Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

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.

Prognostic value and immunological role of CSNK1D in human cancers.

CSNK1D, also known as CK1δ, is a crucial gene involved in various biological processes such as cell cycle, transcriptional regulation, apoptosis, cell polarity, and cell motility. It is associated with different cancers and neurodegenerative diseases. This study aimed to investigate the role of CSNK1D in multiple human cancers, particularly hepatocellular carcinoma (HCC), through in vitro experiments. The research utilized various online resources and databases like UCSC, NCBI, GEPIA2, HPA, cBioPortal, SangerBox, UALCAN, and TCGA for analyzing CSNK1D expression, prognosis significance, immune features, and gene alterations in cancers. RT-PCR was employed to evaluate CSNK1D expression in normal liver and liver cancer cell lines. In vitro experiments, including CCK-8, Edu, wound healing, and Transwell assays, were conducted to assess CSNK1D's biological function in HCC cells. Results demonstrated consistent upregulation of CSNK1D in various tumors. Heightened CSNK1D expression correlated with reduced overall survival and disease-free survival rates in different cancer patient cohorts. Significant associations were found between CSNK1D expression levels and immune cell infiltrations, immune checkpoint inhibitors, tumor mutation burden, and microsatellite instability across multiple malignancies. Notably, statistical analyses using TCGA and ICGC data identified CSNK1D as a robust and independent prognostic biomarker in HCC. Inhibiting CSNK1D expression effectively hindered cell proliferation, migration, and invasion in cellular experiments. In conclusion, this study suggests that CSNK1D may serve as a biomarker for tumor prognosis and immunotherapy. It influences the proliferation and metastasis of HCC cells.

CK1δ and CK1ε Signaling Sustains Mitochondrial Metabolism and Cell Survival in Multiple Myeloma.

Multiple myeloma remains an incurable malignancy due to acquisition of intrinsic programs that drive therapy resistance. Here we report that casein kinase-1δ (CK1δ) and CK1ε are therapeutic targets in multiple myeloma that are necessary to sustain mitochondrial metabolism. Specifically, the dual CK1δ/CK1ε inhibitor SR-3029 had potent in vivo and ex vivo anti-multiple myeloma activity, including against primary multiple myeloma patient specimens. RNA sequencing (RNA-seq) and metabolic analyses revealed inhibiting CK1δ/CK1ε disables multiple myeloma metabolism by suppressing genes involved in oxidative phosphorylation (OxPhos), reducing citric acid cycle intermediates, and suppressing complexes I and IV of the electron transport chain. Finally, sensitivity of multiple myeloma patient specimens to SR-3029 correlated with elevated expression of mitochondrial genes, and RNA-seq from 687 multiple myeloma patient samples revealed that increased CSNK1D, CSNK1E, and OxPhos genes correlate with disease progression and inferior outcomes. Thus, increases in mitochondrial metabolism are a hallmark of multiple myeloma progression that can be disabled by targeting CK1δ/CK1ε. SIGNIFICANCE: CK1δ and CK1ε are attractive therapeutic targets in multiple myeloma whose expression increases with disease progression and connote poor outcomes, and that are necessary to sustain expression of genes directing OxPhos.

Dual Anta-Inhibitors Targeting Protein Kinase CK1δ and A2A Adenosine Receptor Useful in Neurodegenerative Disorders.

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.

CSNK1D-mediated phosphorylation of HNRNPA2B1 induces miR-25-3p/miR-93-5p maturation to promote prostate cancer cell proliferation and migration through m6A-dependent manner.

It has been reported that heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) is highly expressed in prostate cancer (PCa) and associated with poor prognosis of patients with PCa. Nevertheless, the specific mechanism underlying HNRNPA2B1 functions in PCa remains not clear. In our study, we proved that HNRNPA2B1 promoted the progression of PCa through in vitro and in vivo experiments. Further, we found that HNRNPA2B1 induced the maturation of miR-25-3p/miR-93-5p by recognizing primary miR-25/93 (pri-miR-25/93) through N6-methyladenosine (m6A)-dependent manner. In addition, both miR-93-5p and miR-25-3p were proven as tumor promoters in PCa. Interestingly, by mass spectrometry analysis and mechanical experiments, we found that casein kinase 1 delta (CSNK1D) could mediate the phosphorylation of HNRNPA2B1 to enhance its stability. Moreover, we further proved that miR-93-5p targeted BMP and activin membrane-bound inhibitor (BAMBI) mRNA to reduce its expression, thereby activating transforming growth factor ß (TGF-ß) pathway. At the same time, miR-25-3p targeted forkhead box O3 (FOXO3) to inactivate FOXO pathway. These results collectively indicated that CSNK1D stabilized HNRNPA2B1 facilitates the processing of miR-25-3p/miR-93-5p to regulate TGF-ß and FOXO pathways, resulting in PCa progression. Our findings supported that HNRNPA2B1 might be a promising target for PCa treatment.

Structure-Based Development of Isoform-Selective Inhibitors of Casein Kinase 1ε vs Casein Kinase 1δ.

Specific inhibition of a single kinase isoform is a challenging task due to the highly conserved nature of ATP-binding sites. Casein kinase 1 (CK1) δ and ε share 97% sequence identity in their catalytic domains. From a comparison of the X-ray crystal structures of CK1δ and CK1ε, we developed a potent and highly CK1ε-isoform-selective inhibitor (SR-4133). The X-ray co-crystal structure of the CK1δ-SR-4133 complex reveals that the electrostatic surface between the naphthyl unit of SR-4133 and CK1δ is mismatched, destabilizing the interaction of SR-4133 with CK1δ. Conversely, the hydrophobic surface area resulting from the Asp-Phe-Gly motif (DFG)-out conformation of CK1ε stabilizes the binding of SR-4133 in the ATP-binding pocket of CK1ε, leading to the selective inhibition of CK1ε. The potent CK1ε-selective agents display nanomolar growth inhibition of bladder cancer cells and inhibit the phosphorylation of 4E-BP1 in T24 cells, which is a direct downstream effector of CK1ε.

Casein kinase 1δ/ε phosphorylates fused in sarcoma (FUS) and ameliorates FUS-mediated neurodegeneration.

Aberrant cytoplasmic accumulation of an RNA-binding protein, fused in sarcoma (FUS), characterizes the neuropathology of subtypes of ALS and frontotemporal lobar degeneration, although the effects of post-translational modifications of FUS, especially phosphorylation, on its neurotoxicity have not been fully characterized. Here, we show that casein kinase 1δ (CK1δ) phosphorylates FUS at 10 serine/threonine residues in vitro using mass spectrometric analyses. We also show that phosphorylation by CK1δ or CK1ε significantly increased the solubility of FUS in human embryonic kidney 293 cells. In transgenic Drosophila that overexpress wt or P525L ALS-mutant human FUS in the retina or in neurons, we found coexpression of human CK1δ or its Drosophila isologue Dco in the photoreceptor neurons significantly ameliorated the observed retinal degeneration, and neuronal coexpression of human CK1δ extended fly life span. Taken together, our data suggest a novel regulatory mechanism of the assembly and toxicity of FUS through CK1δ/CK1ε-mediated phosphorylation, which could represent a potential therapeutic target in FUS proteinopathies.

Casein Kinase 1δ Inhibitors as Promising Therapeutic Agents for Neurodegenerative Disorders.

Casein kinase 1 (CK1) belongs to the serine-threonine kinase family and is expressed in all eukaryotic organisms. At least six human isoforms of CK1 (termed α, γ1-3, δ and ε) have been cloned and characterized. CK1δ isoform modulates several physiological processes, including DNA damage repair, circadian rhythm, cellular proliferation and apoptosis. Therefore, CK1δ dysfunction may trigger diverse pathologies, such as cancer, inflammation and central nervous system disorders. Overexpression and aberrant activity of CK1δ have been connected to hyperphosphorylation of key proteins implicated in the development of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases and Amyotrophic Lateral Sclerosis. Thus, CK1δ inhibitors have attracted attention as potential drugs for these pathologies and several compounds have been synthesized or isolated from natural sources to be evaluated for their CK1δ inhibitory activity. Here we report a comprehensive review on the development of CK1δ inhibitors, with a particular emphasis on structure-activity relationships and computational studies, which provide useful insight for the design of novel inhibitors.

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

C5-Iminosugar modification of casein kinase 1δ lead 3-(4-fluorophenyl)-5-isopropyl-4-(pyridin-4-yl)isoxazole promotes enhanced inhibitor affinity and selectivity.

The quest for isoform-selective and specific ATP-competitive protein kinase inhibitors is of great interest, as inhibitors with these qualities will come with reduced toxicity and improved efficacy. However, creating such inhibitors is very challenging due to the high molecular similarity of kinases ATP active sites. To achieve selectivity for our casein kinase (CK) 1 inhibitor series, we elected to endow our previous CK1δ-hit, 3-(4-fluorophenyl)-5-isopropyl-4-(pyridin-4-yl)isoxazole (1), with chiral iminosugar scaffolds. These scaffolds were attached to C5 of the isoxazole ring, a position deemed favorable to facilitate binding interactions with the ribose pocket/solvent-open area of the ATP binding pocket of CK1δ. Here, we describe the synthesis of analogs of 1 ((-)-/(+)-34, (-)-/(+)-48), which were prepared in 13 steps from enantiomerically pure ethyl (3R,4S)- and ethyl (3S,4R)-1-benzyl-4-[(tert-butyldimethylsilyl)oxy]-5-oxopyrrolidine-3-carboxylate ((-)-11 and (+)-11), respectively. The synthesis involved the coupling of Weinreb amide-activated chiral pyrrolidine scaffolds with 4- and 2-fluoro-4-picoline and reaction of the resulting 4-picolyl ketone intermediates ((-)-/(+)-40 and (-)-/(+)-44) with 4-fluoro-N-hydroxybenzenecarboximidoyl chloride to form the desired isoxazole ring. The activity of the compounds against human CK1δ, -ε, and -α was assessed in recently optimized in vitro assays. Compound (-)-34 was the most active compound with IC50 values (CK1δ/ε) of 1/8 µM and displayed enhanced selectivity toward CK1δ.

CK1δ stimulates ubiquitination-dependent proteasomal degradation of ATF4 to promote chemoresistance in gastric Cancer.

Chemoresistance remains a major obstacle to successful cancer therapy, especially for advanced cancers. It used to be recognised as a stable outcome resulting from genetic changes. However, recent studies showed that chemoresistance can also be unstable and reversible with the involvement of non-genetic alterations. In the present study, we found that activating transcription factor 4 (ATF4) is downregulated in chemoresistant gastric cancer cells. The over-expression of ATF4 reversed chemoresistance by activating CHOP transcription to enhance drug-induced apoptosis, and vice versa. Moreover, casein kinase 1 delta (CK1δ) was identified as the kinase responsible for ATF4-S219 phosphorylation, which triggered ßTrCP-mediated ATF4 polyubiquitination to promote its proteasomal degradation subsequently. Interestingly, drug withdrawal gradually restored chemosensitivity as well as ATF4 expression in chemoresistant cells, highlighting the dependence of dynamic drug resistance on ATF4 protein expression. In line with these findings, the inhibition of ATF4 protein degradation by CK1δ or proteasome inhibitors overcame chemoresistance both in vitro and in vivo. Taken together, these results indicate that CK1δ stimulates ßTrCP-dependent ATF4 polyubiquitination and subsequent proteasomal degradation to promote chemoresistance in gastric cancer. Stabilisation of the ATF4 protein with bortezomib (BTZ), an anticancer drug that inhibits proteasomal degradation, might be a rational strategy to improve chemotherapeutic efficacy in gastric cancer.

Cyclical phosphorylation of LRAP35a and CLASP2 by GSK3ß and CK1δ regulates EB1-dependent MT dynamics in cell migration.

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-3ß (GSK3ß) 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 GSK3ß 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.

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.

CK1δ-Derived Peptides as Novel Tools Inhibiting the Interactions between CK1δ and APP695 to Modulate the Pathogenic Metabolism of APP.

Alzheimer's disease (AD) is the major cause of dementia, and affected individuals suffer from severe cognitive, mental, and functional impairment. Histologically, AD brains are basically characterized by the presence of amyloid plaques and neurofibrillary tangles. Previous reports demonstrated that protein kinase CK1δ influences the metabolism of amyloid precursor protein (APP) by inducing the generation of amyloid-ß (Aß), finally contributing to the formation of amyloid plaques and neuronal cell death. We therefore considered CK1δ as a promising therapeutic target and suggested an innovative strategy for the treatment of AD based on peptide therapeutics specifically modulating the interaction between CK1δ and APP. Initially, CK1δ-derived peptides manipulating the interactions between CK1δ and APP695 were identified by interaction and phosphorylation analysis in vitro. Selected peptides subsequently proved their potential to penetrate cells without inducing cytotoxic effects. Finally, for at least two of the tested CK1δ-derived peptides, a reduction in Aß levels and amyloid plaque formation could be successfully demonstrated in a complex cell culture model for AD. Consequently, the presented results provide new insights into the interactions of CK1δ and APP695 while also serving as a promising starting point for further development of novel and highly innovative pharmacological tools for the treatment of AD.

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.

The CK1δ/ε-AES axis regulates tumorigenesis and metastasis in colorectal cancer.

Background: Amino-terminal enhancer of split (AES) has been identified as a tumor and metastasis suppressor in some cancers including colorectal cancer (CRC), but very little is known about the regulation of AES expression. Methods: Bioinformatics analysis was used to investigate the expression patterns of AES, CK1δ and CK1ε. The co-immunoprecipitation, GST pull-down, Western Blot, real-time PCR and immunohistochemistry were performed to study the mechanism underlying the regulation of AES expression by CK1δ/ε. The biological function was assessed by in vitro colony formation, transwell, sphere formation, tumor organoids, in vivo tumor metastasis model and patient-derived colorectal tumor xenografts (PDTX) model. Results: A strong inverse relationship was observed between the expression of AES and the expression of CK1δ/ε. Mechanically, AES could interact with CK1δ/ε and SKP2 using its Q domain. SKP2 mediated the ubiquitination and degradation of AES in a CK1δ/ε-dependent manner. CK1δ/ε phosphorylated AES at Ser121 and accelerated the SKP2-mediated ubiquitination and degradation of AES. In colon cancer cells, CK1δ/ε antagonized the effect of wild-type AES but not that of its mutant (S121A) on Wnt and Notch signaling, leading to an increase in the expression of Wnt target genes and Notch target genes. By downregulating the expression of AES, CK1δ/ε enhanced anchorage-independent growth, migration, invasion and sphere formation in colon cancer cells. CK1δ/ε also promoted the growth of APCmin/+ colorectal tumor organoids and liver metastasis in colon cancer mouse models through the regulation of AES degradation. Furthermore, CK1 inhibitor SR3029 treatment suppressed tumor growth via stabilizing AES in APCmin/+ colorectal tumor organoids and patient-derived colorectal tumor xenografts (PDTX). Conclusions: Our results revealed that the CK1δ/ε-AES axis is important for CRC tumorigenesis and metastasis, and targeted inhibition of this axis may be a potential therapeutic strategy for CRC.

ß-Trcp and CK1δ-mediated degradation of LZTS2 activates PI3K/AKT signaling to drive tumorigenesis and metastasis in hepatocellular carcinoma.

Distant metastasis is the leading cause of treatment failure in patients with hepatocellular carcinoma (HCC). However, the underlying mechanisms have not been fully elucidated. Here, we report that Leucine zipper tumor suppressor 2 (LZTS2) is downregulated and correlated with poor prognosis in HCC. Furthermore, we provide evidence that LZTS2 associates with p85 to inhibit the activation of PI3K/AKT signaling and impairs HCC tumorigenesis and metastasis in vitro and in vivo. Moreover, we identify LZTS2 as a bona fide substrate of the E3 ligase ß-Trcp and protein kinase CK1δ, which are responsible for the ubiquitination and degradation of LZTS2. Importantly, we show that the ß-Trcp and CK1δ-mediated degradation of LZTS2 promotes HCC progression and metastasis by activating PI3K/AKT signaling. Collectively, our study not only illustrates the roles of LZTS2 in regulating HCC tumorigenesis and metastasis but also reveals a novel posttranslational modification of LZTS2 by ß-Trcp and CK1δ, indicating that the ß-Trcp/CK1δ/LZTS2/PI3K axis may be a novel oncogenic driver involved in HCC progression and metastasis.

Kinase and GPCR polypharmacological approach for the identification of efficient anticancer medicines.

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.

Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder Cancer Cells to Gemcitabine Treatment by Upregulating Deoxycytidine Kinase.

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.

Exosomes derived from hucMSC attenuate renal fibrosis through CK1δ/ß-TRCP-mediated YAP degradation.

Exosomes from human umbilical cord mesenchymal stem cells (hucMSC-Ex) have been suggested as novel nanomaterials for regenerative medicine. Here we explored the roles of hucMSC-Ex through regulating Yes-associated protein (YAP) in renal injury repair by using rat unilateral ureteral obstruction (UUO) models. Our study identified mechanical stress induced YAP nucleus expression and stimulated collagen deposition and interstitial fibrosis in the kidney. Then, infusion with hucMSC-Ex promoted YAP nuclear cytoplasmic shuttling and ameliorated renal fibrosis in UUO model. Interestingly, hucMSC-Ex delivered casein kinase 1δ (CK1δ) and E3 ubiquitin ligase ß-TRCP to boost YAP ubiquitination and degradation. Knockdown of CK1δ and ß-TRCP in hucMSC decreased the repairing effects of hucMSC-Ex on renal fibrosis. Our results suggest that hucMSC-Ex attenuates renal fibrosis through CK1δ/ß-TRCP inhibited YAP activity, unveiling a new mechanism for the therapeutic effects of hucMSC-Ex on tissue injury and offering a potential approach for renal fibrosis treatment.