Phenotypic Proteomic Profiling Reveals Convergent Kinase Targets for Circadian Clock Modulators

Determining the mechanisms of action of drug molecules that modulate circadian rhythms is critical to develop novel compounds to treat clock disorders. Here we employed Phenotypic Proteomic Profiling (PPP) integrating multipronged proteomics approaches including global proteome, phosphoproteome, kinome mapping, and proteome-wide profiling of thermal stability (TPP) to systematically determine convergent molecular targets of four circadian period lengthening compounds (Longdaysin, Roscovitine, Purvalanol A, and SP600125) in human cells. We demonstrate convergent changes in phosphorylation level and activity of several proteins and kinases involved in vital signaling pathways including MAPK, NGF, BCR, AMPK, and mTOR signaling by the compounds. Kinome profiling using desthiobiotin-ATP enrichment quantitative proteomics and radiometric assays further indicated inhibition of CKId, ERK1/2, CDK2/7, TNIK and STK26 activity as a common mechanism of action for the compounds. Pharmacological or genetic inhibition of several convergent kinases resulted in circadian period lengthening, establishing them as novel bone fide circadian targets. TPP analysis using live cells revealed binding of these drugs to clock regulatory kinases, signaling molecules, and ubiquitination mediator (F-box) proteins. Phenotypic proteomic profiling thus establishes a set of novel circadian clock effectors.