2022/10/27 更新

写真a

ヒロタ ツヨシ
廣田 毅
HIROTA Tsuyoshi
所属
トランスフォーマティブ生命分子研究所 特任准教授
大学院担当
大学院理学研究科
職名
特任准教授

学位 1

  1. 博士(理学) ( 2003年3月   東京大学 ) 

研究分野 1

  1. ライフサイエンス / 機能生物化学

現在の研究課題とSDGs 1

  1. 概日時計の分子機構の解析

経歴 9

  1. 科学技術振興機構   さきがけ研究員(兼任)

    2014年10月 - 2018年3月

  2. 名古屋大学   トランスフォーマティブ生命分子研究所   特任准教授

    2014年5月 - 現在

  3. 南カリフォルニア大学   研究員

    2013年7月 - 2014年4月

  4. カリフォルニア大学サンディエゴ校   研究員

    2012年7月 - 2013年7月

  5. カリフォルニア大学サンディエゴ校   研究員

    2008年10月 - 2012年6月

  6. カリフォルニア大学サンディエゴ校   研究員

    2007年9月 - 2008年9月

  7. スクリプス研究所   研究員

    2007年4月 - 2007年9月

  8. 東京大学   大学院理学系研究科   特任助教

    2003年4月 - 2007年3月

  9. 日本学術振興会   特別研究員 (DC1)

    2000年4月 - 2003年3月

▼全件表示

学歴 1

  1. 東京大学   大学院理学系研究科   生物化学専攻

    1998年4月 - 2003年3月

      詳細を見る

    国名: 日本国

 

論文 62

  1. CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy. 査読有り

    Miller S, Kesherwani M, Chan P, Nagai Y, Yagi M, Cope J, Tama F, Kay SA, Hirota T

    Proceedings of the National Academy of Sciences of the United States of America   119 巻 ( 40 ) 頁: e2203936119   2022年10月

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences of the United States of America  

    The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.

    DOI: 10.1073/pnas.2203936119

    Scopus

    PubMed

  2. Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation. 査読有り 国際誌

    Miller S, Srivastava A, Nagai Y, Aikawa Y, Tama F, Hirota T

    Proceedings of the National Academy of Sciences of the United States of America   118 巻 ( 26 )   2021年6月

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences of the United States of America  

    The circadian clock is a biological timekeeper that operates through transcription–translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations (“out” for CRY1 and “in” for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic “out” and “in” conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

    DOI: 10.1073/pnas.2026191118

    Web of Science

    Scopus

    PubMed

  3. Reversible modulation of circadian time with chronophotopharmacology. 査読有り 国際誌

    Kolarski D, Miró-Vinyals C, Sugiyama A, Srivastava A, Ono D, Nagai Y, Iida M, Itami K, Tama F, Szymanski W, Hirota T, Feringa BL

    Nature communications   12 巻 ( 1 ) 頁: 3164 - 3164   2021年5月

     詳細を見る

    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.

    DOI: 10.1038/s41467-021-23301-x

    Web of Science

    Scopus

    PubMed

  4. Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock. 査読有り

    Kolarski D, Miller S, Oshima T, Nagai Y, Aoki Y, Kobauri P, Srivastava A, Sugiyama A, Amaike K, Sato A, Tama F, Szymanski W, Feringa BL, Itami K, Hirota T

    Journal of the American Chemical Society   143 巻 ( 4 ) 頁: 2078 - 2087   2021年1月

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of the American Chemical Society  

    CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop"located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

    DOI: 10.1021/jacs.0c12280

    Web of Science

    Scopus

    PubMed

  5. An Isoform-Selective Modulator of Cryptochrome 1 Regulates Circadian Rhythms in Mammals. 査読有り

    Miller S, Aikawa Y, Sugiyama A, Nagai Y, Hara A, Oshima T, Amaike K, Kay SA, Itami K, Hirota T

    Cell chemical biology   27 巻 ( 9 ) 頁: 1192 - 1198.e5   2020年6月

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Cell Chemical Biology  

    Cryptochrome 1 (CRY1) and CRY2 are core regulators of the circadian clock, and the development of isoform-selective modulators is important for the elucidation of their redundant and distinct functions. Here, we report the identification and functional characterization of a small-molecule modulator of the mammalian circadian clock that selectively controls CRY1. Cell-based circadian chemical screening identified a thienopyrimidine derivative KL201 that lengthened the period of circadian rhythms in cells and tissues. Functional assays revealed stabilization of CRY1 but not CRY2 by KL201. A structure-activity relationship study of KL201 derivatives in combination with X-ray crystallography of the CRY1-KL201 complex uncovered critical sites and interactions required for CRY1 regulation. KL201 bound to CRY1 in overlap with FBXL3, a subunit of ubiquitin ligase complex, and the effect of KL201 was blunted by knockdown of FBXL3. KL201 will facilitate isoform-selective regulation of CRY1 to accelerate chronobiology research and therapeutics against clock-related diseases.

    DOI: 10.1016/j.chembiol.2020.05.008

    Web of Science

    Scopus

    PubMed

  6. Isoform-selective regulation of mammalian cryptochromes. 査読有り

    Miller S, Son YL, Aikawa Y, Makino E, Nagai Y, Srivastava A, Oshima T, Sugiyama A, Hara A, Abe K, Hirata K, Oishi S, Hagihara S, Sato A, Tama F, Itami K, Kay SA, Hatori M, Hirota T

    Nature chemical biology   16 巻 ( 6 ) 頁: 676 - 685   2020年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Chemical Biology  

    CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY–compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure. [Figure not available: see fulltext.]

    DOI: 10.1038/s41589-020-0505-1

    Web of Science

    Scopus

    PubMed

  7. Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth. 査読有り 国際誌

    Oshima T, Niwa Y, Kuwata K, Srivastava A, Hyoda T, Tsuchiya Y, Kumagai M, Tsuyuguchi M, Tamaru T, Sugiyama A, Ono N, Zolboot N, Aikawa Y, Oishi S, Nonami A, Arai F, Hagihara S, Yamaguchi J, Tama F, Kunisaki Y, Yagita K, Ikeda M, Kinoshita T, Kay SA, Itami K, Hirota T

    Science advances   5 巻 ( 1 ) 頁: eaau9060   2019年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Advances  

    Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type–dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

    DOI: 10.1126/sciadv.aau9060

    Web of Science

    Scopus

    PubMed

  8. Controlling the Circadian Clock with High Temporal Resolution through Photodosing. 査読有り 国際誌

    Kolarski D, Sugiyama A, Breton G, Rakers C, Ono D, Schulte A, Tama F, Itami K, Szymanski W, Hirota T, Feringa BL

    Journal of the American Chemical Society   141 巻 ( 40 ) 頁: 15784 - 15791   2019年

     詳細を見る

    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of the American Chemical Society  

    Circadian clocks, biological timekeepers that are present in almost every cell of our body, are complex systems whose disruption is connected to various diseases. Controlling cellular clock function with high temporal resolution in an inducible manner would yield an innovative approach for the circadian rhythm regulation. In the present study, we present structure-guided incorporation of photoremovable protecting groups into a circadian clock modifier, longdaysin, which inhibits casein kinase I (CKI). Using photodeprotection by UV or visible light (400 nm) as the external stimulus, we have achieved quantitative and light-inducible control over the CKI activity accompanied by an accurate regulation of circadian period in cultured human cells and mouse tissues, as well as in living zebrafish. This research paves the way for the application of photodosing in achieving precise temporal control over the biological timing and opens the door for chronophotopharmacology to deeper understand the circadian clock system.

    DOI: 10.1021/jacs.9b05445

    Web of Science

    Scopus

    PubMed

  9. Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period. 査読有り

    Uehara TN, Nonoyama T, Taki K, Kuwata K, Sato A, Fujimoto KJ, Hirota T, Matsuo H, Maeda AE, Ono A, Takahara TT, Tsutsui H, Suzuki T, Yanai T, Kay SA, Itami K, Kinoshita T, Yamaguchi J, Nakamichi N

    Plant & cell physiology   63 巻 ( 4 ) 頁: 450 - 462   2022年1月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Plant and Cell Physiology  

    The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional-translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

    DOI: 10.1093/pcp/pcac011

    Web of Science

    Scopus

    PubMed

  10. Structural and Chemical Biology Approaches Reveal Isoform-Selective Mechanisms of Ligand Interactions in Mammalian Cryptochromes. 招待有り 査読有り

    Miller S, Hirota T

    Frontiers in physiology   13 巻   頁: 837280   2022年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Frontiers in Physiology  

    Cryptochromes (CRYs) are core components of the circadian feedback loop in mammals, which regulates circadian rhythmicity in a variety of physiological processes including sleep–wake cycles and metabolism. Dysfunction of CRY1 and CRY2 isoforms has been associated with a host of diseases, such as sleep phase disorder and metabolic diseases. Accumulating evidence for distinct roles of CRY1 and CRY2 has highlighted the need for CRY isoform-selective regulation; however, highly conserved sequences in CRY ligand-binding sites have hindered the design of isoform-selective compounds. Chemical biology approaches have been identifying small-molecule modulators of CRY proteins, which act in isoform-non-selective and also isoform-selective manners. In this review, we describe advances in our understanding of CRY isoform selectivity by comparing X-ray crystal structures of mammalian CRY isoforms in apo form and in complexes with compounds. We discuss how intrinsic conformational differences in identical residues of CRY1 and CRY2 contribute to unique interactions with different compound moieties for isoform selectivity.

    DOI: 10.3389/fphys.2022.837280

    Web of Science

    Scopus

    PubMed

  11. A methylbenzimidazole derivative regulates mammalian circadian rhythms by targeting Cryptochrome proteins. 招待有り 査読有り

    Yagi M, Miller S, Nagai Y, Inuki S, Sato A, Hirota T

    F1000Research   11 巻   頁: 1016   2022年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:F1000Research  

    Background: Impairment of the circadian clock has been associated with numerous diseases, including sleep disorders and metabolic disease. Although small-molecules that modulate clock function may form the basis of drug discovery of clock-related diseases, only a few compounds that selectively target core clock proteins have been identified. Three scaffolds were previously discovered as small-molecule activators of the clock protein Cryptochrome (CRY), and they have been providing powerful tools to understand and control the circadian clock system. Identifying new scaffolds will expand the possibilities of drug discovery. Methods: A methylbenzimidazole derivative TH401 identified from cell-based circadian screens was characterized. Effects of TH401 on circadian rhythms were evaluated in cellular assays. Functional assays and X-ray crystallography were used to elucidate the effects of the compound on CRY1 and CRY2 isoforms. Results: TH401 lengthened the period of circadian rhythms and stabilized both CRY1 and CRY2. The compound repressed Per2 reporter activity, which was reduced by Cry1 or Cry2 knockout and abolished by Cry1/Cry2 double knockout, indicating the dependence on CRY isoforms. Thermal shift assays showed slightly higher interaction of TH401 with CRY2 over CRY1. The crystal structure of CRY1 in complex with TH401 revealed a conformational change of the gatekeeper W399, which is involved in isoform-selectivity determination. Conclusions: The present study identified a new small-molecule TH401 that targets both CRY isoforms. This compound has expanded the chemical diversity of CRY activators, and will ultimately aid in the development of therapeutics against circadian clock-related disorders.

    DOI: 10.12688/f1000research.124658.1

    Scopus

    PubMed

  12. Cell-Based Phenotypic Screens to Discover Circadian Clock-Modulating Compounds. 招待有り 査読有り 国際誌

    Hatori M, Hirota T

    Methods in molecular biology (Clifton, N.J.)   2482 巻   頁: 95 - 104   2022年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Methods in Molecular Biology  

    There is increasing demand to control circadian clock functions in a conditional manner for deeper understanding of the circadian system as well as for potential treatment of clock-related diseases. Small-molecule compounds provide powerful tools to reveal novel functions of target proteins in the circadian clock mechanism, and can be great therapeutic candidates. Here we describe the detailed methods of measuring cellular circadian rhythms in a high-throughput manner for chemical screening to identify compounds that affect circadian rhythms by targeting clock-related proteins.

    DOI: 10.1007/978-1-0716-2249-0_6

    Scopus

    PubMed

  13. Preface 招待有り

    Hirota T., Hatori M., Panda S.

    Neuromethods   186 巻   2022年

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Neuromethods  

    Scopus

  14. Modulation of circadian clock by crude drug extracts used in Japanese Kampo medicine. 査読有り 国際誌

    Zhang M, Kobayashi K, Atsumi H, Katada Y, Nakane Y, Chen J, Nagano R, Kadofusa N, Nishiwaki-Ohkawa T, Kon N, Hirota T, Sato A, Makino T, Yoshimura T

    Scientific reports   11 巻 ( 1 ) 頁: 21038 - 21038   2021年10月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.

    DOI: 10.1038/s41598-021-00499-w

    Web of Science

    Scopus

    PubMed

  15. Effects of cryptochrome-modulating compounds on circadian behavioural rhythms in zebrafish. 招待有り 査読有り 国際誌

    Iida M, Nakane Y, Yoshimura T, Hirota T

    Journal of biochemistry   171 巻 ( 5 ) 頁: 501 - 507   2021年9月

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of biochemistry  

    The circadian clock controls daily rhythms of various physiological processes, and impairment of its function causes many diseases including sleep disorders. Chemical compounds that regulate clock function are expected to be applied for treatment of circadian clock-related diseases. We previously identified small-molecule compounds KL001, KL101 and TH301 that lengthen the period of cellular circadian clock by directly targeting clock proteins cryptochromes (CRYs) in mammals. KL001 targets both CRY1 and CRY2 isoforms, while KL101 and TH301 are isoform-selective compounds and require CRY C-terminal region for their effects. For further application of these compounds, the effects on locomotor activity rhythms at the organismal level need to be investigated. Here we used zebrafish larvae as an in vivo model system and found that KL001 lengthened the period of locomotor activity rhythms in a dose-dependent manner. In contrast, KL101 and TH301 showed no effect on the period. The amino acid sequences of CRY C-terminal regions are diverged in zebrafish and mammals, supporting the importance of this region for the effects of KL101 and TH301. This study demonstrated efficacy of CRY modulation for controlling circadian behavioural rhythms in organisms and suggested species-dependent differences in the effects of isoform-selective CRY-modulating compounds.

    DOI: 10.1093/jb/mvab096

    Web of Science

    Scopus

    PubMed

  16. 体内時計のケミカルバイオロジー研究と創薬の可能性 招待有り

    廣田 毅, 松田 智宏

    MEDCHEM NEWS   31 巻 ( 2 ) 頁: 62 - 67   2021年5月

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:公益社団法人 日本薬学会  

    概日時計は地球上の生物に普遍的なシステムであり、生体内の1日周期のリズムを支配する。概日時計を構成する時計タンパク質の発現、翻訳後修飾、分解のサイクルにより、多様な生理機能がリズミカルな制御を受けている。現代社会において、シフトワークや社会的な時差ぼけなどによる概日リズムの乱れが深刻化しており、睡眠障害だけでなく、がんや代謝性疾患などのさまざまな疾患につながることが報告されている。概日時計に関連する疾患の分子メカニズム解明や治療に向け、時計タンパク質を標的とする低分子化合物は有用なツールとなる。本稿では、これまでに同定されたユニークな時計調節化合物と、創薬の可能性について解説する。

    DOI: 10.14894/medchem.31.2_62

    CiNii Research

  17. Comparing the efficacy and selectivity of Ck2 inhibitors. A phosphoproteomics approach. 査読有り

    Borgo C, Cesaro L, Hirota T, Kuwata K, D'Amore C, Ruppert T, Blatnik R, Salvi M, Pinna LA

    European journal of medicinal chemistry   214 巻   頁: 113217 - 113217   2021年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:European Journal of Medicinal Chemistry  

    CK2 (an acronym derived from the misnomer “casein kinase 2”) denotes a ubiquitous, highly pleiotropic protein kinase which has been implicated in global human pathologies, with special reference to cancer. A large spectrum of fairly selective, cell permeable CK2 inhibitors are available, one of which, CX4945 is already in clinical trials for the treatment of neoplasia. Another recently developed CK2 inhibitor, GO289, displays in vitro potency and selectivity comparable to CX4945. Here the cellular efficiency of these two inhibitors has been evaluated by treating C2C12 myoblasts for 5 h with each of them at 4 μM concentration and running a quantitative phosphoproteomics analysis of phosphosites affected by the two compounds. A small but significant proportion of the quantified phosphosites is decreased by treatment with CX4945 and, even more with GO289. This figure substantially increases if a subset of quantified phosphosites conforming to the CK2 consensus (pS/pT-x-x-D/E/pS/pT) is considered. Also in this case GO289 is more effective than CX4945. By adopting stringent criteria two shortlists of 70 and 35 sites whose phosphorylation is decreased >50% by GO289 and CX4945, respectively, have been generated. All these phosphosites conform to the consensus of CK2 with just sporadic exceptions. Their WebLogos are indistinguishable from that of bona fide CK2 phosphosites and their Two-Sample Logos rule out any significant contribution of Pro-directed and basophilic protein kinases to their generation. To sum up, we can conclude that by treating C2C12 cells for 5 h with either CX4945 or GO289 off-target effects are negligible since almost all the phosphosites undergoing a substantial reduction are attributable to CK2, with a higher inhibitory efficacy displayed by GO289. CX4945 and GO289 provide highly selective tools to control the CK2-dependent phosphoproteome compared with previously developed CK2 inhibitors.

    DOI: 10.1016/j.ejmech.2021.113217

    Web of Science

    Scopus

    PubMed

  18. Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythms. 査読有り

    Kolarski D, Sugiyama A, Rodat T, Schulte A, Peifer C, Itami K, Hirota T, Feringa BL, Szymanski W

    Organic & biomolecular chemistry   19 巻 ( 10 ) 頁: 2312 - 2321   2021年

     詳細を見る

    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Organic and Biomolecular Chemistry  

    Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of thecis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity together with the accompanying modulation of cellular circadian rhythms in which CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.

    DOI: 10.1039/D1OB00014D

    Web of Science

    Scopus

    PubMed

  19. A N-terminally deleted form of the CK2α' catalytic subunit is sufficient to support cell viability. 査読有り

    Borgo C, D'Amore C, Cesaro L, Itami K, Hirota T, Salvi M, Pinna LA

    Biochemical and biophysical research communications   531 巻 ( 3 ) 頁: 409 - 415   2020年10月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Biochemical and Biophysical Research Communications  

    Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual “CK2-like” activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2α′ on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immuno-precipitates with the β-subunit being downregulated by specific CK2α′ targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2α′ mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2α/α’−/− cells relies on this deleted form of CK2α′, whose discovery provides novel perspectives about the biological role of CK2.

    DOI: 10.1016/j.bbrc.2020.07.112

    Web of Science

    Scopus

    PubMed

  20. Small Molecules Modulating Mammalian Biological Clocks: Exciting New Opportunities for Synthetic Chemistry 査読有り

    Amaike Kazuma, Oshima Tsuyoshi, Skoulding Nicola Stephanie, Toyama Yoshifumi, Hirota Tsuyoshi, Itami Kenichiro

    CHEM   6 巻 ( 9 ) 頁: 2186 - 2198   2020年

     詳細を見る

    担当区分:責任著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chem  

    Challenges and opportunities: • Increasing opportunities for the discovery of new circadian clock modulators, which are attractive drug candidates for clock-related diseases in pharmaceutical industry, and to enhance crop yield in agriculture. • Development of new reactions for rapid access to hit compound derivatives, accelerating structure-activity relationship studies, which are generally time consuming. • New approaches for target identification of clock-modulating small molecules, which is still challenging, enable molecular-level understanding and new structural designs.

    DOI: 10.1016/j.chempr.2020.08.011

    Web of Science

    Scopus

  21. 低分子化合物を用いた概日時計機能の操作 招待有り

    廣田 毅

    日本薬理学会年会要旨集   93 巻 ( 0 ) 頁: 1-S12-3 - S12-3   2020年

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:公益社団法人 日本薬理学会  

    <p>In mammals, circadian rhythms are generated through transcriptional regulatory networks of the clock genes. To search for novel clock modifiers, we applied chemical biology approaches. From hundreds of thousands of small molecules with diverse structure, we identified a number of compounds that potently change the period of the circadian clock in human cells. Among the period lengthening compounds, we previously discovered the first small molecule targeting the core clock protein CRY. The compound KL001 interacts with FAD-binding pocket of CRY and inhibits FBXL3-dependent degradation. By analyzing KL001 derivatives, we found 10 times more potent compound KL044. KL001 and KL044 share carbazole group and act on both CRY1 and CRY2. We further identified novel period lengthening compounds KL101 and TH301 that do not have carbazole group. Surprisingly, we discovered that KL101 is selective against CRY1 while TH301 shows much higher effect on CRY2. To understand molecular basis of the CRY1/CRY2 selectivity, we determined the X-ray crystal structures of CRY1-KL101, CRY1-TH301, CRY2-TH301, and CRY1-KL044 complexes. In this presentation, I will discuss these unique compounds that will enable atomic-level dissection of the functional difference between CRY1 and CRY2 proteins and their selective manipulation.</p>

    DOI: 10.1254/jpssuppl.93.0_1-s12-3

    CiNii Research

  22. Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing. 査読有り 国際誌

    Fribourgh JL, Srivastava A, Sandate CR, Michael AK, Hsu PL, Rakers C, Nguyen LT, Torgrimson MR, Parico GCG, Tripathi S, Zheng N, Lander GC, Hirota T, Tama F, Partch CL

    eLife   9 巻   2020年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:eLife  

    Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24-hour periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

    DOI: 10.7554/eLife.55275

    Web of Science

    Scopus

    PubMed

  23. "Janus" efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms. 査読有り

    D'Amore C, Moro E, Borgo C, Itami K, Hirota T, Pinna LA, Salvi M

    Biochimica et biophysica acta. Molecular cell research   1867 巻 ( 11 ) 頁: 118807   2020年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Biochimica et Biophysica Acta - Molecular Cell Research  

    Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.

    DOI: 10.1016/j.bbamcr.2020.118807

    Web of Science

    Scopus

    PubMed

  24. Pharmacological Interventions to Circadian Clocks and Their Molecular Bases. 招待有り 査読有り

    Miller S, Hirota T

    Journal of molecular biology   432 巻 ( 12 ) 頁: 3498 - 3514   2020年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Molecular Biology  

    Daily physiological rhythms are regulated by the body's internal timekeeper known as the circadian clock. Expression, post-translational modification, and degradation of clock proteins constituting the circadian clock are precisely controlled in a rhythmic manner. Perturbation of these processes by nature and nurture results in physiological dysfunction and diseases. Small-molecule modulators of clock or clock-related proteins can adjust clock functions, and thus represent a promising method of therapeutic treatment for a range of clock-related diseases. In this review, we will introduce the identification and development of small-molecule compounds that target clock proteins, as well as X-ray crystal structures of protein-compound complexes that facilitate the understanding of clock protein regulation and drug derivatization. Furthermore, we describe the effects of these compounds in a diseased setting and discuss the therapeutic potential of clock modulators.

    DOI: 10.1016/j.jmb.2020.01.003

    Web of Science

    Scopus

    PubMed

  25. 化合物を使って体内時計を操る 招待有り

    廣田 毅

    化学   75 巻   頁: 66 - 67   2020年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    CiNii Research

  26. Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen. 査読有り

    Mosser EA, Chiu CN, Tamai TK, Hirota T, Li S, Hui M, Wang A, Singh C, Giovanni A, Kay SA, Prober DA

    Scientific reports   9 巻 ( 1 ) 頁: 12405   2019年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

    DOI: 10.1038/s41598-019-48914-7

    Web of Science

    Scopus

    PubMed

    その他リンク: http://orcid.org/0000-0003-4876-3608

  27. Casein kinase 1 family regulates PRR5 and TOC1 in the Arabidopsis circadian clock. 査読有り 国際誌

    Uehara TN, Mizutani Y, Kuwata K, Hirota T, Sato A, Mizoi J, Takao S, Matsuo H, Suzuki T, Ito S, Saito AN, Nishiwaki-Ohkawa T, Yamaguchi-Shinozaki K, Yoshimura T, Kay SA, Itami K, Kinoshita T, Yamaguchi J, Nakamichi N

    Proceedings of the National Academy of Sciences of the United States of America   116 巻 ( 23 ) 頁: 11528 - 11536   2019年6月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences of the United States of America  

    The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

    DOI: 10.1073/pnas.1903357116

    Web of Science

    Scopus

    PubMed

  28. Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO–E2–E3 Ligase Ternary Complexes 査読有り 国際誌

    Yinfeng Zhang, Tsuyoshi Hirota, Keiko Kuwata, Shunsuke Oishi, Subramanian, G. Gramani, Jeffrey W. Bode

    Journal of the American Chemical Society   141 巻 ( 37 ) 頁: 14742 - 14751   2019年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Chemical Society ({ACS})  

    E2 conjugating enzymes are the key catalytic actors in the transfer of ubiquitin, SUMO, and other ubiquitin-like modifiers to their substrate proteins. Their high rates of transfer and promiscuous binding complicate studies of their interactions and binding partners. To access specific, covalently linked conjugates of the SUMO E2 conjugating enzyme Ubc9, we prepared synthetic variants bearing site-specific non-native modifications including the following: (1) replacement of Cys93 to 2,3-diaminopropionic acid to form the amide-linked stable E2-SUMO conjugate, which is known to have high affinity for E3 ligases; (2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and (3) an N-Terminal biotin for purification and detection. To construct these Ubc9 variants in a flexible, convergent manner, we combined the three leading methods: native chemical ligation (NCL), α-ketoacid-hydroxylamine (KAHA) ligation, and serine/threonine ligation (STL). Using the synthetic proteins, we demonstrated the selective formation of Ubc9-SUMO conjugates and the trapping of an E3 ligase (RanBP2) to form the stable, covalently linked SUMO1-Ubc9-RanBP2 ternary complex. The powerful combination of ligation methods-which minimizes challenges of functional group manipulations-will enable chemical probes based on E2 conjugating enzymes to trap E3 ligases and facilitate the synthesis of other protein classes.

    DOI: 10.1021/jacs.9b06820

    Web of Science

    Scopus

    PubMed

  29. Chemical Control of Mammalian Circadian Behavior through Dual Inhibition of Casein Kinase Iα and δ 査読有り 国際誌

    Lee J.W, Hirota T, Ono D, Honma S, Honma K.-I, Park K, Kay S.A

    Journal of Medicinal Chemistry   62 巻 ( 4 ) 頁: 1989 - 1998   2019年

     詳細を見る

    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Medicinal Chemistry  

    Circadian rhythms are controlled by transcriptional feedback loops of clock genes and proteins. The stability of clock proteins is regulated by post-translational modification, such as phosphorylation by kinases. In particular, casein kinase I (CKI) phosphorylates the PER protein to regulate proteasomal degradation and nuclear localization. Therefore, CKI inhibition can modulate mammalian circadian rhythms. In the present study, we have developed novel CKIα and CKIδ dual inhibitors by extensive structural modification of N9 and C2 position of longdaysin. We identified NCC007 that showed stronger period effects (0.32 μM for 5 h period lengthening) in a cell-based circadian assay. The following in vitro kinase assay showed that NCC007 inhibited CKIα and CKIδ with an IC 50 of 1.8 and 3.6 μM. We further demonstrated that NCC007 lengthened the period of mouse behavioral rhythms in vivo. Thus, NCC007 is a valuable tool compound to control circadian rhythms through CKI inhibition.

    DOI: 10.1021/acs.jmedchem.8b01541

    Web of Science

    Scopus

    PubMed

  30. 時計遺伝子から考える脳機能と食 招待有り

    羽鳥 恵, 廣田 毅

    FOOD STYLE 21   23 巻   頁: 80 - 83   2019年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    CiNii Research

  31. Conformational dynamics of human protein kinase CK2α and its effect on function and inhibition 査読有り

    Srivastava A, Hirota T, Irle S, Tama F

    Proteins: Structure, Function and Bioinformatics   86 巻 ( 3 ) 頁: 344 - 353   2018年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proteins: Structure, Function and Bioinformatics  

    Protein kinase, casein kinase II (CK2), is ubiquitously expressed and highly conserved protein kinase that shows constitutive activity. It phosphorylates a diverse set of proteins and plays crucial role in several cellular processes. The catalytic subunit of this enzyme (CK2α) shows remarkable flexibility as evidenced in numerous crystal structures determined till now. Here, using analysis of multiple crystal structures and long timescale molecular dynamics simulations, we explore the conformational flexibility of CK2α. The enzyme shows considerably higher flexibility in the solution as compared to that observed in crystal structure ensemble. Multiple conformations of hinge region, located near the active site, were observed during the dynamics. We further observed that among these multiple conformations, the most populated conformational state was inadequately represented in the crystal structure ensemble. The catalytic spine, was found to be less dismantled in this state as compared to the “open” hinge/αD state crystal structures. The comparison of dynamics in unbound (Apo) state and inhibitor (CX4945) bound state exhibits inhibitor induced suppression in the overall dynamics of the enzyme. This is especially true for functionally important glycine-rich loop above the active site. Together, this work gives novel insights into the dynamics of CK2α in solution and relates it to the function. This work also explains the effect of inhibitor on the dynamics of CK2α and paves way for development of better inhibitors.

    DOI: 10.1002/prot.25444

    Web of Science

    Scopus

    PubMed

  32. Nuclear receptor HNF4A transrepresses CLOCK:BMAL1 and modulates tissue-specific circadian networks. 査読有り

    Qu M, Duffy T, Hirota T, Kay SA

    Proceedings of the National Academy of Sciences of the United States of America   115 巻 ( 52 ) 頁: E12305 - E12312   2018年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences of the United States of America  

    Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly transrepresses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK: BMAL1 at a wide array of metabolic genes involved in lipid, glucose, and amino acid homeostasis. Taken together, we establish that HNF4A defines a feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.

    DOI: 10.1073/pnas.1816411115

    Web of Science

    Scopus

    PubMed

  33. Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies. 査読有り

    Hatori M, Gronfier C, Van Gelder RN, Bernstein PS, Carreras J, Panda S, Marks F, Sliney D, Hunt CE, Hirota T, Furukawa T, Tsubota K

    NPJ aging and mechanisms of disease   3 巻 ( 1 ) 頁: 9   2017年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:npj Aging and Mechanisms of Disease  

    Mammals receive light information through the eyes, which perform two major functions: image forming vision to see objects and non-image forming adaptation of physiology and behavior to light. Cone and rod photoreceptors form images and send the information via retinal ganglion cells to the brain for image reconstruction. In contrast, nonimage-forming photoresponses vary widely from adjustment of pupil diameter to adaptation of the circadian clock. nonimage-forming responses are mediated by retinal ganglion cells expressing the photopigment melanopsin. Melanopsin-expressing cells constitute 1–2% of retinal ganglion cells in the adult mammalian retina, are intrinsically photosensitive, and integrate photic information from rods and cones to control nonimage-forming adaptation. Action spectra of ipRGCs and of melanopsin photopigment peak around 480 nm blue light. Understanding melanopsin function lets us recognize considerable physiological effects of blue light, which is increasingly important in our modern society that uses light-emitting diode. Misalignment of circadian rhythmicity is observed in numerous conditions, including aging, and is thought to be involved in the development of age-related disorders, such as depression, diabetes, hypertension, obesity, and cancer. The appropriate regulation of circadian rhythmicity by proper lighting is therefore essential. This perspective introduces the potential risks of excessive blue light for human health through circadian rhythm disruption and sleep deprivation. Knowing the positive and negative aspects, this study claims the importance of being exposed to light at optimal times and intensities during the day, based on the concept of the circadian clock, ultimately to improve quality of life to have a healthy and longer life.

    DOI: 10.1038/s41514-017-0010-2

    Web of Science

    Scopus

    PubMed

  34. Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBα Degradation 査読有り

    Zhao X, Hirota T, Han X, Cho H, Chong L.-W, Lamia K, Liu S, Atkins A.R, Banayo E, Liddle C, Yu R.T, Yates J.R, Kay S.A, Downes M, Evans R.M

    Cell   165 巻 ( 7 ) 頁: 1644 - 1657   2016年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Cell  

    Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.

    DOI: 10.1016/j.cell.2016.05.012

    Web of Science

    Scopus

    PubMed

  35. C-H activation generates period-shortening molecules that target cryptochrome in the mammalian circadian clock. 査読有り

    Oshima T, Yamanaka I, Kumar A, Yamaguchi J, Nishiwaki-Ohkawa T, Muto K, Kawamura R, Hirota T, Yagita K, Irle S, Kay SA, Yoshimura T, Itami K

    Angewandte Chemie (International ed. in English)   54 巻 ( 24 ) 頁: 7193 - 7197   2015年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Angewandte Chemie - International Edition  

    The synthesis and functional analysis of KL001 derivatives, which are modulators of the mammalian circadian clock, are described. By using cutting-edge CH activation chemistry, a focused library of KL001 derivatives was rapidly constructed, which enabled the identification of the critical sites on KL001 derivatives that induce a rhythm-changing activity along with the components that trigger opposite modes of action. The first period-shortening molecules that target the cryptochrome (CRY) were thus discovered. Detailed studies on the effects of these compounds on CRY stability implicate the existence of an as yet undiscovered regulatory mechanism. A change in rhythm: The first functional analysis of KL001 derivatives, which are mammalian circadian-clock modulators, was enabled by cutting-edge CH activation. The sites of the KL001 derivatives that are critical for their rhythm-changing activity were elucidated, which led to the discovery of the first period-shortening molecules that target the cryptochrome.

    DOI: 10.1002/anie.201502942

    Web of Science

    Scopus

    PubMed

  36. Identification of small-molecule modulators of the circadian clock. 査読有り

    Hirota T, Kay SA

    Methods in enzymology   551 巻   頁: 267 - 282   2015年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Methods in Enzymology  

    Chemical biology or chemical genetics has emerged as an interdisciplinary research area applying chemistry to understand biological systems. The development of combinatorial chemistry and high-throughput screening technologies has enabled large-scale investigation of the biological activities of diverse small molecules to discover useful chemical probes. This approach is applicable to the analysis of the circadian clock mechanisms through cell-based assays to monitor circadian rhythms using luciferase reporter genes. We and others have established cell-based high-throughput circadian assays and have identified a variety of novel small-molecule modulators of the circadian clock by phenotype-based screening of hundreds of thousands of compounds. The results demonstrated the effectiveness of chemical biology approaches in clock research field. This technique will become more and more common with propagation of high-throughput screening facilities. This chapter describes assay development, screening setups, and their optimization for successful screening campaigns.

    DOI: 10.1016/bs.mie.2014.10.015

    Web of Science

    Scopus

    PubMed

  37. Development of Small-Molecule Cryptochrome Stabilizer Derivatives as Modulators of the Circadian Clock. 査読有り

    Lee JW, Hirota T, Kumar A, Kim NJ, Irle S, Kay SA

    ChemMedChem   10 巻 ( 9 ) 頁: 1489 - 1497   2015年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ChemMedChem  

    Small-molecule probes have been playing prominent roles in furthering our understanding of the molecular underpinnings of the circadian clock. We previously discovered a carbazole derivative, KL001 (N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)-N-(furan-2-ylmethyl)methanesulfonamide), as a stabilizer of the clock protein cryptochrome (CRY). Herein we describe an extensive structure-activity relationship analysis of KL001 derivatives leading to the development of a highly active derivative: 2-(9H-carbazol-9-yl)-N-(2-chloro-6-cyanophenyl)acetamide (KL044). Subsequent 3D-QSAR analysis identified critical features of KL001 derivatives and provided a molecular-level understanding of their interaction with CRY. The electron-rich carbazole, amide/hydroxy linker, sulfonyl group, and electron-withdrawing nitrile moieties contribute to greater biological activity. The hydrogen bonding interactions with Ser394 and His357 as well as stronger CH-π interactions with Trp290 make KL044 a better binder than KL001. KL044 lengthened the circadian period, repressed Per2 activity, and stabilized CRY in reporter assays with roughly tenfold higher potency than KL001. Altogether, KL044 is a powerful chemical tool to control the function of the circadian clock through its action on CRY. Circadian modulators: An extensive SAR study of KL001, a stabilizer of the clock protein CRY, resulted in the development of a highly active derivative, KL044. Subsequent 3D-QSAR analysis provided molecular-level understanding of the regulatory mechanism of CRY function. KL044 is a powerful chemical tool to control circadian rhythms by targeting CRY.

    DOI: 10.1002/cmdc.201500260

    Web of Science

    Scopus

    PubMed

  38. In vivo role of phosphorylation of cryptochrome 2 in the mouse circadian clock. 査読有り

    Hirano A, Kurabayashi N, Nakagawa T, Shioi G, Todo T, Hirota T, Fukada Y

    Molecular and cellular biology   34 巻 ( 24 ) 頁: 4464 - 4473   2014年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1128/MCB.00711-14

    Web of Science

    Scopus

    PubMed

  39. Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock. 査読有り

    St John PC, Hirota T, Kay SA, Doyle FJ 3rd

    Proceedings of the National Academy of Sciences of the United States of America   111 巻 ( 5 ) 頁: 2040 - 2045   2014年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1073/pnas.1323618111

    Web of Science

    Scopus

    PubMed

  40. Biosynthesis and biological actions of pineal neurosteroids in domestic birds. 査読有り

    Tsutsui K, Haraguchi S, Hatori M, Hirota T, Fukada Y

    Neuroendocrinology   98 巻 ( 2 ) 頁: 97 - 105   2013年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1159/000353782

    Web of Science

    Scopus

    PubMed

  41. New biosynthesis and biological actions of avian neurosteroids. 査読有り 国際誌

    Tsutsui K, Haraguchi S, Inoue K, Miyabara H, Ubuka T, Hatori M, Hirota T, Fukada Y

    Journal of experimental neuroscience   7 巻 ( 1 ) 頁: 15 - 29   2013年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.4137/JEN.S11148

    Scopus

    PubMed

  42. Real-time in vivo monitoring of circadian E-box enhancer activity: a robust and sensitive zebrafish reporter line for developmental, chemical and neural biology of the circadian clock. 査読有り

    Weger M, Weger BD, Diotel N, Rastegar S, Hirota T, Kay SA, Strähle U, Dickmeis T

    Developmental biology   380 巻 ( 2 ) 頁: 259 - 273   2013年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.ydbio.2013.04.035

    Web of Science

    Scopus

    PubMed

  43. Identification of small molecule activators of cryptochrome. 査読有り

    Hirota T, Lee JW, St John PC, Sawa M, Iwaisako K, Noguchi T, Pongsawakul PY, Sonntag T, Welsh DK, Brenner DA, Doyle FJ 3rd, Schultz PG, Kay SA

    Science (New York, N.Y.)   337 巻 ( 6098 ) 頁: 1094 - 1097   2012年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1126/science.1223710

    Web of Science

    Scopus

    PubMed

  44. A small molecule modulates circadian rhythms through phosphorylation of the period protein. 査読有り

    Lee JW, Hirota T, Peters EC, Garcia M, Gonzalez R, Cho CY, Wu X, Schultz PG, Kay SA

    Angewandte Chemie (International ed. in English)   50 巻 ( 45 ) 頁: 10608 - 10611   2011年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/anie.201103915

    Web of Science

    Scopus

    PubMed

  45. Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways. 査読有り

    Hatori M, Hirota T, Iitsuka M, Kurabayashi N, Haraguchi S, Kokame K, Sato R, Nakai A, Miyata T, Tsutsui K, Fukada Y

    Proceedings of the National Academy of Sciences of the United States of America   108 巻 ( 12 ) 頁: 4864 - 4869   2011年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1073/pnas.1015959108

    Web of Science

    Scopus

    PubMed

  46. Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis. 査読有り

    Zhang EE, Liu Y, Dentin R, Pongsawakul PY, Liu AC, Hirota T, Nusinow DA, Sun X, Landais S, Kodama Y, Brenner DA, Montminy M, Kay SA

    Nature medicine   16 巻 ( 10 ) 頁: 1152 - U133   2010年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/nm.2214

    Web of Science

    Scopus

    PubMed

  47. High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase 査読有り

    Hirota T, Lee J.W, Lewis W.G, Zhang E.E, Breton G, Liu X, Garcia M, Peters E.C, Etchegaray J.-P, Traver D, Schultz P.G, Kay S.A

    PLoS Biology   8 巻 ( 12 ) 頁: e1000559   2010年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1371/journal.pbio.1000559

    Web of Science

    Scopus

    PubMed

  48. DYRK1A and glycogen synthase kinase 3β, a dual-kinase mechanism directing proteasomal degradation of CRY2 for circadian timekeeping 査読有り

    Kurabayashi N, Hirota T, Sakai M, Sanada K, Fukada Y

    Molecular and Cellular Biology   30 巻 ( 7 ) 頁: 1757 - 1768   2010年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1128/MCB.01047-09

    Web of Science

    Scopus

    PubMed

  49. Transcriptional repressor TIEG1 regulates Bmal1 gene through GC box and controls circadian clockwork. 査読有り

    Hirota T, Kon N, Itagaki T, Hoshina N, Okano T, Fukada Y

    Genes to cells : devoted to molecular & cellular mechanisms   15 巻 ( 2 ) 頁: 111 - 121   2010年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1111/j.1365-2443.2009.01371.x

    Web of Science

    Scopus

    PubMed

  50. A genome-wide RNAi screen for modifiers of the circadian clock in human cells. 査読有り

    Zhang EE, Liu AC, Hirota T, Miraglia LJ, Welch G, Pongsawakul PY, Liu X, Atwood A, Huss JW 3rd, Janes J, Su AI, Hogenesch JB, Kay SA

    Cell   139 巻 ( 1 ) 頁: 199 - 210   2009年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.cell.2009.08.031

    Web of Science

    Scopus

    PubMed

  51. High-throughput screening and chemical biology: new approaches for understanding circadian clock mechanisms. 査読有り

    Hirota T, Kay SA

    Chemistry & biology   16 巻 ( 9 ) 頁: 921 - 927   2009年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.chembiol.2009.09.002

    Web of Science

    Scopus

    PubMed

  52. A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3β 査読有り

    Hirota T, Lewis W.G, Liu A.C, Jae W.L, Schultz P.G, Kay S.A

    Proceedings of the National Academy of Sciences of the United States of America   105 巻 ( 52 ) 頁: 20746 - 20751   2008年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1073/pnas.0811410106

    Web of Science

    Scopus

    PubMed

  53. Activation of TGF-β/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts 査読有り

    Kon N, Hirota T, Kawamoto T, Kato Y, Tsubota T, Fukada Y

    Nature Cell Biology   10 巻 ( 12 ) 頁: 1463 - U197   2008年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/ncb1806

    Web of Science

    Scopus

    PubMed

  54. Circadian proteomics of the mouse retina. 査読有り

    Tsuji T, Hirota T, Takemori N, Komori N, Yoshitane H, Fukuda M, Matsumoto H, Fukada Y

    Proteomics   7 巻 ( 19 ) 頁: 3500 - 3508   2007年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/pmic.200700272

    Web of Science

    Scopus

    PubMed

  55. Phosphorylation of mCRY2 at Ser557 in the hypothalamic suprachiasmatic nucleus of the mouse. 査読有り

    Kurabayashi N, Hirota T, Harada Y, Sakai M, Fukada Y

    Chronobiology international   23 巻 ( 1-2 ) 頁: 129 - 134   2006年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1080/07420520500464478

    Web of Science

    Scopus

    PubMed

  56. Ser-557-phosphorylated mCRY2 is degraded upon synergistic phosphorylation by glycogen synthase kinase-3β 査読有り

    Harada Y, Sakai M, Kurabayashi N, Hirota T, Fukada Y

    Journal of Biological Chemistry   280 巻 ( 36 ) 頁: 31714 - 31721   2005年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.M506225200

    Web of Science

    Scopus

    PubMed

  57. Resetting mechanism of central and peripheral circadian clocks in mammals. 査読有り

    Hirota T, Fukada Y

    Zoological science   21 巻 ( 4 ) 頁: 359 - 368   2004年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.2108/zsj.21.359

    Web of Science

    Scopus

    PubMed

  58. p38 mitogen-activated protein kinase regulates oscillation of chick pineal circadian clock. 査読有り

    Hayashi Y, Sanada K, Hirota T, Shimizu F, Fukada Y

    The Journal of biological chemistry   278 巻 ( 27 ) 頁: 25166 - 25171   2003年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.M212726200

    Web of Science

    Scopus

    PubMed

  59. 哺乳類培養細胞におけるグルコース応答とサーカディアンリズム

    広田 毅, 岡野 俊行, 深田 吉孝

    生物物理   43 巻   頁: S11   2003年

     詳細を見る

    記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:一般社団法人 日本生物物理学会  

    DOI: 10.2142/biophys.43.S11_2

  60. Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts. 査読有り

    Hirota T, Okano T, Kokame K, Shirotani-Ikejima H, Miyata T, Fukada Y

    The Journal of biological chemistry   277 巻 ( 46 ) 頁: 44244 - 44251   2002年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.M206233200

    Web of Science

    Scopus

    PubMed

  61. Chicken pineal clock genes: Implication of BMAL2 as a bidirectional regulator in circadian clock oscillation 査読有り

    Okano, T., Yamamoto, K., Okano, K., Hirota, T., Kasahara, T., Sasaki, M., Takanaka, Y., Fukada, Y.

    Genes to Cells   6 巻 ( 9 ) 頁: 825 - 836   2001年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL SCIENCE LTD  

    Background: In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.
    Results: We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production.
    Conclusion: The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.

    DOI: 10.1046/j.1365-2443.2001.00462.x

    Web of Science

    Scopus

  62. Effect of brefeldin A on melatonin secretion of chick pineal cells 査読有り

    Hirota, T., Kagiwada, S., Kasahara, T., Okano, T., Murata, M., Fukada, Y.

    Journal of Biochemistry   129 巻 ( 1 ) 頁: 51 - 59   2001年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.

    DOI: 10.1093/oxfordjournals.jbchem.a002836

    Web of Science

    Scopus

▼全件表示

書籍等出版物 1

  1. 分子・細胞・個体レベルにおける動物の光環境応答とサーカディアンリズム

    深田 吉孝, 岡野 俊行, 仲村 厚志, 和田 恭高, 広田 毅, 小島 大輔( 担当: 単著)

    深田吉孝  2007年 

     詳細を見る

MISC 9

  1. 細胞外pHのアルカリ性化はDec1遺伝子の誘導を介して概日時計の位相をリセットする

    金 尚宏, 広田 毅, 河本 健, 加藤 幸夫, 坪田 匡史, 深田 吉孝  

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集81回・31回 巻   頁: 4T16 - 4   2008年11月

     詳細を見る

    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

  2. ニワトリ松果体におけるコレステロール合成系酵素群の遺伝子発現解析

    飯塚倫子, 倉林伸博, 羽鳥恵, 広田毅, 原口省吾, 筒井和義, 深田吉孝  

    日本比較生理生化学会大会予稿集30th 巻   頁: 23   2008年

     詳細を見る

    記述言語:日本語  

    J-GLOBAL

  3. Sequential phosphorylation of mouse cryptochrome2

    Nobuhiro Kurabayashi, Tsuyoshi Hirota, Yoko Harada, Mihiko Sakai, Yoshitaka Fukada  

    ZOOLOGICAL SCIENCE23 巻 ( 12 ) 頁: 1194 - 1194   2006年12月

     詳細を見る

    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:ZOOLOGICAL SOC JAPAN  

    Web of Science

  4. 【ここまで分かった生物時計の分子ネットワーク リン酸化による時計タンパク質の制御機構から睡眠・肥満への関与まで】時計タンパク質の多段階リン酸化

    広田 毅, 深田 吉孝  

    実験医学24 巻 ( 4 ) 頁: 452 - 459   2006年3月

     詳細を見る

    記述言語:日本語   出版者・発行元:(株)羊土社  

    概日時計の自律的な発振メカニズムにおいては,時計遺伝子の転写・翻訳とともに時計タンパク質のリン酸化が重要な役割を果たす.いくつかの時計タンパク質は,複数のキナーゼによって段階的にリン酸化され,その分解速度や細胞内局在,転写調節などの機能が緻密に調節されている.本稿では,時計タンパク質をリン酸化するキナーゼの同定,およびリン酸化サイトの決定に関する最近の知見を紹介するとともに,多段階リン酸化による時計タンパク質の制御機構を概説する(著者抄録)

  5. 分子時計の生化学 グルコース投与によるrat-1細胞の時計遺伝子発現における概日リズムの誘導

    広田 毅, 岡野 俊行, 小亀 浩市, 池島 裕子, 谷, 宮田 敏行, 深田 吉孝  

    生化学74 巻 ( 8 ) 頁: 643 - 643   2002年8月

     詳細を見る

    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

  6. ニワトリ松果体の概日時計構成分子cBMAL2

    岡野 俊行, 山本 和幸, 岡野 恵子, 広田 毅, 佐々木 桃子, 高中 陽子, 深田 吉孝  

    日本時間生物学会会誌: Journal of Chronobiology6 巻 ( 2 ) 頁: 87 - 87   2000年10月

     詳細を見る

    記述言語:日本語  

    CiNii Books

  7. ブレフェルジンAを用いたニワトリ松果体細胞のメラトニン分泌機構の解析

    広田 毅, 鍵和田 聡, 笠原 和起, 岡野 俊行, 村田 昌之, 深田 吉孝  

    生化学72 巻 ( 8 ) 頁: 1016 - 1016   2000年8月

     詳細を見る

    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

  8. 松果体とサーカディアンリズム

    深田吉孝, 広田毅  

    Clinical Neuroscience特集「サーカディアンリズムのしくみと働き」18 巻 ( 10 ) 頁: 1147 - 1149   2000年

  9. IDENTIFICATION OF CHICKEN PINEAL CLOCK GENES(Physiology)Proceedings of the Seventy-First Annual Meeting of the Zoological Society of Japan :

    Okano T., Yamamoto K., Okano K., Hirota T., Sasaki M., Takanaka Y., Fukada Y.  

    Zoological science17 巻   頁: 92 - 92   2000年

     詳細を見る

    記述言語:英語   出版者・発行元:Zoological Society of Japan  

    CiNii Books

    その他リンク: http://id.nii.ac.jp/1141/00035547/

▼全件表示

科研費 9

  1. 革新的な機能調節化合物の創製による概日時計システムの統合的な理解と制御

    研究課題/研究課題番号:21H04766  2021年4月 - 2024年3月

    科学研究費助成事業  基盤研究(A)

    廣田 毅

      詳細を見る

    担当区分:研究代表者 

    配分額:42510000円 ( 直接経費:32700000円 、 間接経費:9810000円 )

    睡眠・覚醒など様々な生理機能の日内リズムを支配する概日時計について、私たちが世界に先駆けて発見した独自の時計調節化合物を用い、新たな時計タンパク質の発見や概日リズムの自在な制御を可能にする新技術を開発する。これらの革新的な技術を用いて、分子メカニズム解析から組織・個体における機能制御を統合的に行い、従来の分子遺伝学研究を超えて概日時計システムの作動原理を徹底解剖する。

  2. ケージド時計調節化合物を用いた概日リズムの光制御と細胞間相互作用の解析

    研究課題/研究課題番号:20K21269  2020年7月 - 2022年3月

    科学研究費助成事業  挑戦的研究(萌芽)

    廣田 毅

      詳細を見る

    担当区分:研究代表者 

    配分額:6500000円 ( 直接経費:5000000円 、 間接経費:1500000円 )

    申請者は概日リズムの周期を変化させる新規化合物を発見して鍵となる制御機構を明らかにしてきた。多細胞から成る概日時計システムの理解に向けて解明すべき重要課題が、細胞間の相互作用である。本研究では時計調節化合物のケージド誘導体を用い、狙ったタイミングに狙った細胞で概日時計を定量的に操作する新技術を生み出す。これを概日リズムの1細胞イメージングに応用し、細胞間相互作用の時空間的な解析に挑戦する。
    概日時計は分子から個体の各階層を時間・空間的に統合して多様な生理現象の日内リズムを生み出す。申請者はケミカルバイオロジーの手法を応用し、概日リズムの周期を変化させる新規化合物を発見して鍵となる制御機構を明らかにしてきた。多細胞から成る概日時計システムの理解に向けた次のステップとして解明すべき重要課題が、細胞間の相互作用である。これまでに時計調節化合物longdaysinをもとにケージド誘導体を開発し、時間的な精密制御を実現した。本研究ではこのようなケージド時計調節化合物を用い、狙ったタイミングに狙った細胞で概日時計を定量的に操作する新技術を生み出す。これを概日リズムの1細胞イメージングに応用し、細胞間相互作用の時空間的な解析に挑戦する。本年度は、時計遺伝子Bmal1のプロモーターにルシフェラーゼを連結したレポーターをもつヒトU2OS細胞と、超高感度カメラを搭載した顕微鏡システムを用いて、1細胞レベルの概日リズム測定系をセットアップし、ケージドlongdaysinを用いて光照射条件の検討を開始した。化合物の有無にかかわらず、光源の特性から光照射によって細胞毒性が見られたため、フィルター等の最適化を行っている。並行して、ケージド化合物が不可逆的な変化をもたらすのに対し、可逆的な調節が可能である光スイッチ化合物の開発を行った。光によってトランス体とシス体に構造変化するアゾベンゼンを、longdaysinおよび新規の時計調節化合物TH129に導入した。
    計画通りに実験系をセットアップし、条件検討が順調に進展している。その上、光スイッチ化合物の開発を進めている。
    ケージド化合物および光スイッチ化合物を用いて条件検討を進め、概日リズムの局所的な操作を可能にすることにより、細胞間相互作用を解析する。

  3. ヘムとCOがクロストークする概日リズム調節機構の分子ツールによる解明

    研究課題/研究課題番号:20H02871  2020年4月 - 2023年3月

    科学研究費助成事業  基盤研究(B)

    北岸 宏亮, 廣田 毅

      詳細を見る

    担当区分:研究分担者 

    細胞内には約24時間周期で遺伝子発現のリズムが刻まれており,その制御にはヘムやCOといった生体内小分子が関与していると考えられているが,その詳細は明らかではない。本研究では従来の遺伝子工学的手法では制御が困難なヘムおよびCOに対して,これらの動態をコントロールすることのできるケミカルツール群を開発して活用することにより,ヘムとCOがクロストークする概日リズム制御機構の一端を明らかにすることを目的とする。
    本研究では,細胞内概日リズム制御におけるヘムおよび一酸化炭素の関与について,解明を目標としている。初年度では,細胞内にて代謝分解されるが一酸化炭素を発生しないメチル化ヘム誘導体の合成および細胞内一酸化炭素を送達する化合物の合成に着手し,それぞれ達成した。さらに細胞内において選択的に一酸化炭素を捕捉する化合物hemoCDを利用して,細胞内一酸化炭素の濃度を定量するアッセイを構築した。このアッセイを用いることで,生体内一酸化炭素の臓器分布を測定することに成功した。臓器内COの定量については,過去にガスクロマトグラフィーを用いた計測が確立されているが,我々が精査した結果,ガスクロマトグラフィーよりもhemoCDアッセイの方がより多くのCOを検出できることが明らかになった。さらに外部から吸入したCOの体内分布についても計測した。その結果,組織内に分布したCOは徐々に血液に移行してヘモグロビンに結合し,最終的に肺から排出される経路が存在することがわかった。すなわちヘモグロビンには組織内COの蓄積を緩和するはたらきがあることが,我々のhemoCDアッセイによるCO動態解析により明らかとなった。hemoCDを用いたCO定量アッセイについては特許の出願を行った。さらに上記成果を論文としてまとめて,Nature系オープンアクセス誌Communicatins Biologyに公表した。この成果についてはプレスリリースを行い,広く報道された。
    本プロジェクトの最終目標は,ヘムとCOがクロストークする概日リズム制御機構の解明であるが,初年度においてその実験に必要な化合物の合成が完了した。さらに関連する技術において特許出願および論文公表を達成することができた。
    分担者の廣田氏との共同体制を強固にして,ヘムとCOが関与する細胞内概日リズム制御機構の解明に本格的に取り組む。

  4. 新規化合物を用いて迫る概日時計タンパク質CRY1とCRY2の特異性の分子基盤

    研究課題/研究課題番号:18H02402  2018年4月 - 2021年3月

    科学研究費助成事業  基盤研究(B)

    廣田 毅

      詳細を見る

    担当区分:研究代表者 

    配分額:17550000円 ( 直接経費:13500000円 、 間接経費:4050000円 )

    哺乳類の概日時計を構成するCRYタンパク質には、CRY1とCRY2というよく似たホモログが存在する。これらの機能的な差異の解析は、概日時計がどのように正確に発振するのか、いかに生理機能を調節するのか、という根本的な疑問に対するよいモデル系となる。本研究では、CRY1とCRY2のそれぞれに選択的な新規化合物を見出し、その作用機序を解析することでCRY1とCRY2の特異性を生み出すメカニズムを明らかにした。
    CRY1とCRY2は配列が非常によく似ているため、選択的な化合物のデザインは困難であった。本研究では、配列がほぼ同一である化合物結合ポケットに対して選択性を生み出すユニークな化合物を見出し、それらの作用機構を解明した。概日時計が乱れるリズム障害は現代社会における深刻な問題となっており、本研究による制御機構の理解と特異的な調節ツールの開発は、CRYを利用した疾患治療法の開発に向けた重要な足がかりとなるであろう。

  5. 概日リズムの振幅制御機構の解明に向けたツール化合物の開発と作用機序解析

    研究課題/研究課題番号:18K19171  2018年6月 - 2020年3月

    挑戦的研究(萌芽)

    廣田 毅

      詳細を見る

    担当区分:研究代表者 

    配分額:6240000円 ( 直接経費:4800000円 、 間接経費:1440000円 )

    概日リズムの振幅は肥満や老化によって減少し、生理機能の調節における重要性が知られているものの、分子レベルの理解が遅れている。本研究は概日リズムの振幅の制御機構にケミカルバイオロジーの手法を用いてアプローチすることを試みた。化合物スクリーニングからリズム振幅を増加させる新規化合物を発見し、作用機序を解析した。さらに研究を進めることにより、振幅制御の理解が深まると期待できる。
    本研究により、振幅を増加させる新規化合物を発見した。このようなユニークな化合物が起点となり、将来的には概日リズム疾患の治療への応用に繋がる可能性が期待される。さらに、ケージドlongdaysinを開発して光を用いて概日リズム周期を誘導的かつ精密に調節することを世界で初めて実現し、chronophotopharmacologyという新領域を切り拓いた。

  6. 時計タンパク質制御分子の創製と機能解析から概日時計機構に迫る

    研究課題/研究課題番号:15H05590  2015年4月 - 2018年3月

    廣田 毅

      詳細を見る

    担当区分:研究代表者 

    配分額:23530000円 ( 直接経費:18100000円 、 間接経費:5430000円 )

    概日時計は分子から個体の各階層を時間・空間的に統合して生理現象の日内リズムを生み出す。本研究では概日時計の周期制御に中心的な役割を果たすPERとCRYの安定性および複合体形成に焦点を当て、これらのプロセスを狙った機能調節化合物を探索し、CRY1に選択的に作用する全く新しい化合物を発見した。並行してSUMO化修飾に注目した解析を行い、CRYのSUMO化を見出した。独自のツールを用いて概日時計の発振機構の解明と自在な機能操作を可能にしていく。

  7. 概日時計タンパク質CRYの機能を調節する低分子化合物

    研究課題/研究課題番号:26891011  2014年8月 - 2015年3月

    廣田 毅

      詳細を見る

    担当区分:研究代表者 

    配分額:1430000円 ( 直接経費:1100000円 、 間接経費:330000円 )

    本研究では、時計タンパク質CRYの安定性を変化させる新規の化合物を同定し、それをプローブとしてCRYの機能調節機構を解明することを目指す。そのために本年度は化合物スクリーニング系のセットアップを行った。CRY1とルシフェラーゼの融合タンパク質であるCRY1-LUCを安定発現するHEK293細胞を用い、申請者が発見したCRYを安定化する化合物KL001の効果を指標に細胞数、培地量、化合物添加のタイミング、処理時間などを検討し、384ウェルプレートを用いたアッセイ条件を最適化した。化合物処理によるCRY1-LUCの蓄積量の変化を用いたエンドポイントアッセイとすることで、半減期を測定するよりも手順を簡略化し、ハイスループット化への目途をつけた。コントロールとしてLUCを安定発現する細胞を用い、化合物がルシフェラーゼに与える影響を評価することで、CRYに対する作用を明らかにする。さらに名古屋大学トランスフォーマティブ生命分子研究所の化合物ライブラリーセンターと共同で、384ウェルプレートに対応した細胞分注機およびプレートリーダーを導入した。現在、化合物投与に用いる微量分注機をセットアップしているところである。機器の準備が整い次第、化合物ライブラリーセンターが所有する化合物のうち1万種類をスクリーニングし、CRYの発現量を増加あるいは減少させる化合物がどの程度の頻度で出現するかを見積もる予定である。
    26年度が最終年度であるため、記入しない。
    26年度が最終年度であるため、記入しない。

  8. 哺乳類の末梢組織における概日時計のグルコース応答

    研究課題/研究課題番号:16770091  2004年 - 2005年

    廣田 毅

      詳細を見る

    担当区分:研究代表者 

    配分額:3200000円 ( 直接経費:3200000円 )

    交付申請書に記載した研究実施計画に沿って研究を行い、下記の成果を得た。
    TIEG1蛋白質の部分配列を有する抗原蛋白質を作製し、ウサギに免疫した。得られた抗血清をアフィニティー精製し、抗TIEG1抗体とした。この抗体を用いてウエスタンブロット解析した結果、TIEG1は主に核内に存在する約65kDaの蛋白質で、rat-1細胞における発現量はグルコース刺激によって急速に上昇することが判明した。TIEG1蛋白質量はグルコース投与の2時間後にピークに達し、これはTieg1 mRNAのピークから約1時間遅れていたものの、mRNAと蛋白質の発現プロファイルが互いに似ていたことから、TIEG1蛋白質の合成・分解は比較的速いと考えられた。さらに、生体内においてもTIEG1が時計入力因子として働く可能性を検証するため、マウスを2日間絶食させた後に餌を2時間与え、摂食が肝臓におけるTIEG1蛋白質量に与える効果を解析した。その結果、餌を与えた場合は絶食を続けた場合と比べ、TIEG1蛋白質量が有意に上昇することがわかった。一方、Per1およびBmal1遺伝子の発現量は摂食によって低下したことから、TIEG1は生体内においてもPer1・Bmal1遺伝子の抑制因子として時計入力に関与すると考えられた。
    以上の解析と並行して、時計蛋白質E4BP4およびCRY2のリン酸化動態を解析した。ニワトリE4BP4のSer182およびマウスCRY2のSer557のリン酸化は、各蛋白質のプロテアソームを介した分解を導く。本研究では、Ser182リン酸化型のE4BP4に対する抗体を作製し、ニワトリ松果体においてSer182がリン酸化されていることを見出すとともに、Ser557リン酸化型のCRY2に対する抗体を用い、マウスの肝臓だけでなく視交叉上核においてもSer557のリン酸化量が顕著な概日リズムを示すことを明らかにした。

  9. 分子・細胞・個体レベルにおける動物の光環境応答とサーカディアンリズム

    研究課題/研究課題番号:14104003  2002年 - 2006年

    深田 吉孝

      詳細を見る

    担当区分:研究分担者 

    (1)ニワトリ松果体G11の光活性化が概日時計の位相シフトを導くことを見出した。また、ピノプシン遺伝子の光依存的転写調節に必須の光応答エレメントを同定した。
    (2)網膜桿体のG蛋白質のファルネシル化が、光情報伝達だけでなく明順応に重要な役割を果たす事を示した。一方、ゼブラフィッシュ網膜の錐体の光受容体キナーゼGRK7-1は桿体GRK1Aの数十倍も高い活性を示した。
    (3)ニワトリ松果体やカエル網膜のMAPK活性が夜にピークを持つ概日リズムを示した。一方、マウス視交叉上核では、MAPKの活性化の時刻と光刺激に対する応答パターンが背内側部と中心部において互いに異なる事を示した。時計システムにおいてMAPKによってリン酸化されるBMAL1、CRY1およびCRY2のリン酸化部位とリン酸化に伴う活性変化を同定した。また、SCOPがK-Rasを介してMAPKを制御することを見出した。一方、ニワトリ松果体においてp38キナーゼ/MAPKAPK2経路が昼に時計発振系に入力して位相前進を導く可能性を示した。
    (4)ニワトリ松果体では明期の延長によりE4BP4の発現レベルが高く維持され、Per2の転写抑制を介して位相後退を引き起した。E4BP4はCKIεによってリン酸化され、プロテアソームを介して分解された。
    (5)末梢時計モデルとしてrat-1細胞をグルコース刺激すると概日リズムがリセットされることを見出し、その際、時計遺伝子の発現を調節する分子の候補としてTieg1とVdup1遺伝子を同定した。TIEG1はグルコース投与によって発現量が急上昇し、Bmal1プロモーターに直接作用して転写を抑制した。
    (6)ゼブラフィッシュ松果体の光受容分子エクソロドプシンの松果体特異的な遺伝子発現を担う新規配列PIPEを同定した。酵母のone-hybrid系を用いたスクリーニングにより複数のPIPE結合因子候補を同定した。

▼全件表示