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  • The substitution of serine by an alanine


    The substitution of serine 185 by an alanine led to an increase of EGF-R stabilization along with a better binding of PRK1, a RhoB effector described to be involved in EGF-R intracellular traffic. Overall these data indicated that the phosphate group on serine 185 impaired its binding to the effector and consequently its functions. Moreover we observed that the inhibition of CK1 by IC261 prevented fully the decrease of the EGF-R cellular pool after addition of EGF (data not shown). The other CK1 inhibitor, D4476, displayed a similar effect, slowing down also strongly the EGF-R decrease after addition of EGF (data not shown). These results suggested that CK1 might play a key role in EGF-R stability in mammal cells and thus might regulate receptor trafficking. Besides it was shown that yeast casein kinase I controls a late step in the endocytic trafficking [66]. Some publications have also proposed that mammalian CK1 play a role in membrane trafficking, such as protein traffic through the early secretory pathway [67] and synaptic vesicle exocytosis [68]. The CK1 phosphorylation of RhoB might be also critical for its role in oncogenesis. It is noteworthy that several reports link elevated CK1 activity and/or expression to cancer [56], thus it could be hypothesized that this process contributes to the downregulation of RhoB activity during carcinogenesis in addition to the downregulation of its expression [20], [22], [23].
    Alzheimer’s disease is a devastating illness, which robs patients of the ability to manage their lives on their own. This illness is accompanied by protein BGP-15 in the brain composed of the amyloid-β-peptide (Aβ), which are called amyloid plaques. The amyloid-β-peptide is generated by the subsequent degradation of the amyloid precursor protein (APP), a type I transmembrane protein, by two aspartyl proteases, the β-secretase and the γ-secretase. The γ-secretase is a promising target for therapeutic intervention as it liberates various Aβ-peptides with a length of 38, 40, or 42 amino acids. The toxicity depends on the length: Aβ is the most toxic species while Aβ is regarded to be non-toxic as increased production of Aβ does not diminish cellular viability. Several γ-secretase inhibitors (GSI), which decrease total Aβ levels, and several γ-secretase modulators (GSM), which shift the cleavage-site to the non-toxic Aβ, have been identified so far., , Flajolet et al. reported IC261 () (), a presumably selective ATP-competitive casein kinase 1ε (CK1ε) inhibitor, which is also an equipotent inhibitor to the CK1δ-isoform (CK1δ) (IC=2.57μM in cells). IC261 exerts rather weak GSI activity in comparison to reported potent GSIs., , , IC261 causes a significant reduction of Aβ (68%) and Aβ (61%) levels in N2a cells overexpressing constitutively active CK1ε-271 within 5–50μM concentration BGP-15 at 3h after incubation. The reported increase of Aβ secretion by approximately 35% (Fig. 2A in Ref. ) under overexpression of constitutively active CK1δ in N2a cells and the similarity of IC261 with the known, potent GSM (Sulindac-S (), and Sulindac-sulfon ()) stimulated us to investigate the oxoindole-backbone of IC261 common to many kinase inhibitors and the potential influence of CK1ε/δ inhibitors on γ-secretase activity). H4-cells do neither express constitutively active CK1ε-271 nor do they overexpress CK1ε, which were postulated to be the regulating CK1 isoforms. Actually, Aβ secretion from the utilized H4 cells responded to IC261 treatment five times stronger, suggesting an CK1ε independent effect. A dual structure–activity relationship analysis (SAR) towards γ-secretase activity in H4-cells and CK1δ activity was carried out by systematical variation of the oxoindole substitution utilizing the CK1/IC261 co-crystallized structure (PDB: ). (). The CK1 isoforms differ in the primary structure of the C-terminal non-catalytic domain. However, CK1δ and CK1ε do not differ in the ATP-binding-site for IC261, thus a cell free CK1δ activity assay guided the structure–activity relationship for both CK1 isoforms. This approach is commonly employed for the development of GSK3α/β inhibitors.