Abstract
The circadian clock is a timekeeping system for regulation of numerous biological daily rhythms. One characteristic of the circadian clock is that period length remains relatively constant in spite of environmental fluctuations, such as temperature change. Here, using the curated collection of in-house small molecule chemical library (ITbM chemical library), we show that small molecule 3,4-dibromo-7-azaindole (B-AZ) lengthened the circadian period of Arabidopsis thaliana (Arabidopsis). B-AZ has not previously been reported to have any biological and biochemical activities. Target identification can elucidate the mode of action of small molecules, but we were unable to make a molecular probe of B-AZ for target identification. Instead, we performed other analysis, gene expression profiling that potentially reveals mode of action of molecules. Short-term treatment of B-AZ decreased the expression of four dawn- and morning-phased clock-associated genes, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. Consistently, amounts of PRR5 and TIMING OF CAB EXPRESSION 1 (TOC1) proteins, transcriptional repressors of CCA1, LHY, PRR9 and PRR7 were increased upon B-AZ treatment. B-AZ inhibited Casein Kinase 1 family (CK1) that phosphorylates PRR5 and TOC1 for targeted degradation. A docking study and molecular dynamics simulation suggested that B-AZ interacts with the ATP-binding pocket of human CK1 delta, whose amino acid sequences are highly similar to those of Arabidopsis CK1. B-AZ-induced period-lengthening effect was attenuated in prr5 toc1 mutants. Collectively, this study provides a novel and simple structure CK1 inhibitor that modulates circadian clock via accumulation of PRR5 and TOC1.
Highlights
Circadian clocks are biological timekeeping systems that allow organisms to coordinate their activities with daily fluctuations such as light–dark and warm–cold cycles that originate from earth’s rotation
Fundamental properties of circadian clocks are well conserved among bacteria, fungi, plants and animals, clock components are different among phylogenetic lineages (Nohales and Kay 2016)
We searched for small molecules that could regulate circadian clock, from the ITbM chemical library, our unique chemical library that was enriched with plant hormone mimic molecules for use in plant-based phenotypic screening (Ziadi et al 2017, Toh et al 2018)
Summary
Circadian clocks are biological timekeeping systems that allow organisms to coordinate their activities with daily fluctuations such as light–dark and warm–cold cycles that originate from earth’s rotation. Fundamental properties of circadian clocks (a period of about 24 h under constant conditions can be entrained by the environmental time cues, and period length is robust against environmental fluctuations) are well conserved among bacteria, fungi, plants and animals, clock components are different among phylogenetic lineages (Nohales and Kay 2016). The transcription translation feedback loop controls the circadian rhythms of many physiological processes through directly regulating the expression of key genes in output pathways (Huang et al 2012, Nakamichi et al 2012, Nagel et al 2015, Kamioka et al 2016, Liu et al 2016, Ezer et al 2017, Adams et al 2018). Network architectures are partly conserved among flowering plants (Toda et al 2019), but divergence is important for adaptation (Itoh et al 2019)
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