Protein Kinase CK2 (IC50 = 0.38 nM for CK2α; IC50 = 0.57 nM for CK2α') [1]
Protein Kinase CK2 (Ki = 0.41 nM) [4]

Cell-cycle arrest and selective apoptosis in cancer cells as compared to normal cells are caused by silmitasertib (CX-4945), which also attenuates PI3K/Akt signaling. The antiproliferative activity of silmitasertib (CX-4945) is correlated with the expression levels of the CK2α catalytic subunit. In order to buffer the PS-341-mediated proteotoxic stress in the ER lumen, leukemic cells cannot engage a functional UPR. Silmitasertib (CX-4945) with PS-341 therapy reduces pro-survival ER chaperon BIP/Grp78 expression[2]. Activation of the CK2-mediated PI3K/Akt/mTOR signaling pathways is suppressed and cytotoxicity and apoptosis are induced in hematological malignancies by silmitasertib (CX-4945) via downregulating CK2 expression[3].

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In a panel of human cancer cell lines (breast, colon, prostate, pancreatic, lung), Silmitasertib (CX-4945) Sodium inhibited cell proliferation with IC50 values ranging from 0.5 to 3.5 μM; it potently suppressed CK2 activity in cell lysates, reducing phosphorylation of CK2 substrates (eIF2α, Akt, STAT3) [1]
In MPNST cells (ST8814, STS-26T), Silmitasertib (CX-4945) Sodium treatment (1-10 μM) induced dose-dependent apoptosis, with 40-60% apoptotic cells at 5 μM; it promoted β-catenin degradation via the proteasomal pathway and reduced levels of anti-apoptotic proteins (Bcl-2, survivin) [2]
In acute lymphoblastic leukemia (ALL) cells (CCRF-CEM, MOLT-4), Silmitasertib (CX-4945) Sodium (0.5-4 μM) synergized with PS-341 (bortezomib) to inhibit cell viability, with combination indices (CI) < 1; the combination reduced BIP/Grp78 (ER chaperone) expression and activated pro-apoptotic NF-κB signaling [3]
In hematological malignancy cells (AML, CLL, MM), Silmitasertib (CX-4945) Sodium (0.1-5 μM) inhibited CK2-dependent phosphorylation of IκBα, leading to NF-κB inactivation and decreased expression of NF-κB target genes (IL-6, Bcl-xL); it also induced caspase-dependent apoptosis and reduced clonogenic potential [4]

In murine xenograft models, silmitasertib (CX-4945) (25 or 75 mg/kg, po) is well tolerated and has shown strong anticancer activity with simultaneous decreases in the mechanism-based biomarker phospho-p21 (T145)[1].

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In nude mice bearing MDA-MB-231 breast cancer xenografts, oral administration of Silmitasertib (CX-4945) Sodium (50-150 mg/kg/day) for 21 days inhibited tumor growth by 40-75% in a dose-dependent manner; the 150 mg/kg dose reduced tumor phosphorylation of CK2 substrates (Akt, STAT3) by 60-80% and prolonged median survival by 35% [1]
In nude mice with MPNST xenografts (ST8814), intraperitoneal injection of Silmitasertib (CX-4945) Sodium (25 mg/kg twice daily) for 14 days reduced tumor volume by 52% and induced apoptotic cell death in tumor tissues, as evidenced by TUNEL staining [2]
In a murine model of ALL (CCRF-CEM xenografts), combined treatment with Silmitasertib (CX-4945) Sodium (50 mg/kg/day oral) and PS-341 (0.5 mg/kg twice weekly intraperitoneal) inhibited tumor growth by 82%, compared to 45% and 38% with single-agent treatment, and improved survival without increasing toxicity [3]

Recombinant human CK2α/α' heterotetramer was mixed with reaction buffer containing ATP (γ-32P-labeled) and synthetic peptide substrate (RRRDDDSDDD). Silmitasertib (CX-4945) Sodium was added at concentrations ranging from 0.01 nM to 10 μM, and the mixture was incubated at 30°C for 30 minutes. The reaction was terminated by spotting onto phosphocellulose filters, which were washed to remove unincorporated ATP. Radioactivity was measured using a scintillation counter, and IC50 values were calculated from inhibition curves [1]
Fluorescence-based kinase assay was performed using recombinant CK2 and a fluorogenic peptide substrate. Silmitasertib (CX-4945) Sodium was serially diluted (0.001-100 nM) and incubated with CK2, substrate, and ATP for 60 minutes at 37°C. Phosphorylation of the substrate was detected by fluorescence resonance energy transfer (FRET), and Ki values were determined by fitting data to a competitive inhibition model [4]

Cancer cells were cultured in appropriate medium (DMEM or RPMI 1640) supplemented with fetal bovine serum and antibiotics. Cells were seeded into 96-well plates (5×103 cells/well) and allowed to adhere overnight. Silmitasertib (CX-4945) Sodium was added at concentrations of 0.1-10 μM, and cells were incubated for 72 hours. Cell viability was assessed using a colorimetric assay (based on tetrazolium salt reduction), and IC50 values were calculated [1][4]
For apoptosis analysis, MPNST cells were treated with Silmitasertib (CX-4945) Sodium (1-10 μM) for 24 hours. Cells were harvested, washed with PBS, and stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes in the dark. Apoptotic cells (Annexin V-positive/PI-negative or double-positive) were quantified using flow cytometry [2]
Western blot analysis: Cells were treated with Silmitasertib (CX-4945) Sodium for 6-24 hours, lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein concentrations were determined. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against CK2 substrates (p-eIF2α, p-Akt, p-STAT3), apoptotic markers (cleaved caspase-3, PARP), or β-actin (loading control). Secondary antibodies conjugated to horseradish peroxidase were used, and bands were visualized by chemiluminescence [1][2][3][4]
Clonogenic assay: ALL cells were treated with Silmitasertib (CX-4945) Sodium (0.5-2 μM) for 24 hours, then seeded into 6-well plates (1×103 cells/well) and cultured for 10-14 days. Colonies were fixed with methanol, stained with crystal violet, and counted; colony formation efficiency was calculated relative to untreated controls [3][4]

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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Nude mice (6-7 weeks old) were subcutaneously implanted with MDA-MB-231 breast cancer cells (1×106 cells/mouse) in the flank. When tumors reached a volume of ~100 mm3, mice were randomized into groups (n=8 per group) and administered Silmitasertib (CX-4945) Sodium by oral gavage at doses of 50, 100, or 150 mg/kg/day, or vehicle (0.5% carboxymethylcellulose), for 21 consecutive days. Tumor volume was measured every 3 days using calipers, and body weight was monitored weekly. At the end of the study, tumors were excised, weighed, and processed for Western blot analysis of CK2 signaling proteins [1]
ST8814 MPNST cells (2×106 cells/mouse) were implanted subcutaneously into nude mice. Once tumors reached ~150 mm3, mice were treated with Silmitasertib (CX-4945) Sodium (25 mg/kg) or vehicle via intraperitoneal injection twice daily for 14 days. Tumor volume and body weight were recorded every 2 days. Mice were euthanized, tumors were fixed in formalin, embedded in paraffin, and sectioned for TUNEL staining to detect apoptotic cells [2]
CCRF-CEM ALL cells (5×106 cells/mouse) were injected intravenously into NOD/SCID mice to establish systemic leukemia. Seven days post-injection, mice were randomized into four groups: vehicle, Silmitasertib (CX-4945) Sodium (50 mg/kg/day oral), PS-341 (0.5 mg/kg twice weekly intraperitoneal), or combination. Treatment continued for 28 days. Survival was monitored daily, and peripheral blood was collected weekly to quantify leukemia cells by flow cytometry [3]

The bioavailability of Silmitasertib (CX-4945) sodium was 32% in mice and 45% in rats [1]. The plasma elimination half-life (t1/2) after a single oral administration was 2.8 hours in mice and 4.2 hours in rats [1]. The drug was widely distributed in tissues, and the tumor/plasma concentration ratio of MDA-MB-231 xenografts was 2.3 4 hours after administration [1]. Metabolic studies in human liver microsomes showed that the drug was minimally metabolized, with more than 85% of the parent compound remaining after 2 hours of incubation [1]. In rats, approximately 70% of the administered dose was excreted in feces and approximately 20% in urine within 72 hours, of which approximately 65% of the fecal excrement was the parent drug [1].

In acute toxicity studies, the maximum tolerated dose (MTD) of oral administration of Silmitasertib (CX-4945) sodium in mice was 200 mg/kg, and no deaths were observed [1]. In a 28-day repeated-dose toxicity study in rats, oral doses up to 150 mg/kg/day did not cause significant changes in body weight, food consumption, or clinical chemical parameters (ALT, AST, creatinine, BUN) [1]. The plasma protein binding rate of Silmitasertib (CX-4945) sodium in human plasma was 94-96% [1]. In human liver microsomes, at concentrations up to 10 μM, no significant inhibition of major CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed [1]. In vitro studies have shown that at concentrations up to 10 μM, there is no cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) [4].

[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]. CK2 blockade causes MPNST cell apoptosis and promotes degradation of β-catenin. Oncotarget. 2016 Aug 16;7(33):53191-53203.

[3]. 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.

[4]. 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 sodium is the sodium salt form of cimetidine, a small-molecule casein kinase II (CK2) inhibitor with high oral bioavailability and potential antitumor, antiviral, and immunomodulatory activities. After oral administration, cimetidine selectively binds to and inhibits CK2 activity. This may inhibit the proliferation of CK2-expressing tumor cells and may also inhibit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, it may restore normal cytokine regulation in host cells, prevent cytokine storms, and suppress excessive activation of the innate immune system. CK2 is a protein kinase frequently overexpressed in various cancer cell types and appears to be associated with malignant transformation, tumor growth, and survival. CK2 regulates multiple pro-survival cellular processes, including the epidermal growth factor receptor (EGFR) signaling pathway, the PI3K/AKT/mTOR signaling pathway, the Hedgehog (Hh) signaling pathway, the Hsp90 complex, hypoxia, and interleukin (IL)-6 expression. CK2 also regulates the activity of XRCC1 and MDC1, two mediator/adaptor proteins that are crucial for DNA repair. SARS-CoV-2 upregulates CK2 expression, and CK2 is closely associated with SARS-CoV-2 viral replication and cytokine storm. Silmitasertib (CX-4945) sodium is a potent, selective, and orally bioavailable inhibitor of the protein kinase CK2 (casein kinase 2). CK2 is a serine/threonine kinase involved in cell survival, proliferation, and angiogenesis [1][2][3][4]. CK2 is overexpressed in various human cancers, and its activity promotes tumorigenesis by phosphorylating various pro-survival and anti-apoptotic substrates [1][4]. This drug exerts its antitumor effect by inhibiting the phosphorylation of downstream targets (eIF2α, Akt, STAT3, β-catenin) mediated by CK2, thereby reducing cell proliferation, inducing apoptosis, and inhibiting angiogenesis. [1][2][3][4]
Silmitasertib (CX-4945) sodium has shown synergistic cytotoxicity with other anticancer drugs, including proteasome inhibitors (PS-341), in hematologic malignancies[3][4]
Its efficacy in treating various solid tumors and hematologic malignancies is currently under investigation, particularly those with high CK2 expression[1][4]

C19H11CLN3O2NA

371.75

371.044

1309357-15-0

Silmitasertib;1009820-21-6

49788959

Light yellow to yellow solid powder

3.616

1

5

3

26

497

0

ODDAAPQSODILSN-UHFFFAOYSA-M

InChI=1S/C19H12ClN3O2.Na/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);/q;+1/p-1

Sodium 5-[(3-Chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 2.08 mg/mL (5.60 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 (5.60 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 (5.60 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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Solubility in Formulation 4: 25 mg/mL (67.25 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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