MilliporeSigma
  • Phenotypic proteomic profiling identifies a landscape of targets for circadian clock-modulating compounds.

Phenotypic proteomic profiling identifies a landscape of targets for circadian clock-modulating compounds.

Life science alliance (2019-12-04)
Sandipan Ray, Radoslaw Lach, Kate J Heesom, Utham K Valekunja, Vesela Encheva, Ambrosius P Snijders, Akhilesh B Reddy
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Roscovitine, A potent, reversible, and selective inhibitor of Cdks that exhibits about 10-fold greater efficacy towards p34-cdk1 and p33-cdk2 and 20-fold greater efficacy towards p33-cdk5 relative to Olomoucine.
Sigma-Aldrich
Longdaysin, ≥98% (HPLC)
Sigma-Aldrich
meso-Tetraphenylporphyrin, BioReagent, suitable for fluorescence, ≥99.0% (HPLC)
Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, BioXtra, ≥99% (HPLC), from microbial
Sigma-Aldrich
SP600125, ≥98% (HPLC)