Identification of small molecule synthetic inhibitors of DNA polymerase β by NMR chemical shift mapping
Identification of small molecule synthetic inhibitors of DNA polymerase β by NMR chemical shift mapping
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DOI：
10.1074/jbc.m402842200
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发表时间：
2004-09-17
影响因子：
4.8
通讯作者：
Mullen, GP
中科院分区：
文献类型：
--
作者：
Hu, HY;Horton, JK;Mullen, GP
DNA聚合酶β (β -pol)通过碱基切除修复（BER）途径在受损DNA碱基修复中起核心作用。无β -pol小鼠成纤维细胞的主要表型是对dna甲基化剂甲磺酸甲酯过敏。β -pol的8-kDa结构域中的残基似乎与已知的天然产物β -pol抑制剂koetjapic酸相互作用，通过NMR化学位移作图确定。数据表明，结合袋为螺旋-2和螺旋-4之间的疏水裂缝，提供DNA结合和酶的脱氧核糖磷酸裂解酶活性。然后测试了九种结构相关的合成化合物，它们含有芳香或其他疏水性基团，并与两个羧酸基团结合。它们被发现与酶表面上相同或非常相似的区域结合。在野生型和β -pol缺失小鼠成纤维细胞中，这些化合物增强甲磺酸甲酯细胞毒性（细胞BER容量的指标）的能力随后被确定。这些试剂中最具活性和β -pol特异性的是pamoic酸，它被进一步表征并发现是纯化β -pol在BER底物上的脱氧核糖磷酸裂解酶和DNA聚合酶活性的抑制剂。我们的研究结果表明，基于核磁共振的制图技术可以用于设计小分子酶抑制剂，包括那些在临床环境中有潜在用途的酶抑制剂。
DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.