CK1 Delta Research Articles

Structural Investigations on 2-Amidobenzimidazole Derivatives as New Inhibitors of Protein Kinase CK1 Delta.

Protein kinase CK1δ (CK1δ) is a serine-threonine/kinase that modulates different physiological processes, including the cell cycle, DNA repair, and apoptosis. CK1δ overexpression, and the consequent hyperphosphorylation of specific proteins, can lead to sleep disorders, cancer, and neurodegenerative diseases. CK1δ inhibitors showed anticancer properties as well as neuroprotective effects in cellular and animal models of Parkinson's and Alzheimer's diseases and amyotrophic lateral sclerosis. To obtain new ATP-competitive CK1δ inhibitors, three sets of benzimidazole-2-amino derivatives were synthesized (1-32), bearing different substituents on the fused benzo ring (R) and diverse pyrazole-containing acyl moieties on the 2-amino group. The best-performing derivatives were those featuring the (1H-pyrazol-3-yl)-acetyl moiety on the benzimidazol-2-amino scaffold (13-32), which showed CK1δ inhibitor activity in the low micromolar range. Among the R substituents, 5-cyano was the most advantageous, leading to a compound endowed with nanomolar potency (23, IC50 = 98.6 nM). Molecular docking and dynamics studies were performed to point out the inhibitor-kinase interactions.

Upregulation of Stress-Induced Protein Kinase CK1 Delta is associated with a Poor Prognosis for patients with Hepatocellular Carcinoma.

Objective: This study was designed to analyze the expression of CSNK1D in hepatocellular carcinoma (HCC) and investigate the relationship between the expression of CSNK1D and the prognosis of HCC patients. Methods: The CSNK1D and alpha-fetoprotein (AFP) expression levels in patients with HCC and their corresponding clinical data were downloaded from The Cancer Genome Atlas (TCGA) and sorted with a Perl program. CSNK1D and AFP expression differences in liver tissue and liver cancer were compared and analyzed, based on the online database human cancer metastasis database, the relationships between the expression levels of CSNK1D and AFP and the proliferation and metastases of HCC were explored. The immunohistochemical data obtained from the Human Protein Atlas Database further verified the differences in the expression levels of CSNK1D and AFP in liver tissues and liver cancer tissues. Through Kaplan-Meier survival analysis, the effects of CSNK1D and AFP expression levels on the prognosis of patients with HCC were investigated, and the influences of and patients' gender, age and grades of cancer cells, tumor size, the status of lymph node metastasis, distant metastasis, and tumor stage on the expression of CSNK1D were analyzed with R language. The influence of differential expressions of CSNK1D on survival time was compared and the prognostic factors influencing the survival of HCC patients were statistically explored by univariate analysis and multivariate analysis. The potential influencing mechanism of CSNK1D on the prognosis of HCC patients was explored by Gene Set Enrichment Analysis (GSEA) enrichment. Results: The expression level of CSNK1D and AFP in cancer foci was significantly higher than that in normal tissues, However, in the same patient, the expression levels of AFP in paracarcinoma tissues and cancer tissues showed no significant difference. The expression level of CSNK1D in HCC with distant metastases was higher than that in those without metastasis, but the expression level of AFP in metastatic HCC was lower than that in those HCC without metastases. In immunohistochemical tests, CSNK1D was moderately positive in normal liver tissues, slightly positive in normal bile duct tissues, and highly positive in HCC. AFP was slightly positive in normal liver tissues and negative in HCC, but it was not detected in normal intrahepatic bile duct tissue. Survival analysis results suggested that the higher expression level of CSNK1D corresponded to the shorter survival period, whereas the expression level of AFP showed no significant influence on survival time. The expression level of CSNK1D was not correlated with gender, age, the status of lymph node metastasis status, or distant metastasis of patients. The main factors influencing the expression level of CSNK1D included tumor size, cancer cell grade, and tumor stage. The expression levels of CSNK1D in T2 and T3 were higher than that in T1. The expression levels of CSNK1D in G3 and G4 were higher than that in G1. The expression levels of CSNK1D in Stage II and Stage III were higher than that in Stage I. Univariate analysis suggested that tumor size, cell grade, distant metastasis, clinical stage, and CSNK1D expression level were the prognostic factors influencing the survival of patients. Multivariate analysis suggested that CSNK1D expression level was an independent factor influencing the prognosis of HCC patients. GSEA enrichment analysis indicated that CSNK1D mainly affected the prognosis of HCC patients through cell cycle, WNT signaling pathway, amino acid degradation metabolism, and other pathways. Conclusion: CSNK1D is an independent influencing factor for the prognosis of HCC patients and has the potential to be developed as a potential therapeutic target for HCC, and better than AFP in predicting the prognosis of HCC.

3,4-Dibromo-7-Azaindole Modulates Arabidopsis Circadian Clock by Inhibiting Casein Kinase 1 Activity.

The circadian clock is a timekeeping system for regulation of numerous biological daily rhythms. One characteristic of the circadian clock is that period length remains relatively constant in spite of environmental fluctuations, such as temperature change. Here, using the curated collection of in-house small molecule chemical library (ITbM chemical library), we show that small molecule 3,4-dibromo-7-azaindole (B-AZ) lengthened the circadian period of Arabidopsis thaliana (Arabidopsis). B-AZ has not previously been reported to have any biological and biochemical activities. Target identification can elucidate the mode of action of small molecules, but we were unable to make a molecular probe of B-AZ for target identification. Instead, we performed other analysis, gene expression profiling that potentially reveals mode of action of molecules. Short-term treatment of B-AZ decreased the expression of four dawn- and morning-phased clock-associated genes, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. Consistently, amounts of PRR5 and TIMING OF CAB EXPRESSION 1 (TOC1) proteins, transcriptional repressors of CCA1, LHY, PRR9 and PRR7 were increased upon B-AZ treatment. B-AZ inhibited Casein Kinase 1 family (CK1) that phosphorylates PRR5 and TOC1 for targeted degradation. A docking study and molecular dynamics simulation suggested that B-AZ interacts with the ATP-binding pocket of human CK1 delta, whose amino acid sequences are highly similar to those of Arabidopsis CK1. B-AZ-induced period-lengthening effect was attenuated in prr5 toc1 mutants. Collectively, this study provides a novel and simple structure CK1 inhibitor that modulates circadian clock via accumulation of PRR5 and TOC1.

Abstract 2939: Developing new therapeutic strategy to bladder cancer by targeting CK1 delta

Abstract Bladder cancer is a common malignancy with high recurrent rate, but targeted therapy other than immune checkpoint inhibitors is currently unavailable. Hence, there is an unmet medical need for the discovery of new therapeutic approach to bladder cancer. By investigating Oncomine database, the mRNA level of CK1 delta is higher in bladder cancer compared to normal tissue, which correlates with survival rate. The aim of this study is to develop new CK1 delta inhibitors and examine their therapeutic potential to bladder cancer as well as the pharmacological mechanisms. Cell viability, cell cycle progression, apoptosis, migration and downstream signaling pathway were analyzed. The results showed that treatment of CK1 delta inhibitors decreased cell viability and inhibited wnt/β-catenin pathway in bladder cancer cell lines. CK1 delta inhibitors increased the accumulation of sub-G1 phase by flow cytometry analysis, as well as the activation of caspase-3, 8, 9 and PARP cleavage by western blot, indicating the promotion of cell apoptosis. Meanwhile, necroptosis was also involved as evident by the rescuing effects by knocking down MLKL in bladder cancer cells. In conclusion, this study demonstrates that CK1 delta is a potential target for bladder cancer therapy in the future. Note: This abstract was not presented at the meeting. Citation Format: Yu-Chen Lin, Mei-chuan Chen, Jing-Ping Liou, Chun-Han Chen. Developing new therapeutic strategy to bladder cancer by targeting CK1 delta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2939.

Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-β in Pulmonary Fibrosis.

Transforming growth factor-beta (TGF-β) is a major mediator of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). However, therapeutic global inhibition of TGF-β is limited by unwanted immunosuppression and mitral valve defects. We performed an extensive literature search to uncover a little-known connection between TGF-β signaling and casein kinase (CK) activity. We have examined the abundance of CK1 delta and epsilon (CK1δ/ε) in lung tissue from IPF patients and non-diseased controls, and investigated whether inhibition of CK1δ/ε with PF670462 inhibits pulmonary fibrosis. CK1δ/ε levels in lung tissue from IPF patients and non-diseased controls were assessed by immunohistochemistry. Anti-fibrotic effects of the CK1δ/ε inhibitor PF670462 were assessed in pre-clinical models, including acute and chronic bleomycin mouse models and in vitro experiments on spheroids made from primary human lung fibroblast cells from IPF and control donors, and human A549 alveolar-like adenocarcinoma-derived epithelial cells. Increased expression of CK1δ and ε in IPF lungs compared to non-diseased controls was accompanied by increased levels of the product, phospho-period 2. In vitro, PF670462 prevented TGF-β-induced epithelial-mesenchymal transition. The stiffness of IPF-derived spheroids was reduced by PF670462 and TGF-β-induced fibrogenic gene expression was inhibited. The CK1δ/ε inhibitor PF670462 administered systemically or locally by inhalation prevented both acute and chronic bleomycin-induced pulmonary fibrosis in mice. PF670462 administered in a ‘therapeutic’ regimen (day 7 onward) prevented bleomycin-induced lung collagen accumulation. Elevated expression and activity of CK1 δ and ε in IPF and anti-fibrogenic effects of the dual CK1δ/ε inhibitor, PF670462, support CK1δ/ε as novel therapeutic targets for IPF.

Abstract 537: CK1 delta as a therapeutic target in bladder cancer

Abstract Bladder cancer is a common malignancy worldwide, but targeted therapy is currently unavailable. Therefore, there is an unmet medical need for bladder cancer patients. Oncomine database revealed that gene expression of CK1 delta is higher in bladder cancer compared to normal tissue. The purpose of this study is to investigate the role of CK1 delta in the progression of bladder cancer, and the feasibility of CK1 delta as a therapeutic target. Lentiviral delivery of shRNA and CK1 delta inhibitors was used to modulate gene expression or kinase activity of CK1 delta in bladder cancer cell lines. Cell viability, cell cycle progression, apoptosis, migration and downstream signaling pathway were analyzed. The results showed that knocking down CK1 delta or treatment of CK1 delta inhibitors decreased cell viability and inhibited wnt/β-catenin pathway in bladder cancer cell lines. CK1 delta inhibitors increased the accumulation of sub-G1 phase by flow cytometry analysis, as well as the activation of caspase-3, 8, 9 and PARP cleavage by Western blot, indicating the promotion of cell apoptosis. Knocking down of CK1 delta and the treatment of CK1 delta inhibitors impaired cell migration activity, and altered protein expression of EMT markers, such as E-cadherin, vimentin, snail and slug. In conclusion, this study demonstrates that CK1 delta is a potential target for bladder cancer therapy. Citation Format: Chun-Han Chen. CK1 delta as a therapeutic target in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 537.

The discovery of novel CK1 delta inhibitors for bladder cancer therapy

The discovery of novel CK1 delta inhibitors for bladder cancer therapy

Abstract B064: Therapeutic potential of CK1 delta inhibition in pancreatic and bladder cancer

Abstract Gemcitabine has potent antitumor activity in several human cancers and represents the standard of care for advanced pancreatic ductal adenocarcinoma (PDAC). Nevertheless, despite aggressive treatment, long-term survival in PDAC is rare. Identification of specific drivers of PDAC is required to instruct the development of more targeted therapeutics. Casein kinase 1 delta (CK1δ) and epsilon (CK1ϵ) are serine/threonine kinases that regulate key effectors involved in tumor proliferation and differentiation such as Wnt-ß-catenin axis, PI3K/AKT signaling pathways, and p53 function. Aberrant regulation of the Wnt and p53 pathways occurs frequently in PDAC. Additionally, increased CK1δ expression has been found in bladder cancer, where gemcitabine is also a standard of care. This prompted us to test the antitumor effects of SR-3029, a potent, dual CK1δ/ϵ inhibitor developed in our laboratories in pancreatic and bladder cancer cells, and assess whether the combination of SR-3029 with gemcitabine would result in therapeutic synergy. Inhibition of cell proliferation (EC50) by SR-3029, both as a single agent and in combination with gemcitabine, was tested on a panel of several pancreatic and bladder cancer cells. Potent inhibition of proliferation was observed in a dose- and time-dependent fashion and SR-3029 acted in synergy with gemcitabine, as quantified by the Chou Talalay method. As described in our previous breast cancer studies, shRNAs targeting CK1δ and SR-3029 were sufficient to block colony formation of PDAC and bladder cancer cells. Gemcitabine acts a deoxycytidine analogue, converted into its active metabolite by deoxycytidine kinase (dCK) enzyme through a series of phosphorylation steps. The phosphorylated form of gemcitabine ultimately adds into the DNA strand terminating synthesis. dCK overexpression in pancreatic and bladder cancer tissues is associated with gemcitabine chemosensitivity. Searching for possible mechanisms explaining the synergy between SR-3029 and gemcitabine, we found that, upon treatment with SR-3029 and CK1δ knockdown, both dCK RNA and protein expression increased, possibly sensitizing to the antitumor effects of gemcitabine. We finally expanded our results in vivo and we evaluated the effect of SR-3029 alone and in combination with gemcitabine in an orthotopic mouse model of pancreatic cancer. Consistently, we observed a significant increase of tumor growth delay after gemcitabine plus SR-3029 treatment compared to gemcitabine alone. Importantly, this antitumor effect was accompanied by an increase of dCK expression in the xenografts. In summary, we believe that CK1δ may be a clinically relevant target in both pancreatic and bladder cancer, and in this setting SR-3029 holds promise as a future therapeutic option. Citation Format: Francesca Vena, Simon Bayle, Andrii Monastyrskyi, Derek Duckett, William Roush. Therapeutic potential of CK1 delta inhibition in pancreatic and bladder cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B064.

Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.

Identification of novel protein kinase CK1 delta (CK1δ) inhibitors through structure-based virtual screening

In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimer’s disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1δ inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1δ inhibitors known today (IC 50 = 0.3 and 0.6 μM, respectively).

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo

Background: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. Aims: We analysed the...

994 The Casein kinase 1 delta (CK1 delta) specific inhibitor IC261 impinges growth of pancreatic tumor cells and the expression of CK1delta in healthy young adult BALB/c mice

994 The Casein kinase 1 delta (CK1 delta) specific inhibitor IC261 impinges growth of pancreatic tumor cells and the expression of CK1delta in healthy young adult BALB/c mice

Casein Kinase Iγ Subfamily.: MOLECULAR CLONING, EXPRESSION, AND CHARACTERIZATION OF THREE MAMMALIAN ISOFORMS AND COMPLEMENTATION OF DEFECTS IN THE SACCHAROMYCES CEREVISIAE YCK GENES

Casein kinase I, one of the first protein kinases identified biochemically, is known to exist in multiple isoforms in mammals. Using a partial cDNA fragment corresponding to an isoform termed CK1 gamma, three full-length rat testis cDNAs were cloned that defined three separate members of this subfamily. The isoforms, designated CK1 gamma 1, CK1 gamma 2, and CK1 gamma 3, have predicted molecular masses of 43,000, 45,500, and 49,700. CK1 gamma 3 may also exist in an alternatively spliced form. The proteins are more than 90% identical to each other within the protein kinase domain but only 51-59% identical to other casein kinase I isoforms within this region. Messages for CK1 gamma 1 (2 kilobases (kb)), CK1 gamma 2 (1.5 and 2.4 kb), and CK1 gamma 3 (2.8 kb) were detected by Northern hybridization of testis RNA. Message for CK1 gamma 3 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK1 gamma 1 and CK1 gamma 2 messages were restricted to testis. All three CK1 gamma isoforms were expressed as active enzymes in Escherichia coli and partially purified. The enzymes phosphorylated typical in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4. Phosphorylation of the D4 peptide was activated by heparin whereas phosphorylation of the protein substrates was inhibited. The known casein kinase I inhibitor CK1-7 also inhibited the CK1 gamma s although less effectively than the CK1 alpha or CK1 delta isoforms. All three CK1 gamma s underwent autophosphorylation when incubated with ATP and Mg2+. The YCK1 and YCK2 genes in Saccharomyces cerevisiae encode casein kinase I homologs, defects in which lead to aberrant morphology and growth arrest. Expression of mammalian CK1 gamma 1 or CK1 gamma 3 restored growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins and these CK1 isoforms.