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Exploiting an Asp-Glu “switch” in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia

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Science Translational Medicine  07 Mar 2018:
Vol. 10, Issue 431, eaam8460
DOI: 10.1126/scitranslmed.aam8460
  • Fig. 1 A single amino acid difference in the ATP-binding domain of GSK3α and GSK3β results in structural and topological differences.

    (A) Aligned primary amino acid sequences of GSK3α and GSK3β. Residue differences between the two paralogs are in red. The main regions of the kinases are color-coded: hinge (yellow), backend region (green), P-loop (pink), DFG motif (blue), and activation loop (orange). Key amino acids for autoregulation, S21/9 and Y279/216, are underlined. Amino acids within 6 Å of the ligand are marked by an asterisk. (B) X-ray crystal structure (2.4 Å resolution) of hGSK3β bound to BRD0209, a dual GSK3α/β inhibitor. The inset shows a hydrogen bond network centered on the Asp133 side chain, on the backend region of the hinge connecting the N- and C-lobes of GSK3. The main regions of the kinases are again color-coded. (C) A comparison between the hinge backend and ATP-binding site of apo GSK3β and an MD simulation of apo GSK3α highlight the size difference between their hydrophobic pockets.

  • Fig. 2 Paralog-selective inhibitors of GSK3α and GSK3β were designed and characterized.

    (A) General synthetic scheme for synthesis of the pyrazolo-tetrahydroquinolinone scaffold. TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol. (B) IC50 values for the inhibition of GSK3α and GSK3β were determined at Km of ATP in a motility shift microfluidic assay (Caliper) measuring phosphorylation of a synthetic substrate. Values are average of three or more experiments. Data are shown as IC50 values in μM ± SD. Compounds were tested in duplicate in a 12-point dose curve with threefold dilution starting at 33.3 μM. (C) Kinome-wide selectivity for BRD0705 and BRD3731 represented on a kinome phylogenetic tree. Each inhibitor was screened against 311 kinases at a 10 μM concentration. Kinases with >50% inhibition are depicted (percentage inhibition proportional to size of red dot).

  • Fig. 3 An Asp (D)–to–Glu (E) switch in the enzymatic hinge backend underlies paralog selectivity of GSK3α and GSK3β inhibitors.

    (A) Primary sequence comparison of WT GSK3α, WT GSK3β, and GSK3β (D133E) and GSK3α (E196D) mutants. (B) X-ray structures of WT hGSK3β bound to BRD3731 (panels 1 to 3) and BRD0705 (panels 4 to 6) and hGSK3β (D133E) bound to BRD0705 (panels 7 to 9). The side chain of Asp133 in GSK3β forms a complex hydrogen bond network at the back of the kinase hinge and controls the shape and size of the adjacent selectivity pocket. (C and D) GSK3β (D133E) mutation “switches” compound selectivity. IC50 values for the inhibition of GSK3β (D133E) were determined at Km of ATP in a motility shift microfluidic assay (Caliper) measuring phosphorylation of a synthetic substrate. Representative IC50 curves show the comparison in enzymatic assays using GSK3α (WT, blue), GSK3β (WT, red), and GSK3β (D133E, black) protein constructs (C). Values are the average of three or more experiments. Data are shown as IC50 values in μM ± SD (D). Compounds were tested in duplicate in a 12-point dose curve with threefold dilution starting at 33.3 μM. (E) FLAG-tagged WT and E196D mutant GSK3α were overexpressed in HEK 293T cells. Western immunoblot for p-GSK3α (Tyr279) and total GSK3α after FLAG-GSK3α immunoprecipitation (IP) and treatment with dimethyl sulfoxide (DMSO) or BRD0705 at 10 μM.

  • Fig. 4 GSK3α- and GSK3β-selective inhibitors play a differential role on β-catenin stabilization in a context-dependent manner.

    (A) Biophysical measurement of GSK3 target engagement in HEK 293 cells by BRET signal between a NanoLuc-fused protein target and a small molecule labeled with the NanoBRET acceptor dye. Kd values for each inhibitor are reported as means ± SEM of two replicates. (B) β-Catenin immunofluorescence staining in HEK 293T after treatment with the indicated inhibitor. β-Catenin is shown in red, and 4′,6-diamidino-2-phenylindole is shown in blue. (C) β-Catenin TCF/LEF luciferase reporter assay in HEK 293T after treatment with the indicated inhibitor. P < 0.05 calculated using a Mann-Whitney test in comparison with control conditions. Data are means ± SEM of 10 replicates. (D and E) Western immunoblot for β-catenin and vinculin after treatment with BRD0705 and BRD0320 (D) or BRD3731 and BRD0320 (E) in HL-60 cells. (F) Western immunoblot for β-catenin, phospho–β-catenin (S675), phospho–β-catenin (S33/37/T41), and actin after treatment with the indicated inhibitors in TF-1 cells. (G) β-Catenin TCF/LEF green fluorescent protein (GFP) reporter assay in TF-1 after 24 hours of treatment with the indicated inhibitor. P < 0.05 is calculated using a Welch’s t test in comparison with control conditions. Data are means ± SEM of three replicates. (H and I) Western immunoblots for β-catenin and actin after treatment with the indicated inhibitors in MV4-11 (H) and MLL-AF9 murine leukemic cells (I). BM, bone marrow.

  • Fig. 5 BRD0705 induces differentiation in AML cell lines and primary patient samples through GSK3α-selective inhibition.

    (A and B) Time-response (A) and dose-response (B) Western immunoblots for β-catenin, phospho-GSK3α/β (Tyr279/216), total GSK3α/β, glycogen synthase (GYS), phospho-GYS (S641), and vinculin after treatment with the indicated inhibitors in U937 cells. (C) Dose-response Western immunoblot for β-catenin, phospho-GSK3α/β (Tyr279/216), total GSK3α/β, and vinculin after treatment with the indicated inhibitors in primary AML patient samples. (D) May-Grunwald-Giemsa staining after BRD0705 treatment for 6 days. Scale bar, 10 μm. (E) Fluorescence-activated cell sorting (FACS) analysis of the expression of CD11b, CD11c, and CD14 cell surface markers after BRD0705 treatment. *P < 0.05 calculated using a Welch’s t test in comparison with the control condition. Data are means ± SEM of three replicates. (F) FACS analysis of the expression of CD14 and CD117 cell surface markers in five primary AML samples treated with BRD0705. *P < 0.05 calculated using nonparametric Mann-Whitney test in comparison with control condition. Data are means ± SEM of five primary AML samples. (G) Western immunoblot for total GSK3α/β and vinculin in U937 after GSK3α CRISPR KO. (H) FACS analysis of the expression of CD11b cell surface marker in U937 GSK3α-WT, GSK3α-KO#1, and GSK3α-KO#2 treated with the indicated inhibitor. P < 0.05 calculated using a Welch’s t test in comparison with the DMSO condition for each clone (#) or the DMSO condition in the GSK3α-WT clone (*). Data are means ± SEM of three replicates.

  • Fig. 6 GSK3α- and GSK3β-selective inhibitors trigger differential transcriptional programs.

    (A and B) Venn diagrams (A) and scatterplots (B) of genes modulated after BRD0705, BRD3731, or BRD0320 treatment. Significantly depleted or enriched genes after BRD3731 or BRD0705 treatment are highlighted in blue and red, respectively (log2FC < −0.5 or > 0.5 and P ≤ 0.05). (C) Heatmaps of the top 30 genes found to be differentially expressed by genomic profiling upon BRD0705, BRD3731, or BRD0320 treatment in the U937 cell line. Depleted and enriched genes are in blue and red, respectively. Data are presented as row-normalized. Each column corresponds to one of three replicates for each treatment condition. The BRD0705, BRD3731, and BRD0320 signatures identified by RNA-seq were then interrogated in a functional enrichment analysis across the MSigDB database. (D to F) GSEA plots for β-catenin signaling pathway induction after treatment with BRD0705 (D), BRD3731 (E), or BRD0320 (F). NES, normalized enrichment score.

  • Fig. 7 BRD0705 impairs colony formation in AML cell lines and patient cells and shows in vivo efficacy in multiple AML mouse models.

    (A to C) Colony formation assay of the indicated AML cell lines (A), five primary AML samples (B), and human CD34 cells (C) after BRD0705 treatment. Data are represented as means ± SEM of three replicates. (D) MLL-AF9 cells (250,000) injected into secondary recipient mice after 24 hours of pretreatment with DMSO, BRD0705, BRD3731, or BRD0320. Kaplan-Meier curves are shown (n = 5 per group). (E) MLL-AF9 cells injected into secondary recipient mice in a serially diluted manner (500,000, 250,000, 100,000, or 50,000 cells). Frequency of LICs calculated using ELDA software. (F) MLL-AF9 AML cells injected into secondary recipient mice 3 days before treatment with vehicle (black) or BRD0705 at 30 mg/kg (red). Kaplan-Meier curves for each group of mice (n = 5 per group). (G) HL-60–Luc cells injected into recipient mice and treated with vehicle (black) or BRD0705 at 15 mg/kg (red) or 30 mg/kg (blue). Bioluminescence was quantified. (H) Kaplan-Meier curves for each group of mice treated as in (G) (n = 5 per group). (I) MV4-11–Luc cells injected into recipient mice and treated with vehicle (black), BRD0705 (red), BRD3731 (blue), or BRD0320 (dashed blue). Bioluminescence was quantified. Data are means ± SEM. *P value calculated for the latest time point. (J) Kaplan-Meier curves for each group of mice (n = 10 per group). P < 0.05 determined by log-rank (Mantel-Cox) test in comparison with vehicle.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/431/eaam8460/DC1

    Materials and Methods

    Fig. S1. GSK3 conservation across species.

    Fig. S2. Compounds’ electron density maps.

    Fig. S3. X-ray crystal structure of GSK3 from U. maydis.

    Fig. S4. Schematic of backend interactions.

    Fig. S5. Hinge backend interaction measurements and energy plots.

    Fig. S6. Synthesis of pyrazolodihydroquinolinones.

    Fig. S7. Spectra for compounds in Fig. 2B.

    Fig. S8. X-ray crystal structures of hGSK3B bound to BRD3731 and BRD0705 and hGSK3B (D133E) bound to BRD0705.

    Fig. S9. Live cell target engagement analysis for GSK3α and GSK3β using NanoBRET.

    Fig. S10. Heterogeneous effects of BRD3731 on differentiation in AML cell lines.

    Fig. S11. Differentiation and impaired stemness and mitochondria transcriptional programs triggered by BRD0705.

    Fig. S12. Heterogeneous effects of BRD3731 on colony formation in AML cell lines.

    Fig. S13. Pharmacokinetic, efficacy, and tolerability properties of BRD0705.

    Fig. S14. Glycogen accumulation induced by BRD0705 in AML.

    Table S1. X-ray data reduction statistics for x-ray co-crystal structures of hGSK3β with BRD0209, BRD3731, and BRD0705.

    Table S2. Crystallographic refinement statistics for x-ray co-crystal structures of hGSK3β with BRD0209, BRD3731, and BRD0705.

    Table S3. Kinome selectivity for BRD0320 (Carna Biosciences).

    Table S4. Kinome selectivity for BRD5648 (Carna Biosciences).

    Table S5. Kinome selectivity for BRD0705 (Carna Biosciences).

    Table S6. Kinome selectivity for BRD3731 (Carna Biosciences).

    Table S7. Final diffraction statistics for the mutant hGSK3β (D133E)/BRD0705 complex crystal used in structure determination.

    Table S8. Final refinement statistics for the mutant hGSK3β (D133E)/BRD0705 complex structure.

    Table S9. Lists of genes differentially expressed between control and BRD0705-treated U937 cells.

    Table S10. Lists of genes differentially expressed between control and BRD3731-treated U937 cells.

    Table S11. Lists of genes differentially expressed between control and BRD0320-treated U937 cells.

    Table S12. Top enriched gene sets from the functional groups predicted in BRD0705 versus DMSO enrichment map.

    Table S13. Cytogenetics and molecular and clinical characteristics of patient samples used in this study.

  • Supplementary Material for:

    Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia

    Florence F. Wagner,* Lina Benajiba, Arthur J. Campbell, Michel Weïwer, Joshua R. Sacher, Jennifer P. Gale, Linda Ross, Alexandre Puissant, Gabriela Alexe, Amy Conway, Morgan Back, Yana Pikman, Ilene Galinsky, Daniel J. DeAngelo, Richard M. Stone, Taner Kaya, Xi Shi, Matthew B. Robers, Thomas Machleidt, Jennifer Wilkinson, Olivier Hermine, Andrew Kung, Adam J. Stein, Damodharan Lakshminarasimhan, Michael T. Hemann, Edward Scolnick, Yan-Ling Zhang, Jen Q. Pan, Kimberly Stegmaier,* Edward B. Holson

    *Corresponding author. Email: fwagner{at}broadinstitute.org (F.F.W.); kimberly_stegmaier{at}dfci.harvard.edu (K.S.)

    Published 7 March 2018, Sci. Transl. Med. 10, eaam8460 (2018)
    DOI: 10.1126/scitranslmed.aam8460

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. GSK3 conservation across species.
    • Fig. S2. Compounds’ electron density maps.
    • Fig. S3. X-ray crystal structure of GSK3 from U. maydis.
    • Fig. S4. Schematic of backend interactions.
    • Fig. S5. Hinge backend interaction measurements and energy plots.
    • Fig. S6. Synthesis of pyrazolodihydroquinolinones.
    • Legend for fig. S7
    • Fig. S8. X-ray crystal structures of hGSK3B bound to BRD3731 and BRD0705 and hGSK3B (D133E) bound to BRD0705.
    • Fig. S9. Live cell target engagement analysis for GSK3α and GSK3β using NanoBRET.
    • Fig. S10. Heterogeneous effects of BRD3731 on differentiation in AML cell lines.
    • Fig. S11. Differentiation and impaired stemness and mitochondria transcriptional programs triggered by BRD0705.
    • Fig. S12. Heterogeneous effects of BRD3731 on colony formation in AML cell lines.
    • Fig. S13. Pharmacokinetic, efficacy, and tolerability properties of BRD0705.
    • Fig. S14. Glycogen accumulation induced by BRD0705 in AML.
    • Table S1. X-ray data reduction statistics for x-ray co-crystal structures of hGSK3β with BRD0209, BRD3731, and BRD0705.
    • Table S2. Crystallographic refinement statistics for x-ray co-crystal structures of hGSK3β with BRD0209, BRD3731, and BRD0705.
    • Table S7. Final diffraction statistics for the mutant hGSK3β (D133E)/BRD0705 complex crystal used in structure determination.
    • Table S8. Final refinement statistics for the mutant hGSK3β (D133E)/BRD0705 complex structure.
    • Legends for tables S3 to S6 and S9 to 13

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Fig. S7 (.pdf format). Spectra for compounds in Fig. 2B.
    • Table S3 (Microsoft Excel format). Kinome selectivity for BRD0320 (Carna Biosciences).
    • Table S4 (Microsoft Excel format). Kinome selectivity for BRD5648 (Carna Biosciences).
    • Table S5 (Microsoft Excel format). Kinome selectivity for BRD0705 (Carna Biosciences).
    • Table S6 (Microsoft Excel format). Kinome selectivity for BRD3731 (Carna Biosciences).
    • Table S9 (Microsoft Excel format). Lists of genes differentially expressed between control and BRD0705-treated U937 cells.
    • Table S10 (Microsoft Excel format). Lists of genes differentially expressed between control and BRD3731-treated U937 cells.
    • Table S11 (Microsoft Excel format). Lists of genes differentially expressed between control and BRD0320-treated U937 cells.
    • Table S12 (Microsoft Excel format). Top enriched gene sets from the functional groups predicted in BRD0705 versus DMSO enrichment map.
    • Table S13 (Microsoft Excel format). Cytogenetics and molecular and clinical characteristics of patient samples used in this study.

    [Download Fig. S7]

    [Download Supplementary Tables ]

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