Identification and Validation of Cryptochrome Inhibitors That Modulate the Molecular Circadian Clock

Circadian rhythms, biological oscillations with a period of about 24 h, are maintained by a genetically determined innate time-keeping system called the molecular circadian clockwork. Despite the physiological and clinical importance of the circadian clock, the development of small molecule modulators that directly target the core clock machinery has only been recently initiated. In the present study, we aimed to identify novel small molecule modulators influencing the molecular feedback loop of the circadian clock by applying our two-step cell-based screening strategy based on E-box-mediated transcriptional activity to test more than 1000 drug-like compounds. A derivative of 2-ethoxypropanoic acid designated as compound <b>15</b> was selected as the most promising candidate in terms of both efficacy and potency. We then performed pull-down assays with the biotinylated compound and find out that both cryptochrome (CRY)­1 and 2 (CRY1/2), key negative components of the mammalian circadian clock, as molecular targets of compound <b>15</b>. In accordance with the binding property, compound <b>15</b> enhanced E-box-mediated transcription in a CRY1/2-dependent manner, and more importantly, it attenuated the circadian oscillation of Per2-Luc and Bmal1-dLuc activities in cultured fibroblasts, indicating that compound <b>15</b> can functionally inhibit the effects of CRY1/2 in the molecular circadian clockwork. In conclusion, the present study describes the first novel chemical inhibitor of CRY1/2 that inhibits the repressive function of CRY1/2, thereby activating CLOCK-BMAL1-evoked E-box-mediated transcription. Further optimizations and subsequent functional studies of this compound may lead to development of efficient therapeutic strategies for a variety of physiological and metabolic disorders with circadian natures

Meal Time Shift Disturbs Circadian Rhythmicity along with Metabolic and Behavioral Alterations in Mice

<div><p>In modern society, growing numbers of people are engaged in various forms of shift works or trans-meridian travels. Such circadian misalignment is known to disturb endogenous diurnal rhythms, which may lead to harmful physiological consequences including metabolic syndrome, obesity, cancer, cardiovascular disorders, and gastric disorders as well as other physical and mental disorders. However, the precise mechanism(s) underlying these changes are yet unclear. The present work, therefore examined the effects of 6 h advance or delay of usual meal time on diurnal rhythmicities in home cage activity (HCA), body temperature (BT), blood metabolic markers, glucose homeostasis, and expression of genes that are involved in cholesterol homeostasis by feeding young adult male mice in a time-restrictive manner. Delay of meal time caused locomotive hyperactivity in a significant portion (42%) of subjects, while 6 h advance caused a torpor-like symptom during the late scotophase. Accordingly, daily rhythms of blood glucose and triglyceride were differentially affected by time-restrictive feeding regimen with concurrent metabolic alterations. Along with these physiological changes, time-restrictive feeding also influenced the circadian expression patterns of low density lipoprotein receptor (LDLR) as well as most LDLR regulatory factors. Strikingly, chronic advance of meal time induced insulin resistance, while chronic delay significantly elevated blood glucose levels. Taken together, our findings indicate that persistent shifts in usual meal time impact the diurnal rhythms of carbohydrate and lipid metabolisms in addition to HCA and BT, thereby posing critical implications for the health and diseases of shift workers.</p> </div

Weekly food and water consumption profiles.

<p>Young adult male mice were first entrained to a 12∶12 LD photoperiodic cycle for 1 week, fed time-restrictively for 4 weeks, and then fed <i>ad libitum</i> for 2 weeks as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044053#pone-0044053-g001" target="_blank">Figure 1</a>. Weekly consumption of food and water measured regularly at the end of each week. (A) Weekly food consumption profiles during the whole experimental period (left) and total food consumption during the 4 weeks of time restrictive feeding period (right). (B) Weekly water consumption profiles during the whole experimental period (left) and total water consumption during the 4 weeks of time restrictive feeding period (right). Data are expressed as mean ± S.E.M. (n = 4), *p<0.05 <i>vs</i>. other groups.</p

ANOVA <i>F</i> and <i>p</i> values for BT and HCA rhythms.

<p>Significant differences (****<i>p</i><0.0001) are indicated in bold type.</p><p>ANOVA, analysis of variance.</p

Effects of one week of time-restrictive feeding on the phases of Per1, LDLR, and LDLR regulatory factors gene expression in the mouse liver.

<p>Young adult male mice, entrained to a 12∶12 photoperiodic cycle, were fed time-restrictively for seven consecutive days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044053#pone-0044053-g001" target="_blank">Figure 1</a>. On the 8^th day, mice were sacrificed at the indicated zeitgeber time (ZT) and liver samples were obtained. RNA isolation, reverse transcription, and real-time polymerase chain reaction were performed to measure specific messages for mouse <i>Per1</i>, <i>ldlr</i>, and LDLR regulatory factors. All mRNA levels were normalized to <i>tbp</i> mRNA levels. Data are expressed as mean ± S.E.M. (n = 4).</p

Daily rhythms of blood glucose and some metabolic parameters related to cholesterol homeostasis in mice fed time-restrictively.

<p>Young adult male C57BL/6J mice were first entrained to a 12∶12 LD photoperiodic cycle for two weeks. Then mice were fed time-restrictively for seven consecutive days as denoted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044053#pone-0044053-g001" target="_blank">Figure 1</a>. Mice were sacrificed by cervical dislocation at the indicated ZT and whole blood samples were collected. Total cholesterol (A), HDL cholesterol (B), plasma triglyceride (C), blood glucose (D) levels were determined by specific kits obtained from Callegari^TM. All data are expressed as mean ± S.E.M. (n = 4–8). Statistical analyses are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044053#pone-0044053-t002" target="_blank">Table 2</a>.</p

ANOVA <i>F</i> and <i>p</i> values for each physiological index.

<p>Significant differences (*<i>p</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 and ****<i>p</i><0.0001) are indicated in bold type.</p><p>ANOVA, analysis of variance; ns, not significant.</p

Daily rhythms of body temperature in young adult male mice under time-restrictive feeding regimen.

<p>Young adult male mice, surgically implanted with E-mitter probes, were first entrained to a 12∶12 LD photoperiodic cycle for two weeks with food and water available <i>ad libitum</i>. Then mice were fed time-restrictively as schematized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044053#pone-0044053-g001" target="_blank">Figure 1</a> for 4 weeks and returned to being fed <i>ad libitum</i>. Body temperature (BT) was continuously recorded for 7 subsequent weeks (See <i>M&M</i>). (A) Representative double-plot actograms of BT in AF, DF, and NF mice. Dark rectangles above the actograms indicate the 12 h scotophase maintained throughout the experiment. (B) Daily patterns of BT during the time-restrictive feeding. To generate the daily pattern of BT, monitoring results for the whole time-restrictive period were averaged as 1 h bins and the resulting 28 day profiles were pooled according to the indicated ZT to generate the averaged daily pattern (mean ± S.E.M.). Statistical analyses are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044053#pone-0044053-t001" target="_blank">Table 1</a>.</p

Effects of chronic time-restrictive feeding on body weight, fasting blood glucose, glucose tolerance, and response to insulin.

<p>Young adult male mice were first entrained to a 12∶12 LD photoperiodic cycle for 2 weeks and then fed time-restrictively for 9 consecutive weeks. After 16 h fasting, mice were weighed (A) and fasting glucose levels were measured (B) at the 5^th week. Then oral glucose tolerance test was performed. Blood glucose levels over time in response to an oral glucose load (C) and area under the curve for OGTT (D) are shown. At the 9^th week, mice were weighed after 16 h fasting, and fasting glucose levels were measured. Then insulin tolerance test was performed. Blood glucose levels over time in response to insulin (E) and area under the curve (F) were determined. All data are expressed as mean ± S.E.M. (n = 3–4), *p<0.05 <i>vs</i>. control AF group.</p

Daily and circadian expressions of LDLR and some LDLR regulatory factors in the mouse liver.

<p>To determine daily expression patterns, young adult C57BL/6J male mice were entrained to a 12L:12D cycle for two weeks and liver samples were quickly obtained at the indicated ZT. For circadian sampling, mice were entrained to a 12L:12D cycle for two weeks and released to constant darkness (DD). On the second day after light-off, liver samples were obtained at the indicated circadian time (CT). RNA isolation, reverse transcription, and real-time polymerase chain reaction to measure specific messages for mouse <i>ldlr</i>, and LDLR regulatory factors. All mRNA levels were normalized to <i>tbp</i> mRNA levels. Data are expressed as mean ± S.E.M. (n = 8).</p