RT Journal Article SR Electronic T1 Phenotypic proteomic profiling identifies a landscape of targets for circadian clock–modulating compounds JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e201900603 DO 10.26508/lsa.201900603 VO 2 IS 6 A1 Sandipan Ray A1 Radoslaw Lach A1 Kate J Heesom A1 Utham K Valekunja A1 Vesela Encheva A1 Ambrosius P Snijders A1 Akhilesh B Reddy YR 2019 UL https://www.life-science-alliance.org/content/2/6/e201900603.abstract AB Determining the exact targets and mechanisms of action of drug molecules that modulate circadian rhythms is critical to develop novel compounds to treat clock-related disorders. Here, we have used phenotypic proteomic profiling (PPP) to systematically determine molecular targets of four circadian period–lengthening compounds in human cells. We demonstrate that the compounds cause similar changes in phosphorylation and activity of several proteins and kinases involved in vital pathways, including MAPK, NGF, B-cell receptor, AMP-activated protein kinases (AMPKs), and mTOR signaling. Kinome profiling further indicated inhibition of CKId, ERK1/2, CDK2/7, TNIK, and MST4 kinases as a common mechanism of action for these clock-modulating compounds. Pharmacological or genetic inhibition of several convergent kinases lengthened circadian period, establishing them as novel circadian targets. Finally, thermal stability profiling revealed binding of the compounds to clock regulatory kinases, signaling molecules, and ubiquitination proteins. Thus, phenotypic proteomic profiling defines novel clock effectors that could directly inform precise therapeutic targeting of the circadian system in humans.