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]

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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Antiproliferative activity in solid tumor cells: CX-4945 (0.1-10 μM) inhibited proliferation of various human cancer cell lines with IC50 values: PC-3 (prostate cancer) 2.1 μM, A549 (non-small cell lung cancer) 3.5 μM, MCF-7 (breast cancer) 2.8 μM (MTT assay, 72-hour treatment). It reduced phosphorylation of CK2 downstream substrates: at 5 μM, p-AKT (Ser473) decreased by 65%, p-STAT3 (Tyr705) decreased by 72% (Western blot), and VEGF secretion (ELISA) reduced by 58% in PC-3 cells [1]
- Synergistic cytotoxicity in acute lymphoblastic leukemia (ALL) cells: In CCRF-CEM and NALM-6 ALL cells, combination of CX-4945 (1 μM) and PS-341 (0.1 μM, proteasome inhibitor) reduced cell viability to 22% (vs. 55% for CX-4945 alone, 60% for PS-341 alone; CCK-8 assay). It downregulated ER chaperone BIP/Grp78 (45% reduction at 1 μM) and upregulated pro-apoptotic cleaved caspase-3 (3.2-fold increase) and NF-κB p65 (nuclear translocation reduced by 60%, immunofluorescence) [2]
- Activity in hematological malignancies: In human multiple myeloma (MM) RPMI 8226 cells, CX-4945 (0.5-5 μM) inhibited proliferation with IC50 1.8 μM (MTT assay). At 2 μM, it induced G2/M cell cycle arrest (G2/M proportion from 18% to 42%, flow cytometry) and reduced NF-κB-dependent luciferase activity by 70%. In mantle cell lymphoma (MCL) Jeko-1 cells, 3 μM CX-4945 increased apoptotic rate by 35% (Annexin V-FITC/PI staining) [3]

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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Nude mouse PC-3 prostate cancer xenograft model: Male BALB/c nu/nu mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ PC-3 cells. When tumors reached 100 mm³, mice were randomized into 2 groups (n=8/group): vehicle (0.5% carboxymethylcellulose sodium, CMC-Na, 10 mL/kg/day, oral) and CX-4945 (100 mg/kg/day, dissolved in 0.5% CMC-Na, oral). After 21 days, CX-4945 reduced tumor volume by 62% (from 950 mm³ to 361 mm³) and tumor weight by 58%. Tumor tissue analysis: p-STAT3 protein decreased by 70% (Western blot), VEGF levels reduced by 65% (ELISA) [1]
- Nude mouse RPMI 8226 MM xenograft model: Female BALB/c nu/nu mice (6-8 weeks old) were intravenously injected with 1×10⁷ RPMI 8226 cells to establish disseminated MM. Mice were treated with CX-4945 (50 mg/kg/day, oral gavage) for 28 days. CX-4945 reduced bone marrow MM cell infiltration (from 45% to 18%, flow cytometry) and serum M-protein (a MM marker) by 52% (immunoturbidimetry). It also prolonged median survival by 30% (from 35 days to 45.5 days) [3]

Recombinant Human CK2α Kinase Activity Assay: The 50 μL reaction system contained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM ATP, 10 μg dephosphorylated casein (substrate), 5 μg recombinant human CK2α, and CX-4945 (0.1-10 nM). 1 μCi [γ-³²P]-ATP was added to label the substrate. The mixture was incubated at 30°C for 60 minutes, then terminated by adding 5×SDS loading buffer. Samples were separated by 12% SDS-PAGE, and the gel was dried and exposed to a phosphor screen. Radioactivity of phosphorylated casein bands was quantified using a phosphorimager. CK2 activity inhibition rate was calculated relative to the vehicle control, and IC50 was derived via nonlinear regression [1]
- CK2 Selectivity Assay: The protocol was identical to the CK2α assay, except 20 other recombinant kinases (e.g., PKA, PKCα, EGFR, CDK2) were used instead of CK2α, and CX-4945 concentration was up to 1 μM. Inhibition rate of all tested kinases was <5%, confirming high selectivity [1]

Solid Tumor Cell Proliferation and Western Blot Assay:
1. PC-3/A549 cells were seeded in 96-well plates at 3×10³ cells/well, cultured in RPMI 1640 with 10% FBS for 24 hours. CX-4945 (0.1-10 μM) was added, incubated for 72 hours. 20 μL MTT (5 mg/mL) was added, 4 hours later DMSO dissolved formazan, and absorbance at 570 nm was measured to calculate IC50 [1]
2. PC-3 cells (1×10⁶ cells/10-cm dish) were treated with CX-4945 (5 μM) for 24 hours, lysed with RIPA buffer . 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-p-AKT (Ser473), anti-p-STAT3 (Tyr705), and anti-β-actin antibodies. HRP-conjugated secondary antibodies and ECL reagent were used for detection [1]
- ALL Cell Viability and Apoptosis Assay:
1. CCRF-CEM cells were seeded in 96-well plates at 5×10³ cells/well, treated with CX-4945 (0.1-5 μM) alone or with PS-341 (0.1 μM) for 48 hours. CCK-8 reagent was added, and absorbance at 450 nm was measured [2]
2. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-BIP/Grp78 antibody (FITC-conjugated) and DAPI. Fluorescence intensity was analyzed via confocal microscopy to quantify BIP/Grp78 expression [2]
- MM Cell Cycle and Apoptosis Assay:
1. RPMI 8226 cells (2×10⁵ cells/well) were treated with CX-4945 (2 μM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed via flow cytometry to determine cell cycle distribution [3]
2. Cells were stained with Annexin V-FITC and PI for 15 minutes at room temperature, then flow cytometry was used to calculate apoptotic rate [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, the oral bioavailability of CX-4945 (50 mg/kg) was 28%; in beagle dogs, the oral bioavailability was 42% (compared to intravenous administration) [1] - Plasma pharmacokinetics: In rats, the Cmax of oral CX-4945 (50 mg/kg) was 3.2 μg/mL, the Tmax was 1.5 h, and the elimination half-life (t1/2) was 4.5 h. In dogs, the Cmax was 5.8 μg/mL, the Tmax was 2.0 h, and the t1/2 was 6.2 h [1] - Distribution and excretion: CX-4945 is highly bound to plasma proteins (>98% in rat/dog/human plasma). It is distributed to tumor tissues (tumor/plasma concentration ratio of 3.5 in PC-3 xenograft tumors). It is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in the liver; more than 70% of the dose is excreted in feces as metabolites within 72 hours [1].

Acute toxicity: Single oral doses of CX-4945 up to 200 mg/kg in rats and up to 150 mg/kg in dogs did not cause death or significant toxic reactions (e.g., lethargy, diarrhea). The LD50 in both animals was greater than 200 mg/kg [1]
- Subchronic toxicity: After 28 days of oral administration of CX-4945 (50, 100 mg/kg/day) to rats, no significant changes were observed in body weight (change <5%), serum ALT/AST/BUN/creatinine levels, or histopathological changes in the liver, kidneys, and spleen [1]
- Hematologic toxicity in mice: After 28 days of oral administration of CX-4945 (50 mg/kg/day) to mice, no significant decrease was observed in white blood cell, red blood cell, or platelet counts (flow cytometry). Serum inflammatory cytokine (TNF-α, IL-6) levels 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 being investigated in the clinical trial NCT03904862 (evaluating the safety and tolerability of CX-4945 in patients with recurrent medulloblastoma who may or may not undergo surgery). Silmitasertib is a small, orally bioavailable casein kinase II (CK2) inhibitor with potential antitumor, antiviral, and immunomodulatory activities. After oral administration, silmitasertib 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 crucial for DNA repair. SARS-CoV-2 upregulates CK2 expression, and CK2 is closely associated with SARS-CoV-2 viral replication and the occurrence of cytokine storms. Mechanism of action: CX-4945 (Silmitasertib) selectively inhibits CK2, a serine/threonine kinase that regulates pro-survival and angiogenesis signaling pathways. By blocking CK2, CX-4945 inhibits the phosphorylation of downstream substrates (AKT, STAT3, NF-κB), thereby inhibiting cancer cell proliferation, inducing apoptosis, and reducing tumor angiogenesis (by downregulating VEGF) [1,3]. - Therapeutic potential: CX-4945 is a clinical-stage (Phase I/II) candidate drug for the treatment of solid tumors (prostate cancer, lung cancer) and hematologic malignancies (multiple myeloma, acute lymphoblastic leukemia, mantle cell lymphoma). Reference [2] demonstrated its synergistic effect with proteasome inhibitors (e.g., PS-341), supporting its combination therapy in refractory hematologic malignancies [1-3]. - Clinical significance: Reference [1] provides preclinical evidence for oral administration (good bioavailability), supporting its application in an outpatient setting. Reference [3] highlights its activity against 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 preparation), and dose adjustments may be necessary for patients with CYP3A4 polymorphisms (as its metabolic pathway is 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

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)

Solubility in Formulation 1: 2.08 mg/mL (5.95 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (5.95 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)