Cell-permeable dual inhibitors of protein kinases CK2 and PIM-1: structural features and pharmacological potential

Abstract

It has been proposed that dual inhibitors of protein kinases CK2 and PIM-1 are tools particularly valuable to induce apoptosis of cancer cells, a property, however, implying cell permeability, which is lacking in the case of selective CK2/PIM-1 inhibitors developed so far. To fill this gap, we have derivatized the scaffold of the promiscuous CK2 inhibitor TBI with a deoxyribose moiety, generating TDB, a selective, cell-permeable inhibitor of CK2 and PIM-1. Here, we shed light on the structural features underlying the potency and narrow selectivity of TDB by exploiting a number of TDB analogs and by solving the 3D structure of the TDB/CK2 complex at 1.25 Å resolution, one of the highest reported so far for this kinase. We also show that the cytotoxic efficacy of TDB is almost entirely due to apoptosis, is accompanied by parallel inhibition of cellular CK2 and PIM-1, and is superior to both those observed combining individual inhibitors of CK2 and PIM-1 and by treating cells with the CK2 inhibitor CX4945. These data, in conjunction with the observations that cancer cells are more susceptible than non-cancer cells to TDB and that such a sensitivity is maintained in a multi-drug resistance background, highlight the pharmacological potential of this compound.

Fig. 1

Inhibition of CK2 (a) and PIM-1 (b) by TDB is competitive with respect to ATP. CK2 and PIM-1 activities were determined as described in the Experimental section in the presence of the indicated concentrations of inhibitor. K _i was calculated from the K _m/V _max against inhibitor concentration [i]; replot is shown in theinset. Results are means of experiments run in triplicate, with the SEM never exceeding 10 %

Fig. 2

a CK2α (cyan) with bound TDB (salmon). 2Fo-Fc map contoured at 1σ is shown in blue, anomalous map contoured at 5σ is shown in pink. b TDB binds to CK2α ATP pocket. The interacting residues are shown as sticks and the bridging water molecule as a sphere. Halogen and hydrogen bonds are shown as dotted lines. c TDB binding to CK2α in orientation II compared to K68 (violet) and DRB (yellow) in orientation I. The deoxyribofuranosyl moiety of TDB is closer to the hinge region compared to the DRB ribofuranosyl group and not compatible with the closed hinge conformation observed in the DRB-bound CK2α (grey)

Fig. 3

Molecular docking complex between the crystal structure of PIM1 (green, PDB code: 4DTK) and TDB (yellow)

Fig. 4

Inhibition profile of a panel of 124 kinases by TDB (1 μM). Conditions are described or referenced in the Experimental section. Residual activities are listed in the Supplementary data section (Table S1)

Fig. 5

Effects of TDB on CK2 and Pim-1 endocellular activity. a HEK-293T cells were transfected with Akt in order to better detect the CK2 target site Ser129 of Akt [45]. Transfected cells were treated for 6 h with increasing concentrations of TDB. Cell lysate proteins (10 μg) were analyzed by Western blot with the indicated antibodies. Tubulin was used as loading control. b CEM cells were treated for 24 h with increasing concentrations of TDB. Cell lysate proteins (20 μg) were analyzed by Western blot with the indicated antibodies. Tubulin was used as loading control

Fig. 6

Effect of TDB on cell viability. a CEM cells were treated for 24 h with increasing concentrations of the CK2 inhibitor quinalizarin (Q), or the Pim-1 inhibitor TCS, or both (Q+TCS), or TDB. Cell viability was detected by the MTT method. Mean ± SEM values of four independent experiments are shown. b CEM cells were treated with 5 μM TDB for the indicated times, then apoptosis was assessed by analyzing PARP cleavage by Western blot of 10 μg of cell lysate proteins; tubulin was used as loading control. Representative results of at least three experiments are shown. c CEM cells were treated for 4 h with the indicated concentrations of TDB. The Cell Death Detection Elisa kit (Roche) was used for detecting nucleosome release in the cytosol (apoptosis) and in the extracellular medium (necrosis). Mean ± SEM of four experiments are shown

Fig. 7

Sensitivity to TDB of different cell lines. Cell viability was measured by the MTT method after cell treatment for 24 h with increasing concentrations of TDB. 100 % cell viability was assigned to the vehicle-treated control cells. At least three experiments were performed for each cell line; mean ± SEM values are shown. a Comparison between tumor and non-tumor cell lines. b Comparison between a multidrug-resistant cell variant (R-CEM) and its parental line (CEM). c DC₅₀ values indicate the TDB concentrations (±SEM), which induce 50 % of cell death in 24 h