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Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth

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Science Advances  23 Jan 2019:
Vol. 5, no. 1, eaau9060
DOI: 10.1126/sciadv.aau9060
  • Fig. 1 GO289 lengthens circadian period.

    (A) Chemical structure of GO289. (B and C) Effect of GO289 on circadian rhythms in Bmal1-dLuc and Per2-dLuc U2OS cells (B) and cells differentiated from Per2::LucSV knock-in ES cells (C). Luminescence rhythms were monitored in the presence of various concentrations of GO289 and shown in the left (Bmal1-dLuc) and middle (Per2-dLuc) panels of (B) and the left panel of (C) (mean of n = 4). Period changes compared to a dimethyl sulfoxide (DMSO) control are plotted in the right panel of (B) and (C) (n = 4). ****P < 0.0001 and ***P < 0.001 against the DMSO control. (D) General synthetic scheme for GO289 derivatives. (E) Period-lengthening activity of GO289 derivatives. Luminescence rhythms of Bmal1-dLuc cells were monitored in the presence of various concentrations (threefold, 12-point dilution series) of GO289 derivatives (n ≥ 2), and the concentration required for half-maximal period lengthening is shown as logEC50. Modified part of the compound is shown in color. C4 and C3 positions of the benzene ring at R6 correspond to the para and meta positions, respectively. (F) Summary of the SAR study.

  • Fig. 2 GO289 interacts with CK2.

    (A) Chemical structure of GO457. (B) Effect of GO457 on circadian period in Bmal1-dLuc U2OS cells. ****P < 0.0001 and ***P < 0.001 against the DMSO control (n = 4). (C) GO289-interacting proteins. Agarose-conjugated GO457 was incubated with a U2OS cell lysate in the presence of 0 and 50 μM GO289 [competition (−) and (+), respectively]. Affinity-purified proteins were analyzed by LC-MS/MS. Proteins identified by ≥3 tandem MS spectra from ≥2 unique peptides (numbers in parentheses) and showed ≥5-fold signal reduction upon competition with GO289 [ratio (+)/(−) ≤ 0.2] in the first analysis are shown (columns 2 to 4). Enrichment with the affinity probe was estimated by comparing with the MS spectra number in the input sample (columns 5 and 6). Low enrichment efficiency (recovery ≤ 10%) is indicated in italics. The result of the second analysis focusing on 20- to 55-kDa proteins is shown in columns 8 to 10. Proteins fulfilling the criteria in both analyses are indicated in red. MW, molecular weight. (D and E) Interaction of GO289 with CK2. Affinity-purified proteins from a U2OS cell lysate (D) or recombinant CK2 (E) were analyzed by immunoblotting with specific antibodies. The asterisk indicates a nonspecific protein that interacted with the probe, independent of GO289.

  • Fig. 3 GO289 potently and selectively inhibits CK2.

    (A to D) Effect of GO289 on kinase activity in vitro. Activity of CK2 (A) and PIM2 (C) was analyzed in the presence of GO289 at various concentrations (n = 2). A panel of 60 kinases (B) and DYRK, HIPK, and PIM family kinases (D) were screened with 5 μM GO289 (n = 2). In (D), the effect of GO289 on multiple kinases is compared to published values for CK2 inhibitors TBB, DMAT, and CX-4945. ND, not determined. (E) Effects of CK2 inhibitors on circadian period and reporter signal intensity in Bmal1-dLuc U2OS cells. Changes in period (left) and luminescence intensity (right) compared to the DMSO control are plotted (n = 4). P values are summarized in table S3. (F and G) Effect of GO289 on cellular CK2 activity. HEK293T cells (F) or U2OS cells (G) were treated with GO289 at various concentrations for 24 hours and subjected to immunoblotting with anti-phosphorylated (anti-phospho) CK2 substrate antibody. The membrane was reprobed with anti-phospho PKA (protein kinase A) substrate and anti–β-actin antibodies (F) or stained with CBB (Coomassie Brilliant Blue) (G).

  • Fig. 4 GO289 affects phosphorylation of clock proteins.

    (A) Phosphorylation sites inhibited by GO289. HEK293T cells expressing Flag-tagged mouse clock proteins and CK2α were treated with GO289. Immunoprecipitated clock proteins were subjected to LC-MS/MS analysis. Phosphorylation level was calculated by dividing spectra number of phosphorylated peptides with total peptides (ratio). Effect of GO289 was defined as the ratio of the GO289-treated sample to the control sample (GO289/control). Sites abundantly phosphorylated (ratio of control sample ≥ 0.1) and inhibited by GO289 (GO289/control ≤ 0.5) are shown. (B and C) Phosphorylation of PER2(680-705) peptide by CK2. (B) Activity of CK2 and CKIδ was analyzed by measuring ATP levels (n = 3 to 6). (C) Ser/Thr residues were mutated to Ala (n = 2 to 4), and ATP half-life is plotted in the right panel. ****P < 0.0001, ***P < 0.001, **P < 0.01 against no peptide control. (D) Effect of GO289 on circadian rhythms of Bmal1-Luc reporter in wild-type and Per2 knockout cells. Period changes compared to the DMSO control are plotted in the right panel (n = 3 to 4). P values are against wild-type cells. (E) Effect of GO289 on BMAL1 S90 phosphorylation. Mouse NIH-3T3 cells were treated with 10 μM GO289 for different times and subjected to immunoprecipitation/immunoblotting with specific antibodies.

  • Fig. 5 GO289 inhibits cancer cell growth.

    (A and C) Effect of GO289 on growth of human RCC lines (A) and mouse AML MLL-AF9 cells or normal LSK cells (C). Cell numbers are plotted in the left panel by setting the DMSO control to 1 [n = 6 in (A) and n = 3 in (C)]. For (A), pIC50 values are plotted on the right, and P values are summarized in table S3. (B) Correlation of growth inhibition by GO289 with Bmal1 reporter induction. Degree of Bmal1 reporter induction (40) was calculated by dividing intensity of the peak with time 0 and plotted against pIC50 values from (A). (D) Effect of GO289 on circadian period and reporter signal intensity in spleen explants of MLL-AF9 mice. Luminescence rhythms of the Per2::Luc knock-in reporter were monitored in the presence of GO289 and indicated in the top left (MLL-AF9 mice, mean of n = 11) and the bottom left (control mice, mean of n = 10 to 20). Changes in intensity (top right) and period (bottom right) compared to the DMSO control are plotted. ****P < 0.0001, **P < 0.01 against the DMSO control.

  • Fig. 6 X-ray crystal structure of the CK2-GO289 complex.

    (A) Overall structure of CK2α in complex with GO289. (B) Surface of the GO289 binding pocket. GO289 stacked flat in the binding site with a slight rotation of the phenyl ring forming a stacking interaction with H160. (C) Interactions of GO289 with CK2α. Hydrogen bond, halogen bond, and stacking interactions are shown as dotted lines in blue, yellow, and green, respectively. (D) Period-lengthening activity of GO289 derivatives. Luminescence rhythms of Bmal1-dLuc cells were monitored in the presence of various concentrations (threefold, 12-point dilution series) of GO289 derivatives (n ≥ 2), and the concentration for half-maximal period lengthening is shown as logEC50. The modified part of the compound is shown in red. (E) Structure-based sequence alignment of the binding pocket. Data for CK2α, PIM1, PIM2, CLK2, and DYRK2 are from published x-ray crystal structures, while those for HIPK1 are based on its primary sequence. Conserved residues are shown in blue. (F) Comparison of the binding mode of GO289, TBB, DMAT, and CX-4945. DMAT and CX-4945 interact directly with V116 of the hinge region.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau9060/DC1

    Fig. S1. Effect of GO289 on circadian period, CKI activity, and CRY stability.

    Fig. S2. Effect of GO289 on kinase activity.

    Fig. S3. Effect of CKI inhibitor LH846 and GSK-3 inhibitor CHIR-99021 on the immunoreactivity of an anti-phospho CK2 substrate antibody.

    Fig. S4. Effect of CK2 inhibition on the growth and circadian rhythms of cancer cells.

    Fig. S5. Effect of GO289 derivatives on CK2 activity.

    Fig. S6. Structural features of the CK2α-GO289 complex.

    Fig. S7. Mouse Per2 gene knockout with CRISPR-Cas9.

    Table S1. Effect of GO289 on the phosphorylation of PER2 residues previously reported to be phosphorylated by CK2.

    Table S2. Data collection and refinement statistics.

    Table S3. Statistical analysis of Figs. 3E and 5A.

    Data file S1. Compound synthesis.

    Data file S2. Compound charts.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Effect of GO289 on circadian period, CKI activity, and CRY stability.
    • Fig. S2. Effect of GO289 on kinase activity.
    • Fig. S3. Effect of CKI inhibitor LH846 and GSK-3 inhibitor CHIR-99021 on the immunoreactivity of an anti-phospho CK2 substrate antibody.
    • Fig. S4. Effect of CK2 inhibition on the growth and circadian rhythms of cancer cells.
    • Fig. S5. Effect of GO289 derivatives on CK2 activity.
    • Fig. S6. Structural features of the CK2α-GO289 complex.
    • Fig. S7. Mouse Per2 gene knockout with CRISPR-Cas9.
    • Table S1. Effect of GO289 on the phosphorylation of PER2 residues previously reported to be phosphorylated by CK2.
    • Table S2. Data collection and refinement statistics.
    • Table S3. Statistical analysis of Figs. 3E and 5A.
    • Data file S1. Compound synthesis.

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