Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site.
Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site.
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DOI：
10.1038/nchembio.2148
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发表时间：
2016-10
影响因子：
14.8
通讯作者：
Grasby JA
中科院分区：
文献类型：
--
作者：
Exell JC;Thompson MJ;Finger LD;Shaw SJ;Debreczeni J;Ward TA;McWhirter C;Siöberg CL;Martinez Molina D;Abbott WM;Jones CD;Nissink JW;Durant ST;Grasby JA
结构特异性核酸酶人瓣核酸内切酶 1 (hFEN1) 在 DNA 复制和修复中发挥着关键作用，并且可能作为肿瘤学靶点而受到关注。我们展示了抑制剂结合的 hFEN1 的第一个晶体结构，并显示了与两个镁离子配位的活性位点结合的环状 N-羟基脲。三种此类化合物具有相似的 IC50 值，但作用方式略有不同。其中一种对蛋白质和蛋白质-底物复合物具有相当的亲和力，并通过与活性位点催化金属离子结合来阻止反应，从而阻止反应所需的底物 DNA 不配对。其他化合物与底物更具竞争力。细胞热转变数据显示，两种抑制剂类型与细胞中的 hFEN1 结合，治疗后明显激活 DNA 损伤反应。然而，细胞 EC50 显着高于体外抑制常数，并且讨论了这对于利用 hFEN1 作为药物靶点的影响。
The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed.