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 (IC₅₀ CK1δ = 4 nM, IC₅₀ CK1ε = 25 nM), 12a (IC₅₀ CK1δ = 19 nM, IC₅₀ CK1ε = 227 nM), and 16b (IC₅₀ CK1δ = 8 nM, IC₅₀ 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 (EC₅₀ = 3.5 µM) and Panc89 (EC₅₀ = 1.5 µM). SAR is substantiated by X-ray crystallographic analysis of 16b in CK1δ and 11b in p38α.

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.

Protein kinases of the Casein Kinase 1 family play a vital role in the regulation of numerous cellular processes. Apart from functions associated with regulation of proliferation, differentiation, or apoptosis, localization of several Casein Kinase 1 isoforms to the centrosome and microtubule asters also implicates regulatory functions in microtubule dynamic processes. Being localized to the spindle apparatus during mitosis Casein Kinase 1 directly modulates microtubule dynamics by phosphorylation of tubulin isoforms. Additionally, site-specific phosphorylation of microtubule-associated proteins can be related to the maintenance of genomic stability but also microtubule stabilization/destabilization, e.g., by hyper-phosphorylation of microtubule-associated protein 1A and RITA1. Consequently, approaches interfering with Casein Kinase 1-mediated microtubule-specific functions might be exploited as therapeutic strategies for the treatment of cancer. Currently pursued strategies include the development of Casein Kinase 1 isoform-specific small molecule inhibitors and therapeutically useful peptides specifically inhibiting kinase-substrate interactions.

A main pathological event in Alzheimer’s disease is the generation of neurofibrillary tangles originating from hyperphosphorylated and subsequently aggregated tau proteins. Previous reports demonstrated the critical involvement of members of the protein kinase family CK1 in the pathogenesis of Alzheimer’s disease by hyperphosphorylation of tau. However, precise mechanisms and effects of CK1-mediated tau phosphorylation are still not fully understood. In this study, we analyzed recombinant tau441 phosphorylated by CK1δ in vitro via mass spectrometry and identified ten potential phosphorylation sites, five of them are associated to Alzheimer’s disease. To confirm these results, in vitro kinase assays and two-dimensional phosphopeptide analyses were performed with tau441 phosphomutants confirming Alzheimer’s disease-associated residues Ser68/Thr71 and Ser289 as CK1δ-specific phosphorylation sites. Treatment of differentiated human neural progenitor cells with PF-670462 and Western blot analysis identified Ser214 as CK1δ-targeted phosphorylation site. The use of an in vitro tau aggregation assay demonstrated a possible role of CK1δ in tau aggregation. Results obtained in this study highlight the potential of CK1δ to be a promising target in the treatment of Alzheimer’s disease.

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC₅₀ value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.