Circadian clocks exist in almost all types of living organism and play a fundamental role in regulating daily physiological and behavioural processes. Drugs or small molecules that are capable of modulating circadian rhythms, could be important in developing therapeutic approaches directed towards diverse types of human diseases associated with circadian dysfunctions, such as shift-work disorder, jet lag, and sleep disorders. In this study, we carried out an integrated global proteome and phosphoproteome analysis, thermal proteome profiling (TPP), and kinome profiling to decipher the molecular targets of seven circadian period modulating drugs in the human osteosarcoma cell line – a robust and well-characterized circadian model system. Luminescence rhythms of Per2-dluc cells indicated that Longdaysin drastically lengthened circadian period. Roscovitine, Purvalanol A, and SP600125 also introduced an increase in period length, while treatment with Chir99021, 1-Azakenpaullone, and Indirubin-3′ oxime resulted shorter circadian periods. TMT-based quantitative proteomics analysis revealed increased expressions of kinase inhibitors and reduced levels of cyclin-dependent and other kinases (Casein kinases, Calcium/calmodulin-dependent protein kinases, Serine/threonine protein kinases and GSK-3β) after treatment with these drugs, which were validated further by kinase enrichment assays using desthiobiotin nucleotide (ATP) probes. An integrated phosphoproteomics analysis indicated altered phosphorylation levels of several proteins associated with diverse signalling pathways and circadian rhythmicity. Moreover, TPP analysis revealed direct binding of some of these drugs with clock regulatory kinases and their modulators. Notably, radioactivity-based biochemical assays indicated inhibition of CAMK1/2, CDK2/7, CK1d, TNIK and STK26 activity as a common mechanism for the circadian period lengthening drugs. Taken together, our findings reveal that these compounds modulate circadian period through inhibition of multiple kinases. We anticipate that this comprehensive study will contribute towards a better understanding of the mechanism of action of these circadian period altering drugs. This is critical in clearly defining molecular targets in order to modulate daily rhythms for therapeutic benefit.
Dr. Ray is a postdoctoral research associate at the UCL Institute of Neurology and a visiting scientist at The Francis Crick Institute, UK. He is working in the Biological Clocks and Sleep Group headed by Prof. Akhilesh B. Reddy. He has substantial expertise in cutting-edge proteomics technologies and big-data analysis skills with a very solid record of scientific publications in reputed journals. His current research interests are to understand the cross-talks among circadian clocks, sleep-wake cycles, and diverse signaling networks using systems-level approaches. By combining multiplexed quantitative proteomics and Thermal Proteome Profiling (TPP) he is investigating mechanisms of action of circadian period altering drugs in mammalian cells. This is critical in clearly defining molecular targets in order to modulate daily rhythms for therapeutic benefit (e.g. in shift work disorder, jet lag and sleep disorders). He is a 2018 TMT Research Award recipient (Silver Level).