Department of Molecular Biology and Genetics, Koc University, Rumelifeneri Yolu, Sariyer, Istanbul, Turkey.
Department of Chemical and Biological Engineering, Koc University, Rumelifeneri Yolu, Sariyer, Istanbul, Turkey.
J Biol Chem. 2020 Mar 13;295(11):3518-3531. doi: 10.1074/jbc.RA119.011332. Epub 2020 Feb 4.
Proper function of many physiological processes requires a robust circadian clock. Disruptions of the circadian clock can result in metabolic diseases, mood disorders, and accelerated aging. Therefore, identifying small molecules that specifically modulate regulatory core clock proteins may potentially enable better management of these disorders. In this study, we applied a structure-based molecular-docking approach to find small molecules that specifically bind to the core circadian regulator, the transcription factor circadian locomotor output cycles kaput (CLOCK). We identified 100 candidate molecules by virtual screening of ∼2 million small molecules for those predicted to bind closely to the interface in CLOCK that interacts with its transcriptional co-regulator, Brain and muscle Arnt-like protein-1 (BMAL1). Using a mammalian two-hybrid system, real-time monitoring of circadian rhythm in U2OS cells, and various biochemical assays, we tested these compounds experimentally and found one, named CLK8, that specifically bound to and interfered with CLOCK activity. We show that CLK8 disrupts the interaction between CLOCK and BMAL1 and interferes with nuclear translocation of CLOCK both and Results from further experiments indicated that CLK8 enhances the amplitude of the cellular circadian rhythm by stabilizing the negative arm of the transcription/translation feedback loop without affecting period length. Our results reveal CLK8 as a tool for further studies of CLOCK's role in circadian rhythm amplitude regulation and as a potential candidate for therapeutic development to manage disorders associated with dampened circadian rhythms.
许多生理过程的正常功能都需要一个稳健的生物钟。生物钟的紊乱可能导致代谢疾病、情绪障碍和加速衰老。因此，鉴定出专门调节核心生物钟蛋白的小分子，可能会更好地治疗这些疾病。在这项研究中，我们应用基于结构的分子对接方法，寻找专门与核心生物钟调节剂转录因子 circadian locomotor output cycles kaput (CLOCK)结合的小分子。我们通过对大约 200 万个小分子进行虚拟筛选，预测出那些与 CLOCK 界面紧密结合的小分子，这些小分子与 CLOCK 的转录共调节剂 Brain and muscle Arnt-like protein-1 (BMAL1)相互作用，从而鉴定出 100 种候选分子。我们使用哺乳动物双杂交系统、U2OS 细胞实时监测昼夜节律和各种生化测定，对这些化合物进行了实验测试，发现了一种名为 CLK8 的小分子，它可以特异性地结合并干扰 CLOCK 的活性。我们发现 CLK8 破坏了 CLOCK 和 BMAL1 之间的相互作用，并干扰了 CLOCK 的核转位，无论是在 还是 实验结果进一步表明，CLK8 通过稳定转录/翻译反馈环的负臂来增强细胞昼夜节律的振幅，而不影响周期长度。我们的结果揭示了 CLK8 作为进一步研究 CLOCK 在昼夜节律振幅调节中的作用的工具，以及作为治疗与昼夜节律减弱相关疾病的潜在候选药物。
