation at room temperature, the reaction is stopped by adding 15 μL of 50 mM EDTA. The reaction is further incubated for 60 min at room temperature with the addition of detection reagent (15 μL) in buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% w/v BSA) containing antiphosphoserine-IκBα-32/36 monoclonal antibody 12C2, labeled with W-1024 europium chelate. Using a Packard Discovery plate reader, the amount of phosphorylation of GST-IκBα is calculated as the ratio of a specific 665-nm energy transfer signal to a reference 620-nm europium signal.

Ten microliters of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from stock solution (10 mg/mL) are added to each well of 96-well plates containing glioma cells, and the mixture is then incubated at 37 °C for 2-4 h. Plating is carried out at 37 °C for 4 hours in a humid environment after adding 100 μL of 10% sodium dodecyl sulfate (SDS) in 0.01 N HCL to solubilize the oxidized MTT. At 570 nm, a plate reader reads plates.

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Cells at 50%–80% confluence in 6-well plates were treated with various concentrations of BMS-345541, TPCA-1, or IFNα, either separately or in combination for 24 h, followed by infection with vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) for 1.5 h at a multiplicity of infection of ∼0.1 plaque-forming unit per cell. The VSV yield in the medium was assayed by plaque formation at 24 h postinfection on Vero cells (Yang and others 2000). The EMCV yield in the medium was assayed by quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR) using an EMCV 3D gene-specific primer pair: 5′-CCCTACCTCACGGAATGGGGCAAA-3′ (forward), 5′-GGTGAGAGCAAGCCTCGCAAAGAC-3′ (reverse) (Perez and Diaz de Arce 2009). Viral RNA from 200 μL of medium was isolated using TRIZol reagent. Data were normalized to the viral RNA sample isolated from EMCV stock with known viral titer. [2]

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Immunofluorescence staining[2]
Cells were grown in 48-well plates, pretreated with BMS-345541 or TPCA-1 for 2 h, and stimulated with recombinant TNF-α for 30 min or IFN for 1 h. Cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton×100. After blocking with 5% goat serum, cells were incubated with anti-p65, anti-STAT2, or anti-pSTAT1, and subsequently stained with Alex 594-labeled goat anti-rabbit IgG.

Murine collagen-induced arthritis
3, 10, or 20 mg/kg
Administered via i.p. or b.i.d.

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BALB/c female nude mice were purchased from Vital Rival. All experiments were performed in the Animal Center of Gansu University of Traditional Chinese Medicine. Six-week-old nude mice were injected subcutaneously with HCC827 cells (5 × 106). HCC827 cells were suspended in serum-free RPMI 1640. When tumor volumes were reached approximately 80 mm3, mice were randomized to groups of 6 animals to receive either vehicle control, TPCA-1 alone, gefitinib alone, or TPCA-1 and gefitinib together. Gefitinib was suspended in 0.5% (w/v) methylcellulose and administered once daily by oral gavage (2 mg/kg). TPCA-1 was dissolved in PBS and administered by intraperitoneally at a daily dosage of 10 mg/kg. Mice in the untreated group were given the same volumes of PBS by injection and 0.5% (w/v) methylcellulose by oral gavage. Tumor size was measured every 2 days using calipers. The average tumor volume was calculated according to the equation: tumor volume = 0.5 × (large diameter) × (small diameter)2. Tumor weight was measured at the endpoints of this study.[3]

[1]. J Pharmacol Exp Ther . 2005 Jan;312(1):373-81.

[2]. J Interferon Cytokine Res . 2012 Aug;32(8):368-77.

[3]. Mol Cancer Ther . 2014 Mar;13(3):617-29.

2-(carbamoylamino)-5-(4-fluorophenyl)-3-thiophenecarboxamide is a member of thiophenes and an aromatic amide.
TPCA-1 is a selective inhibitor of human IκB kinase 2 (IKK-2).

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emonstration that IkappaB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-kappaB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC(50) = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-alpha, IL-6, and IL-8 with an IC(50) = 170 to 320 nM. Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.[1]

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The nuclear factor-kappa B (NFκB) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NFκB activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NFκB regulates various aspects of the cellular response to interferon (IFN). However, the role of the upstream regulator of the NFκB signaling pathway, the inhibitor of κB kinase (IKK) complex, on IFN function has not been examined. In the present study, we examined the effects of 2 IKK inhibitors, N-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)-1,2-ethanediamine hydrochloride (BMS-345541) and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), on IFN action in several human glioma cell lines. IKK inhibitors inhibit glioma cell proliferation, as well as TNF-induced RelA (p65) nuclear translocation and NFκB-dependent IL8 gene expression. Importantly, BMS-345541 and TPCA-1 differentially inhibit IFN-induced gene expression, completely suppressing MX1 and GBP1 gene expression, while having only a minor effect on ISG15 expression. Furthermore, these IKK inhibitors displayed marked differences in blocking IFN-induced antiviral action against cytopathic effects and replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). Our results show that the IKK complex plays an important function in IFN-induced gene expression and antiviral activity. Since VSV and EMCV are oncolytic viruses used in cancer therapy, our results indicate the potential synergy in combining IKK inhibitors with oncolytic viruses.[2]

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Epidermal growth factor receptor (EGFR) is a clinical therapeutic target to treat a subset of non-small cell lung cancer (NSCLC) harboring EGFR mutants. However, some patients with a similar kind of EGFR mutation show intrinsic resistance to tyrosine kinase inhibitors (TKI). It indicates that other key molecules are involved in the survival of these cancer cells. We showed here that 2-[(aminocarbonyl)amino]-5 -(4-fluorophenyl)-3- thiophenecarboxamide (TPCA-1), a previously reported inhibitor of IκB kinases (IKK), blocked STAT3 recruitment to upstream kinases by docking into SH2 domain of STAT3 and attenuated STAT3 activity induced by cytokines and cytoplasmic tyrosine kinases. TPCA-1 is an effective inhibitor of STAT3 phosphorylation, DNA binding, and transactivation in vivo. It selectively repressed proliferation of NSCLC cells with constitutive STAT3 activation. In addition, using pharmacologic and genetic approaches, we found that both NF-κB and STAT3 could regulate the transcripts of interleukin (IL)-6 and COX-2 in NSCLC harboring EGFR mutations. Moreover, gefitinib treatment only did not efficiently suppress NF-κB and STAT3 activity. In contrast, we found that treatment with TKIs increased phosho-STAT3 level in target cells. Inhibiting EGFR, STAT3, and NF-κB by combination of TKIs with TPCA-1 showed increased sensitivity and enhanced apoptosis induced by gefitinib. Collectively, in this work, we identified TPCA-1 as a direct dual inhibitor for both IKKs and STAT3, whereas treatment targeting EGFR only could not sufficiently repress NF-κB and STAT3 pathways for lung cancers harboring mutant EGFR. Therefore, synergistic treatment of TPCA-1 with TKIs has potential to be a more effective strategy for cancers.[3]

C12H10FN3O2S

279.29

279.047

C, 51.61; H, 3.61; F, 6.80; N, 15.05; O, 11.46; S, 11.48

507475-17-4

9903786

White to gray solid powder

1.5±0.1 g/cm3

442.6±45.0 °C at 760 mmHg

221.5±28.7 °C

0.0±1.1 mmHg at 25°C

1.686

2.72

3

4

3

19

361

0

S1C(=C(C(N([H])[H])=O)C([H])=C1C1C([H])=C([H])C(=C([H])C=1[H])F)N([H])C(N([H])[H])=O

SAYGKHKXGCPTLX-UHFFFAOYSA-N

InChI=1S/C12H10FN3O2S/c13-7-3-1-6(2-4-7)9-5-8(10(14)17)11(19-9)16-12(15)18/h1-5H,(H2,14,17)(H3,15,16,18)

2-(carbamoylamino)-5-(4-fluorophenyl)thiophene-3-carboxamide

GW683965; TPCA-1; GW-683965; TPCA1; TPCA-1; 507475-17-4; 5-(4-Fluorophenyl)-2-ureidothiophene-3-carboxamide; IKK-2 Inhibitor IV; TPCA1; 2-(carbamoylamino)-5-(4-fluorophenyl)thiophene-3-carboxamide; [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide; IKK 2 Inhibitor IV; TPCA 1; GW 683965

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: ≥ 7.5 mg/mL (26.85 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 75.0 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: ≥ 7.5 mg/mL (26.85 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 75.0 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.

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Solubility in Formulation 3: ≥ 7.5 mg/mL (26.85 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 75.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% Cremophor EL, 2% N,N-dimethylacetamide: 15 mg/mL

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