CX-4945 (Silmitasertib) is a highly selective inhibitor of casein kinase 2 (CK2), with an IC50 of 1.3 nM against recombinant human CK2α (catalytic subunit) and a Ki of 0.4 nM (competitive inhibition with ATP). It shows no significant inhibition of other kinases (e.g., PKA, PKC, EGFR) at concentrations up to 1 μM, demonstrating strict kinase selectivity [1]

与正常细胞相比，癌细胞的细胞周期停滞和选择性凋亡是由 silmitasertib (CX-4945) 引起的，它还能减弱 PI3K/Akt 信号传导。 silmitasertib (CX-4945) 的抗增殖活性与 CK2α 催化亚基的表达水平相关。为了缓冲 ER 腔中 PS-341 介导的蛋白毒性应激，白血病细胞无法参与功能性 UPR。 Silmitasertib (CX-4945) 与 PS-341 联合治疗可降低促存活 ER 伴侣 BIP/Grp78 表达 [2]。 silmitasertib (CX-4945) 通过下调 CK2 表达来抑制 CK2 介导的 PI3K/Akt/mTOR 信号通路的激活，并在血液恶性肿瘤中诱导细胞毒性和细胞凋亡 [3]。

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实体瘤细胞抗增殖活性：CX-4945（0.1-10 μM）对多种人癌细胞系的增殖有抑制作用，IC50分别为：PC-3（前列腺癌）2.1 μM、A549（非小细胞肺癌）3.5 μM、MCF-7（乳腺癌）2.8 μM（MTT法，处理72小时）。5 μM浓度下，它可降低CK2下游底物的磷酸化水平：p-AKT（Ser473）降低65%、p-STAT3（Tyr705）降低72%（Western blot），并使PC-3细胞的VEGF分泌减少58%（ELISA）[1]
- 与PS-341联用对急性淋巴细胞白血病（ALL）细胞的协同细胞毒性：在CCRF-CEM和NALM-6 ALL细胞中，CX-4945（1 μM）与PS-341（0.1 μM，蛋白酶体抑制剂）联用，细胞活力降至22%（CX-4945单药组为55%，PS-341单药组为60%；CCK-8法）。它可下调ER伴侣蛋白BIP/Grp78（1 μM时降低45%），上调促凋亡的活化型caspase-3（增加3.2倍），并减少NF-κB p65的核转位（降低60%，免疫荧光）[2]
- 血液系统肿瘤活性：在人多发性骨髓瘤（MM）RPMI 8226细胞中，CX-4945（0.5-5 μM）抑制增殖，IC50为1.8 μM（MTT法）。2 μM时诱导G2/M期细胞周期阻滞（G2/M期比例从18%升至42%，流式细胞术），并使NF-κB依赖性荧光素酶活性降低70%。在套细胞淋巴瘤（MCL）Jeko-1细胞中，3 μM CX-4945使凋亡率增加35%（Annexin V-FITC/PI染色）[3]

在小鼠异种移植模型中，silmitasertib (CX-4945)（25 或 75 mg/kg，口服）具有良好的耐受性，并显示出强大的抗癌活性，同时降低基于机制的生物标志物磷酸-p21 (T145)[1]。

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裸鼠PC-3前列腺癌异种移植模型：6-8周龄雄性BALB/c nu/nu裸鼠皮下注射5×10⁶个PC-3细胞，待肿瘤达100 mm³时随机分为2组（每组n=8）：溶媒组（0.5%羧甲基纤维素钠，CMC-Na，10 mL/kg/天，口服）和CX-4945组（100 mg/kg/天，溶于0.5% CMC-Na，口服）。处理21天后，CX-4945使肿瘤体积减少62%（从950 mm³降至361 mm³），肿瘤重量减少58%。肿瘤组织分析：p-STAT3蛋白降低70%（Western blot），VEGF水平降低65%（ELISA）[1]
- 裸鼠RPMI 8226 MM播散模型：6-8周龄雌性BALB/c nu/nu裸鼠尾静脉注射1×10⁷个RPMI 8226细胞，建立播散性MM模型。小鼠接受CX-4945（50 mg/kg/天，口服灌胃）处理28天。CX-4945使骨髓MM细胞浸润率从45%降至18%（流式细胞术），血清M蛋白（MM标志物）降低52%（免疫比浊法），并使中位生存期延长30%（从35天延长至45.5天）[3]

重组人CK2α激酶活性实验：50 μL反应体系包含25 mM Tris-HCl（pH 7.5）、10 mM MgCl₂、1 mM ATP、10 μg去磷酸化酪蛋白（底物）、5 μg重组人CK2α及CX-4945（0.1-10 nM），加入1 μCi [γ-³²P]-ATP标记底物。30°C孵育60分钟后，加入5×SDS上样缓冲液终止反应，12% SDS-PAGE电泳分离样品，凝胶干燥后曝光于磷屏，磷成像仪定量磷酸化酪蛋白条带的放射性。以溶媒对照组为基准计算CK2活性抑制率，非线性回归推导IC50[1]
- CK2选择性实验：实验方案与CK2α激酶活性实验一致，差异在于用20种其他重组激酶（如PKA、PKCα、EGFR、CDK2）替代CK2α，且CX-4945浓度最高达1 μM。所有测试激酶的抑制率均<5%，证实其高选择性[1]

实体瘤细胞增殖与Western blot实验：
1. PC-3/A549细胞以3×10³细胞/孔接种于96孔板，含10% FBS的RPMI 1640培养24小时，加入CX-4945（0.1-10 μM）孵育72小时。加入20 μL MTT（5 mg/mL），4小时后DMSO溶解甲瓒，检测570 nm吸光度计算IC50[1]
2. PC-3细胞（1×10⁶细胞/10 cm培养皿）用CX-4945（5 μM）处理24小时，含蛋白酶/磷酸酶抑制剂的RPMI缓冲液裂解细胞。30 μg蛋白经10% SDS-PAGE电泳后转移至PVDF膜，一抗孵育p-AKT（Ser473）、p-STAT3（Tyr705）及β-肌动蛋白，HRP标记二抗结合后ECL显色[1]
- ALL细胞活力与凋亡实验：
1. CCRF-CEM细胞以5×10³细胞/孔接种于96孔板，用CX-4945（0.1-5 μM）单药或与PS-341（0.1 μM）联用处理48小时，加入CCK-8试剂，检测450 nm吸光度[2]
2. 细胞用4%多聚甲醛固定，0.1% Triton X-100透化，FITC标记的抗BIP/Grp78抗体与DAPI染色，共聚焦显微镜分析荧光强度以定量BIP/Grp78表达[2]
- MM细胞周期与凋亡实验：
1. RPMI 8226细胞（2×10⁵细胞/孔）用CX-4945（2 μM）处理24小时，70%乙醇固定，碘化丙啶（PI）染色，流式细胞术分析细胞周期分布[3]
2. 细胞用Annexin V-FITC与PI室温染色15分钟，流式细胞术计算凋亡率[3]

Dissolved in DMSO, and diluted in PBS; 25 or 75 mg/kg; Oral gavage
Female immunocompromised mice CrTac:Ncr-Foxn1nu injected with BxPC-3 or BT-474 cells

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PC-3 Prostate Cancer Xenograft Model: Male BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle). Mice were subcutaneously injected with 5×10⁶ PC-3 cells (suspended in 100 μL PBS + 50 μL Matrigel) into the right flank. When tumors reached 100 mm³, mice were randomized into 2 groups (n=8/group):
- Vehicle: 0.5% CMC-Na, oral gavage once daily (10 mL/kg);
- CX-4945: 100 mg/kg/day CX-4945 (dissolved in 0.5% CMC-Na), oral gavage once daily (10 mL/kg).
Treatment lasted 21 days. Tumor volume was measured every 3 days (volume = length × width² / 2). On day 21, mice were euthanized, tumors were harvested for Western blot and VEGF ELISA [1]
- RPMI 8226 MM Disseminated Model: Female BALB/c nu/nu mice (6-8 weeks old) were intravenously injected with 1×10⁷ RPMI 8226 cells via the tail vein. Three days later, mice were divided into 2 groups (n=10/group):
- Vehicle: 0.5% CMC-Na, oral gavage once daily (10 mL/kg);
- CX-4945: 50 mg/kg/day CX-4945 (dissolved in 0.5% CMC-Na), oral gavage once daily (10 mL/kg).
Treatment lasted 28 days. Mouse survival was recorded daily. On day 28, mice were euthanized: bone marrow cells were collected for flow cytometry (MM cell quantification), and serum was collected for M-protein detection [3]

Oral bioavailability: In rats, oral administration of CX-4945 (50 mg/kg) showed a bioavailability of 28%; in beagle dogs, oral bioavailability was 42% (compared to intravenous administration) [1]
- Plasma pharmacokinetics: In rats, oral CX-4945 (50 mg/kg) had a Cmax of 3.2 μg/mL, Tmax of 1.5 hours, and elimination half-life (t1/2) of 4.5 hours. In dogs, Cmax was 5.8 μg/mL, Tmax was 2.0 hours, t1/2 was 6.2 hours [1]
- Distribution and excretion: CX-4945 is highly bound to plasma proteins (>98% in rat/dog/human plasma). It distributes to tumor tissues (tumor/plasma concentration ratio = 3.5 in PC-3 xenografts). Metabolism is primarily via cytochrome P450 3A4 (CYP3A4) in the liver; >70% of the dose is excreted via feces (as metabolites) within 72 hours [1]

Acute toxicity: Single oral administration of CX-4945 up to 200 mg/kg in rats and 150 mg/kg in dogs caused no mortality or obvious toxicity (e.g., lethargy, diarrhea). LD50 was >200 mg/kg in both species [1]
- Subchronic toxicity: Rats treated with CX-4945 (50, 100 mg/kg/day, oral) for 28 days showed no significant changes in body weight (<5% variation), serum ALT/AST/BUN/creatinine, or histopathology of liver, kidney, and spleen [1]
- Hematological toxicity in mice: Mice treated with CX-4945 (50 mg/kg/day, oral) for 28 days had no significant reduction in white blood cells, red blood cells, or platelets (flow cytometry). Serum inflammatory cytokines (TNF-α, IL-6) remained unchanged [3]

[1]. CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res. 2010 Dec 15;70(24):10288-98.

[2]. Synergistic cytotoxic effects of PS-341 and CK2 inhibitor CX-4945 in acute lymphoblastic leukemia: turning off the prosurvival ER chaperone BIP/Grp78 and turning on the pro-apoptotic NF-κB. Oncotarget. 2016 Jan 12;7(2):1323-40.

[3]. The casein kinase 2 inhibitor, CX-4945, as an anti-cancer drug in treatment of human hematological malignancies. Front Pharmacol. 2015 Mar 31;6:70.

Silmitasertib is under investigation in clinical trial NCT03904862 (Testing the Safety and Tolerability of CX-4945 in Patients With Recurrent Medulloblastoma Who May or May Not Have Surgery).
Silmitasertib is an orally bioavailable small-molecule inhibitor of the enzyme casein kinase II (CK2), with potential antineoplastic, anti-viral and immunomodulatory activities. Upon oral administration, silmitasertib selectively binds to and inhibits the activity of CK2. This may inhibit proliferation of CK2-expressing tumor cells, and may also inhibit the replication of severe acute respiratory syndrome coronavirus-2 (SARS-COV-2). In addition, this may restore normal host cell cytokine regulation, prevent cytokine storm and suppress the hyperactivation of the innate immune system. CK2, a protein kinase often overexpressed in a variety of cancer cell types, appears to be correlated with malignant transformation, tumor growth and survival. CK2 regulates a diverse array of pro-survival cellular processes including epidermal growth factor receptor (EGFR) signaling, PI3K/AKT/mTOR signaling, hedgehog (Hh) signaling, Hsp90 machinery, hypoxia, and interleukin (IL)-6 expression. CK2 also regulates the activity of XRCC1 and MDC1, two mediator/adaptor proteins that are essential for DNA repair. CK2 is upregulated by SARS-COV-2 and is associated with SARS-COV-2 viral replication and the development of cytokine storm.

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Mechanism of action: CX-4945 (Silmitasertib) selectively inhibits CK2, a serine/threonine kinase that regulates prosurvival and angiogenic signaling pathways. By blocking CK2, it suppresses phosphorylation of downstream substrates (AKT, STAT3, NF-κB), inhibiting cancer cell proliferation, inducing apoptosis, and reducing tumor angiogenesis (via VEGF downregulation) [1,3]
- Therapeutic potential: CX-4945 is a clinical-stage (Phase I/II) candidate for solid tumors (prostate, lung cancer) and hematological malignancies (MM, ALL, MCL). Literature [2] demonstrated its synergistic effect with proteasome inhibitors (e.g., PS-341), supporting combination therapy in refractory hematological cancers [1-3]
- Clinical relevance: Literature [1] provided preclinical evidence for oral administration (favorable bioavailability), supporting its use in outpatient settings. Literature [3] highlighted its activity in bone marrow-resident MM cells, addressing a key challenge in MM treatment (minimal residual disease) [1,3]
- Limitations: CX-4945 has moderate solubility in water (requires CMC-Na or DMSO for formulation) and may require dose adjustment in patients with CYP3A4 polymorphisms (due to metabolism via CYP3A4) [1]

C19H12CLN3O2

349.77

349.061

1009820-21-6

Silmitasertib sodium salt;1309357-15-0

24748573

Yellow to orange solid powder

1.5±0.1 g/cm3

568.5±50.0 °C at 760 mmHg

297.6±30.1 °C

0.0±1.6 mmHg at 25°C

1.789

3.29

75.11

2

5

3

25

491

0

MUOKSQABCJCOPU-UHFFFAOYSA-N

InChI=1S/C19H12ClN3O2/c20-12-2-1-3-13(9-12)22-18-15-6-7-21-10-16(15)14-5-4-11(19(24)25)8-17(14)23-18/h1-10H,(H,22,23)(H,24,25)

5-(3-chloroanilino)benzo[c][2,6]naphthyridine-8-carboxylic acid

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: 2.08 mg/mL (5.95 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 20.8 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: 2.08 mg/mL (5.95 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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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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。