Selective Inhibition of DNA Polymerase β by a Covalent Inhibitor.
Selective Inhibition of DNA Polymerase β by a Covalent Inhibitor.
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DOI：
10.1021/jacs.1c02453
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发表时间：
2021-06-02
影响因子：
15
通讯作者：
Greenberg MM
中科院分区：
文献类型：
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作者：
Yuhas SC;Laverty DJ;Lee H;Majumdar A;Greenberg MM
DNA聚合酶β（Pol β）在DNA修复过程中起着重要作用，与肿瘤的发生密切相关。缺乏这种酶的选择性抑制剂。受抑制Pol β的抗肿瘤药物产生的DNA损伤的启发，我们已经着手开发这种酶的共价小分子抑制剂。使用涉及化学合成文库的两阶段过程，我们鉴定了Pol β的强效不可逆抑制剂（14）（KI = 1.8 ± 0.45 μM，kinact =（7.0 ± 1.0）× 10−3 s−1）。抑制剂14相对于其他DNA聚合酶选择性地使Pol β失活。对用14处理的Pol β的胰蛋白酶解酶进行的LC-MS/MS分析鉴定了聚合酶结合位点内的两个共价修饰的赖氨酸，其中一个先前确定在DNA结合中起作用。荧光各向异性实验表明，用14预处理Pol β阻止DNA结合。在野生型小鼠胚胎成纤维细胞（MEF）中使用前抑制剂（pro-14）的实验表明，抑制剂（5 μM）本身无细胞毒性，但与DNA烷化剂甲磺酸甲酯（MMS）协同作用，杀死细胞。此外，Pol β null MEF中的实验表明pro-14对靶酶具有选择性。最后，pro-14还与MMS和博来霉素协同作用以杀死HeLa细胞。结果表明，pro-14在研究Pol β在疾病中的作用中是一个潜在有用的工具。
DNA polymerase β (Pol β) plays a vital role in DNA repair and has been closely linked to cancer. Selective inhibitors of this enzyme are lacking. Inspired by DNA lesions produced by antitumor agents that inactivate Pol β, we have undertaken the development of covalent small-molecule inhibitors of this enzyme. Using a two-stage process involving chemically synthesized libraries, we identified a potent irreversible inhibitor (14) of Pol β (KI = 1.8 ± 0.45 μM, kinact = (7.0 ± 1.0) × 10−3 s−1). Inhibitor 14 selectively inactivates Pol β over other DNA polymerases. LC-MS/MS analysis of trypsin digests of Pol β treated with 14 identified two lysines within the polymerase binding site that are covalently modified, one of which was previously determined to play a role in DNA binding. Fluorescence anisotropy experiments show that pretreatment of Pol β with 14 prevents DNA binding. Experiments using a pro-inhibitor (pro-14) in wild type mouse embryonic fibroblasts (MEFs) indicate that the inhibitor (5 μM) is itself not cytotoxic but works synergistically with the DNA alkylating agent, methylmethanesulfonate (MMS), to kill cells. Moreover, experiments in Pol β null MEFs indicate that pro-14 is selective for the target enzyme. Finally, pro-14 also works synergistically with MMS and bleomycin to kill HeLa cells. The results suggest that pro-14 is a potentially useful tool in studies of the role of Pol β in disease.
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影响因子：
3.7
作者：
Braithwaite EK;Kedar PS;Stumpo DJ;Bertocci B;Freedman JH;Samson LD;Wilson SH
通讯作者：
Wilson SH
DNA聚合酶β的结构和机制。
影响因子：
2.9
作者：
Beard WA;Wilson SH
通讯作者：
Wilson SH
影响因子：
15
作者：
Laverty, Daniel J.;Mortimer, Ifor P.;Greenberg, Marc M.
通讯作者：
Greenberg, Marc M.
影响因子：
4.8
作者：
Donigan, Katherine A.;Sun, Ka-Wai;Sweasy, Joann B.
通讯作者：
Sweasy, Joann B.
DOI：
10.1038/nrc3185
发表时间：
2012-01-12
期刊：
Nature reviews. Cancer
影响因子：
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作者：
通讯作者：
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