Analysis of the phosphoproteome of CK2α(-/-)/Δα' C2C12 myoblasts compared to the wild-type cells.

CK2 is a Ser/Thr protein kinase composed of two catalytic (α/α') subunits and a non-catalytic ß-subunit dimer, whose activity is often abnormally high in cancer cells. The concept that CK2 may be dispensable for cell survival has been challenged by the finding that viable CK2α/α' knock-out myoblast clones still express small amounts of an N-terminally deleted α' subunit generated during the CRISPR/Cas9 procedure. Here we show that, although the overall CK2 activity of these CK2α(-/-)/Δα' (KO) cells is less than 10% compared to wild-type (WT) cells, the number of phosphosites with the CK2 consensus is comparable to that of WT cells. A more in-depth analysis, however, reveals that the two phosphoproteomes are not superimposable according to a number of criteria, notably a functional analysis of the phosphoproteome found in the two types of cells, and variable sensitivity of the phosphosites to two structurally unrelated CK2 inhibitors. These data support the idea that a minimal CK2 activity, as in KO cells, is sufficient to perform basic housekeeping functions essential for cell survival, but not to accomplish several specialized tasks required upon cell differentiation and transformation. From this standpoint, a controlled downregulation of CK2 would represent a safe and valuable anti-cancer strategy.

CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy.

The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.

Modulation of circadian clock by crude drug extracts used in Japanese Kampo medicine.

Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.

Reversible modulation of circadian time with chronophotopharmacology.

The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.

Comparing the efficacy and selectivity of Ck2 inhibitors. A phosphoproteomics approach.

CK2 (an acronym derived from the misnomer "casein kinase 2") denotes a ubiquitous, highly pleiotropic protein kinase which has been implicated in global human pathologies, with special reference to cancer. A large spectrum of fairly selective, cell permeable CK2 inhibitors are available, one of which, CX4945 is already in clinical trials for the treatment of neoplasia. Another recently developed CK2 inhibitor, GO289, displays in vitro potency and selectivity comparable to CX4945. Here the cellular efficiency of these two inhibitors has been evaluated by treating C2C12 myoblasts for 5 h with each of them at 4 µM concentration and running a quantitative phosphoproteomics analysis of phosphosites affected by the two compounds. A small but significant proportion of the quantified phosphosites is decreased by treatment with CX4945 and, even more with GO289. This figure substantially increases if a subset of quantified phosphosites conforming to the CK2 consensus (pS/pT-x-x-D/E/pS/pT) is considered. Also in this case GO289 is more effective than CX4945. By adopting stringent criteria two shortlists of 70 and 35 sites whose phosphorylation is decreased >50% by GO289 and CX4945, respectively, have been generated. All these phosphosites conform to the consensus of CK2 with just sporadic exceptions. Their WebLogos are indistinguishable from that of bona fide CK2 phosphosites and their Two-Sample Logos rule out any significant contribution of Pro-directed and basophilic protein kinases to their generation. To sum up, we can conclude that by treating C2C12 cells for 5 h with either CX4945 or GO289 off-target effects are negligible since almost all the phosphosites undergoing a substantial reduction are attributable to CK2, with a higher inhibitory efficacy displayed by GO289. CX4945 and GO289 provide highly selective tools to control the CK2-dependent phosphoproteome compared with previously developed CK2 inhibitors.

Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythms.

Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of the cis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity together with the accompanying modulation of cellular circadian rhythms in which CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.

"Janus" efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms.

Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.

A N-terminally deleted form of the CK2α' catalytic subunit is sufficient to support cell viability.

Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual "CK2-like" activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2α' on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immuno-precipitates with the ß-subunit being downregulated by specific CK2α' targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2α' mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2α/α'-/- cells relies on this deleted form of CK2α', whose discovery provides novel perspectives about the biological role of CK2.

An Isoform-Selective Modulator of Cryptochrome 1 Regulates Circadian Rhythms in Mammals.

Cryptochrome 1 (CRY1) and CRY2 are core regulators of the circadian clock, and the development of isoform-selective modulators is important for the elucidation of their redundant and distinct functions. Here, we report the identification and functional characterization of a small-molecule modulator of the mammalian circadian clock that selectively controls CRY1. Cell-based circadian chemical screening identified a thienopyrimidine derivative KL201 that lengthened the period of circadian rhythms in cells and tissues. Functional assays revealed stabilization of CRY1 but not CRY2 by KL201. A structure-activity relationship study of KL201 derivatives in combination with X-ray crystallography of the CRY1-KL201 complex uncovered critical sites and interactions required for CRY1 regulation. KL201 bound to CRY1 in overlap with FBXL3, a subunit of ubiquitin ligase complex, and the effect of KL201 was blunted by knockdown of FBXL3. KL201 will facilitate isoform-selective regulation of CRY1 to accelerate chronobiology research and therapeutics against clock-related diseases.

Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies.

Mammals receive light information through the eyes, which perform two major functions: image forming vision to see objects and non-image forming adaptation of physiology and behavior to light. Cone and rod photoreceptors form images and send the information via retinal ganglion cells to the brain for image reconstruction. In contrast, nonimage-forming photoresponses vary widely from adjustment of pupil diameter to adaptation of the circadian clock. nonimage-forming responses are mediated by retinal ganglion cells expressing the photopigment melanopsin. Melanopsin-expressing cells constitute 1-2% of retinal ganglion cells in the adult mammalian retina, are intrinsically photosensitive, and integrate photic information from rods and cones to control nonimage-forming adaptation. Action spectra of ipRGCs and of melanopsin photopigment peak around 480 nm blue light. Understanding melanopsin function lets us recognize considerable physiological effects of blue light, which is increasingly important in our modern society that uses light-emitting diode. Misalignment of circadian rhythmicity is observed in numerous conditions, including aging, and is thought to be involved in the development of age-related disorders, such as depression, diabetes, hypertension, obesity, and cancer. The appropriate regulation of circadian rhythmicity by proper lighting is therefore essential. This perspective introduces the potential risks of excessive blue light for human health through circadian rhythm disruption and sleep deprivation. Knowing the positive and negative aspects, this study claims the importance of being exposed to light at optimal times and intensities during the day, based on the concept of the circadian clock, ultimately to improve quality of life to have a healthy and longer life.

Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBα Degradation.

Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.

Identification of small molecule activators of cryptochrome.

Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compounds have been identified that selectively target core clock proteins. From an unbiased cell-based circadian phenotypic screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, our studies using KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001-mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes.

High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase.

The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ∼120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.

Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis.

During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)--two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)α. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.

A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta.

The circadian clock controls daily oscillations of gene expression at the cellular level. We report the development of a high-throughput circadian functional assay system that consists of luminescent reporter cells, screening automation, and a data analysis pipeline. We applied this system to further dissect the molecular mechanisms underlying the mammalian circadian clock using a chemical biology approach. We analyzed the effect of 1,280 pharmacologically active compounds with diverse structures on the circadian period length that is indicative of the core clock mechanism. Our screening paradigm identified many compounds previously known to change the circadian period or phase, demonstrating the validity of the assay system. Furthermore, we found that small molecule inhibitors of glycogen synthase kinase 3 (GSK-3) consistently caused a strong short period phenotype in contrast to the well-known period lengthening by lithium, another presumed GSK-3 inhibitor. siRNA-mediated knockdown of GSK-3beta also caused a short period, confirming the phenotype obtained with the small molecule inhibitors. These results clarify the role of GSK-3beta in the period regulation of the mammalian clockworks and highlight the effectiveness of chemical biology in exploring unidentified mechanisms of the circadian clock.