TPCA-1 (GW-683965; GW683965)

STAT3 ; NF-κB; IKK2 (IC50 = 17.9 nM)

体外活性：在时间分辨荧光共振能量转移测定中，TPCA-1 抑制人 IKK-2 活性，IC50 为 17.9 nM。此外，TPCA-1 被证明具有 ATP 竞争性。此外，TPCA-1 对 IKK-1 和 JNK3 的 IC50 值分别为 400 nM 和 3600 nM。 TPCA-1 以浓度依赖性方式抑制 TNF-α、IL-6 和 IL-8 的产生，IC50 值分别为 170、290 和 320 nM。 TPCA-1 抑制神经胶质瘤细胞增殖，以及 TNF 诱导的 RelA (p65) 核转位和 NFκB 依赖性 IL8 基因表达。重要的是，TPCA-1 抑制 IFN 诱导的基因表达，完全抑制 MX1 和 GBP1 基因表达，而对 ISG15 表达只有很小的影响。激酶测定：重组人 IKK-2（残基 1-756）在杆状病毒中表达为 N 末端 GST 标记的融合蛋白，并使用时间分辨荧光共振能量转移测定评估其活性。简而言之，将用测定缓冲液（50 mM HEPES、10 mM MgCl2、1 mM CHAPS，pH 7.4，含 1 mM DTT 和 0.01% w/v BSA）稀释的 IKK-2 添加到含有不同浓度的孔中化合物或二甲基亚砜 (DMSO) 载体（最终 3%）。通过添加总体积为 30 μL 的 GST-IκBα 底物（最终 25 nM）/ATP（最终 1 μM）来启动反应。反应在室温下孵育 30 分钟，然后添加 15 μL 50 mM EDTA 终止。检测试剂 (15 µL) 溶于缓冲液（100 mM HEPES、pH 7.4、150 mM NaCl 和 0.1% w/v BSA）中，含有抗磷酸丝氨酸-IκBα-32/36 单克隆抗体 12C2（用 W-1024 铕螯合物标记）和添加别藻蓝蛋白标记的抗GST抗体，并将反应物进一步在室温下孵育60分钟。使用 Packard Discovery 酶标仪，以特定 665 nm 能量转移信号与参考铕 620 nm 信号的比率来测量 GST-IκBα 的磷酸化程度。细胞测定：将储备溶液 (10 mg/mL) 中的 10 微升 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 添加到含有神经胶质瘤的 96 孔板的每个孔中细胞并在 37°C 下孵育 2-4 小时。通过添加 100 μL 10% 十二烷基硫酸钠 (SDS) 的 0.01 N HCL 溶液溶解氧化 MTT，并将板在加湿室中于 37 °C 下孵育 4 小时。在酶标仪上于 570 nm 处读取酶标板。

以 3、10 或 20 mg/kg、ip、bid 预防性施用 TPCA-1 可导致小鼠胶原诱导性关节炎 (CIA) 严重程度呈剂量依赖性降低。 TPCA-1以10mg/kg，ip，bid给药所导致的疾病严重程度的显着降低和疾病发作的延迟与抗风湿药物依那西普（etanercept）以4mg/kg，ip，每天预防性给药时的效果相当。另一天。在TPCA-1和依那西普治疗的小鼠的爪组织中，p65的核定位以及IL-1β、IL-6、TNF-α和干扰素-γ的水平显着降低。此外，体内给予TPCA-1可显着降低胶原诱导的离体T细胞增殖。 TPCA-1 20 mg/kg（而非 3 或 10 mg/kg，腹膜内，每日两次）的治疗性给药可显着降低 CIA 的严重程度，依那西普每隔一天 12.5 mg/kg，腹膜内给药也能显着降低 CIA 的严重程度。

时间分辨荧光共振能量转移测定用于测定在杆状病毒中表达为 N 末端 GST 标记融合蛋白的重组人 IKK-2（残基 1-756）的活性。简而言之，将在测定缓冲液（50 mM HEPES、10 mM MgCl2、1 mM CHAPS，pH 7.4，含 1 mM DTT 和 0.01% w/v BSA）中稀释的 IKK-2（最终 5 nM）添加到含有各种物质的孔中。该物质或二甲基亚砜 (DMSO) 载体的浓度（最终浓度为 3%）。在总体积 30 L 中，添加 GST-IB 底物（最终 25 nM）和 ATP（最终 1 μM）以开始反应。室温孵育 30 分钟后，添加 15 μL 50 mM EDTA 终止反应。将反应物在室温下进一步孵育 60 分钟，并添加含有抗磷酸丝氨酸-IκBα-32/的缓冲液（100 mM HEPES、pH 7.4、150 mM NaCl 和 0.1% w/v BSA）中的检测试剂（15 μL）。 36 单克隆抗体 12C2，用 W-1024 铕螯合物标记。使用 Packard Discovery 酶标仪，GST-IκBα 的磷酸化量计算为特定 665 nm 能量转移信号与参考 620 nm 铕信号的比率。

将来自储备溶液 (10 mg/mL) 的 10 微升 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 添加到含有神经胶质瘤细胞的 96 孔板的每个孔中，并且然后将混合物在 37°C 下孵育 2-4 小时。添加 100 μL 10%十二烷基硫酸钠 (SDS) 的 0.01 N HCL 溶液以溶解氧化的 MTT 后，在潮湿环境下于 37 °C 进行电镀 4 小时。酶标仪在 570 nm 处读取酶标板。

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将6孔板中50%-80%合流的细胞分别或联合使用不同浓度的BMS-345541、TPCA-1或IFNα处理24小时，然后用水泡性口炎病毒（VSV）或脑心肌炎病毒（EMCV）感染1.5小时，感染次数为每个细胞约0.1个斑块形成单位。通过感染后24小时在Vero细胞上形成斑块来检测VSV在培养基中的产量（Yang等人2000年）。使用EMCV 3D基因特异性引物对：5 ' -CCCTACCTCACGGAATGGGGCAAA-3 ‘（正向），5 ’ -GGTGAGAGCAAGCCTCGCAAAGAC-3 '（反向），通过定量实时逆转录聚合酶链反应（qRT-PCR）检测培养基中的EMCV产量（Perez and Diaz de Arce 2009）。用TRIZol试剂从200 μL培养基中分离病毒RNA。数据归一化为从已知病毒滴度的EMCV stock中分离的病毒RNA样本。 [2]

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免疫荧光染色[2]
细胞在48孔板中培养，用BMS-345541或TPCA-1预处理2小时，重组TNF-α刺激30分钟或IFN刺激1小时。用PBS洗涤细胞，用4%多聚甲醛固定，并用0.1% Triton×100渗透。5%山羊血清阻断后，细胞与抗p65、抗stat2或抗pstat1孵育，随后用Alex 594标记的山羊抗兔IgG染色。

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

126.45

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)

配方 1 中的溶解度: ≥ 7.5 mg/mL (26.85 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 75.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 7.5 mg/mL (26.85 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 75.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 7.5 mg/mL (26.85 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 75.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2% Cremophor EL, 2% N,N-dimethylacetamide: 15 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。