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

@entity1 @entity6495 @entity19 @entity3963 @entity71874 @entity17342 @entity3392 @entity307
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 @entity1 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 @entity19 suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound @entity71874 . @entity71874 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 @entity17342  , @entity6495 , and @entity3963 as targets of @entity71874 responsible for the observed effect on circadian period. Although individual knockdown of @entity17342  , @entity6495 , and @entity3963 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to @entity71874 treatment. We characterized the role of @entity6495 in the clock mechanism and found that @entity6495 -mediated phosphorylation stimulated degradation of a clock protein @entity307 , similar to the function of @entity17342  . @entity71874 treatment inhibited @entity307 degradation, providing insight into the mechanism of @entity71874 -dependent period lengthening. Using larval @entity3392 , we further demonstrated that @entity71874 drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed @entity6495 as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. @entity71874 provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.
