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

We describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms, whereas HTH-01-015 inhibits only NUAK1. These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases tested including 10 AMPK family members. WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterised substrate namely MYPT1 that is phosphorylated by NUAK1 at Ser445. We identify a mutation that does not affect basal NUAK1 activity but renders it resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating phosphorylation of MYPT1 we find that in cells overexpressing drug resistant NUAK1, 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 mouse embryonic fibroblasts (MEFs) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1 knock-out. 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 knock-down. WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knock-down. Our data indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAKs .

Source: BJ Signal - October 30, 2013 Category: Biochemistry Authors: S Banerjee, S J. Buhrlage, H Huang, X Deng, W Zhou, J Wang, R Traynor, A R. Prescott, D R. Alessi, N S. Gray Tags: BJ ChemBio Source Type: research