Development of a potent and selective chemical probe for the pleiotropic kinase CK2

Abstract

Building on the pyrazolopyrimidine CK2 (casein kinase 2) inhibitor scaffold, we designed a small targeted library. Through comprehensive evaluation of inhibitor selectivity, we identified inhibitor 24 (SGC-CK2-1) as a highly potent and cell-active CK2 chemical probe with exclusive selectivity for both human CK2 isoforms. Remarkably, despite years of research pointing to CK2 as a key driver in cancer, our chemical probe did not elicit a broad antiproliferative phenotype in >90% of >140 cell lines when tested in dose-response. While many publications have reported CK2 functions, CK2 biology is complex and an available high-quality chemical tool such as SGC-CK2-1 will be indispensable in deciphering the relationships between CK2 function and phenotypes.

Keywords: CK2; IDG; cancer; casein kinase 2; chemical probe; crystal structure; kinase; nanoBRET; proliferation; small molecule.

Figure 1.

Summary of Biological Pathways Mediated by CK2

Figure 2.. Structures of Reported and Synthesized Pyrazolopyrimidine CK2 Inhibitors

(A) Structures of literature-reported CK2 inhibitors used in studies aimed at interrogating CK2 function. (B) Structures of non-exemplified pyrazolopyrimidines synthesized.

Figure 3.. X-Ray Crystallographic Structure of Human CK2α in Complex with Pyrazolopyrimidines and Kinome-wide Selectivity of the Library

(A) and (B) 24 (A, PDB code: 6Z83) is shown in yellow and 29 (B, PDB code: 6Z84) in teal stick representation, respectively. The hinge region is colored brown, the αC helix blue, the DWG motif green, and water molecules are shown as red spheres. The pink P-loop was made transparent for better view of the interaction. Hydrogen bonds are indicated as black dashed lines. The insert on the upper left corner of each panel shows the electron density map (2F_o−F_c) of the bound ligand contoured at 1σ. (C) Heat map of kinome-wide selectivity of all analogs and CX-4945, ranked from most (top) to least (bottom) selective, when profiled against 403 wild-type kinases at 1 μM in the DiscoverX scanMAX platform. (D) Kinome-wide selectivity of SGC-CK2–1 (24) displayed as % control and ranked from most (left) to least (right) inhibited when profiled against 403 wild-type kinases at 1 μM in the DiscoverX scanMAX platform.

Figure 4.. SGC-CK2–1 (24) Evaluated for Antiproliferative Activity

(A) Potency data for 140 cell lines grouped by origin. (B) Growth inhibited cell lines within 140 panel with an IC₅₀ value <1 μM. (C) Proliferation data as IC₅₀ values for all 140 cancer cell lines profiled. (D) Overlapping cell lines in antiproliferative panels. (E) NCI60 results for SCG-CK2–1 when tested at 10 μM. Bars above zero indicate growth inhibition, while bars below zero indicate lethality. (F) Cell lines with growth inhibited percentage (GIPRCNT) <10 when treated with 10 μM SGC-CK2–1. (G) NCI60 results for SCG-CK2–1N when tested at 10 μM. All bars reflect growth inhibition. (H) Cell lines with growth inhibited percentage (GIPRCNT) <65 when treated with 10 μM SGC-CK2–1N.

Figure 5.. Western Blot Analysis and Quantification in Response to SGC-CK2–1 (24)

(A) Western blot for HCT-116 cell protein extracts (20 μg) treated with decreasing doses of SGC-CK2–1 and CX-4945 as positive control (from left to right: CX-4945 10 μM, DMSO 0.1%, SGC-CK2–1 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, and 0.01 μM). The inhibition of phosphorylated AKT (S129) and AKT1 was analyzed after 3h (A1) and 24h (A2) of inhibitor treatment. GAPDH were used as a loading control. (B) Intensity levels of phosphorylated AKT (S129) normalized against GAPDH loading control of western blot analysis after 3h (B1) and 24h (B2) of compound treatment. (C) Phosphorylation of EIF2S2 in FT-U2OS cells expressing wild-type (WT) or double mutant (DM, V66A/I174A) CK2α. Expression of exogenous CK2α was induced with tetracycline for 48h and cells were then treated for 24h with decreasing doses of CX-4945 or SGC-CK2–1: CX-4945 (30, 20, 10, 5, 1 μM), SGC-CK2–1 (10, 7.5, 5, 2.5, 1, 0.5 μM). As controls, cells were also subjected to treatment with DMSO (vehicle control) or were analyzed either without induction of exogenous CK2α or without any treatment as indicated. (C1) Phosphorylation of EIF2S2 was assessed by immunoblot analysis (top panels) together with analysis of total levels of EIF2S2 (middle panels) and GAPDH (bottom panels) as loading controls. (C2) Residual CK2 activity reflects phosphorylation of EIF2S2 expressed as the percentage of the level of phosphorylation detected in the absence of the inhibitor.