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Casein kinase 1δ (CK1δ) controls essential biological processes including circadian rhythms and wingless-related integration site (Wnt) signaling, but how its activity is regulated is not well understood. CK1δ is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, δ1 and δ2, are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C termini (XCT), but with marked changes in potential phosphorylation sites. Here, we test whether the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and hydrogen/deuterium exchange mass spectrometry, we show that the δ1 XCT is preferentially phosphorylated by the kinase and the δ1 tail makes more extensive interactions across the kinase domain. Mutation of δ1-specific XCT phosphorylation sites increases kinase activity both in vitro and in cells and leads to changes in the circadian period, similar to what is reported in vivo. Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. δ1 autoinhibition relies on conserved anion-binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1δ. These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase., Competing Interests: Competing interests statement:The authors declare no competing interest.

CK1δ is a serine-threonine kinase involved in several pathological conditions including neuroinflammation and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. Specifically, it seems that an inhibition of CK1δ could have a neuroprotective effect in these conditions. Here, a series of [1,2,4]triazolo[1,5-a][1,3,5]triazines were developed as ATP-competitive CK1δ inhibitors. Both positions 2 and 5 have been explored leading to a total of ten compounds exhibiting IC 50 s comprised between 29.1 µM and 2.08 µM. Three of the four most potent compounds (IC 50  < 3 µM) bear a thiophene ring at the 2 position. All compounds have been submitted to computational studies that identified the chain composed of at least 2 atoms (e.g., nitrogen and carbon atoms) at the 5 position as crucial to determine a key bidentate hydrogen bond with Leu85 of CK1δ. Most potent compounds have been tested in vitro, resulting passively permeable to the blood-brain barrier and, safe and slight neuroprotective on a neuronal cell model. These results encourage to further structural optimize the series to obtain more potent CK1δ inhibitors as possible neuroprotective agents to be tested on models of the above-mentioned neurodegenerative diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

Alzheimer's disease (AD) is an enigmatic neurological illness that offers few treatment options. Recent exploration has highlighted the crucial connection of the Wnt signaling pathway in AD pathogenesis, shedding light on potential therapeutic targets. The present study focuses on the dual targeting of glycogen synthase kinase-3β (GSK-3β) and casein kinase-1δ (CK-1δ) within the framework of the Wnt signaling pathway as a possible technique for AD intervention. GSK-3β and CK-1δ are multifunctional kinases known for their roles in tau hyperphosphorylation, amyloid processing, and synaptic dysfunction, all of which are major hallmarks of Alzheimer's disease. They are intricately linked to Wnt signaling, which plays a pivotal part in sustaining neuronal function and synaptic plasticity. Dysregulation of the Wnt pathway in AD contributes to cognitive decline and neurodegeneration. This review delves into the molecular mechanisms by which GSK-3β and CK-1δ impact the Wnt signaling pathway, elucidating their roles in AD pathogenesis. We discuss the potential of small-molecule inhibitors along with their SAR studies along with the multi-targetd approach targeting GSK-3β and CK-1δ to modulate Wnt signaling and mitigate AD-related pathology. In summary, the dual targeting of GSK-3β and CK-1δ within the framework of the Wnt signaling pathway presents an innovative and promising avenue for future AD therapies, offering new hope for patients and caregivers in the quest to combat this challenging condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Protein kinases are responsible for healthy cellular processes and signalling pathways, and their dysfunction is the basis of many pathologies. There are numerous small molecule inhibitors of protein kinases that systemically regulate dysfunctional signalling processes. However, attaining selectivity in kinase inhibition within the complex human kinome is still a challenge that inspires unconventional approaches. One of those approaches is photopharmacology, which uses light-controlled bioactive molecules to selectively activate drugs only at the intended space and time, thereby avoiding side effects outside of the irradiated area. Still, in the context of kinase inhibition, photopharmacology has thus far been rather unsuccessful in providing light-controlled drugs. Here, we present the discovery and optimisation of a photoswitchable inhibitor of casein kinase 1δ (CK1δ), important for the control of cell differentiation, circadian rhythm, DNA repair, apoptosis, and numerous other signalling processes. Varying the position at which the light-responsive azobenzene moiety has been introduced into a known CK1δ inhibitor, LH846, revealed the preferred regioisomer for efficient photo-modulation of inhibitory activity, but the photoswitchable inhibitor suffered from sub-optimal (photo)chemical properties. Replacement of the bis-phenyl azobenzene group with the arylazopyrazole moiety yielded a superior photoswitch with very high photostationary state distributions, increased solubility and a 10-fold difference in activity between irradiated and thermally adapted samples. The reasons behind those findings are explored with molecular docking and molecular dynamics simulations. Results described here show how the evaluation of privileged molecular architecture, followed by the optimisation of the photoswitchable unit, is a valuable strategy for the challenging design of the photoswitchable kinase inhibitors.

Mammalian cell cytoskeletal reorganization for efficient directional movement requires tight coordination of actomyosin and microtubule networks. In this study, we show that LRAP35a potentiates microtubule stabilization by promoting CLASP2/EB1 interaction besides its complex formation with MRCK/MYO18A for retrograde actin flow. The alternate regulation of these two networks by LRAP35a is tightly regulated by a series of phosphorylation events that dictated its specificity. Sequential phosphorylation of LRAP35a by Protein Kinase A (PKA) and Glycogen Synthase Kinase-3β (GSK3β) initiates the association of LRAP35a with CLASP2, while subsequent binding and further phosphorylation by Casein Kinase 1δ (CK1δ) induce their dissociation, which facilitates LRAP35a/MRCK association in driving lamellar actomyosin flow. Importantly, microtubule dynamics is directly moderated by CK1δ activity on CLASP2 to regulate GSK3β phosphorylation of the SxIP motifs that blocks EB1 binding, an event countered by LRAP35a interaction and its competition for CK1δ activity. Overall this study reveals an essential role for LRAP35a in coordinating lamellar contractility and microtubule polarization in cell migration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fragment-Based Drug Discovery (FBDD) has become, in recent years, a consolidated approach in the drug discovery process, leading to several drug candidates under investigation in clinical trials and some approved drugs. Among these successful applications of the FBDD approach, kinases represent a class of targets where this strategy has demonstrated its real potential with the approved kinase inhibitor Vemurafenib. In the Kinase family, protein kinase CK1 isoform δ (CK1δ) has become a promising target in the treatment of different neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the present work, we set up and applied a computational workflow for the identification of putative fragment binders in large virtual databases. To validate the method, the selected compounds were tested in vitro to assess the CK1δ inhibition.

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.

Protein kinase CK1δ expression and activity is involved in different pathological situations that include neuroinflammatory and neurodegenerative diseases. For this reason, protein kinase CK1δ has become a possible therapeutic target for these conditions. 5,6-fused bicyclic heteroaromatic systems that resemble adenine of ATP represent optimal scaffolds for the development of a new class of ATP competitive CK1δ inhibitors. In particular, a new series of [1,2,4]triazolo[1,5-c]pyrimidines and [1,2,4]triazolo[1,5-a][1,3,5]triazines was developed. Some crucial interactors have been identified, such as the presence of a free amino group able to interact with the residues of the hinge region at the 5- and 7- positions of the [1,2,4]triazolo[1,5-c]pyrimidine and [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffolds, respectively; or the presence of a 3-hydroxyphenyl or 3,5-dihydroxyphenyl moiety at the 2- position of both nuclei. Molecular modeling studies identified the key interactions involved in the inhibitor-protein recognition process that appropriately fit with the outlined structure-activity relationship. Considering the fact that the CK1 protein kinase is involved in various pathologies in particular of the central nervous system, the interest in the development of new inhibitors permeable to the blood-brain barrier represents today an important goal in the pharmaceutical field. The best potent compound of the series is the 5-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)benzen-1,3-diol (compound 51, IC 50  = 0.18 μM) that was predicted to have an intermediate ability to cross the membrane in our in vitro assay and represents an optimal starting point to both studies the therapeutic value of protein kinase CK1δ inhibition and to develop new more potent derivatives., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Discovery of an anticancer medicine using a single target protein has often been unsuccessful due to the complexity of pathogenic mechanisms as well as the presence of redundant signaling pathways. In this work, we attempted to find promising anticancer drug candidates by simultaneously targeting casein kinase 1 delta (CK1δ) and muscarinic acetylcholine receptor M3 (M3R). Through the structure-based virtual screening and de novo design with the modified potential function for protein-ligand binding, a series of benzo[4,5]imidazo[1,2-a][1,3,5]triazine-2-amine (BITA) derivatives were identified as CK1δ inhibitors and also as M3R antagonists. The biochemical potencies of these bifunctional molecules reached the nanomolar and low-micromolar levels with respect to CK1δ and M3R, respectively. A common interaction feature in the calculated CK1δ-inhibitor and M3R-antagonist complexes is that the BITA moiety is well-stabilized in the orthosteric site of M3R and the hinge region of CK1δ through the establishment of the three hydrogen bonds and the hydrophobic contacts in the vicinity. The computational and experimental results found in this work exemplify the efficiency of kinase and GPCR polypharmacology in developing anticancer medicines.

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression., (©2020 American Association for Cancer Research.)

Bladder cancer is the second most common genitourinary malignancy in the world. However, only immune-checkpoint inhibitors and erdafitinib are available to treat advanced bladder cancer. Our previous study reported that 4-((4-(4-ethylpiperazin-1-yl) phenyl)amino)-N-(3,4,5-trichlorophenyl)-7H-pyrrolo-[2, 3-d]pyrimidine-7-carboxamide hydrochloride (13i HCl) is a potent CK1δ inhibitor showing significant anti-bladder cancer activity. In this study, we elucidated the pharmacological mechanisms underlying 13i HCl's inhibition of human bladder cancer. Our results demonstrate that expression of the CSNK1D gene, which codes for CK1δ, is upregulated in superficial and infiltrating bladder cancer patients in two independent datasets. CK1δ knockdown decreased β-catenin expression in bladder cancer cells and inhibited their growth. Additionally, 13i HCl suppressed bladder cancer cell proliferation and increased apoptosis. We also observed that inhibition of CK1δ using 13i HCl or PF-670462 triggers necroptosis in bladder cancer cells. Finally, 13i HCl inhibited bladder cancer cell migration and reversed their mesenchymal characteristics. These findings suggest further development of 13i HCl as a potential therapeutic agent to treat bladder cancer is warranted.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide that responds poorly to existing therapies. The Casein kinase 1 (CK1) isoforms CK1δ and CK1ε are reported to be highly expressed in several tumor types, and both genetic and pharmacological inhibition of CK1δ/ε activity has deleterious effects on tumor cell growth. IC261, an CK1δ/ε selectively inhibitor, shows anti-tumor effect against pancreatic tumor and glioblastoma, but its role in HCC remains poorly characterized. In our research, IC261 displayed time- and dose-dependent inhibition of HCC cell proliferation, and induced G2/M arrest and cell apoptosis in vitro. However, the anti-tumor effects of IC261 was independent of CK1δ/ε. Additionally, IC261 was verified to induce centrosome fragmentation during mitosis independent of CK1δ status, and intraperitoneal injection of IC261 to HCCLM3 xenograft models inhibited tumor growth. Taken together, our data indicated that IC261 has therapeutic potential for HCC., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2020. Published by Elsevier Inc.)

Pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease where no treatment exists, involves the compartmentalization of the nuclear protein TDP-43 (TAR DNA-binding protein 43) in the cytoplasm which is promoted by its aberrant phosphorylation and others posttranslational modifications. Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis (phosphorylation and localization) in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the in vivo modulation of TDP-43 toxicity by CK-1δ inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1δ inhibitors in a future disease-modifying treatment for ALS.

Casein kinase 1δ (CK1δ) and casein kinase 1ε (CK1ε) have been proposed to be involved in DNA replication, differentiation and apoptosis, thus participating in the regulation of tumorigenesis. However, their functions in colon cancer and the underlying mechanism remain unclear. Here, we found that the expression of CK1ε and CK1δ increased significantly in cancer tissues and the upregulation of CK1ε and CK1δ were closely related to poor differentiation, advanced TNM stage and poor prognosis of colon cancer. CK1δ/ε inhibitor IC261 could induce a decrease in cell survival and proliferation, and an increase in apoptosis in colon cancer cells. Interestingly, IC261 increased the level of aerobic glycolysis in colon cancer cells. Meanwhile, IC261 caused the decrease of p53 protein level and the misregulation of glycolysis related genes (TIGAR, G6PD, GLUT1) which are closely related to the regulation of glycolysis by p53. Inhibiting p53 by siRNA or inhibitor could significantly attenuate the upregulation of aerobic glycolysis induced by IC261. Finally, inhibition of aerobic glycolysis can further increase the cytotoxicity induced by IC261. Collectively, our results revealed that IC261 could inhibit the growth of colon cancer cells and increase the level of aerobic glycolysis, which is regulated by p53-dependent manner. This result suggested that targeting CK1δ/ε and glycolysis might be a valuable strategy treatment and combination therapies for colon cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

PF-05251749 is a dual inhibitor of casein kinase 1 δ/ε, key regulators of circadian rhythm. As a result of its mechanism of action, PF-05251749 may also change the heart rate corrected QT (QTc) circadian rhythm, which may confound detection of drug-induced QTc prolongation. In this analysis, a nonlinear mixed effect model including a multioscillator function was developed in addition to fitting the prespecified linear mixed effect concentration-QTc model, to identify QTc liability of PF-05251749 in the presence of potential circadian rhythm change. The modeling results suggested lack of clinically meaningful QTc prolongation (upper bound of 90% confidence interval for ∆∆QTc < 10 milliseconds) and that the drug-induced QTc circadian rhythm change was not present. However, simulation results indicated that inference of drug-induced QTc prolongation could be misleading if the drug effect on QTc circadian rhythm is not properly addressed. The modeling and simulation results suggest that prespecification of the concentration-QTc model should be reconsidered for drugs with circadian rhythm modulation potential., (© 2019 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Protein kinases of the CK1 family can be involved in numerous physiological and pathophysiological processes. Dysregulated expression and/or activity as well as mutation of CK1 isoforms have previously been linked to tumorigenesis. Among all neoplastic diseases, colon and rectal cancer (CRC) represent the fourth leading cause of cancer related deaths. Since mutations in CK1δ previously found in CRC patients exhibited increased oncogenic features, inhibition of CK1δ is supposed to have promising therapeutic potential for tumors, which present overexpression or mutations of this CK1 isoform. Therefore, it is important to develop new small molecule inhibitors exhibiting higher affinity toward CK1δ mutants. In the present study, we first characterized the kinetic properties of CK1δ mutants, which were detected in different tumor entities. Subsequently, we characterized the ability of several newly developed IWP-based inhibitors to inhibit wild type and CK1δ mutants and we furthermore analyzed their effects on growth inhibition of various cultured colon cancer cell lines. Our results indicate, that these compounds represent a promising base for the development of novel CRC therapy concepts.

The molecular clock network in mast cells has been shown to be a factor responsible for circadian regulation of allergic inflammation. PF670462 is a selective inhibitor of casein kinase 1δ and ε (CK1δ/ε) that control the posttranslational modification of clock proteins. The aims of this study were to evaluate the effects of PF670462 on gene and protein expression of FcεRI, the high-affinity IgE receptor, in canine mast cells and on IgE-mediated immediate-type cutaneous reactions in dogs. PF670462 decreased mRNA expression of FcεRIα and β, but not γ, and protein expression of FcεRI in a canine mast cell line. Furthermore, PF670462 suppressed IgE-mediated immediate-type cutaneous erythema in dogs. These findings indicate that CK1δ/ε function as regulators for FcεRI expression and IgE-mediated cutaneous reactions in dogs.

Members of the highly conserved pleiotropic CK1 family of serine/threonine-specific kinases are tightly regulated in the cell and play crucial regulatory roles in multiple cellular processes from protozoa to human. Since their dysregulation as well as mutations within their coding regions contribute to the development of various different pathologies, including cancer and neurodegenerative diseases, they have become interesting new drug targets within the last decade. However, to develop optimized CK1 isoform-specific therapeutics in personalized therapy concepts, a detailed knowledge of the regulation and functions of the different CK1 isoforms, their various splice variants and orthologs is mandatory. In this review we will focus on the stress-induced CK1 isoform delta (CK1δ), thereby addressing its regulation, physiological functions, the consequences of its deregulation for the development and progression of diseases, and its potential as therapeutic drug target., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sleep disturbances and memory impairment are common symptoms of Alzheimer's disease (AD). Given that the circadian clock regulates sleep, hippocampal function, and neurodegeneration, it represents a therapeutic target against AD. Casein kinase 1δ/ε (CK1δ/ε) are clock regulators and overexpressed in AD brains, making them viable targets to improve sleep and cognition. In this study, we evaluated the therapeutic potential of a small molecule CK1δ/ε inhibitor (PF-670462) in a triple transgenic mouse model of AD (3xTg-AD). Mass spectrometry-based proteomic analyses revealed that PF-670462 administration in 3xTg-AD mice reversed hippocampal proteomic alterations in several AD-related and clock-regulated pathways, including synaptic plasticity and amyloid precursor protein processing. Furthermore, PF-670462 administration rescued working memory deficits and normalized behavioral circadian rhythm disturbances in 3xTg-AD mice. Our study provides in vivo proof of concept for CK1δ/ε inhibition against AD-associated hippocampal proteomic changes, memory impairment, and circadian disturbances.

In mammals, the master circadian clock synchronizes daily rhythms of physiology and behavior with the day-night cycle. Failure of synchrony, which increases the risk for numerous chronic diseases, can be treated by phase adjustment of the circadian clock pharmacologically, for example, with melatonin, or a CK1δ/ε inhibitor. Here, using in silico experiments with a systems pharmacology model describing molecular interactions, and pharmacokinetic and behavioral experiments in cynomolgus monkeys, we find that the circadian phase delay caused by CK1δ/ε inhibition is more strongly attenuated by light in diurnal monkeys and humans than in nocturnal mice, which are common preclinical models. Furthermore, the effect of CK1δ/ε inhibition strongly depends on endogenous PER2 protein levels, which differs depending on both the molecular cause of the circadian disruption and the patient's lighting environment. To circumvent such large interindividual variations, we developed an adaptive chronotherapeutics to identify precise dosing regimens that could restore normal circadian phase under different conditions. Our results reveal the importance of photosensitivity in the clinical efficacy of clock-modulating drugs, and enable precision medicine for circadian disruption., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors.

In this study, we report on the modification of a 3,4-diaryl-isoxazole-based CK1 inhibitor with chiral pyrrolidine scaffolds to develop potent and selective CK1 inhibitors. The pharmacophore of the lead structure was extended towards the ribose pocket of the adenosine triphosphate (ATP) binding site driven by structure-based drug design. For an upscale compatible multigram synthesis of the functionalized pyrrolidine scaffolds, we used a chiral pool synthetic route starting from methionine. Biological evaluation of key compounds in kinase and cellular assays revealed significant effects of the scaffolds towards activity and selectivity, however, the absolute configuration of the chiral moieties only exhibited a limited effect on inhibitory activity. X-ray crystallographic analysis of ligand-CK1δ complexes confirmed the expected binding mode of the 3,4-diaryl-isoxazole inhibitors. Surprisingly, the original compounds underwent spontaneous Pictet-Spengler cyclization with traces of formaldehyde during the co-crystallization process to form highly potent new ligands. Our data suggests chiral "ribose-like" pyrrolidine scaffolds have interesting potential for modifications of pharmacologically active compounds.

Glycogen synthase kinase 3β (GSK-3β) and casein kinase 1δ (CK-1δ) are emerging targets for the treatment of neuroinflammatory disorders, including Parkinson's disease. An inhibitor able to target these two kinases was developed by docking-based design. Compound 12, 3-(7-amino-5-(cyclohexylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)-2-cyanoacrylamide, showed combined inhibitory activity against GSK-3β and CK-1δ [IC 50 (GSK-3β)=0.17 μm; IC 50 (CK-1δ)=0.68 μm]. In particular, classical ATP competition was observed against CK-1δ, and a co-crystal of compound 12 inside GSK-3β confirmed a covalent interaction between the cyanoacrylamide warhead and Cys199, which could help in the development of more potent covalent inhibitors of GSK-3β. Preliminary studies on in vitro models of Parkinson's disease revealed that compound 12 is not cytotoxic and shows neuroprotective activity. These results encourage further investigations to validate GSK-3β/CK-1δ inhibition as a possible new strategy to treat neuroinflammatory/degenerative diseases., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Riluzole, approved by the US Food and Drug Administration (FDA) in 1995, is the most widespread oral treatment for the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). The drug, whose mechanism of action is still obscure, mitigates progression of the illness, but unfortunately with only limited improvements. Herein we report the first demonstration, using a combination of computational and in vitro studies, that riluzole is an ATP-competitive inhibitor of the protein kinase CK1 isoform δ, with an IC 50 value of 16.1 μm. This allows us to rewrite its possible molecular mechanism of action in the treatment of ALS. The inhibition of CK1δ catalytic activity indeed links the two main pathological hallmarks of ALS: transactive response DNA-binding protein of 43 kDa (TDP-43) proteinopathy and glutamate excitotoxicity, exacerbated by the loss of expression of glial excitatory amino acid transporter-2 (EAAT2)., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The tumor promoter 12- O -tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/β-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/β-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/β-catenin signaling in a casein kinase 1 (CK1) ε/δ-dependent manner. TPA stabilized CK1ε and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1ε-LRP6-axin1 complex, leading to an increase in cytosolic β-catenin. Moreover, TPA increased the association of β-catenin with TCF4E in a CK1ε/δ-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1ε/δ inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/β-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/β-catenin signaling., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wt CK1δ. IWPs also strongly inhibited the gatekeeper mutant M82F CK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.

Casein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1-induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition., (© 2018 by The American Society of Hematology.)

Casein kinase 1δ/ε have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1δ/ε inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Described herein is the design, synthesis and biological evaluation of a series of N-(1H-pyrazol-3-yl)quinazolin-4-amines against a panel of eight disease relevant protein kinases. The kinase inhibition results indicated that two compounds inhibited casein kinase 1δ/ε (CK1δ/ε) with some selectivity over related kinases, namely CDK5/p25, GSK-3α/β, and DYRK1A. Docking studies with 3c and 3d revealed the key interactions with desired amino acids in the ATP binding site of CK1δ. Furthermore, compound 3c also elicited selective cytotoxic activity against the pancreas ductal adenocarcinoma (PANC-1) cell line. Taken together, the results of this study establish N-(1H-pyrazol-3-yl)quinazolin-4-amines especially 3c and 3d as valuable lead molecules with great potential for CK1δ/ε inhibitor development targeting neurodegenerative disorders and cancer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The involvement of protein kinase CK1δ in the pathogenesis of severe disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, familial advanced sleep phase syndrome, and cancer has dramatically increased interest in the development of effective small molecule inhibitors for both therapeutic application and basic research. Unfortunately, the design of CK1 isoform-specific compounds has proved to be highly complicated due to the existence of six evolutionarily conserved human CK1 members that possess similar, different, or even opposite physiological and pathophysiological implications. Consequently, only few potent and selective CK1δ inhibitors have been reported so far and structurally divergent approaches are urgently needed in order to establish SAR that might enable complete discrimination of CK1 isoforms and related p38α MAPK. In this study we report on design and characterization of optimized 4,5-diarylimidazoles as highly effective ATP-competitive inhibitors of CK1δ with compounds 11b (IC 50 CK1δ = 4 nM, IC 50 CK1ε = 25 nM), 12a (IC 50 CK1δ = 19 nM, IC 50 CK1ε = 227 nM), and 16b (IC 50 CK1δ = 8 nM, IC 50 CK1ε = 81 nM) being among the most potent CK1δ-targeting agents published to date. Inhibitor compound 11b , displaying potential as a pharmacological tool, has further been profiled over a panel of 321 protein kinases exhibiting high selectivity. Cellular efficacy has been evaluated in human pancreatic cancer cell lines Colo357 (EC 50 = 3.5 µM) and Panc89 (EC 50 = 1.5 µM). SAR is substantiated by X-ray crystallographic analysis of 16b in CK1δ and 11b in p38α.

Background: Tar DNA binding protein 43 (TDP-43) hyperphosphorylation, caused by Casein kinase 1 (CK-1) protein isoforms, is associated with the onset and progression of Amyotrophic Lateral Sclerosis (ALS). Among the reported isoforms and splice variants of CK-1 protein superfamily, CK-1δ is known to phosphorylate different serine and threonine sites on TDP-43 protein in vitro and thus qualifies as a potential target for ALS treatment., Results: The developed GQSAR (group based quantitative structure activity relationship) model displayed satisfactory statistical parameters for the dataset of experimentally reported N-Benzothiazolyl-2-Phenyl Acetamide derivatives. A combinatorial library of molecules was also generated and the activities were predicted using the statistically sound GQSAR model. Compounds with higher predicted inhibitory activity were screened against CK-1δ that resulted in to the potential novel leads for CK-1δ inhibition., Conclusions: In this study, a robust fragment based QSAR model was developed on a congeneric set of experimentally reported molecules and using combinatorial library approach, a series of molecules were generated from which we report two top scoring, CK-1δ inhibitors i.e., CHC (6-benzyl-2-cyclopropyl-4-{[(4-cyclopropyl-6-ethyl-1,3-benzothiazol-2-yl)carbamoyl]methyl}j-3-fluorophenyl hydrogen carbonate) and DHC (6-benzyl-4-{[(4-cyclopropyl-6-ethyl-1,3-benzothiazol-2-yl)carbamoyl]methyl}-2-(decahydronaphthalen-1-yl)-3-hydroxyphenyl hydrogen carbonate) with binding energy of -6.11 and -6.01 kcal/mol, respectively.

The halogenated alkaloid chloromethylhalicyclamine B (1), together with the known natural compound halicyclamine B (2), was isolated from the extract of the sponge Acanthostrongylophora ingens. The structure of compound 1 was determined by spectroscopic means, and it was shown that 1 is produced by reaction of 2 with CH 2 Cl 2 used for extraction. Compound 1 was a selective CK1δ/ε inhibitor with an IC 50 of 6 μM, while the natural compound 2 was inactive. The absolute configuration of 1 was determined by quantum mechanical calculation of its ECD spectrum, and this also determined the previously unknown absolute configuration of the parent halicyclamine B (2). Computational studies, validated by NOESY data, showed that compound 1 can efficiently interact with the ATP-binding site of CK1δ in spite of its globular structure, very different from the planar structure of known inhibitors of CK1δ. This opens the way to the design of a new structural type of CK1δ/ε inhibitors.

Background: Mutations in the progranulin gene (GRN) are the most common cause of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). TDP-43 pathology is characterized by the hyperphosphorylation of the protein at Serine 409/410 residues. Casein kinase-1δ (CK-1δ) was reported to phosphorylate TDP-43 directly. Previous works from our laboratory described the presence of CDK6/pRb-dependent cell cycle alterations, and cytosolic accumulation of TDP-43 protein in lymphoblast from FTLD-TDP patients carriers of a loss-of function mutation in GRN gene (c.709-1G > A). In this work, we have investigated the effects of two brain penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27) designed and synthetized in our laboratory on cell proliferation, TDP-43 phosphorylation and subcellular localization, as well as their effects on the known nuclear TDP-43 function repressing the expression of CDK6., Results: We report here that both CK-1δ inhibitors (IGS-2.7 and IGS-3.27) normalized the proliferative activity of PGRN-deficient lymphoblasts by preventing the phosphorylation of TDP-43 fragments, its nucleo-cytosol translocation and the overactivation of the CDK6/pRb cascade. Moreover, ours results show neuroprotective effects of CK-1δ inhibitors in a neuronal cell model of induced TDP-43 phosphorylation., Conclusions: Our results suggest that modulating CK-1δ activity could be considered a novel therapeutic approach for the treatment of FTLD-TDP and other TDP-43 proteinopathies.

Background: The circadian clock temporally gates signaling through the high-affinity IgE receptor (FcεRI) in mast cells, thereby generating a marked day/night variation in allergic reactions. Thus manipulation of the molecular clock in mast cells might have therapeutic potential for IgE-mediated allergic reactions., Objective: We determined whether pharmacologically resetting the molecular clock in mast cells or basophils to times when FcεRI signaling was reduced (ie, when core circadian protein period 2 [PER2] is upregulated) resulted in suppression of IgE-mediated allergic reactions., Methods: We examined the effects of PF670462, a selective inhibitor of the key clock component casein kinase 1δ/ε, or glucocorticoid, both of which upregulated PER2 in mast cells, on IgE-mediated allergic reactions both in vitro and in vivo., Results: PF670462 or corticosterone (or dexamethasone) suppressed IgE-mediated allergic reactions in mouse bone marrow-derived mast cells or basophils and passive cutaneous anaphylactic reactions in mice in association with increased PER2 levels in mast cells or basophils. PF670462 or dexamethasone also ameliorated allergic symptoms in a mouse model of allergic rhinitis and downregulated allergen-specific basophil reactivity in patients with allergic rhinitis., Conclusion: Pharmacologically resetting the molecular clock in mast cells or basophils to times when FcεRI signaling is reduced can inhibit IgE-mediated allergic reactions. The results suggest a new strategy for controlling IgE-mediated allergic diseases. Additionally, this study suggests a novel mechanism underlying the antiallergic actions of glucocorticoids that relies on the circadian clock, which might provide a novel insight into the pharmacology of this drug in allergic patients., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Identification of specific drivers of human cancer is required to instruct the development of targeted therapeutics. We demonstrate that CSNK1D is amplified and/or overexpressed in human breast tumors and that casein kinase 1δ (CK1δ) is a vulnerability of human breast cancer subtypes overexpressing this kinase. Specifically, selective knockdown of CK1δ, or treatment with a highly selective and potent CK1δ inhibitor, triggers apoptosis of CK1δ-expressing breast tumor cells ex vivo, tumor regression in orthotopic models of triple-negative breast cancer, including patient-derived xenografts, and tumor growth inhibition in human epidermal growth factor receptor 2-positive (HER2(+)) breast cancer models. We also show that Wnt/β-catenin signaling is a hallmark of human tumors overexpressing CK1δ, that disabling CK1δ blocks nuclear accumulation of β-catenin and T cell factor transcriptional activity, and that constitutively active β-catenin overrides the effects of inhibition or silencing of CK1δ. Thus, CK1δ inhibition represents a promising strategy for targeted treatment in human breast cancer with Wnt/β-catenin involvement., (Copyright © 2015, American Association for the Advancement of Science.)

We have previously demonstrated that the circadian clock protein period (Per)1 coordinately regulates multiple genes involved in Na(+) reabsorption in renal collecting duct cells. Consistent with these results, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. The proximal tubule is responsible for a majority of Na(+) reabsorption. Previous work has demonstrated that expression of Na(+)/H(+) exchanger 3 (NHE3) oscillates with a circadian pattern and Na(+)-glucose cotransporter (SGLT)1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of the present study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3 but not SGLT2 in the renal cortex of mice. Per1 small interfering RNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA suggested that the effects of Per1 on NHE3 and SGLT1 expression occurred at the level of transcription. Per1 and the circadian protein CLOCK were detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na(+)-K(+)-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney., (Copyright © 2015 the American Physiological Society.)

In this study as the first attempt; comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA) and AutoGPA-based 3D-QSAR methods were applied on a set of 47 recently reported Ck1d inhibitors, in order to gain an insight into the structural requirements which providing guidelines for the design of next generation compounds with enhanced bioactivity. The results of 3D-QSAR analyses indicated that hydrophobic and negatively charged groups at 6th position of benzothiazole ring and positively charged and bulky groups at ortho position of phenyl ring are favorable for high activity. Moreover, molecular docking studies with GOLD protocol revealed that this chemical series has two different orientations in CK1d active site: orientation 1, in which the benzothiazole ring of the compounds is the closet to the hydrophobic area created by Ile23 and 37, Ala36 Lys 38, Met80, 82 and Val81, and orientation 2, in which the benzene ring of the compounds is directed toward the hydrophobic center. Molecular docking result of the riluzole, the only drug approved by FDA for amyotrophic lateral sclerosis (ALS), indicated that the orientation 2 is preferred due to the presence of OCF3 group in R(1) situation at 6th position of benzothiazole ring, while with replacement of OCF3 group by CF3, the orientation 1 is observed. At the end, to find similar analogs by virtual screening, a two-stage approach: pharmacophore-based screening using generated AutoGPA-based 3D-QSAR model followed by structure-based virtual screening using molecular docking was employed. Visual inspection of the docking results of virtually obtained hits revealed two different binding orientations, in which compounds with high GOLD fitness scores produced binding modes, which were the same as the one observed in compounds with orientation 1, whereas the binding modes of the structures with low GOLD fitness scores were in agreement with orientation 2. Further, the drug-like properties of the obtained seven hits with the highest GOLD scores were investigated as a tool to optimize the selection of the most suitable candidates for drug development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Circadian rhythms are generated through a transcription-translation feedback loop involving clock genes and the casein kinases CSNK1D and CSNK1E. In this study, we investigated the effects of the casein kinase inhibitor PF-670462 (50 mg/kg) on rhythmic expression of clock genes in the liver, pancreas and suprachiasmatic nucleus (SCN) as well as plasma corticosterone, melatonin and running behaviour in rats and compared them to the responses to a 4 h extension of the light phase. PF-670462 acutely phase delayed the rhythmic transcription of Bmal1, Per1, Per2 and Nr1d1 in both liver and pancreas by 4.5 ± 1.3 and 4.5 ± 1.2 h, respectively, 1 day after administration. In the SCN, the rhythm of Nr1d1 and Dbp mRNA expression was delayed by 4.2 and 4 h, respectively. Despite these changes, the time of peak plasma melatonin secretion was not delayed, although the plasma corticosterone rhythm and onset of wheel-running activity were delayed by 2.1 and 1.1 h, respectively. These changes are in contrast to the effects of the 4 h light extension, which resulted in delays in peak expression of the clock genes of less than 1 h and no change in the melatonin or corticosterone rhythms. The ability of the casein kinase inhibitor to bring about large phase shifts in the rhythms of major metabolic target tissues may lead to new drugs being developed to rapidly phase adjust circadian rhythms to alleviate the metabolic impact of shift work.

Deregulation of CK1 (casein kinase 1) activity can be involved in the development of several pathological disorders and diseases such as cancer. Therefore, research interest in identifying potent CK1-specific inhibitors is still increasing. A previously published potent and selective benzimidazole-derived CK1δ/ε-specific inhibitor compound with significant effects on several tumor cell lines was further modified to difluoro-dioxolo-benzoimidazole derivatives displaying remarkable inhibitory effects and increased intracellular availability. In the present study, we identified two heterocyclic molecules as new CK1-specific inhibitor compounds with favorable physicochemical properties and notable selectivity in a kinome-wide screen. Being compared to other CK1 isoforms, these compounds exhibited advanced isoform selectivity toward CK1δ. Moreover, newly designed compounds showed increased growth inhibitory activity in a panel of different tumor cell lines as determined by analyses of cell viability and cell cycle distribution. In summary, presented lead optimization resulted in new highly selective CK1δ-specific small molecule inhibitors with increased biological activity.

The synthesis and structure-activity relationships of novel 4-(4'-fluorophenyl)imidazoles as selective p38α MAPK, CK1δ and JAK2 inhibitors with improved water solubility are described. Microwave-assisted multicomponent reactions afforded 4-fluorophenyl-2,5-disubstituted imidazoles. Carboxylate and phosphonate groups were introduced via 'click' reactions. The kinase selectivity was influenced by the heteroaryl group at imidazole C-5 and the position of a carboxylic acid or tetrazole at imidazole C-2. For example, pyrimidines 15 and 34 inhibited p38α MAPK with IC50=250 nM and 96 nM, respectively. Pyridine 3 gave CK1δ inhibition with IC50=89 nM and pyridin-2-one 31 gave JAK2 inhibition with IC50=62 nM., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inhibition of casein kinase 1 delta (CK1δ) blocks primary ciliogenesis in human telomerase reverse transcriptase immortalized retinal pigmented epithelial and mouse inner medullary collecting duct cells-3. Mouse embryonic fibroblasts (MEFs) and retinal cells from Csnk1d (CK1δ)-null mice also exhibit ciliogenesis defects. CK1δ catalytic activity and centrosomal localization signal (CLS) are required to rescue cilia formation in MEFs(Csnk1d null). Furthermore, expression of a truncated derivative containing the CLS displaces full-length CK1δ from the centrosome and decreases ciliary length in control MEFs, suggesting that centrosomal CK1δ has a role in ciliogenesis. CK1δ inhibition also alters pericentrosomal or ciliary distribution of several proteins involved in ciliary transport, including Ras-like in rat brain-11A, Ras-like in rat brain-8A, centrosomal protein of 290 kDa, pericentriolar material protein 1, and polycystin-2, as well as the Golgi distribution of its binding partner, A-kinase anchor protein 450 (AKAP450). As reported for AKAP450, CK1δ was required for microtubule nucleation at the Golgi and maintenance of Golgi integrity. Overexpression of an AKAP450 fragment containing the CK1δ-binding site inhibits Golgi-derived microtubule nucleation, Golgi distribution of intraflagellar transport protein 20 homologue, and ciliogenesis. Our results suggest that CK1δ mediates primary ciliogenesis by multiple mechanisms, one involving its centrosomal function and another dependent on its interaction with AKAP450 at the Golgi, where it is important for maintaining Golgi organization and polarized trafficking of multiple factors that mediate ciliary transport., (© 2014 Greer et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.

The development of a series of potent and highly selective casein kinase 1δ/ε (CK1δ/ε) inhibitors is described. Starting from a purine scaffold inhibitor (SR-653234) identified by high throughput screening, we developed a series of potent and highly kinase selective inhibitors, including SR-2890 and SR-3029, which have IC₅₀ ≤ 50 nM versus CK1δ. The two lead compounds have ≤100 nM EC50 values in MTT assays against the human A375 melanoma cell line and have physical, in vitro and in vivo PK properties suitable for use in proof of principle animal xenograft studies against human cancer cell lines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Zebrafish have become a popular model for studies of the circadian timing mechanism. Taking advantage of its rapid development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines, much knowledge has been gained about the circadian clock system in this species. However, the post-translational modifications of clock proteins, and in particular the phosphorylation of PER proteins by Casein kinase I delta and epsilon (CK1δ and CK1ε), have so far not been examined in the zebrafish. Using pharmacological inhibitors for CK1δ and CK1ε, a pan-CK1δ/ε inhibitor PF-670462, and a CK1ε -selective inhibitor PF-4800567, we show that CK1δ activity is crucial for the functioning of the circadian timing mechanism of zebrafish, while CK1ε plays a minor role. The CK1δ/ε inhibitor disrupted circadian rhythms of promoter activity in the circadian clock-containing zebrafish cell line, PAC-2, while the CK1ε inhibitor had no effect. Zebrafish larvae that were exposed to the CK1δ/ε inhibitor showed no rhythms of locomotor activity while the CK1ε inhibitor had only a minor effect on locomotor activity. Moreover, the addition of the CK1δ/ε inhibitor disrupted rhythms of aanat2 mRNA expression in the pineal gland. The pineal gland is considered to act as a central clock organ in fish, delivering a rhythmic hormonal signal, melatonin, which is regulated by AANAT2 enzymatic activity. Therefore, CK1δ plays a key role in the circadian timing system of the zebrafish. Furthermore, the effect of CK1δ inhibition on rhythmic locomotor activity may reflect its effect on the function of the central clock in the pineal gland as well as its regulation of peripheral clocks.

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function and blood pressure. We previously showed that the circadian clock protein Period (Per)1, positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (αENaC), which contributes to blood pressure regulation. Casein kinases 1δ and 1ε (CK1δ/ε) are critical regulators of clock proteins. CK1δ/ε must phosphorylate the circadian clock protein Per1 in order for the latter to enter the nucleus. We used a commercially available CK1δ/ε inhibitor, PF670462, to test the effect of CK1δ/ε blockade and inhibited Per1 nuclear entry on αENaC in a model of the renal cortical collecting duct (mpkCCD(c14) cells). CK1δ/ε blockade prevented Per1 and Clock from interacting with an E-box from the αENaC promoter. CK1δ/ε inhibition reduced αENaC mRNA levels by <60%. A similar decrease in αENaC mRNA was observed following siRNA-mediated CK1δ/ε knock-down. Inhibition of CK1δ/ε effectively prevented the transcriptional response of αENaC to aldosterone, suggesting an interaction between the circadian clock and aldosterone-mediated regulation of αENaC. CK1δ/ε inhibition significantly reduced αENaC but increased Caveolin-1 membrane protein levels; transepithelial current, a measure of ENaC activity, was decreased. Importantly, single channel analysis in amphibian renal cells demonstrated a dramatic decrease in the number of patches with observable ENaC current following CK1δ/ε inhibition. The present study shows for the first time that CK1δ/ε inhibition and impaired Per1 nuclear entry results in decreased αENaC expression and ENaC activity, providing further support for direct control of ENaC by the circadian clock.

In this study we identified two heterocyclic compounds (5 and 6) as potent and specific inhibitors of CK1δ (IC(50) = 0.040 and 0.042 μM, respectively). Whereas compound 5 exhibited fivefold higher affinity towards CK1δ than to CK1ε (IC(50) CK1ε = 0.199 μM), compound 6 also inhibited CK1ε (IC(50) = 0.0326 μM) in the same range as CK1δ. Selected compound 5 was screened over 442 kinases identifying 5 as a highly potent and selective inhibitor of CK1δ. X-ray analysis of 5 bound to CK1δ demonstrated its binding mode. In addition, characterization of 5 and 6 in a cell biological approach revealed the ability of both compounds to inhibit proliferation of tumor cell lines in a dose and cell line specific manner. In summary, our optimizations lead to the development of new highly selective CK1δ and ε specific inhibitors with biological activity.

During the past decade, it has been shown that circadian clock genes have more than a simple circadian time-keeping role. Clock genes also modulate motivational processes and have been implicated in the development of psychiatric disorders such as drug addiction. Recent studies indicate that casein-kinase 1ε/δ (CK1ε/δ)--one of the components of the circadian molecular clockwork-might be involved in the etiology of addictive behavior. The present study was initiated to study the specific role of CK1ε/δ in alcohol relapse-like drinking using the 'Alcohol Deprivation Effect' model. The effect of CK1ε/δ inhibition was tested on alcohol consumption in long-term alcohol-drinking rats upon re-exposure to alcohol after deprivation using a four-bottle free-choice paradigm with water, 5%, 10%, and 20% ethanol solutions, as well as on saccharin preference in alcohol-naive rats. The inhibition of CK1ε/δ with systemic PF-670462 (0, 10, and 30 mg/kg) injections dose-dependently decreased, and at a higher dosage prevented the alcohol deprivation effect, as compared with vehicle-treated rats. The impact of the treatment was further characterized using nonlinear regression analyses on the daily profiles of drinking and locomotor activity. We reveal that CK1ε/δ inhibition blunted the high daytime alcohol intake typically observed upon alcohol re-exposure, and induced a phase shift of locomotor activity toward daytime. Only the highest dose of PF-670462 shifted the saccharin intake daily rhythm toward daytime during treatment, and decreased saccharin preference after treatment. Our data suggest that CK1 inhibitors may be candidates for drug treatment development for alcoholism.

Bipolar disorder is a terrible and debilitating disease with limited treatment options. Circadian rhythm disruptions are prominent in bipolar subjects, and studies have shown that rhythm stabilization through psychosocial interventions can improve their symptoms. Furthermore, mice with a mutation in one of the central circadian proteins, CLOCK, have severely disrupted rhythms along with a behavioral profile that closely resembles human mania. A compound has been developed (CK01, similar to PF-670462) that inhibits the activity of casein kinase 1 (CK1), a critical protein involved in the timing of the molecular clock. Previous studies have shown that PF-670462 and other similar compounds are capable of entraining and stabilizing rhythms in arrhythmic animals. Here we show that chronic administration of CK01 leads to a reversal of the anxiety-related behavior, and partial reversal of the depression-related phenotypes of the Clock mutant mouse. This drug had no significant effects on the behavior of wild-type mice at the doses tested. These results suggest that CK1ε/δ inhibitors could be viable drugs for the treatment of bipolar disorder.