Effect of Natural and Semisynthetic Pseudoguianolides on the Stability of NF-κB: DNA Complex Studied by Agarose Gel Electrophoresis
Effect of Natural and Semisynthetic Pseudoguianolides on the Stability of NF-κB: DNA Complex Studied by Agarose Gel Electrophoresis
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DOI：
10.1371/journal.pone.0115819
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发表时间：
2015-01-23
期刊：
影响因子：
3.7
通讯作者：
Linares-Pasten, Javier A.
中科院分区：
文献类型：
--
作者：
Villagomez, Rodrigo;Hatti-Kaul, Rajni;Linares-Pasten, Javier A.
核因素。B （NF-kappa B）是一个很有前景的药物发现靶点。nf - κ B是RelA和p50亚基的异二聚体复合物，与DNA相互作用，调节多种基因的表达；它的失调会引发多种疾病，包括炎症、免疫缺陷和癌症。有一些基于全细胞研究的实验证据表明，天然倍半萜内酯（Sls）可以通过Michael添加将RelA亚基烷基化来抑制NF-kappa B与DNA的相互作用。在本研究中，利用NF-kappa B异源二聚体、伪guianolides和含有两个NF-kappa B识别序列的1000 bp回文DNA片段设计生化试验，筛选了28种天然和半合成的假guianolides作为NF-kappa B的潜在抑制剂。通过比较在常规琼脂糖凝胶电泳中游离DNA片段与nf - κ B -DNA复合物的相对数量，估计化合物对复合物的不稳定作用。实验结果和随后的结构-活性关系研究允许鉴定假愈木酚内酯骨架中的几个相关结构特征，这些特征对于增强NF-kappa B-DNA复合物的解离能力是必要的。最活跃的化合物被取代在C-3 （α -羰基）上，除了具有α -亚甲基- γ -内酯部分，这是RelA烷基化所必需的。
The nuclear factor.B (NF-kappa B) is a promising target for drug discovery. NF-kappa B is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-kappa B with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-kappa B in a biochemical assay that was designed using pure NF-kappa B heterodimer, pseudoguianolides and a similar to 1000 bp palindromic DNA fragment harboring two NF-kappa B recognition sequences. By comparing the relative amount of free DNA fragment to the NF-kappa B -DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-kappa B-DNA complex. The most active compounds are substituted at C-3 (alpha-carbonyl), in addition to having the alpha-methylene-gamma-lactone moiety which is essential for the alkylation of RelA.