2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 2. Structure-Based Optimization and Investigation of Effects Specific to the Allosteric Mode of Action

Abstract

Protein CK2 has gained much interest as an anticancer drug target in the past decade. We had previously described the identification of a new allosteric site on the catalytic α-subunit, along with first small molecule ligands based on the 4-(4-phenylthiazol-2-ylamino)benzoic acid scaffold. In the present work, structure optimizations guided by a binding model led to the identification of the lead compound 2-hydroxy-4-((4-(naphthalen-2-yl)thiazol-2-yl)amino)benzoic acid (27), showing a submicromolar potency against purified CK2α (IC₅₀ = 0.6 μM). Furthermore, 27 induced apoptosis and cell death in 786-O renal cell carcinoma cells (EC₅₀ = 5 μM) and inhibited STAT3 activation even more potently than the ATP-competitive drug candidate CX-4945 (EC₅₀ of 1.6 μM vs 5.3 μM). Notably, the potencies of our allosteric ligands to inhibit CK2 varied depending on the individual substrate. Altogether, the novel allosteric pocket was proved a druggable site, offering an excellent perspective to develop efficient and selective allosteric CK2 inhibitors.

Figure 1.

Chemical structures of CX-4945 and previously described allosteric inhibitors (1-4). The IC₅₀s against human CK2α are indicated.

Figure 2.

Enzymatic characterization of compound 27. (A) Lineweaver-Burk inhibition plot of human recombinant CK2α by compound 27 at various concentrations: 0, 0.5, 1, 1.5, 2.5 μM. K_i was determined by plotting the slopes at varying inhibitor concentration from three independent experiments. (B) CK2α and CK2α₂β₂ activity with two peptide substrates in the presence of increasing concentrations of compound 27.

Figure 3.

Thermodynamic characterization of binding of the allosteric inhibitor 27 to CK2α by ITC. The top panel shows the raw heat signal for successive injections of dissolved compound 27 into a CK2α solution at 21 °C. The bottom panel shows the integrated heats of injections corrected for heats of dilution, with the solid line corresponding to the best fit of the data to a bimolecular binding model. Shown is one experiment out of two that gave similar values.

Figure 4.

Percentage of inhibition of GST-CK2α (wild type (WT) or single alanine mutant) in the presence of compound 27 (1 μM); see Table S1 (Supporting Information) for explicite values. The error bars indicate ± S.D.

Figure 5.

Predicted binding mode of the naphthalen-2-yl-thiazolamine derivatives in the allosteric pocket. Compounds 19 (A) and 27 (B) (cyan) were docked into the hydrophobic pocket between the glycine–rich loop and the αC-helix in the crystallized inactive conformation of CK2α (PDB entry 3FWQ) using MOE. Residues that are potentially relevant to the binding are labeled. Residues that caused a drop of 27 inhibitory potency when mutated to alanine are colored in magenta. The carboxylate forms salt bridges with Lys74 and Lys77, while the naphthalene ring is buried in the mostly hydrophobic cavity, additionally anchored by cation–π (with Lys71) and/ or CH–π interactions (with Gly177/Leu178). A further CH–π interaction was consistently observed between Val73 and the benzoic acid ring. Interactions are indicated by dashed lines (H-bonds/salt bridges: blue; CH–π interactions: brown). The green dashed line denotes the shortest distance between His160 and Tyr50.

Figure 6.

Compound 27 inhibits CK2–dependent substrate phosphorylations in cells and induces apoptosis. (A) Cellular inhibition of CK2 by compound 27. 786-O cells were plated and incubated for 24 h in the presence of various concentrations of 27. The phosphorylation status of the CK2 protein substrates were measured by Western Blot analysis of cell extracts. Band intensities were quantified using ImageJ and normalized using anti-GAPDH antibody signals as loading control. ^aEC₅₀ calculated assuming that phosphorylation levels will approach zero with increasing concentrations of 27; ^bEC₅₀ calculated assuming a basal level of non-responsive P-α-catenin of 50% of the DMSO control. (B) Apoptosis induction upon compound 27 treatment. Appearance of cleaved PARP and decrease of survivin levels were observed by Western Blot analysis of cell extracts. (C) 27 shows a higher potency to inhibit the STAT3 activation in 786-O cells than CX-4945.

Figure 7.

The potency of 4 but not of CX-4945 to inhibit CK2–catalyzed phosphorylations in cells depends on the substrate. 786-O cells were incubated with various concentrations of 4 (A, B) or CX-4945 (C, D) for 24 h, and the phosphorylation status of two protein substrates of CK2 was measured by Western Blot analysis of the cell extracts. GAPDH was probed as loading control. (B, D) Western Blot quantification was performed using ImageJ and fitted to a sigmoid equation using SigmaPlot to determine the EC₅₀s. ^aHalf-maximum decrease relating to the basal level of non-responsive α-catenin phosphorylation (ca. 50% of the control).

Figure 8.

Differential effects of the allosteric inhibitor 27 and the ATP-site directed inhibitor CX-4945 on the CK2-catalyzed phosphorylation of nucleolin and cellular consequences. (A) Phophosphorylation of substrate proteins (● nucleolin, ■ GST-Six1, ▲ CK2β auto-phosphorylation) by CK2α₂β₂ in the presence of various concentrations of compound 27 (upper panel) or the ATP-competitive inhibitor CX-4945 (lower panel). Graphs represent the average of two measurements. (B) HeLa pEGFP-CK2α cell imaging showing the subcellular localization of CK2α after 12 h treatment with DMSO, compound 27 (15 μM) or CX-4945 (8 μM). Nuclei were stained with Hoechst-33342 and merged images as well as GFP single channel were depicted for clarity.

Scheme 1.

Hantzsch-type synthesis. Reagents and conditions: i) Br₂, CHCl₃, 40 °C, 5 min or Br₂, HBr 32% in AcOH, MeOH, 60 °C, 4h. ii) CS₂, Et₃N in THF/H₂O 1/1, RT, 24 h, then I₂ in THF, 0 °C to RT, 3 h. iii) NH₄OH 30%, RT, 6 h. iv) EtOH, reflux, 3–12 h.

Scheme 2.

Preparation of compound 30 through aromatic nucleophilic substitution. Reagents and conditions: i) HCl_(aq) 37%, 1,4-dioxane, 90 °C, 48 h.

Scheme 3.

Syntheses of esters and amides 31–36. Reagents and conditions: i) H₂SO₄ 95% 3 drops, MeOH, reflux, 2 h. ii) 32: HATU, DIPEA, dry DMF, 30 min; NaH, RSO₂NH₂, dry THF, RT, 30 min; then RT, 4 h. 33: EDC·HCl, DMAP, RSO₂NH₂, CH₂Cl₂, RT, 36 h. iii) Bromomethyl acetate, Et₃N, dry DMF, RT, 12 h.