Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Department of Brain and Cognitive Sciences, Scranton College, Ewha Womans University, Seoul, Republic of Korea.
Life Sci. 2018 May 1;200:49-55. doi: 10.1016/j.lfs.2018.03.022. Epub 2018 Mar 10.
We have previously identified a chemical scaffold possessing 2-ethoxypropanoic acid (designated as KS15) that directly binds to the C-terminal region of cryptochromes (CRYs: CRY1 and CRY2) and enhances E-box-mediated transcription. However, it is still unclear how KS15 impairs the feedback actions of the CRYs and which chemical moieties are functionally important for its actions.
The E-box-mediated transcriptional activities were mainly used to examine the effects of KS15 and its derivatives. Co-immunoprecipitation assays accompanied by immunoblotting were employed to monitor protein-protein associations. We also examined the effects of KS15 and selected derivatives on circadian molecular rhythms in cultured cells.
The present study shows that KS15 inhibits the interaction between CRYs and Brain-Muscle-Arnt-Like protein 1 (BMAL1), thereby impairing the feedback actions of CRYs on E-box-dependent transcription by CLOCK:BMAL1 heterodimer, an indispensable transcriptional regulator of the mammalian circadian clock. Subsequent structure-activity relationship analyses using a well-designed panel of derivatives identified the structural requirements for the effects of KS15 on CRY-evoked regulation of E-box-mediated transcription. We found that KS15 and several derivatives significantly reduce the amplitude and delayed the phase of molecular circadian rhythms in fibroblast cultures.
Taken together, our results provide valuable information on the molecular mode-of-action as well as the chemical components of the CRYs inhibitor that pharmacologically impact on the transcriptional activity of the CLOCK:BMAL1 heterodimer.
我们之前发现了一种含有 2-乙氧基丙酸（命名为 KS15）的化学支架，它可以直接与隐色体（CRY1 和 CRY2）的 C 端区域结合，并增强 E 盒介导的转录。然而，目前尚不清楚 KS15 如何损害 CRY 的反馈作用，以及哪些化学部分对其作用具有功能重要性。
主要使用 E 盒介导的转录活性来检查 KS15 及其衍生物的作用。我们还采用免疫共沉淀测定法结合免疫印迹法来监测蛋白-蛋白相互作用。我们还检查了 KS15 和选定的衍生物对培养细胞中昼夜分子节律的影响。
本研究表明 KS15 抑制 CRY 与脑-肌肉-芳香烃受体核转录因子样蛋白 1（BMAL1）之间的相互作用，从而损害 CRY 对 CLOCK:BMAL1 异二聚体依赖性 E 盒转录的反馈作用，CLOCK:BMAL1 异二聚体是哺乳动物昼夜节律钟的必需转录调节剂。随后使用设计良好的衍生物进行的结构-活性关系分析确定了 KS15 对 CRY 诱导的 E 盒介导转录调节的影响的结构要求。我们发现 KS15 和几种衍生物显著降低了成纤维细胞培养物中分子昼夜节律的振幅并延迟了其相位。
总之，我们的研究结果提供了有价值的信息，阐明了 CRY 抑制剂的分子作用模式和化学组成，这些抑制剂对 CLOCK:BMAL1 异二聚体的转录活性具有药理学影响。
