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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.

Obesity is associated with low-grade chronic inflammation, altered levels of adipocytokines, and impaired regulation of gastrointestinal hormones. Secreted, these factors exert immunostimulatory functions directly influencing peripheral immune cells. In the realm of this study, we aimed to investigate the composition and activation status of peripheral blood immune cells in female patients with morbid obesity compared to lean controls using high-dimensional mass cytometry. Besides, we also assessed the influence of bariatric surgery with respect to its ability to reverse obesity-associated alterations within the first-year post-surgery. Patients with morbid obesity showed typical signs of chronic inflammation characterized by increased levels of CRP and fibrinogen. Apart from that, metabolic alterations were characterized by increased levels of leptin and resistin as well as decreased levels of adiponectin and ghrelin compared to the healthy control population. All these however, except for ghrelin levels, rapidly normalized after surgery with regard to control levels. Furthermore, we found an increased population of monocytic CD14 , HLA-DR , CD11b , CXCR3 cells in patients with morbid obesity and an overall reduction of the HLA-DR monocytic expression compared to the control population. Although CD14 , HLA-DR , CD11b , CXCR3 decreased after surgery, HLA-DR expression did not recover within 9 - 11 months post-surgery. Moreover, compared to the control population, patients with morbid obesity showed a perturbed CD4+ T cell compartment, characterized by a strongly elevated CD127 memory T cell subset and decreased naïve T cells, which was not recovered within 9 - 11 months post-surgery. Although NK cells showed an activated phenotype, they were numerically lower in patients with morbid obesity when compared to healthy controls. The NK cell population further decreased after surgery and did not recover quantitatively within the study period. Our results clearly demonstrate that the rapid adaptions in inflammatory parameters and adipocytokine levels that occur within the first year post-surgery do not translate to the peripheral immune cells. Apart from that, we described highly affected, distinct immune cell subsets, defined as CD127 memory T cells and monocytic CD14 , HLA-DR, CD11b , CXCR3 cells, that might play a significant role in understanding and further decoding the etiopathogenesis of morbid obesity.

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.

Thoracic traumas with extra-thoracic injuries result in an immediate, complex host response. The immune response requires tight regulation and can be influenced by additional risk factors such as obesity, which is considered a state of chronic inflammation. Utilizing high-dimensional mass and regular flow cytometry, we define key signatures of obesity-related alterations of the immune system during the response to the trauma. In this context, we report a modification in important components of the splenic response to the inflammatory reflex in obese mice. Furthermore, during the response to trauma, obese mice exhibit a prolonged increase of neutrophils and an early accumulation of inflammation associated CCR2 + CD62L + Ly6C hi monocytes in the blood, contributing to a persistent inflammatory phase. Moreover, these mice exhibit differences in migration patterns of monocytes to the traumatized lung, resulting in decreased numbers of regenerative macrophages and an impaired M1/M2 switch in traumatized lungs. The findings presented in this study reveal an attenuation of the inflammatory reflex in obese mice, as well as a disturbance of the monocytic compartment contributing to a prolonged inflammation phase resulting in fewer phenotypically regenerative macrophages in the lung of obese mice.

Introduction: The CK1 family is involved in a variety of physiological processes by regulating different signaling pathways, including the Wnt/β-catenin, the Hedgehog and the p53 signaling pathways. Mutations or dysregulation of kinases in general and of CK1 in particular are known to promote the development of cancer, neurodegenerative diseases and inflammation. There is increasing evidence that CK1 isoform specific small molecule inhibitors, including CK1δ- and CK1ε-specific inhibitors of Wnt production (IWP)-based small molecules with structural similarity to benzimidazole compounds, have promising therapeutic potential. Methods: In this study, we investigated the suitability of the zebrafish model system for the evaluation of such CK1 inhibitors. To this end, the kinetic parameters of human CK1 isoforms were compared with those of zebrafish orthologues. Furthermore, the effects of selective CK1δ inhibition during zebrafish embryonic development were analyzed in vivo . Results: The results revealed that zebrafish CK1δA and CK1δB were inhibited as effectively as human CK1δ by compounds G2-2 with IC 50 values of 345 and 270 nM for CK1δA and CK1δB versus 503 nM for human CK1δ and G2-3 exhibiting IC 50 values of 514 and 561 nM for zebrafish CK1δA and B, and 562 nM for human CK1δ. Furthermore, the effects of selective CK1δ inhibition on zebrafish embryonic development in vivo revealed phenotypic abnormalities indicative of downregulation of CK1δ. Treatment of zebrafish embryos with selected inhibitors resulted in marked phenotypic changes including blood stasis, heart failure, and tail malformations. Conclusion: The results suggest that the zebrafish is a suitable in vivo assay model system for initial studies of the biological relevance of CK1δ inhibition.

Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania . This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the presence of Leishmania casein kinase 1 (L-CK1.2), a signalling kinase. L-CK1.2 is essential for parasite survival and thus might be essential for host subversion. To get insights into the functions of L-CK1.2 in the macrophage, the systematic identification of its host substrates is crucial, we thus developed an easy method to identify substrates, combining phosphatase treatment, in vitro kinase assay and Stable Isotope Labelling with Amino acids in Cell (SILAC) culture-based mass spectrometry. Implementing this approach, we identified 225 host substrates as well as a potential novel phosphorylation motif for CK1. We confirmed experimentally the enrichment of our substratome in bona fide L-CK1.2 substrates and showed they were also phosphorylated by human CK1δ. L-CK1.2 substratome is enriched in biological processes such as “viral and symbiotic interaction,” “actin cytoskeleton organisation” and “apoptosis,” which are consistent with the host pathways modified by Leishmania upon infection, suggesting that L-CK1.2 might be the missing link. Overall, our results generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogens adapted for intracellular survival.

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease first described in 1906 by the pathologist Alois Alzheimer. Amyloid plaques and intracellular neurofibrillary tangles (NFTs) are widely accepted and have been extensively described as hallmarks of AD. The presence and distribution of amyloid plaques, NFTs and synaptic degeneration correlate with the course and degree of cognitive decline. Remarkably, both histopathological hallmarks, amyloid plaques and NFTs, are related to phosphorylation events. More precisely, amyloid plaques are composed of Aβ peptides, whose production is modulated by the phosphorylation of amyloid precursor protein (APP) mediated by kinases, whereas NFTs are a result of hyperphosphorylation events of tau. Even though several studies proposed that the family of casein kinase (CK)1 is involved in the pathogenesis of AD, so far, not much is known about CK1δ-specific phosphorylation of AD-associated key proteins like APP and tau.As reported, members of the CK1 family are critically involved in the pathogenesis of AD, especially by the hyperphosphorylation of tau. Precise effects and mechanisms of CK1δ-mediated tau phosphorylation are only weakly understood. In the first part of the study recombinantly produced tau441 was phosphorylated by CK1δ in vitro and analyzed via mass spectrometry resulting in ten potential phosphorylation sites. Among them, five potential phosphorylation sites are known to be associated with AD. To verify these results, several biochemical approaches (such as in vitro kinase assay and two-dimensional phosphopeptide analysis) were carried out with generated tau441 phospho mutants. By using these biochemical approaches, the AD-associated phosphorylation sites Ser68/Thr71 and Ser289 were confirmed as targets for CK1δ. To confirm the biochemical approaches, the “Alzheimer’s-in-a-dish” model was established by viral transduction of human neural progenitor cells (hNPCs) with mutant forms APP and presenilin, which are relevant in early-onset familiar AD. Treatment of transduced and differentiated hNPCs with the CK1δ-specific inhibitor PF-670462 and following Western blot analysis detected Ser214 as CK1δ-targeted physiological relevant phosphorylation site. By using an in vitro tau aggregation assay, a possible role of CK1δ-mediated phosphorylation on tau aggregation could be demonstrated. These results provide evidence that CK1δ represents an interesting point for therapeutic intervention in the development of AD, which was further investigated through the inhibition of CK1δ in general (via CK1δ-specific inhibitors) or alternatively, the intervention of CK1δ activity at the level of CK1δ-substrate interactions via CK1δ-derived peptides.Since the majority of previously developed CK1 inhibitors do not selectively inhibit CK1 isoforms, further chemical improvements are being sought to achieve greater selectivity (and potency). In cooperation with external partners, further developed compounds including benzimidazole derivatives, inhibitors of Wnt production (IWP)- derived compounds and isoxazole derivatives were tested for their selectivity and potency towards CK1δ under established standard conditions. Highly selective and potent inhibitors (383, 384 and 394) and their related lead structures were tested for their cytotoxic effects and for their biological effect in the complex “Alzheimer’s-in-a-dish” model mimicking AD pathology. According to the results obtained from the “Alzheimer’s-in-a-dish” model the IWP-related compound 384 and to a lesser extent the chemically related inhibitor Liu-20 seem to have a beneficial effect on tau pathology, but not Aβ pathology. However, potential off-target effects, which might be involved in the beneficial therapeutic effect on tau pathology, have to be investigated.As an alternative to CK1δ-specific inhibitors probably exerting off-target effects, a novel therapeutic approach using CK1δ-derived peptides was investigated. Initially, CK1δ-derived peptides manipulating the protein-protein interaction of CK1δ and APP and/or tau were characterized by interaction and phosphorylation analysis in vitro (via streptavidin-linked assays, surface plasmon resonance and in vitro kinase assays). The set of selected peptides subsequently demonstrated their potential to enter neuronal cells without inducing cytotoxic effects. For at least four of the identified CK1δ-derived peptides, reduced Aβ levels and amyloid plaque formation (δ-101 and δ-311) or reduced tau hyperphosphorylation and aggregation (δ-31 and δ-281) could be successfully demonstrated in the complex “Alzheimer’s-in-a-dish” model mimicking AD pathology.Consequently, the results obtained in this study provide an interesting starting point for further development and optimization steps of novel potent and/or highly selective small-molecule or peptide based-inhibitors as highly innovative and novel pharmacological tools for the treatment of AD.

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-β (Aβ), 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 Aβ 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.

Protein phosphorylation is one of the most important reversible post-translational modifications. It affects every cellular process including differentiation, metabolism and cell cycle. Eukaryotic protein kinases (ePK) catalyse the transfer of a phosphate from ATP onto proteins, which regulates fast changes in protein activity, structure or subcellular localisation. The systematic identification of substrates is thus crucial to characterise the functions of kinases and determine the pathways they regulate, and even more so when studying the impact of pathogens-excreted kinases on the host cell signal transduction. Several strategies and approaches have been used to identify substrates, but all show important limitations thus calling for the development of new efficient and more convenient approaches for kinase substrate identification. Herein, we present SILAkin, a novel and easy method to identify substrates that is applicable to most kinases. It combines phosphatase treatment, pulse heating, in vitro kinase assay (IVKA) and SILAC (Stable Isotope Labeling with Amino acids in Cell culture)-based quantitative mass spectrometry (MS). We developed SILAkin using the Leishmania casein kinase 1 (L-CK1.2) as experimental model. Leishmania, an intracellular parasite causing Leishmaniasis, releases L-CK1.2 in its host cell. Applying this novel assay allowed us to gain unprecedented insight into host-pathogen interactions through the identification of host substrates phosphorylated by pathogen-excreted kinases. We identified 225 substrates, including 85% previously unknown that represent novel mammalian CK1 targets, and defined a novel CK1 phosphorylation motif. The substratome was validated experimentally by L-CK1.2 and human CK1δ, demonstrating the efficiency of SILAkin to identify new substrates and revealing novel regulatory pathways. Finally, SILAkin was instrumental in highlighting host pathways potentially regulated by L-CK1.2 in Leishmania-infected host cells, described by the GO terms ‘viral & symbiotic interaction’, ‘apoptosis’, ‘actin cytoskeleton organisation’, and ‘RNA processing and splicing’. SILAkin thus can generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogen adapted for intracellular survival.

In a 24-week trial involving patients with rheumatoid arthritis that was refractory to biologic agents, the JAK1 inhibitor upadacitinib was superior to the T-cell costimulation modulator abatacept in reducing disease activity as assessed by a composite measure of joint changes and C-reactive protein level.