TBB (NSC-231634)(NSC-231634) is a highly selective, ATP/GTP-competitive inhibitor of casein kinase-2 (CK2).

PKD1:5.9 μM, PIM1:1.04 μM, PIM2:4.3 μM, CK2:0.4 μM(Ki), DYRK2:0.99 μM, PIM3:0.86 μM, CDK2:14 μM, HIPK2:5.3 μM, CK1:47 μM(Ki), DYRK1A:4.36 μM, HIPK3:4.9 μM, DYRK3:5.3 μM

Investigation of the inhibitory power of TBB against a panel of 33 protein kinases reveals the highest potency for CK2 (casein kinase 2) (human CK2: IC50=1.6 μM at 100 μM ATP). TBB also shows moderate inhibition for CDK2 (IC50=15.6 μM), phosphorylase kinase (IC50=8.7 μM), and glycogen synthase kinase 3β (GSK3β) (IC50=11.2 μM), while IC50 values for all other kinases are 50-fold greater than for CK2[1]. TBB (60 μM) may reduce the viability of androgen-insensitive PC-3 cells either alone or in combination with anticancer agents CPT or TRAIL, when administered with a specific schedule, although this time-dependent activity is not due to its effect on apoptosis in PC-3 cells[2]. TBB is an ATP/GTP competitive inhibitor of CK2 and has been tested against 33 protein kinases, both Ser/Thr- and Tyr-specific. At 10 μM TBB (and 100 μM ATP), CK2 is drastically inhibited (>85%), with moderate inhibition observed for phosphorylase kinase, glycogen synthase kinase 3L, and cyclin-dependent kinase 2/cyclin A, with IC50 values one to two orders of magnitude higher than for CK2 (IC50=0.9 μM). Additionally, TBB inhibits endogenous CK2 in cultured Jurkat cells[3].

In a mouse OIR model, treatment with TBB (60 mg/kg per day for 6 days), the most specific CK2 inhibitor known[4], reduces retinal neovascularization by approximately 60%.

Kinase assay: Total kinase activity of CaMKII, determined in a standard 2 min assay (100 μL), contained 35 mM HEPES, 10 mM MgCl2, 1 mM CaCl2, 10 μg of chicken gizzard myosin 20-kD light chain, 0.1 μM calmodulin, and 10 μM [γ-33]ATP at 30 °C. The kinase reaction is halted by adding 1 mL of 10% trichloroacetic acid.

TBB is dissolved in DMSO and stored, and then diluted with appropriate media before use[2]. PC-3 or HeLa cells are cultured routinely in RPMI-1640 and DMEM media, respectively, which are supplemented with 10% FBS, Penicillin (100 U/mL) and Streptomycin (100 μg/mL) at 37°C in a humidified atmosphere of 5% CO2. Cells are seeded at 5×104 cells/well (PC-3) or 2×104 (HeLa) in 24-wells plates and cultured for 72 h. TBB (final concentration 60 μM), CPT (final concentration 5.8 nM), 2-deoxyglucose (2-DG; final concentration 0.5 mM) or TRAIL (final concentration 13.3 ng/mL) are added to the medium individually or in a combination and the cells are cultured for additional time, indicated on each figure. After treatment, the medium with the agent is removed and 500 μL of MTT mixture (0.5 mg/mL for PC-3 and 5.0 mg/mL for HeLa cells in medium without phenol red) is added to each well and incubated for an additional 1 h at 37°C. The formazan crystals are diluted in 250 μL of DMSO. The absorbance is measured at 570 nm[2].