High-throughput screening and chemical biology: new approaches for understanding circadian clock mechanisms - PubMed (original) (raw)

High-throughput screening and chemical biology: new approaches for understanding circadian clock mechanisms

Tsuyoshi Hirota et al. Chem Biol. 2009.

Abstract

Most organisms exhibit daily changes in physiology and metabolism under the control of a cell-autonomous circadian clock. In the core clock mechanism, clock genes form a transcription factor network to generate circadian rhythms of gene expression. Clock protein phosphorylation and histone modifications are also important for the clock regulation. Pharmacological approaches have been making significant contributions to the clock research, for example, in characterizing the roles of protein kinases CKIdelta, CKIepsilon, CK2, and GSK-3beta. Recently, high-throughput circadian functional assays have been established. Chemical biology approaches utilizing high-throughput compound screening together with RNAi-based genomic screening will open a new way for the circadian clock field. Finding a set of compounds that potently affect the clock function will lead to the identification of novel clock components and form the basis for therapeutic strategies directed toward circadian disorders.

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Figures

Figure 1

Figure 1

Mammalian Circadian Clock Mechanism and High-throughput Circadian Assay (A) Transcription factor feedback loops of the mammalian circadian clock. In the core loop, heterodimers of CLOCK (or NPAS2) and BMAL1 activate transcription from E box element, and PER and CRY proteins inhibit the activation. In addition, DBP (or TEF, HLF) activate and E4BP4 repress D box-mediated regulation, and ROR proteins activate and REV-ERV proteins repress RORE-mediated regulation, forming interlocking loops. These feedback loops generate the rhythmic expression of not only clock genes but also output genes to control the circadian changes in physiology and behavior. (B) Circadian high-throughput screening of compound library. A clonal reporter cell line was established by using the circadian reporter Bmal1-dLuc (top panel). Luminescence intensity of the reporter cells showed circadian rhythm by reflecting Bmal1 promoter activity. The rhythm was monitored in the presence of compounds (final 7 μM). One screening of the compound library LOPAC contained four 384-well plates, and profiles of one 384-well plate are represented in bottom left panel. Each horizontal raster line represents a single well, with elapsed time plotted to right. Luminescence intensity data from each well are normalized for amplitude, and then indicated by gray scale: peak is white and trough is black. Red and blue arrowheads indicate the positions of long and short period compounds, respectively. Note that there are many compounds that change the phase of the rhythm without affecting the period. Bottom right panels indicate representative traces for a long period compound (SP600125) and a short period compound (Indirubin-3′- oxime).

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