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

PLoS Biol. 2010 Dec 14;8(12):e1000559. doi: 10.1371/journal.pbio.1000559.

Abstract

The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ∼120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / analogs & derivatives*
  • Adenine / metabolism
  • Animals
  • Biological Clocks / drug effects*
  • Biological Clocks / genetics
  • CLOCK Proteins / genetics*
  • CLOCK Proteins / metabolism*
  • Casein Kinase I / metabolism
  • Cell Line, Tumor
  • Circadian Rhythm / drug effects*
  • Circadian Rhythm / genetics
  • Circadian Rhythm / physiology*
  • Cyclin-Dependent Kinases / metabolism
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / genetics
  • Gene Knockdown Techniques
  • Genome-Wide Association Study
  • Histones / metabolism
  • Humans
  • Mice
  • Mice, Inbred Strains
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Period Circadian Proteins / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism
  • RNA Interference
  • Transcription Factors / genetics
  • Transcription Factors / physiology
  • Zebrafish / genetics
  • Zebrafish / physiology

Substances

  • Histones
  • Period Circadian Proteins
  • Protein Kinase Inhibitors
  • Transcription Factors
  • longdaysin
  • CLOCK Proteins
  • Casein Kinase I
  • Protein-Serine-Threonine Kinases
  • Cyclin-Dependent Kinases
  • cyclin-dependent kinase 7, mouse
  • MAPK1 protein, human
  • Mitogen-Activated Protein Kinase 1
  • Adenine