Controlling the Circadian Clock with High Temporal Resolution through Photodosing

Abstract

Circadian clocks, biological timekeepers that are present in almost every cell of our body, are complex systems whose disruption is connected to various diseases. Controlling cellular clock function with high temporal resolution in an inducible manner would yield an innovative approach for the circadian rhythm regulation. In the present study, we present structure-guided incorporation of photoremovable protecting groups into a circadian clock modifier, longdaysin, which inhibits casein kinase I (CKI). Using photodeprotection by UV or visible light (400 nm) as the external stimulus, we have achieved quantitative and light-inducible control over the CKI activity accompanied by an accurate regulation of circadian period in cultured human cells and mouse tissues, as well as in living zebrafish. This research paves the way for the application of photodosing in achieving precise temporal control over the biological timing and opens the door for chronophotopharmacology to deeper understand the circadian clock system.

Figure 1

General scheme of the photocleavable approach and putative binding mode of CK1α-longdaysin complex. (A) Schematic representation of the photocleavage approach, where light is used to remove the photoremovable protecting group (PPG) for the release of an active compound. (B) Ligand-binding site of CKIα is characterized by a hinge region (green) and an adjacent cavity formed by the P-loop (yellow area; gray represents P-loop). Docking simulations indicated interaction of the purine scaffold of longdaysin with the hinge region (two hydrogen bonds with Leu93 backbone, indicated by yellow line) (glide XP docking score: −7.39 kcal/mol). The table provides mean estimated ligand binding free energies from molecular docking simulations of longdaysin, DK325, and DK359 with CKIα and CKIδ. SD = standard deviation.

Figure 2

Photodeprotection studies of DK325 and DK359. (A) Photodeprotection of DK325 and DK359 using UV (λ = 365 nm) and violet light (λ = 400 nm). (B) UV–vis spectroscopy analysis of photodeprotection of DK325 and DK359 (40 μM in DMSO, 30 °C) showing clear isosbestic points upon irradiation of DK325 with UV light and DK359 with 400 nm light. (C) UPLC traces for monitoring the deprotection of DK325 (left) and DK359 (right) (40 μM in CKI assay buffer) with UV light. Retention time (min) is shown on the x-axis. Shown are the peaks of longdaysin (11.95 min, black box), DK325 (14.49 min, purple box), and DK359 (14.11 min, blue box). (D) UPLC traces for monitoring the deprotection of DK325 (left) and DK359 (right) (40 μM in cellular assay medium) with 400 nm light. Retention time (min) is shown on the x-axis. Shown are the peaks of longdaysin (11.95 min, black box), DK325 (14.49 min, purple box), and DK359 (14.11 min, blue box).

Figure 3

Inhibition of CK1α in a light-dependent manner. (A) Compounds DK325 and DK359 (40 μM final concentration) were applied to CKIα-reaction mixture. The release of longdaysin was controlled by different irradiation duration (0–60 min) after the reaction was initiated by the addition of ATP and peptide substrate. (B and C) In situ irradiation results. Degree of CK1α inhibition was plotted against irradiation time of UV light, λ = 365 nm (B) and with visible light, λ = 400 nm (C). ATP consumption in DMSO control samples, containing the enzyme and peptide substrate without inhibitor, was set at 100% enzyme activity. (D) Effects of 1 h light irradiation on CKIα activity. Showing a nonirradiated (black), UV-light-irradiated (red; λ = 365 nm) and visible light-irradiated (blue, λ = 400 nm) samples. Results are mean ± SD (n = 2) (B–D).

Figure 4

Irradiation-dependent effects of DK325 and DK359 on circadian rhythms in human U2OS cells. (A) Effect of UV light. Bmal1-dLuc reporter cells were treated with various concentrations of compound (six points of 3-fold dilution series in DMSO) and irradiated with 365 nm light for 0–30 min. Luminescence rhythms were then monitored (the left panel, mean of n = 4). Rhythms of DMSO and longdaysin controls are also shown. Period changes compared to a DMSO control are plotted in the right panels (n = 4); p values are summarized in Table S1. (B, C) Effect of visible light (λ = 400 nm). Bmal1-dLuc reporter cells were treated with compounds and irradiated with 400 nm light for 0–30 min at the beginning (B) or in the middle (C, indicated by arrows) of luminescence monitoring. In (C), period changes pre- and postirradiation are plotted in the top right and bottom right panels, respectively.

Figure 5

Irradiation-dependent effects of DK325 and DK359 on circadian rhythms in mouse tissue explants. Spleen tissue (A–D) and the SCN (E–G) of the Per2::Luc knock-in reporter mice were treated with various concentrations of compound and irradiated with λ = 400 nm light for 0–30 min. Luminescence rhythms were then monitored and shown in (A) and (B) (mean of n = 3–4) and in (E) and (F) (representative result). Period changes compared to a DMSO control are plotted in (C) for concentration-dependent period lengthening, in (D) for irradiation-duration-dependent period lengthening (n = 2–4), and in (G) for the SCN (n = 2). ****p < 0.0001, **p < 0.01, *p < 0.05 against the dark control.

Figure 6

Irradiation-dependent effects of DK359 on circadian rhythms in zebrafish larva. (A) Per3::Luc zebrafish larvae were treated with compound DK359 (4 μM) or DMSO at CT6 and irradiated with λ = 400 nm light for 0 and 10 min. Luminescence rhythms were then monitored for 3 d (mean of n = 3). Data are baseline subtracted for detrending. (B) Period changes compared to a DMSO control for 0, 3, 5, and 10 min light exposure are plotted (n = 13–15). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 against the dark control.