Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases.
Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases.
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DOI：
10.1042/bj20131152
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发表时间：
2014-01-01
期刊：
影响因子：
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通讯作者：
Gray NS
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文献类型：
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作者：
Banerjee S;Buhrlage SJ;Huang HT;Deng X;Zhou W;Wang J;Traynor R;Prescott AR;Alessi DR;Gray NS
相关的NUAK 1和NUAK 2是AMPK（AMP活化蛋白激酶）蛋白激酶家族的成员，由LKB 1（肝激酶B1）肿瘤抑制激酶激活。最近的研究表明，它们在调节关键的生物学过程中发挥重要作用，包括Myc驱动的肿瘤发生，衰老，细胞粘附和神经元极性。在本文中，我们描述了NUAK激酶的第一个高度特异性的蛋白激酶抑制剂，即WZ 4003和HTH-01-015。WZ 4003抑制两种NUAK亚型（NUAK 1的IC 50为20 nM，NUAK 2的IC 50为100 nM），而HTH-01-015仅抑制NUAK 1（IC 50为100 nM）。这些化合物显示出极端的选择性，并且不显著抑制测试的139种其他激酶的活性，包括10种AMPK家族成员。在所有测试的细胞系中，WZ 4003和HTH-01-015抑制唯一充分表征的底物MYPT 1（肌球蛋白磷酸靶向亚基1）的磷酸化，MYPT 1在Ser 445处被NUAK 1磷酸化。我们还鉴定了一种突变（A195 T），其不影响基础NUAK 1活性，但使其对WZ 4003和HTH-01-015的抗性增加约50倍。与NUAK 1介导MYPT 1磷酸化一致，我们发现在过表达耐药NUAK 1 [A195 T]而非野生型NUAK 1的细胞中，MYPT 1在Ser 445处的磷酸化不再被WZ 4003或HTH-01-015抑制。我们还证明，WZ 4003和HTH-01-015给药MEF（小鼠胚胎成纤维细胞）在伤口愈合试验中显著抑制迁移，其程度与NUAK 1敲除相似。WZ 4003和HTH-01-015还抑制MEF的增殖，其程度与NUAK 1敲除细胞和U2 OS细胞相同，与NUAK 1 shRNA敲除细胞相同。我们发现WZ 4003和HTH-01-015在3D细胞侵袭测定中损害U2 OS细胞的侵袭潜力，其程度与NUAK 1敲低相同。本研究的结果表明，WZ 4003和HTH-01-015将作为有用的化学探针来描绘NUAK激酶的生物学作用。我们描述了结构多样的激酶抑制剂的发现，解剖的NUAK亚型的生理作用。我们建议使用一种耐药的NUAK 1 [A195 T]突变体来验证这些化合物的生理效应确实是通过抑制NUAKs介导的
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. We describe the discovery of structurally diverse kinase inhibitors to dissect the physiological roles of the NUAK isoforms. We recommend use of an inhibitor-resistant NUAK1[A195T] mutant to verify that the physiological effects of these compounds is indeed mediated through inhibition of NUAKs