通过共价抑制剂选择性抑制DNA聚合酶β
Shelby C Yuhas1, Daniel J Laverty1, Huijin Lee1
1Johns Hopkins University, Department of Chemistry, 3400 North Charles Street, Baltimore, Maryland 21218, United States.
Journal of the American Chemical Society
|May 20, 2021
概括
研究人员开发了一种新的共价抑制剂,可选择性地向DNA聚合酶β (Pol β),这是DNA修复和癌症中的关键酶. 这种Polβ抑制剂是研究其在疾病和癌症治疗中的作用的有希望的工具.
科学领域:
- 生物化学
- 分子生物学
- 医学化学
背景情况:
- DNA聚合酶β (Pol β) 对于DNA修复至关重要,并与癌症发展有关.
- 目前缺乏对Polβ的选择性抑制剂,这阻碍了研究和治疗开发.
- 产生DNA病变的抗瘤药物可以使Polβ失活,为抑制剂设计提供基础.
研究的目的:
- 开发针对DNA聚合酶β (Polβ) 的新型共价小分子抑制剂.
- 描述已知Polβ抑制剂的选择性和作用机制.
- 在癌症研究的细胞模型中评估一种抑制剂 (pro-14) 的潜力.
主要方法:
- 在两阶段选过程中使用化学合成的库来识别抑制剂.
- 进行了体外酶抑制试验 (KI,kinact) 和对其他DNA聚合酶进行选择性分析.
- 使用液体染色体-双重质谱法 (LC-MS/MS) 来确定Polβ上的共价变异位.
- 使用光异性学来评估该抑制剂对PolβDNA结合的影响.
- 在野生类型和Pol β- null小鼠胚胎纤维细胞 (MEF) 和HeLa细胞中进行了基于细胞的测试,使用pro-14与DNA破坏剂结合使用.
主要成果:
- 一个强大的不可逆转的Polβ抑制剂 (14) 被确定为KI=1. 8±0. 45μM和kinact= (7. 0±1. 0) ×10-3s-1.
- 抑制剂14表现出选择性,对其他DNA聚合酶无活性化Polβ.
- 通过LC-MS/MS分析,发现两种lysine在Polβ结合部位内被抑制剂14对应修饰.
- 用抑制剂14进行预处理阻断了Polβ结合DNA的能力.
- 单独使用的益抑制剂 (pro-14) 不具有细胞毒性,但与甲基甲硫酸盐 (MMS) 和白素协同作用,可以杀死癌细胞.
- 细胞实验证实了pro-14对Polβ的选择性.
结论:
- 一种新型共价抑制剂 (14) 和其前抑制剂 (pro-14) 选择性地向DNA聚合酶β (Polβ).
- 这些抑制剂对Pol β活性位点中的关键氨酸残留物进行共价性修改,防止DNA结合.
- 在癌细胞系中,Pro-14与破坏DNA的药物具有协同作用的细胞毒性.
- 开发的抑制剂是研究DNA修复和疾病中的Polβ功能的有价值工具,具有潜在的治疗意义.
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