Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases

Sourav Banerjee, Sara J Buhrlage, Hai-Tsang Huang, Xianming Deng, Wenjun Zhou, Jinhua Wang, Ryan Traynor, Alan R. Prescott, Dario R. Alessi (Lead / Corresponding author), Nathanael S. Gray (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    18 Citations (Scopus)

    Abstract

    The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser445. We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.
    Original languageEnglish
    Pages (from-to)215-225
    Number of pages11
    JournalBiochemical Journal
    Volume457
    Issue number1
    DOIs
    Publication statusPublished - 1 Jan 2014

    Fingerprint

    Liver
    Tumors
    Phosphotransferases
    Neoplasms
    Phosphorylation
    Myosins
    AMP-Activated Protein Kinases
    Phosphates
    Fibroblasts
    Inhibitory Concentration 50
    Assays
    Biological Phenomena
    HTH-01-015
    WZ4003
    Cell adhesion
    Protein Kinase Inhibitors
    Cell Adhesion
    Wound Healing
    Protein Kinases
    Small Interfering RNA

    Keywords

    • AMP-activated protein kinase (AMPK)
    • AMPK-related kinase (ARK5)
    • Kinase inhibitor
    • kinase profiling
    • liver kinase B1 (LKB1)
    • myosin phosphate-targeting subunit (MYPT1)
    • sucrose non-fermenting protein kinase/AMPK related prtein kinase (SNARK)

    Cite this

    Banerjee, S., Buhrlage, S. J., Huang, H-T., Deng, X., Zhou, W., Wang, J., ... Gray, N. S. (2014). Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. Biochemical Journal, 457(1), 215-225. https://doi.org/10.1042/BJ20131152
    Banerjee, Sourav ; Buhrlage, Sara J ; Huang, Hai-Tsang ; Deng, Xianming ; Zhou, Wenjun ; Wang, Jinhua ; Traynor, Ryan ; Prescott, Alan R. ; Alessi, Dario R. ; Gray, Nathanael S. / Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. In: Biochemical Journal. 2014 ; Vol. 457, No. 1. pp. 215-225.
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    Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. / Banerjee, Sourav; Buhrlage, Sara J; Huang, Hai-Tsang; Deng, Xianming; Zhou, Wenjun; Wang, Jinhua; Traynor, Ryan; Prescott, Alan R.; Alessi, Dario R. (Lead / Corresponding author); Gray, Nathanael S. (Lead / Corresponding author).

    In: Biochemical Journal, Vol. 457, No. 1, 01.01.2014, p. 215-225.

    Research output: Contribution to journalArticle

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    AU - Banerjee, Sourav

    AU - Buhrlage, Sara J

    AU - Huang, Hai-Tsang

    AU - Deng, Xianming

    AU - Zhou, Wenjun

    AU - Wang, Jinhua

    AU - Traynor, Ryan

    AU - Prescott, Alan R.

    AU - Alessi, Dario R.

    AU - Gray, Nathanael S.

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    AB - The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser445. We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.

    KW - AMP-activated protein kinase (AMPK)

    KW - AMPK-related kinase (ARK5)

    KW - Kinase inhibitor

    KW - kinase profiling

    KW - liver kinase B1 (LKB1)

    KW - myosin phosphate-targeting subunit (MYPT1)

    KW - sucrose non-fermenting protein kinase/AMPK related prtein kinase (SNARK)

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    DO - 10.1042/BJ20131152

    M3 - Article

    VL - 457

    SP - 215

    EP - 225

    JO - Biochemical Journal

    JF - Biochemical Journal

    SN - 0264-6021

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    ER -