Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening

Abstract

The circadian clock coordinates daily oscillations of essential physiological and behavioral processes. Conversely, aberrant clocks with damped amplitude and/or abnormal period have been associated with chronic diseases and aging. To search for small molecules that perturb or enhance circadian rhythms, we conducted a high-throughput screen of approximately 200,000 synthetic compounds using Per2∷lucSV reporter fibroblast cells and validated 11 independent classes of molecules with Bmal1:luciferase reporter cells as well as with suprachiasmatic nucleus and peripheral tissue explants. Four compounds were found to lengthen the period in both central and peripheral clocks, including three compounds that inhibited casein kinase I ε in vitro and a unique benzodiazepine derivative acting through a non-GABA A receptor target. In addition, two compounds acutely induced Per2∷lucSV reporter bioluminescence, delayed the rhythm, and increased intracellular cAMP levels, but caused rhythm damping. Importantly, five compounds shortened the period of peripheral clocks; among them, four compounds also enhanced the amplitude of central and/or peripheral reporter rhythms. Taken together, these studies highlight diverse activities of drug-like small molecules in manipulating the central and peripheral clocks. These small molecules constitute a toolbox for probing clock regulatory mechanisms and may provide putative lead compounds for treatment of clock-associated diseases.

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

“…The cells were grown as previously described (25). The catalytic subunits of DNA-dependent protein kinase (DNA-PKcs) WT and KO MEF were grown in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin and cultured at 37°C in a humidified incubator with 5% CO 2 .…”

The intricate dance of post-translational modifications in the rhythm of life

“…Chemical inhibition with nonspecific CKI inhibitors increases PER stability and lengthens the circadian period [13][14][15][16]18,19 . CKII also triggers degradation of mammalian (m) PER2 by phosphorylation at Ser53.…”

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

“…Much of what we know about the biochemistry and cell biology of the clock mechanism is based on three cellular models: 3T3 mouse fibroblasts 16,17 , U2OS human osteosarcoma cells 10,11,19,28 , and mouse fibroblasts derived from mice 9,13,34 . The lentiviral vector system permits efficient delivery and stable integration into the host genome of both dividing and non-dividing mammalian cells, and therefore is not limited to certain cell types as in transient transfection.…”

“…HTS assay development mainly focuses on the following two aspects: 1) better reporter cells with brighter luciferase and/or higher reporter expression (see below), and 2) highly sensitive recording devices that accommodate multi-well plates. We and others have used several microplate readers such as Synergy SL2 (BioTek), Infinite M200 (Tecan) 19 , TopCount (PerkinElmer) 28 , and EnVision (Perkin Elmer) 34 . In our lab, both the LumiCycle and Synergy devices are placed in a temperature-controlled and light-tight incubator.…”

“…Cell-based screens, in combination with functional genomics approaches, have been effectively carried out using high-throughput recording systems to screen genome-wide siRNA and shRNA libraries for identification of novel clock factors 10,28 . Similarly, one can employ chemical biological approaches and screen for diverse small molecules to study their effects on clock function 12,19,34,49 . Although major clock genes and their functions have been identified, these screens have identified additional components or modifiers that are involved in regulation or modulation of the clock.…”