Hu Hong-Yu, Horton Julie K, Gryk Michael R, Prasad Rajendra, Naron Jana M, Sun Di-An, Hecht Sidney M, Wilson Samuel H, Mullen Gregory P
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
J Biol Chem. 2004 Sep 17;279(38):39736-44. doi: 10.1074/jbc.M402842200. Epub 2004 Jul 15.
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.
DNA聚合酶β(β-pol)在碱基切除修复(BER)途径修复受损DNA碱基的过程中发挥核心作用。β-pol基因敲除小鼠成纤维细胞的主要表型是对DNA甲基化剂甲磺酸甲酯高度敏感。通过核磁共振化学位移图谱鉴定了β-pol 8 kDa结构域中似乎与已知天然产物β-pol抑制剂柯萜酸相互作用的残基。数据表明结合口袋是螺旋2和螺旋4之间的疏水裂缝,该裂缝提供了该酶的DNA结合和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,这些化合物含有芳香族或其他疏水基团以及两个羧基。发现它们与酶表面的相同或非常相似的区域结合。接下来确定这些化合物在野生型和β-pol基因敲除小鼠成纤维细胞中增强甲磺酸甲酯细胞毒性(细胞BER能力的指标)的能力。这些试剂中活性最高且对β-pol具有特异性的帕莫酸进一步进行了表征,发现它是纯化的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。我们的结果表明,基于核磁共振的图谱技术可用于设计小分子酶抑制剂,包括那些可能在临床环境中使用的抑制剂。