研究者詳細 - 廣田　毅

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

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

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 Cï£¿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 Cï£¿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

Identification of small-molecule modulators of the circadian clock. 査読有り国際誌

Hirota T, Kay SA

Methods in enzymology 551 巻頁： 267 - 282 2015年

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

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

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

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

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