Silmitasertib (CX-4945)

Biological Activity

CX-4945 (Silmitasertib) is the first orally available small molecule inhibitor of protein CK2 in clinical trials for cancer. CX-4945 (Silmitasertib) selectively binds to and inhibits the enzyme casein kinase II (CK2). Protein kinase CK2 (CK2) is involved in a variety of roles essential to the maintenance of cellular homeostasis. CK2 promotes signaling in the Akt pathway and CX-4945 suppresses the phosphorylation of Akt as well as other key downstream mediators of the pathway such as p21. CX-4945 has been reported to show broad spectrum anti-proliferative activity in multiple cancer cell lines. CX-4945 induced apoptosis and caused cell cycle arrest in cancer cells in vitro. CX-4945 exhibited a dose-dependent antitumor activity in a xenograft model of PC3 prostate cancer model and was well tolerated. In vivo time-dependent reduction in the phosphorylation of the biomarker p21 at T145 was observed by immunohistochemistry.

Product Citations

• 

  Sci Rep. 2017 Dec 14;7(1):17569.

  CK2 modulates adipocyte insulinsignaling and is up-regulated in human obesity
  Silmitasertib (CX-4945) purchased from AbMole

• 

  Biomed Res Int. 2015;601543.

  Phosphorylation, Signaling, and Cancer: Targets and Targeting.
  Silmitasertib (CX-4945) purchased from AbMole

• 

  Growth Factors. 2015 Sep 04;259-266.

  Effects of CK2 inhibition in cultured fibroblasts from Type 1 Diabetic patients with or without nephropathy.
  Silmitasertib (CX-4945) purchased from AbMole

• 

  Biochim Biophys Acta. 2015 Jul;1853(7):1693-701.

  Protein kinase CK2 potentiates translation efficiency by phosphorylating eIF3j at Ser127.
  Silmitasertib (CX-4945) purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Sci Rep (2017). Figure 4. CX-4945 (AbMole BioScience, Hong Kong, China)
	Method	ip inject
	Cell Lines	C57BL6/6 J (B6) male mice
	Concentrations	20 mg/kg
	Incubation Time	2 h
	Results	CK2 activity and Akt-signaling were then evaluated in insulin-target tissues. Insulin-treatment did not affect the basal kinase activity of different tissues, while CX-4945 injection strikingly inhibited CK2 in VAT-E, SAT and liver

	Source	Sci Rep (2017). Figure 2. CX-4945 (AbMole BioScience, Hong Kong, China)
	Method	Weatern Blot
	Cell Lines	human adipocyte
	Concentrations	2.5 μM
	Incubation Time	1 h
	Results	Results obtained in 3T3-L1 cells were confirmed in human adipocyte primary cultures, where CX-4945 and DMAT pre-treatment substantially inhibited the insulin-stimulated glucose uptake

	Source	Sci Rep (2017). Figure 1. CX-4945 (AbMole BioScience, Hong Kong, China)
	Method	Weatern Blot
	Cell Lines	human adipocyte
	Concentrations	2.5 μM
	Incubation Time	1 h
	Results	Conversely, a great reduction of insulin-induced glucose uptake was observed in 3T3-L1 mature adipocytes, pre-treated for 1 h with the CK2-inhibitor CX-494528 and then stimulated with increasing insulin doses for 30 min

	Source	Biomed Res Int (2015). Figure 5. CX-4945 (Abmole Bioscience)
	Method	Wound-Healing Assays
	Cell Lines	U2OS cells
	Concentrations	3 µM
	Incubation Time	24 h
	Results	Both CX-4945 and TDB were able to inhibit cell motility in 24–48 h assayswhen cellswere constantly exposed to them (upper part of the figure).

	Source	Biomed Res Int (2015). Figure 4. CX-4945 (Abmole Bioscience)
	Method	Wound-Healing Assays
	Cell Lines	U2OS cells
	Concentrations	3 µM
	Incubation Time	24 h
	Results	We found that TDB-treated cells were much less prone than vehicle-treated cells to forming spheroids, while CX-4945 was similarly effective than TDB when assessed at 24h but has much weaker effect at longer times after the inhibitor removal.

	Source	Biomed Res Int (2015). Figure 3. CX-4945 (Abmole Bioscience)
	Method	Wound-Healing Assays
	Cell Lines	U2OS cells
	Concentrations	3 µM
	Incubation Time	24 h
	Results	The results, shown in Figure 3, indicate that TDB is much more effective than CX-4945 in preventing clone formation and survival.

	Source	Biomed Res Int (2015). Figure 2. CX-4945 (Abmole Bioscience)
	Method	Western Blot
	Cell Lines	CEM or U2OS cells
	Concentrations	3 µM
	Incubation Time	24h, 48h, 72h, 96h
	Results	The results, shown in Figure 2(b), demonstrated that the phosphorylation of Akt1 Ser129 is similarly reduced by the two inhibitors immediately after treatment, while it remains significantly lower than the control only with TDB in case of inhibitor removal, thus confirming the more permanent blockage of the kinase activity by TDB than by CX-4945.

	Source	Biomed Res Int (2015). Figure 1. CX-4945 (Abmole Bioscience)
	Method	Comparison of CX-4945 and TDB efficacy on cellular CK2 activity.
	Cell Lines	CEM or U2OS cells
	Concentrations	1 µM
	Incubation Time	4 h
	Results	To compare the inhibitory efficacy in cells of the two compounds, CX-4945 and TDB, we analyzed their effects on two cell lines, one deriving from a blood tumor (CEM, T-cell lymphoblastoma) and the other from a solid tumor (U2OS, osteosarcoma). Their efficacy was quite similar in inhibiting cellular CK2.

	Source	Growth Factors (2015) . Figure 6.CX-4945 was from AbMole Bioscience (Houston, TX).
	Method	western blot
	Cell Lines	fibroblasts from N, T1DM , and T1DM+
	Concentrations	10 µM
	Incubation Time	72h
	Results	Although we cannot strictly confirm that all the bands shown in the WB of Figure 6 are direct targets of CK2, our observations suggest that the three cell groups might differ in some regulatory mechanisms which depend on CK2 and can be affected by its blockage.

	Source	Growth Factors (2015) . Figure 5.CX-4945 was from AbMole Bioscience (Houston, TX).
	Method	western blot
	Cell Lines	fibroblasts from N, T1DM , and T1DM+
	Concentrations	10 µM
	Incubation Time	72h
	Results	"we did not find any significant difference of phosphorylation level of a CK2 crucial target, Akt Ser129 among the three cell groups. Also the expression of the total Akt protein was very similar among groups (Figure 5). However, as shown in Figure 5, both CX-4945 and TBB treatment produced a reduction of Sp129-Akt phosphorylation to a level that was very similar among the three groups of cells."

	Source	Growth Factors (2015) . Figure 3.CX-4945 was from AbMole Bioscience (Houston, TX).
	Method	Cell viability assay
	Cell Lines	fibroblasts from N, T1DM , and T1DM+
	Concentrations	10 and 20 µM
	Incubation Time	48 h and 72 h
	Results	The results substantially confirmed those obtained with TBB, showing in addition that the fibroblasts from T1DM+ patients were the most sensitive among the subjects’ groups (Figure 3a), their viability being significantly reduced already at the lower dose (10 mM of CX-4945) within 48 h.

	Source	Biochimica et Biophysica Acta (2015) . Figure 4.CX-4945 was purchased from AbMole BioScience (Hong Kong, China.
	Method	western blot and Immunoprecipitation(IP)
	Cell Lines	HEK293
	Concentrations	2.5µM
	Incubation Time	5 h
	Results	CK2 inhibitor CX-4945 induces the dissociation of a large amount of j-subunit from the eIF3 complex both bound or unbound to the 40S subunits and its shift toward the low-density region of the gradient, where CK2α and CK2β are also detectable (fractions 11–14).

	Source	Biochimica et Biophysica Acta (2015) . Figure 3.CX-4945 was purchased from AbMole BioScience (Hong Kong, China.
	Method	western blot and Immunoprecipitation(IP)
	Cell Lines	HEK293
	Concentrations	2.5µM
	Incubation Time	5 h
	Results	CX-4945 and Q1 strongly reduce the phosphorylation extent of Akt1 Ser129, a specific target of CK2 (about 80% and 65%, respectively), while they do not change the protein-level of CK2α, CK2β and eIF3j(wb).CX-4945 or quinalizarin does not affect the interaction of the kinase with the eIF3 subunits eIF3b and eIF3d, it causes an about 3-fold increase of eIF3j co-immuniprecipitation(IP).

	Source	Biochimica et Biophysica Acta (2015) . Figure 1.CX-4945 was purchased from AbMole BioScience (Hong Kong, China.
	Method	Immunoprecipitation(IP)
	Cell Lines	HEK293, Hela, LAMA84
	Concentrations	0.5µM
	Incubation Time	15 min
	Results	The strong inhibition of the 33P-phosphorylation induced by in vitro addition of the CK2 selective and potent inhibitor CX-4945. The kinase co-immunoprecipitated with eIF3j specifically phosphorylates the subunit as demonstrated by the strong reduction of the eIF3j 33P phosphorylation caused by in vitro addition of the CK2 inhibitor CX-4945.

Protocol (for reference only)

Cell Experiment
Cell lines	BT-474 (breast) or BxPC-3 (pancreatic) cancer or HUVEC cells
Preparation method	A 5 mmol/L stock solution in dimethyl sulfoxide was prepared and stored at - 70 ℃. The drug was diluted directly into growth media immediately prior to use.Various cell lines were seeded at a density of 3,000 cells per well 24 hours prior to treatment, in appropriate media, and then treated with indicated concentrations of CX-4945. Suspensions cells were seeded and treated on the same day.Following 4 days of incubation, Alamar Blue (20 mL, 10% of volume per well) was added and the cells were further incubated at 37 C for 4–5 hours. Fluorescence with excitation wavelength at 530–560 nm and emission wavelength at 590 nm was measured.
Concentrations	0, 0.1, 1.0, 5.0, 10 µM
Incubation time	24 h

Animal Experiment
Animal models	Female immunocompromised mice CrTac:Ncr-Foxn1nu (5–7 weeks old) BT474 or BxPC-3 cells Xenografts
Formulation	Solubilized in DMSO and diluted with PBS containing 10% dimethylacetamide (Sigma-Aldrich) and 6% Solutol (Sigma-Aldrich).
Dosages	twice daily at 25 or 75 mg/kg for 31 and 35 consecutive days
Administration	oral gavage

Chemical Information

Molecular Weight	349.77
Formula	C19H12ClN3O2
CAS Number	1009820-21-6
Solubility (25°C)	DMSO ≥ 46 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Siddiqui-Jain et al. Mol Cancer Ther. CK2 inhibitor CX-4945 suppresses DNA repair response triggered by DNA-targeted anticancer drugs and augments efficacy: mechanistic rationale for drug combination therapy.

[2] Pierre et al. Mol Cell Biochem. Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer.

[3] Ferguson et al. FEBS Lett. Structural basis of CX-4945 binding to human protein kinase CK2.