S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Development of circadian clock modulator and its application
세포기반분석법을 통한 생체시계 조절자의 개발 및 적용 연구
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2014. 2. 김경진.
- In mammals, circadian rhythms are found in almost all of biological processes. Based on the physiological and clinical importance of circadian rhythms, identification of novel drugs that can modulate circadian clock is an immerging field. However, small molecule modulators that enhance E box-mediated transcriptional activity have not been reported yet. In Chapter 1, I identified and characterized a novel small molecule KS15 that directly binds to cryptochromes (CRYs) and inhibits its repressive function. In Chapter 2, I investigated the effect of KS15 on proliferation, chemosensitivity and metastatic activity of human breast cancer cells as an application study.
1. In Chapter 1, I aimed to identify novel small molecule modulator influencing the molecular feedback loop of the circadian clock. I designed two-step cell-based screening strategy based on E-box-mediated transcriptional activity to test more than 1,000 drug-like compounds. As a result, KS15 was selected as the most promising drug candidate. I then performed pull-down assays with the biotinylated KS15 and found that both cryptochrome 1 and 2 (CRY1/2), are molecular targets of KS15. In accordance with the binding property, KS15 enhanced E-box-mediated transcription in a CRY1/2-dependent manner, and more importantly, it attenuated the circadian oscillation of Per2-Luc and Bmal1-dLuc activities in cultured fibroblasts, indicating that KS15 can functionally inhibit the effects of CRY1/2 in the molecular circadian clockwork. Finally, intracerebral injection of KS15 in vivo shows delayed phase and lengthened period of circadian rhythm. The present study describes the first novel chemical inhibitor of CRY1/2 that inhibits the repressive function of CRY1/2, thereby modulates circadian clock both in vitro and in vivo.
2. In Chapter 2, based on recent studies that CRY1/2 are involved in the onset of human breast cancer, I evaluated the anti-tumor activity of KS15 on human breast cancer cells in vitro. First, I treated KS15 in various concentrations to human breast cancer cells (MCF-7 and MDA-MB-231) as well as to a normal mammary epithelial cell (MCF10A). As a result, KS15 inhibited the proliferation of MCF-7 cell line in a dose-dependent manner, but not in other cell lines. Also, sensitivities for the anti-tumor drugs were enhanced only in MCF-7 when co-treated with KS15. On the other hand, adhesion ability on extracellular matrix and metastatic activity were not significantly affected by treatment of KS15. These findings suggest that pharmacological inhibition of CRYs has an anti-proliferative effect and increasing chemosensitivity to anti-tumor drugs on a specific type of breast cancer. It appears that KS15 has the potential to be a candidate for anti-tumor drugs or an adjuvant to treatment of breast cancer.
In conclusion, I discovered the first novel chemical inhibitor (KS15) of CRY1/2 that inhibits the repressive function of CRY1/2, thereby activating E-box-mediated transcription regulated by CLOCK:BMAL1 heterodimer. As an application study, I evaluated anti-tumor activity of KS15 on proliferation and chemosensitivity of MCF-7 cells. Further investigation on the detailed mechanism and optimization of this anti-tumor activity of KS15 in needed to develop a novel therapeutic drug for breast cancer.