Pharmacological modulators of the circadian clock as potential therapeutic drugs

Abstract

Circadian clocks are molecular time-keeping systems that underlie daily fluctuations in multiple physiological and biochemical processes. It is well recognized now that dysfunctions of the circadian system (both genetically and environmentally induced) are associated with the development of various pathological conditions. Here we describe the application of high throughput screening approach designed to search for small molecules capable of pharmacological modulation of the molecular clock. We provide evidence for the feasibility and value of this approach for both scientific and therapeutic purposes.

Fig. 1

Schematic diagram of the circadian transcription/translation feedback loops showing the CLOCK/BMAL1 complex as an integral regulatory component. See text for details.

Fig. 2

High throughput screening of chemical libraries

Fig. 3

Cell-based readout systems for targeting the components of the molecular clock. (A) Search for modulators of basic circadian parameters (period, amplitude and phase of the oscillations); (B) Search for modulators of the steady-state activity of circadian proteins independent of the oscillation.

Fig. 4

A typical example of an assay plate. Cells were seeded in 96-well clear-bottom luciferase assay plates in phenol red-free DMED supplemented with 10% FBS at a density of 3×10⁴ cells/well (150μl/well). The compounds dissolved in DMSO were added to 80 wells (columns 2–11) in a volume of ~200 nl using pin tool (V&P Scientific, Inc) and automated liquid handling workstation JANUS by PerkinElmer. Columns 1 and 12 of each plate were used as a control; shown in lavender - had 200 nl DMSO added; shown in yellow - Actynomycin D (column 1 at 4mg/ml; column 12 at 1 mg/ml final concentration). After 24-hr incubation, 30 μl of BrightGlo luciferase substrate (Promega) was added, and after 5-min incubation, luciferase signal was measured using GloMax microplate luminometer (Promega). The compounds that decrease luciferase signal 4 standard deviations below the mean of a control were selected as inhibitors of CLOCK/BMAL1-mediated transactivation (shown in red); compounds that increased luciferase signal 4 standard deviations above the mean of a control were selected as activators (shown in green).