IKK-2 (IC50 = 0.3 μM); IKK-1 (IC50 = 4 μM)

体外活性：BMS-345541 剂量依赖性地抑制 THP-1 单核细胞中 TNF-α 刺激的 IκBα 磷酸化，IC50 约为 4 μM。 BMS-345541 抑制 THP-1 细胞中脂多糖刺激的肿瘤坏死因子 α、白细胞介素 1β、白细胞介素 8 和白细胞介素 6，IC50 值在 1 至 5 μM 范围内。 BMS-345541 以相互排斥的方式与对应于 IκBα 氨基酸 26 - 42（其中 Ser-32 和 Ser-36 变为天冬氨酸）的肽抑制剂结合，并且以非相互排斥的方式与 ADP 结合。 BMS-345541 与 IKK-1 和 IKK-2 上相似的变构位点结合，从而对亚基的活性位点产生不同的影响。 BMS-345541 影响多种有丝分裂细胞周期转变，包括有丝分裂进入、中期到后期进展和胞质分裂。将 BMS-345541 添加到 G 期停滞释放的细胞中，可阻断 Aurora A、B 和 C 的激活、Cdk1 的激活和组蛋白 H3 的磷酸化。用 BMS-345541 处理有丝分裂细胞会导致细胞周期蛋白 B1 和 securin 过早降解、染色体分离缺陷和胞质分裂不当。 BMS-345541 还被发现可以覆盖诺考达唑抑制细胞中的纺锤体检查点。这些作用并非主要归因于 BMS-345541 对有丝分裂激酶（如 Cdk1、Aurora A 或 B、Plk1 或 NEK2）的直接抑制作用。 BMS-345541 (10 μM) 在 72 小时时分别抑制正常人表皮黑色素细胞和转移性黑色素瘤细胞（SK-MEL-5、A375 和 Hs 294T）的生长 96% 和 99%。将 100 μM BMS-345541 应用到 SK-MEL-5 细胞培养物中，通过不依赖 caspase 和 AIF 依赖的线粒体介导的方式，24 小时可导致 87% 的细胞凋亡。 BMS-345541 处理 (10 μM) 导致 IKK 活性和 NF-kB 活性以及 CXCL1 产量减少 76% 和 95%。激酶测定：通过在 30℃下将酶（终浓度为 0.5 μg/mL）添加到 100 μg/mL GST-IκBα 和 5 μM [33P]ATP 的溶液中，进行测量酶催化的 GST-IκBα 磷酸化的测定。 40 mM Tris HCl，pH 7.5，含有 4 mM MgCl2、34mM 磷酸钠、3 mM NaCl、0.6 mM 磷酸钾、1 mM KCl、1 mM 二硫苏糖醇、3% (w/v) 甘油和 250 μg/mL 牛血清白蛋白。测定中使用的[33P]ATP 的比活度为100 Ci/mmol。 5分钟后，加入2×Laemmli样品缓冲液终止激酶反应，并在90℃下热处理1分钟。然后将样品加载到 NuPAGE 10% BisTris 凝胶上。完成 SDS-PAGE 后，将凝胶在平板凝胶干燥机上干燥。然后使用 445Si PhosphorImager 检测条带，并使用 ImageQuant 软件量化放射性。在这些条件下，GST-IκBα 的磷酸化程度与时间和酶浓度呈线性关系。细胞测定：将每孔 1×105 个细胞（转移性人黑色素瘤细胞 SK-MEL-5）铺在含有 10% 胎牛血清培养基的六孔板中过夜，以使细胞粘附。将细胞在含有BMS-345541的培养基中培养72小时。用血细胞计数器对细胞进行计数。

BMS-345541 以剂量依赖性方式有效抑制黑色素瘤生长。与接受治疗的对照动物相比，接受 75 mg/kg BMS-345541 治疗的荷瘤小鼠可有效抑制 SK-MEL-5、A375 和 Hs 294T 肿瘤的生长，分别达 86%、69% 和 67%单独与车辆。 BMS-345541 以 100 mg/kg 剂量口服给药，可降低小鼠葡聚糖硫酸钠诱导的结肠炎的严重程度，其体重比、结肠临床评分、平均损伤评分和平均炎症评分为 0.86（相比于载体组为 0.77） 、1.0（相对于车辆组的 2.5）、5.66（相对于车辆组的 8.52）、6.82（相对于车辆组的 12.33）。 BMS-345541（100 mg/kg），从第一次胶原蛋白免疫开始，每日一次用水口服灌胃给药，可抑制小鼠 CIA 模型中的疾病临床体征（媒介物组为 0 vs ~8），同时伴随通过减少爪子肿胀。 BMS-345541 (100 mg/kg) 将累积关节炎损伤评分从 4.4 降低至 0，同时降低胫跗关节退化以及炎症、滑膜增生、骨吸收和软骨侵蚀的严重程度。在动物的关节中没有观察到明显的损伤，这在组织学上与年龄匹配、无疾病的对照动物的关节没有区别。 BMS-345541 剂量依赖性地抑制 IL-1β 信息，100 mg/kg 剂量组的动物表现出与无病对照动物相当的水平。

通过在 30℃下将酶（终浓度为 0.5 μg/mL）添加到 100 μg/mL GST-IκBα 和 5 μM [33P]ATP 的 40 mM Tris 溶液中，进行测量酶催化的 GST-IκBα 磷酸化的测定。 HCl，pH 7.5，含有 4 mM MgCl2、34mM 磷酸钠、3 mM NaCl、0.6 mM 磷酸钾、1 mM KCl、1 mM 二硫苏糖醇、3% (w/v) 甘油和 250 μg/mL 牛血清白蛋白。测定中使用的 [33P]ATP 的比活性为 100 Ci/mmol。 5 分钟后，通过添加 2× Laemmli 样品缓冲液停止激酶反应，然后在 90 °C 下加热 1 分钟。之后，将样品置于 NuPAGE 10% BisTris 凝胶上。 SDS-PAGE 后，将凝胶在平板凝胶干燥机上干燥。然后使用 445Si PhosphorImager 检测条带，并使用 ImageQuant 软件量化放射性。在这些条件下，GST-IκBα 的磷酸化程度与时间和酶浓度呈线性关系。然后使用 445Si PhosphorImager 识别条带，并使用 ImageQuant 软件测量放射性。在这种情况下，酶浓度和GST-IκBα磷酸化速率呈线性相关。

简而言之，BMS-345541 以不同浓度或不同时间长度应用于 SK-MEL-5 细胞。用胰蛋白酶消化收集细胞，然后将其在冰上固定在70％乙醇中2小时，并用PI溶液（含有2μg/mL PI、0.1％Triton X-100和125单位/mL RNase A的PBS）染色37°C 30 分钟。使用流式细胞术和具有 488 nm 激发和 620 nm 发射的滤光片，测量细胞荧光。使用软件 MultiCycle 分析所得数据。

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将含有10%胎牛血清培养基的六孔板每孔加入1×105个细胞过夜，以促进细胞粘附。将细胞在含有BMS-345541 的培养基中培养 72 小时。使用血细胞计数器对细胞进行计数。

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BMS-345541对人脐静脉内皮细胞粘附分子表达的影响[3]
在用10ng/mL TNFa刺激4小时之前，用BMS-345541预处理0.1mL体积的5000个细胞/孔的96孔板中的HUVEC 1小时。我们使用识别ICAM-1或VCAM-1的小鼠单克隆抗体，然后用山羊抗小鼠HRP检测。

BMS-345541 is created as a 2 mg/mL solution in water with 3% Tween 80. Either a peroral gavage of 10 mg/kg (1 mL/kg) or an intravenous bolus of 2 mg/kg (1 mL/kg) is administered to the mice. Individual mice are given whole blood samples at 0, 0.05, 0.25, 0.5, 1.0, 3.0, 6.0, and 8.0 h after dosing by means of an orbital bleed and a cardiac puncture. Centrifuging whole blood for five minutes at 20×103 g. Before being analyzed, serum is kept at 20°C.

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Dextran sulfate sodium-induced murine model of inflammatory bowel disease [3]
Swiss-Webster mice were given 6% DSS in their drinking water for 7 days to induce intestinal inflammation. Aqueous solutions of test compounds (e.g. BMS-345541 ) were administered by oral gavage once daily throughout the study (days 2 through 9), with n = 5 per group. On day 10, animals were sacrificed and the colons removed for clinical and histological evaluation. Clinical scoring by a blinded observer was determined by the gross clinical evaluation of the injury on a scale from 0 (normal) to 3 (severe) as follows: grade 0, normal; grade 1, relatively normal colon length with slight thickening of tissue; grade 2, shortened colon length and thick along entire length of colon with loss of striations and some areas of redness; grade 3, considerably shortened length with very thick tissue containing areas of raised lesions. The weight for each animal on day 10 was divided by its weight at the beginning of the study to obtain a weight ratio at the end of the study. Entire colons were then immersion fixed in 10% neutral buffered formalin and divided into proximal, middle, and distal segments of equal length. Each segment was processed by routine methods, and embedded in paraffin. Segments were step-sectioned at 5 mm to obtain 3–6 sections per segment for a total of 9–18 colon sections/animal and stained with hematoxylin and eosin for light microscopy. Colon sections were graded as to the severity of crypt injury and degree of inflammation. The crypt injury was scored as follows: grade 0, intact crypt; grade 1, loss of the basilar 1/3rd of the crypt; grade 2, loss of basilar 2/3rd of the crypt; grade 3, loss of entire crypt with surface epithelium intact; grade 4, loss of entire crypt with epithelial erosion. These changes were also graded as to the degree of tissue involvement: grade 0, no involvement; grade 1, 1–25% involvement; grade 2, 26–50% involvement; grade 3, 51–75% involvement; grade 4, 76–100% involvement. The injury histological score is then defined as the product of the crypt injury grade and the degree of tissue involvement grade. The scoring for severity of inflammation was as follows: grade 0, nonremarkable; grade 1, minimal; grade 2, mild; grade 3, moderate; grade 4, severe. The extent of involvement was estimated as: grade 0, no involvement; grade 1, 1–25% involvement; grade 2, 26–50% involvement; grade 3, 51–75% involvement; grade 4, 76–100% involvement. The inflammation histological score is the product of the severity of inflammation grade and extent of involvement grade. Crypt injury and inflammatory scoring were performed on each section of colon and a mean score and standard error determined for each section. Cumulative crypt injury and inflammatory scores for each group were determined. Statistical analysis was performed using ANOVA with Tukey’s post hoc analysis. Significance was considered at a P < 0.05 level.

[1]. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J Biol Chem, 2003, 278(3), 1450-1456.

[2]. BMS-345541 targets inhibitor of kappaB kinase and induces apoptosis in melanoma: involvement of nuclear factor kappaB and mitochondria pathways. Clin Cancer Res, 2006, 12(3 Pt 1), 950-960.

[3]. An inhibitor of IkappaB kinase, BMS-345541, blocks endothelial cell adhesion molecule expression and reduces the severity of dextran sulfate sodium-induced colitis in mice. Inflamm Res, 2003, 52(12), 508-511.

The signal-inducible phosphorylation of serines 32 and 36 of I kappa B alpha is critical in regulating the subsequent ubiquitination and proteolysis of I kappa B alpha, which then releases NF-kappa B to promote gene transcription. The multisubunit I kappa B kinase responsible for this phosphorylation contains two catalytic subunits, termed I kappa B kinase (IKK)-1 and IKK-2. BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKK-2 IC(50) = 0.3 microm, IKK-1 IC(50) = 4 microm). The compound failed to inhibit a panel of 15 other kinases and selectively inhibited the stimulated phosphorylation of I kappa B alpha in cells (IC(50) = 4 microm) while failing to affect c-Jun and STAT3 phosphorylation, as well as mitogen-activated protein kinase-activated protein kinase 2 activation in cells. Consistent with the role of IKK/NF-kappa B in the regulation of cytokine transcription, BMS-345541 inhibited lipopolysaccharide-stimulated tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and interleukin-6 in THP-1 cells with IC(50) values in the 1- to 5-microm range. Although a Dixon plot of the inhibition of IKK-2 by BMS-345541 showed a non-linear relationship indicating non-Michaelis-Menten kinetic binding, the use of multiple inhibition analyses indicated that BMS-345541 binds in a mutually exclusive manner with respect to a peptide inhibitor corresponding to amino acids 26-42 of I kappa B alpha with Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive manner with respect to ADP. The opposite results were obtained when studying the binding to IKK-1. A binding model is proposed in which BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which then affects the active sites of the subunits differently. BMS-345541 was also shown to have excellent pharmacokinetics in mice, and peroral administration showed the compound to dose-dependently inhibit the production of serum tumor necrosis factor alpha following intraperitoneal challenge with lipopolysaccharide. Thus, the compound is effective against NF-kappa B activation in mice and represents an important tool for investigating the role of IKK in disease models.[1]

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Purpose: Constitutive activation of inhibitor of kappaB kinase (IKK) confers melanoma resistance to apoptosis and chemotherapy. Whether IKK is able to serve as a therapeutic target in melanoma is unknown. We explored the possibility of exploiting IKK as a therapeutic target in melanoma by using BMS-345541, a novel compound with a highly selective IKKbeta inhibitory activity, to trigger melanoma cell apoptosis. Experimental design: Three human melanoma cell lines (SK-MEL-5, Hs 294T, and A375), all of which have high constitutive IKK activities, served as in vitro and in vivo melanoma models for treatment with BMS-345541. Two known antitumor drugs (temozolomide and bortezomib) were used as parallel controls for evaluation of the therapeutic efficiency and toxicity of BMS-345541. The effects of BMS-345541 on nuclear factor kappaB (NF-kappaB) signaling and on the apoptosis machinery were investigated. Results: Inhibition of constitutive IKK activity by BMS-345541 resulted in the reduction of NF-kappaB activity, CXCL1 chemokine secretion by cultured melanoma cells and melanoma cell survival in vitro and in vivo. The effect of BMS-345541 on tumor cell growth was through mitochondria-mediated apoptosis, based on the release of apoptosis-inducing factor, dissipation of mitochondrial membrane potential, and reduced ratio of B cell lymphoma gene-2 (Bcl-2)/Bcl-associated X protein (Bax) in mitochondria. The BMS-345541 execution of apoptosis was apoptosis-inducing factor-dependent, but largely caspase-independent. Conclusion: BMS-345541 down-regulation of IKK activity results in mitochondria-mediated apoptosis of tumor cells because the programmed cell death machinery in melanoma cells is highly regulated by NF-kappaB signaling. Therefore, IKK may serve as a potential target for melanoma therapy.[2]

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Objective: Inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are characterized by chronic relapsing inflammation. The transcription of many of the proteins which mediate the pathogenesis in inflammatory bowel disease (e.g., TNFalpha, ICAM-1, VCAM-1) is NF-kappaB-dependent. IkappaB kinase is critical in transducing the signal-inducible activation of NF-kappaB and, therefore, represents a potentially promising target for the development of novel agents to treat inflammatory bowel disease and other inflammatory diseases. Results: Here we show that BMS-345541, a highly selective inhibitor of IkappaB kinase, inhibited the TNFalpha-induced expression of both ICAM-1 and VCAM-1 in human umbilical vein endothelial cells at the same concentration range as cytokine expression is inhibited in monocytic cells (IC(50) congruent with 5 microM). Against dextran sulfate sodium-induced colitis in mice, BMS-345541 administered orally at doses of 30 and 100 mg/kg was effective in blocking both clinical and histological endpoints of inflammation and injury. Conclusion: This represents the first example of an inhibitor of IkappaB kinase with anti-inflammatory activity in vivo and indicates that inhibitors of IkB kinase show the promise of being highly efficacious in inflammatory disorders such as inflammatory bowel disease.[3]

C₁₄H₁₈CLN₅

291.78

291.125

C, 57.63; H, 6.22; Cl, 12.15; N, 24.00

547757-23-3

445430-58-0;547757-23-3 (HCl);445430-59-1 (2HCl);2320261-79-6 (TFA);

9926054

White to off-white solid powder

449.5ºC at 760 mmHg

225.6ºC

3.445

68.24

3

4

3

20

310

0

CC1=CN=C2C(NCCN)=NC3=CC=C(C=C3N21)C.Cl

MIDKPVLYXNLFGZ-UHFFFAOYSA-N

InChI=1S/C14H17N5.ClH/c1-9-3-4-11-12(7-9)19-10(2)8-17-14(19)13(18-11)16-6-5-15;/h3-4,7-8H,5-6,15H2,1-2H3,(H,16,18);1H

N'-(1,8-dimethylimidazo[1,2-a]quinoxalin-4-yl)ethane-1,2-diamine;hydrochloride

UNII-BXU277OCN5; BMS345541; BMS-345541 hydrochloride; BMS-345541 HCl; BMS-345541 (hydrochloride); 1,2-Ethanediamine, N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-, monohydrochloride; BXU277OCN5; BMS 345541; BMS-345541

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~50 mg/mL (~171.4 mM)
DMSO: ~20 mg/mL (~68.5 mM)

配方 1 中的溶解度: ≥ 2 mg/mL (6.85 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 20.0 mg/mL澄清DMSO储备液加入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: ≥ 2 mg/mL (6.85 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 20.0mg/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: ≥ 2 mg/mL (6.85 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 20.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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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。