Newly Developed CK1-Specific Inhibitors Show Specifically Stronger Effects on CK1 Mutants and Colon Cancer Cell Lines

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.


Introduction
CK1δ, a member of the CK1 family (formerly named casein kinase 1 family), is involved in regulation of different cellular processes-among them cell cycle, mitosis, DNA damage signaling, and developmental pathways. Dysregulation of CK1δ expression and/or activity has been observed in different types of disorders, including cancer. So far, different CK1δ mutations have been already identified and seem to have a higher oncogenic potential in comparison to CK1δ wild type [1,2]. Recently, the CK1δ T67S mutant has been identified in colon and rectal cancer (CRC) and this mutant showed increased in vitro kinetics and is associated with increased cell proliferation and tumor growth in both cell culture and in a subcutaneous tumor xenotransplantation mouse model [2]. Another  [5,6]. Positions of the respective mutations in the CK1δ protein and their frequency of detection are indicated. Missense mutations are displayed in green, truncating mutations in black, and other types of mutations (e.g., protein fusion mutations) in purple. Highlighted mutations were selected for further analysis. Abbreviations: A, alanine; aa, amino acids, E, glutamic acid; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; P, proline; Q, glutamine; R, arginine; S, serine; V, valine; W, tryptophan; Y, tyrosine; *, stop codon. Mutations of CSNK1D selected for further analysis after performing a database query at cBioPortal for Cancer Genomics are listed together with information about the type(s) of cancer in which the respective mutations have been identified [5,6]. Abbreviations: A, alanine; E, glutamic acid; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; P, proline; Q, glutamine; R, arginine; S, serine; V, valine; W, tryptophan; Y, tyrosine; *, stop codon.
Basically, kinetic data revealed substrate-specific differences, which were most significant for mutant R115H, displaying remarkably decreased kinase activity compared to wild type CK1δ for substrates α-casein and GST-β-catenin 1−181 (5.4-and 2.7-fold lower, respectively) while kinase activity was remarkably increased for substrate GST-p53 1−64 (3.2-fold increased) . For almost half of the tested mutants (eight out of 17), at least ten-fold lower kinase activity compared to wild type CK1δ could be observed ( Figure 2). Interestingly, except for R168H, CK1δ mutations between amino acids Ile-148 and Leu-252 presented lower V max and k cat compared to the wild type. Mutants E247K and L252P even showed almost no residual kinase activity and presented very low V max and k cat values of 0.0007 and 0.0005-fold less, respectively, compared to the wild type (Table S1). In contrast, hyperactive mutants were also identified, presenting significantly higher values for V max and elevated k cat compared to the wild type. These include mutants A36V, R115H, R127L, R127Q, R168H, R299Q, and Q399 *. Michaelis-Menten kinetics have been analyzed for CK1δ wild type and mutants using either α-casein, GST-β-catenin 1−181 , or GST-p53 1−64 as substrate. The kinetic parameters Vmax (a) and kcat (b) were normalized toward the respective parameters determined for CK1δ wild type. Data is presented as mean values and standard deviation (SD) for experiments performed in triplicate. Abbreviations: A, alanine; E, glutamic acid; G, glycine; GST, glutathione S-transferase; H, histidine; I, isoleucine; K, lysine; kcat, turnover number; L, leucine; M, methionine; P, proline; Q, glutamine; R, arginine; S, serine; V, valine; Vmax, maximum enzyme reaction velocity; W, tryptophan; WT, wild type; Y, tyrosine; *, stop codon.

Hyperactive CK1δ Mutants Are More Sensitive to Different CK1-Specific Inhibitors
As already described in previous studies, mutations in the coding sequence for CK1δ cannot only influence the activity of the enzyme but also the binding and efficacy of small molecule inhibitors [2,21]. Therefore, the sensitivity of CK1δ hyperactive mutants toward different CK1specific inhibitors was analyzed and residual kinase activities upon inhibitor treatment are summarized in Table 2. Interestingly, all tested inhibitors , Richter-2 [17], IWP-2, and IWP-4 [15,22]) showed significantly stronger inhibitory effects on CK1δ R127Q compared to CK1δ WT , leading to a residual activity between 19% and 37%. In addition, kinase activity of CK1δ R168H was also influenced stronger by the benzimidazole-based compounds Bischof-5 and Richter-2. Remarkably stronger inhibition than for CK1δ WT could also be observed for CK1δ R115H applying Bischof-5 as well as for CK1δ R299Q when using IWP-2. Being most sensitive to benzimidazole-based inhibitor compounds and IWPs, mutants CK1δ R115H , CK1δ R127Q , and CK1δ R168H were selected for subsequent analysis.  Michaelis-Menten kinetics have been analyzed for CK1δ wild type and mutants using either α-casein, GST-β-catenin 1−181 , or GST-p53 1−64 as substrate. The kinetic parameters V max (a) and k cat (b) were normalized toward the respective parameters determined for CK1δ wild type. Data is presented as mean values and standard deviation (SD) for experiments performed in triplicate. Abbreviations: A, alanine; E, glutamic acid; G, glycine; GST, glutathione S-transferase; H, histidine; I, isoleucine; K, lysine; k cat , turnover number; L, leucine; M, methionine; P, proline; Q, glutamine; R, arginine; S, serine; V, valine; V max , maximum enzyme reaction velocity; W, tryptophan; WT, wild type; Y, tyrosine; *, stop codon.

Hyperactive CK1δ
Mutants Are More Sensitive to Different CK1-Specific Inhibitors As already described in previous studies, mutations in the coding sequence for CK1δ cannot only influence the activity of the enzyme but also the binding and efficacy of small molecule inhibitors [2,21]. Therefore, the sensitivity of CK1δ hyperactive mutants toward different CK1-specific inhibitors was analyzed and residual kinase activities upon inhibitor treatment are summarized in Table 2. Interestingly, all tested inhibitors , Richter-2 [17], IWP-2, and IWP-4 [15,22]) showed significantly stronger inhibitory effects on CK1δ R127Q compared to CK1δ WT , leading to a residual activity between 19% and 37%. In addition, kinase activity of CK1δ R168H was also influenced stronger by the benzimidazole-based compounds Bischof-5 and Richter-2. Remarkably stronger inhibition than for CK1δ WT could also be observed for CK1δ R115H applying Bischof-5 as well as for CK1δ R299Q when using IWP-2. Being most sensitive to benzimidazole-based inhibitor compounds and IWPs, mutants CK1δ R115H , CK1δ R127Q , and CK1δ R168H were selected for subsequent analysis. Kinase activity of CK1δ WT and mutants was tested in presence of the CK1-specific inhibitors Bischof-5, Richter-2, IWP-2, and IWP-4, which were used in the IC 50 concentration previously determined for CK1δ WT [14,15,17]. Residual activity is presented as normalized values using the respective DMSO control as a reference (100%). Values were adjusted in order to obtain half-maximal inhibition values for CK1δ WT (IC 50 ; 50%). Data is represented as mean values ± SD of normalized triplicates. Residual kinase activities lower than 50% are highlighted in bold. Abbreviations: A, alanine; V, valine; R, arginine; H, histidine; L, leucine; Q, glutamine; WT, wild type; *, stop codon.

Modeling of ATP Binding to CK1δ Mutants
Modeling of MgATP in the mutated CK1δ structures (R115H, R127Q, and R168H) was performed to evaluate differences in binding of MgATP compared to the original binding pose within the ATP active site of wild type CK1δ. In fact, all tested mutations seemed to have no effect on binding of MgATP itself, as they were not situated in close proximity to the ATP-binding pocket. In Figure 3, the respective mutations are highlighted with regard to the apo structure. In line with this notion, performing SiteMap calculations at these mutated structures indicated that all three mutations were located at a potential protein substrate-binding region. Kinase activity of CK1δ WT and mutants was tested in presence of the CK1-specific inhibitors Bischof-5, Richter-2, IWP-2, and IWP-4, which were used in the IC50 concentration previously determined for CK1δ WT [14,15,17]. Residual activity is presented as normalized values using the respective DMSO control as a reference (100%). Values were adjusted in order to obtain half-maximal inhibition values for CK1δ WT (IC50; 50%). Data is represented as mean values ± SD of normalized triplicates. Residual kinase activities lower than 50% are highlighted in bold. Abbreviations: A, alanine; V, valine; R, arginine; H, histidine; L, leucine; Q, glutamine; WT, wild type; *, stop codon.

Modeling of ATP Binding to CK1δ Mutants
Modeling of MgATP in the mutated CK1δ structures (R115H, R127Q, and R168H) was performed to evaluate differences in binding of MgATP compared to the original binding pose within the ATP active site of wild type CK1δ. In fact, all tested mutations seemed to have no effect on binding of MgATP itself, as they were not situated in close proximity to the ATP-binding pocket. In Figure 3, the respective mutations are highlighted with regard to the apo structure. In line with this notion, performing SiteMap calculations at these mutated structures indicated that all three mutations were located at a potential protein substrate-binding region.  R127Q , and (c) CK1δ R168H with MgATP (red/white atoms) bound in the ATP-binding pocket. The mutated residues are highlighted in purple and are indicated with white arrowheads. Induced fit calculations were performed based on a homology model of 4twc [14] on 1csn [23] CK1δ crystal structures (obtained from protein data bank (PDB) [24]) in order to realize the adjustment of the P-loop.
The significantly increased inhibition of CK1δ R127Q by compounds Bischof-5, Richter-2, IWP-2, and IWP-4 suggests an effect of the amino acid exchange toward the active site such as a spatial adjustment of the ATP-binding pocket. However, and in contrast to the wet lab data, our modeling data does not reflect this effect regarding inhibitor binding poses.
In the case of CK1δ R168H , an impact toward the active site is implied as this mutation is located within the activation loop. However, in order to define these influences on inhibitor binding in more detail, further molecular dynamic simulations would be required. In order to illustrate the situation, the result of inhibitor docking of Bischof-5 in CK1δ R168H is shown in Figure S1.

Synthesis of New IWP-Based Inhibitor Compounds
Previously, more profound inhibitory effects on a CK1δ gatekeeper mutant (CK1δ M82F ) in comparison to CK1δ wild type have been determined for several IWP compounds [15]. In order to identify IWP-based inhibitor compounds stronger inhibiting CK1δ mutants than CK1δ WT , new compounds were designed based on the IWP backbone structure. Chan-Lam coupling of compounds  R127Q , and (c) CK1δ R168H with MgATP (red/white atoms) bound in the ATP-binding pocket. The mutated residues are highlighted in purple and are indicated with white arrowheads. Induced fit calculations were performed based on a homology model of 4twc [14] on 1csn [23] CK1δ crystal structures (obtained from protein data bank (PDB) [24]) in order to realize the adjustment of the P-loop.
The significantly increased inhibition of CK1δ R127Q by compounds Bischof-5, Richter-2, IWP-2, and IWP-4 suggests an effect of the amino acid exchange toward the active site such as a spatial adjustment of the ATP-binding pocket. However, and in contrast to the wet lab data, our modeling data does not reflect this effect regarding inhibitor binding poses.
In the case of CK1δ R168H , an impact toward the active site is implied as this mutation is located within the activation loop. However, in order to define these influences on inhibitor binding in more detail, further molecular dynamic simulations would be required. In order to illustrate the situation, the result of inhibitor docking of Bischof-5 in CK1δ R168H is shown in Figure S1.

Synthesis of New IWP-Based Inhibitor Compounds
Previously, more profound inhibitory effects on a CK1δ gatekeeper mutant (CK1δ M82F ) in comparison to CK1δ wild type have been determined for several IWP compounds [15]. In order to identify IWP-based inhibitor compounds stronger inhibiting CK1δ mutants than CK1δ WT , new compounds were designed based on the IWP backbone structure. Chan-Lam coupling of compounds The synthesis of the benzothiazole-linker was already described by García-Reyes and Witt et al. [15]. The coupling-conditions of compounds 8−14 with the benzothiazole-linker 15 were the same as for previous IWP derivatives (Scheme 2) [15,30].

Characterization of Newly Developed IWP-Derivatives
Newly developed IWP-derivatives were first tested in vitro for their inhibitory potency and selectivity among wild type CK1 isoforms α, γ, δ, and ε ( Figure 4a). All tested compounds (16, 17, 18, and 19) strongly inhibited kinase activity of CK1δ and ε, while activity of CK1α and γ was not affected. Subsequently, IC50 values for CK1δ were determined for all new compounds in order to characterize the inhibitory effects on CK1δ in more detail. Among this set of inhibitors, 18 showed The synthesis of the benzothiazole-linker was already described by García-Reyes and Witt et al. [15]. The coupling-conditions of compounds 8−14 with the benzothiazole-linker 15 were the same as for previous IWP derivatives (Scheme 2) [15,30].  [25]. Instead of using an oxygen filled balloon, compressed air was passed through the solvent. Compound 1 was already described by Abd El Kader et al. [26]. Benzyl compounds 5−7 were synthesized based on Roopan et al. using nano iron under basic conditions to provide selective N-substitution [27]. Introduction of the thioxo group (compounds 8−14) followed the procedure of Xu and Yadan using phenyl thionochloroformate [28]. Compound 8 as well has been described in the literature [29]. The synthesis of the benzothiazole-linker was already described by García-Reyes and Witt et al. [15]. The coupling-conditions of compounds 8−14 with the benzothiazole-linker 15 were the same as for previous IWP derivatives (Scheme 2) [15,30].

Characterization of Newly Developed IWP-Derivatives
Newly developed IWP-derivatives were first tested in vitro for their inhibitory potency and selectivity among wild type CK1 isoforms α, γ, δ, and ε ( Figure 4a). All tested compounds (16, 17, 18, and 19) strongly inhibited kinase activity of CK1δ and ε, while activity of CK1α and γ was not affected. Subsequently, IC50 values for CK1δ were determined for all new compounds in order to characterize the inhibitory effects on CK1δ in more detail. Among this set of inhibitors, 18 showed

Characterization of Newly Developed IWP-Derivatives
Newly developed IWP-derivatives were first tested in vitro for their inhibitory potency and selectivity among wild type CK1 isoforms α, γ, δ, and ε ( Figure 4a). All tested compounds (16, 17, 18, and 19) strongly inhibited kinase activity of CK1δ and ε, while activity of CK1α and γ was not affected. Subsequently, IC 50 values for CK1δ were determined for all new compounds in order to characterize the inhibitory effects on CK1δ in more detail. Among this set of inhibitors, 18 showed the strongest In addition to wild type CK1δ the new compounds were also tested for inhibition of mutants CK1δ R127Q and CK1δ T168H . Moreover, the previously published hyperactive mutant CK1δ T67S was also included. Interestingly, inhibitors 16, 17, and 18 inhibit the hyperactive mutant CK1δ T67S slightly stronger when compared to wild type CK1δ (Figure 5a). Kinase activity of CK1δ R127Q was also significantly stronger inhibited by 16 (Figure 5b), while strongest effects on CK1δ R168H activity could be observed for compound 18 ( Figure 5c).  In addition to wild type CK1δ the new compounds were also tested for inhibition of mutants CK1δ R127Q and CK1δ T168H . Moreover, the previously published hyperactive mutant CK1δ T67S was also included. Interestingly, inhibitors 16, 17, and 18 inhibit the hyperactive mutant CK1δ T67S slightly stronger when compared to wild type CK1δ ( Figure 5a). Kinase activity of CK1δ R127Q was also significantly stronger inhibited by 16 (Figure 5b), while strongest effects on CK1δ R168H activity could be observed for compound 18 (Figure 5c).  In addition to wild type CK1δ the new compounds were also tested for inhibition of mutants CK1δ R127Q and CK1δ T168H . Moreover, the previously published hyperactive mutant CK1δ T67S was also included. Interestingly, inhibitors 16, 17, and 18 inhibit the hyperactive mutant CK1δ T67S slightly stronger when compared to wild type CK1δ ( Figure 5a). Kinase activity of CK1δ R127Q was also significantly stronger inhibited by 16 (Figure 5b), while strongest effects on CK1δ R168H activity could be observed for compound 18 ( Figure 5c).  . CK1δ WT and selected hyperactive CK1δ mutants are inhibited by newly developed CK1-specific inhibitors. Inhibition of CK1δ WT and mutants CK1δ T67S (a), CK1δ R127Q (b), or CK1δ R168H (c) by IWP-derivatives 16, 17, 18, and 19 at a concentration of 10 µM has been determined by in vitro kinase assays. Please note that the Y-axis in (a) is set to a maximum of 80%, while in (b,c) it is set to 40%. Residual kinase activity has been normalized toward the respective DMSO control activity (100%). Results are presented as mean values of experiments performed in triplicate. Error bars represent the SD. Statistical analysis was done by performing two-way ANOVA using following levels of significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant. Abbreviations: H, histidine; Q, glutamine; R, arginine; S, serine; T, threonine; TV, transcription variant; WT, wild type.

IWP-Derivative 18 Selectively Inhibits Cellular CK1δ and Shows Stronger Effects on HeLa Cells Expressing Hyperactive CK1δ Mutants
Following biochemical characterization, the effects of newly designed IWP-based compounds on cell viability were analyzed for cells being deficient for CK1δ (HeLa CK1δ −/− ) and cells expressing CK1δ (either parental HeLa or HeLa CK1δ −/− being stably transfected with CK1δ, termed HeLa CK1δ +/+ ) ( Figure 6a). Interestingly, compound 18 and to a lesser extent also compound 19 seemed to have a CK1δ-dependent effect on cell viability. In fact, cell viability of the CK1δ-expressing cells (HeLa parental or CK1δ +/+ ) was more reduced by treatment with compound 18 or 19 compared to CK1δ-deficient cells (HeLa CK1δ −/− ) (Figure 6a). Moreover, 50% effective concentration (EC 50 ) values were determined for HeLa CK1δ +/+ and CK1δ −/− cells, demonstrating that viability of cells expressing CK1δ was significantly stronger affected by treatment with 18 ( Figure 6b). In contrast, effects on cell viability observed for parental cells treated with 16 or 17 were less pronounced than for compounds 18 and 19, and the effects on cell viability were not reduced for CK1δ −/− cells compared to parental and/or CK1δ +/+ cells (Figure 6a).

IWP-Derivative 18 Selectively Inhibits Cellular CK1δ and Shows Stronger Effects on HeLa Cells Expressing Hyperactive CK1δ Mutants
Following biochemical characterization, the effects of newly designed IWP-based compounds on cell viability were analyzed for cells being deficient for CK1δ (HeLa CK1δ −/− ) and cells expressing CK1δ (either parental HeLa or HeLa CK1δ −/− being stably transfected with CK1δ, termed HeLa CK1δ +/+ ) (Figure 6a). Interestingly, compound 18 and to a lesser extent also compound 19 seemed to have a CK1δ-dependent effect on cell viability. In fact, cell viability of the CK1δ-expressing cells (HeLa parental or CK1δ +/+ ) was more reduced by treatment with compound 18 or 19 compared to CK1δ-deficient cells (HeLa CK1δ −/− ) (Figure 6a). Moreover, 50% effective concentration (EC50) values were determined for HeLa CK1δ +/+ and CK1δ −/− cells, demonstrating that viability of cells expressing CK1δ was significantly stronger affected by treatment with 18 ( Figure 6b). In contrast, effects on cell viability observed for parental cells treated with 16 or 17 were less pronounced than for compounds 18 and 19, and the effects on cell viability were not reduced for CK1δ −/− cells compared to parental and/or CK1δ +/+ cells (Figure 6a).

2.7.A Second Set of IWP-Derivatives Shows Improved Inhibition of CK1δ In Vitro and Cell Line-Specific Effects on Colon Cancer Cell Lines
The tested IWP-derivatives, 16, 17, 18, and 19, demonstrated remarkable inhibition of CK1 isoforms δ and ε in vitro and especially for compounds 18 and 19 significant effects on the viability of CK1δ mutant-expressing cells could be observed. Therefore, in order to obtain additional compounds with improved inhibitory potential, a second set of IWP-derived compounds was developed and initially tested in our in vitro screening procedure. In contrast to the phenylsubstituted compounds of the first set, compounds of the second set carried a benzyl-substituted pyrimidinone.
All tested compounds of this second set (20, 21, and 22) strongly inhibited kinase activity of CK1δ and ε. While activity of CK1α was also reduced by at least 50% by all tested compounds, kinase activity of CK1γ was not affected (Figure 8a). Also for the second set of inhibitor compounds, the IC50 values for CK1δ were determined. Among this set of inhibitors, significantly lower IC50 values could be determined in comparison to the first set. The strongest effects and the lowest IC50 values of 0.086 and 0.120 μM could be determined for compounds 20 and 21, respectively (Figure 8b).

A Second Set of IWP-Derivatives Shows Improved Inhibition of CK1δ In Vitro and Cell Line-Specific Effects on Colon Cancer Cell Lines
The tested IWP- derivatives, 16, 17, 18, and 19, demonstrated remarkable inhibition of CK1 isoforms δ and ε in vitro and especially for compounds 18 and 19 significant effects on the viability of CK1δ mutant-expressing cells could be observed. Therefore, in order to obtain additional compounds with improved inhibitory potential, a second set of IWP-derived compounds was developed and initially tested in our in vitro screening procedure. In contrast to the phenyl-substituted compounds of the first set, compounds of the second set carried a benzyl-substituted pyrimidinone.
All tested compounds of this second set (20, 21, and 22) strongly inhibited kinase activity of CK1δ and ε. While activity of CK1α was also reduced by at least 50% by all tested compounds, kinase activity of CK1γ was not affected (Figure 8a). Also for the second set of inhibitor compounds, the IC 50 values for CK1δ were determined. Among this set of inhibitors, significantly lower IC 50 values could be determined in comparison to the first set. The strongest effects and the lowest IC 50 values of 0.086 and 0.120 µM could be determined for compounds 20 and 21, respectively (Figure 8b).
Because compound 20 showed most remarkable inhibition of CK1δ in vitro, this compound was also tested for its effects on parental and CK1δ-deficient (CK1δ −/− ) HeLa cells as well as on CK1δ-deficient HeLa cells being stably transfected with CK1δ (HeLa CK1δ +/+ ) in order to demonstrate CK1δ-selective effects. Although cell viability of HeLa CK1δ −/− was significantly less affected compared to parental cells or HeLa CK1δ +/+ cells, observed effects of compound 20 were not as selective for CK1δ as previously determined for compound 18 (and compound 19) (Figure 9a). Furthermore, testing of compound 20 on HeLa CK1δ −/− cells stably expressing CK1δ mutants T67S, R127Q, or R168H revealed no mutant-specific effects (Figure 9b−d).
With regard to the potential future use of CK1 isoform-specific inhibitors in therapeutic strategies for cancer treatment, the influence of compounds 20, 21, and 22 on cell viability of various established colon cancer cell lines was tested by performing MTT viability assays. In an initial screening using all inhibitor compounds at a concentration of 10 µM, most of the inhibitors of the second set only showed weak effects. For cell line HCT-116, all compounds showed a reduction of cell viability by 30% to 40% (Figure 10a). For cell lines, HT-29 and SW480 inhibitors 20 and 22 also reduced cell viability by approximately 35%, whereas treatment with 21 only resulted in minor changes of viability. Strongest effects among all colon cancer cell lines could be observed for SW620. Treatment with 20 and 21 resulted in a decrease of about 60% and 50%, respectively, while a reduction of only 25% was obtained for 22 (Figure 10a).

pyrimidinone.
All tested compounds of this second set (20, 21, and 22) strongly inhibited kinase activity of CK1δ and ε. While activity of CK1α was also reduced by at least 50% by all tested compounds, kinase activity of CK1γ was not affected (Figure 8a). Also for the second set of inhibitor compounds, the IC50 values for CK1δ were determined. Among this set of inhibitors, significantly lower IC50 values could be determined in comparison to the first set. The strongest effects and the lowest IC50 values of 0.086 and 0.120 μM could be determined for compounds 20 and 21, respectively (Figure 8b). Because compound 20 showed most remarkable inhibition of CK1δ in vitro, this compound was also tested for its effects on parental and CK1δ-deficient (CK1δ −/− ) HeLa cells as well as on CK1δdeficient HeLa cells being stably transfected with CK1δ (HeLa CK1δ +/+ ) in order to demonstrate CK1δselective effects. Although cell viability of HeLa CK1δ −/− was significantly less affected compared to parental cells or HeLa CK1δ +/+ cells, observed effects of compound 20 were not as selective for CK1δ as previously determined for compound 18 (and compound 19) (Figure 9a). Furthermore, testing of compound 20 on HeLa CK1δ −/− cells stably expressing CK1δ mutants T67S, R127Q, or R168H revealed no mutant-specific effects (Figure 9b−d).   For a more detailed characterization, the two best inhibitors per cell line were analyzed for dosedependent effects using different concentrations ranging from 0.625 to 20 μM ( Figure S2). For HCT-116 cells, treatment with inhibitor 20 as well as 22 resulted in a reduction of cell viability by approximately 30%. This could already be observed for low inhibitor concentrations of 0.625 or 1.25 μM, respectively. However, this effect is only very slightly extended by increasing inhibitor concentration and, even at a concentration of 10 μM, only 45% of viability reduction could be determined for both compounds. For HT-29 and SW480 cells, the observed effects were even less remarkable. In addition, effects of 21 on SW620 were only visible at the highest concentrations tested, whereas clear dose-dependent effects could be observed for the treatment of SW620 cells with inhibitor 20. Obtained data even allowed for the determination of an EC50 value of 3 μM for compound 20 and cell line SW620 (Figure 10b and Figure S2).

Discussion
Due to the involvement of CK1 isoforms in numerous cellular processes, dysregulation of CK1 can be linked to the development of different cancers as well as to neurodegenerative diseases. Apart from an increase of expression levels, elevated CK1δ kinase activity can also be caused by mutations occurring in the genomic sequence coding for CK1δ. In order to pharmacologically control CK1δ activity, several small molecule inhibitors are already available. However, CK1 isoform-specificity and pharmacokinetic parameters for most of these inhibitors are not satisfactory and new inhibitor compounds with improved properties are required. Furthermore, specific targeting of CK1δ mutants occurring in certain tumor entities would provide a broad therapeutic window for the treatment of the respective tumor without inducing severe off-target effects in healthy tissue. In order to deal with these issues, in the present study we (i) characterized enzyme kinetic properties of CK1δ mutants identified in various cancer entities including the colon and rectum and (ii) we characterized the CK1δ wild type-and mutant-specific inhibitory potential of a small set of CK1δ-specific small molecule inhibitors in vitro and on four colon cancer cell lines.
So far, only few CK1δ mutants have been described in the literature, which could be linked to the pathogenesis of certain diseases. For instance, mutation CK1δ S97C has been associated with the development of breast cancer and ductal carcinoma [1]. Mutation CK1δ R324H has been linked to For a more detailed characterization, the two best inhibitors per cell line were analyzed for dose-dependent effects using different concentrations ranging from 0.625 to 20 µM ( Figure S2). For HCT-116 cells, treatment with inhibitor 20 as well as 22 resulted in a reduction of cell viability by approximately 30%. This could already be observed for low inhibitor concentrations of 0.625 or 1.25 µM, respectively. However, this effect is only very slightly extended by increasing inhibitor concentration and, even at a concentration of 10 µM, only 45% of viability reduction could be determined for both compounds. For HT-29 and SW480 cells, the observed effects were even less remarkable. In addition, effects of 21 on SW620 were only visible at the highest concentrations tested, whereas clear dose-dependent effects could be observed for the treatment of SW620 cells with inhibitor 20. Obtained data even allowed for the determination of an EC 50 value of 3 µM for compound 20 and cell line SW620 (Figure 10b and Figure S2).

Discussion
Due to the involvement of CK1 isoforms in numerous cellular processes, dysregulation of CK1 can be linked to the development of different cancers as well as to neurodegenerative diseases. Apart from an increase of expression levels, elevated CK1δ kinase activity can also be caused by mutations occurring in the genomic sequence coding for CK1δ. In order to pharmacologically control CK1δ activity, several small molecule inhibitors are already available. However, CK1 isoform-specificity and pharmacokinetic parameters for most of these inhibitors are not satisfactory and new inhibitor compounds with improved properties are required. Furthermore, specific targeting of CK1δ mutants occurring in certain tumor entities would provide a broad therapeutic window for the treatment of the respective tumor without inducing severe off-target effects in healthy tissue. In order to deal with these issues, in the present study we (i) characterized enzyme kinetic properties of CK1δ mutants identified in various cancer entities including the colon and rectum and (ii) we characterized the CK1δ wild type-and mutant-specific inhibitory potential of a small set of CK1δ-specific small molecule inhibitors in vitro and on four colon cancer cell lines.
So far, only few CK1δ mutants have been described in the literature, which could be linked to the pathogenesis of certain diseases. For instance, mutation CK1δ S97C has been associated with the development of breast cancer and ductal carcinoma [1]. Mutation CK1δ R324H has been linked to increased oncogenic potential and was detected in a patient with large and multiple colonic polyps. In an experimental setting, CK1δ R324H has been shown to potently transform RKO colon cancer cells [3]. Although both mutants are not associated with an immediate increase of kinase activity, at least for CK1δ R324H , a mechanism has been detected leading to Wnt/β-catenin-independent morphogenetic movements in early Xenopus development [3]. Furthermore, a hyperactive mutant (CK1δ T67S ) has been found in colon and rectal cancer and expression of CK1δ T67S resulted in higher oncogenic potential in cell culture experiments as well as in a mouse xenotransplantation model [2].
Elevated kinase activity could be determined for mutants GST-CK1δ R127L and GST-CK1δ R127Q , which can be explained by changes in the electrical charge of the protein resulting from the exchange of the positively charged amino acid arginine to hydrophobic leucine or polar, but uncharged, glutamine. These changes in protein charge might result in conformational changes affecting kinase activity. Moreover, the catalytically essential residue Arg-128 is located in direct proximity to the mutated position 127, and the entire sequence between amino acids 128-133 belongs to the catalytic loop of CK1δ. Therefore, mutations within this sequence or in close proximity to it could be linked to alteration of kinase activity.
Remarkably decreased kinase activity could be observed for GST-CK1δ mutants carrying mutations I148M, R160H, R160P, R160S, R168H, R178W, E247K, or L252P. Interestingly, for mutants CK1δ E257K and CK1δ L252P , nearly no residual kinase activity could be determined in enzyme kinetic analysis. Therefore, both mutants can be considered kinase-dead mutants. So far, also mutant CK1δ K38M , bearing an amino acid change at residue Lys-38, which is involved in binding of ATP, has been described as kinase-dead mutant of CK1δ [31,32].
Mutations located in the C-terminus of CK1δ (ranging from amino acid 296 to 415 for human CK1δ transcription variant 1 [33]) resulted in different effects on phosphorylation of α-casein in kinetic analysis. While increased activity was determined for GST-CK1δ R299Q and GST-CK1δ Q399* , decreased activity was observed for GST-CK1δ R316G and GST-CK1δ H414Y . In general, several C-terminal residues of CK1δ are involved in regulation of kinase activity, mostly by phosphorylation events mediated by intramolecular autophosphorylation or by phosphorylation by upstream kinases [21,[34][35][36]. Consequently, if this domain is affected by conformational changes due to amino acid exchanges also regulation of kinase activity might be affected.
In general, mutations in the CK1δ protein might not only affect kinase activity, but can also result in different binding of small molecule inhibitors. This has previously been shown for the hyperactive mutant CK1δ T67S as well as for different phosphorylation-site mutants [2,21]. Considering a potential use in therapeutic strategies for personalized medicine, stronger binding of inhibitor compounds to hyperactive CK1δ mutants could be of outstanding benefit. By selectively inhibiting CK1δ mutants expressed in tumor tissue, a wider therapeutic window is opened and side effects on tissues expressing wild type CK1δ can be limited or even avoided. In order to identify differences in sensitivity toward inhibition by specific small molecule inhibitors, certain inhibitor compounds were tested for their effects on CK1δ mutants. Compared to wild type CK1δ, the hyperactive GST-CK1δ R127Q mutant could be stronger inhibited by all selected inhibitor compounds. Inhibitor-specific effects determined for incubation of GST-CK1δ R127L and GST-CK1δ R299Q with IWP-2 were also stronger than for wild type CK1δ and similar significant observations could as well be made for inhibition of GST-CK1δ A36V and GST-CK1δ R115H with Bischof-5. Mutant GST-CK1δ R168H was significantly stronger inhibited by both Bischof-5 and Richter-2 when being compared to inhibitory effects observed on wild type CK1δ. In contrast, mutations in CK1δ can also lead to reduced inhibition by CK1δ-specific inhibitors. This could be mainly observed for Richter-2 and GST-CK1δ mutants A36V, R115H, and R127L, as well as for IWP-4 and mutants R299Q and Q399*.
Expression of hyperactive CK1δ mutants has been shown to be associated with increased oncogenic potential, while decreased activity of CK1δ results in reduced oncogenic functions [2,4]. Consequently, by treating tumor cells expressing hyperactive CK1δ mutants with CK1δ-specific inhibitors, beneficial effects could be obtained. With the purpose to screen for these beneficial effects, CK1δ mutant-expressing cells were treated with CK1δ-specific inhibitors. For being analyzed in this experimental approach, three CK1δ mutants were selected: mutant CK1δ R127Q presented the highest activity among all tested mutants, and mutant CK1δ R168H represented a unique hyperactive mutation located between Ile-148 and Leu-252. In addition, the previously described mutant CK1δ T67S , being characterized by increased kinase activity and high oncogenic potential, was tested in the selected cell culture model.
In order to identify inhibitors selectively targeting wild type and/or mutant CK1 isoforms, two sets of newly developed inhibitor compounds were tested within the present study. These compounds were designed based on previously reported IWP-derivatives, which demonstrated high potency and selectivity for CK1δ as well as increased affinity to a CK1δ M82F gatekeeper mutant [15]. Initial screening of the IWP-derived compounds 16, 17, 18, and 19 indicated that all compounds strongly inhibit CK1δ (and ε) with IC 50 values within the one-digit micromolar range. While, in general, inhibition of the hyperactive CK1δ mutants T67S, R127Q, and R168H was within the same range as observed for wild type CK1δ, for few of the tested inhibitors slightly stronger inhibition of the mutants could be detected.
Analysis of inhibitory effects of the newly designed inhibitors on cultured cells was performed using HeLa cells. By also using HeLa cells, being deficient for CK1δ (HeLa CK1δ −/− ), the target selectivity of the tested inhibitor compounds could be analyzed. Interestingly, treatment with compound 18 and to a lesser extent also with compound 19 (or compound 20, which was tested in a later stage of the study) showed stronger effects on CK1δ-expressing cells than on HeLa CK1δ −/− cells. We therefore conclude that at least compound 18 selectively targets CK1δ. This conclusion is supported by EC 50 data generated for compound 18, showing a significantly increased EC 50 value for HeLa CK1δ −/− cells. However, the effects observed for HeLa CK1δ −/− cells are still obvious, which might be caused by simultaneous inhibition of other CK1 isoforms like the highly related isoform CK1ε. Furthermore, the effects obtained for compounds 16 and 17 might result from off-target effects on other CK1 isoforms or other cellular kinases in general. Significantly decreased effects on cell viability of HeLa CK1δ +/+ cells in comparison to parental cells could be explained by overexpression of exogenous CK1δ in HeLa CK1δ +/+ cells, potentially leading to highly increased intracellular target concentration.
If the hyperactive CK1δ mutants T67S, R127Q, and R168H were expressed in HeLa CK1δ −/− cells, inhibitory effects mediated by compound 18 were slightly stronger. This finding underlines the marginally increased affinity of compound 18 to CK1δ mutants in comparison to wild type CK1δ. Interestingly, for all three mutant-expressing cell lines, dose-dependently different effects could be observed, thereby demonstrating the need for proper dose-response investigation.
With the aim to identify more potent CK1δ-specific inhibitors, further IWP-derived compounds of a second set were also analyzed. Compared to the first tested set, all of these compounds resulted in remarkable inhibition of CK1δ (and ε), and determined IC 50 values for CK1δ were at least ten orders of magnitude lower than for compounds 16, 17, 18, or 19. In comparison to the originally tested IWP-compounds (IWP-2 and IWP-4; [15]), inhibitors 20, 21, and 22 could be significantly improved concerning their inhibitory effects on CK1δ. Because highly potent inhibitors of CK1 isoforms could be used for the treatment of proliferative diseases, the highly potent inhibitors 20, 21, and 22 were also tested for their effects on established tumor cell lines. Previously, CK1-specific inhibitors already showed promising effects on colon cancer cell lines and therefore, also inhibitors 20, 21, and 22 were subsequently tested on colon cancer cell lines HCT-116, HT-29, SW480, and SW620. While the mutation status of CK1δ (and the highly related isoform ε) in these cell lines remains to be determined and confirmed, in a previous study, we already demonstrated robust expression of CK1δ and ε as well as CK1-specific kinase activity in SW480 and SW620 cells, while HT-29 cells showed remarkably reduced expression of CK1δ as well as reduced CK1-specific kinase activity [2]. Consequently, cell line-specific differences could be observed for the tested inhibitors in our current study. The effects obtained for the treatment of HCT-116, HT-29, and SW480 cells were quite similar, although low expression of CK1δ has previously been demonstrated for HT-29 [2]. The reduction of cell viability could therefore also be mediated via the inhibition of other CK1 isoforms or targeting of other proteins in general. Furthermore, in comparison to previously described difluoro-dioxolo-benzoimidazole-derived compounds like Richter-2 [17], but also in comparison to the original IWP-compound IWP-4 [15], the cellular effects mediated by inhibitors 20, 21, and 22 fell short of expectations. Inadequate ability to cross the cell membrane or low target affinity within the cell might be possible reasons for the obtained results. In contrast to 21 and 22, at least treatment with compound 20 led to comparably strong effects on viability of SW620 colon cancer cells. Keeping in mind that cell lines SW480 and SW620 derived from primary tumor (SW480) and metastasis (SW620) of the same patient, this finding is of particular interest [37]. SW480 and SW620 represent different stages of colon cancer development, and in our investigation compound 20 showed stronger effects on the metastasis compared to the primary tumor. Survival of colon and rectal cancer patients is increasing continuously and, since the prognosis of patients is mainly determined by the occurrence of liver and lung metastases with a reported 5-year overall survival of only 12% [18], successful treatment of distant metastases is desperately warranted to further improve outcome of colon and rectal cancer. With this regard, compound 20 may be an attractive agent for systemic treatment of metastatic disease. Individual screening and molecular subtyping may help in the future to identify possible candidates for such a targeted therapy [19,20].
In order to develop novel treatment strategies, detailed characterization of CK1δ (mutant)-associated functions will enable for (i) the deepened understanding of CK1δ-driven pathogenic processes and (ii) the design of new mutant-specific inhibitors with decreased off-target effects for being used in potential future therapies for various tumor entities, including colon and rectal cancer.

Generation of Expression Vectors
In order to introduce point mutations in CK1δ, site-directed mutagenesis using QuikChange II Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) was performed on pGEX6-P3 or pcDNA3.1 expression vectors, respectively, coding for GST-CK1δ or His-CK1δ. Mutagenesis was performed according to the manufacturer's instructions using the primers listed in Table S2. Generation of pcDNA3.1 expression vectors coding for His-CK1δ wild type or T67S mutant was described by Richter and colleagues [2].

Expression and Purification of GST Fusion Proteins
Overexpression and purification of GST-CK1δ fusion proteins, either wild type or mutant, were performed as described previously [38].

General Procedure for the Synthesis of the Phenyl Pyrimidinone-Derivatives
Molecular sieve (4 Å) was dried in vacuo. 4-(3H)-pyrimidinone, the appropriate phenylboronic acid, and a copper catalyst were dissolved in DMSO and pyridine was added. The reaction mixture was heated to 90 • C for 4 h and compressed air was added through a small tube into the reaction mixture. After cooling, the molecular sieve and the catalyst were filtered off; the filtrate was then quenched with water and extracted with ethyl acetate. The organic phase was washed with water and brine, dried over sodium sulfate, and the solvent was removed under reduced pressure. If necessary, the product was purified by column flash-chromatography on silica gel.
3-Phenylpyrimidin-4(3H)-one (1)   Under nitrogen-atmosphere iron (nano) and the appropriate benzyl chloride were added. The mixture was heated at 110 • C for 2 h, decanted in ice and acidified with 1 M HCl. The aqueous phase was extracted with DCM. The organic phase was washed with water, dried over sodium sulfate and the solvent was removed under reduced pressure. The crude products were purified by column flash-chromatography on silica gel (gradient EE/PE).

General Procedure for the Synthesis of the Phenyl/benzyl-2-thioxo-Pyrimidinone-Derivatives
The appropriate phenyl/benzyl-pyrimidinone derivative and NaHCO 3 were dissolved in a mixture of organic solvent and deionized water under vigorous stirring. Phenyl thionochloroformate was added dropwise and the mixture was stirred at room temperature for 5-16 h. After extraction with ethyl acetate the organic phase was washed three times with brine, dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was then dissolved in methanol. TEA was added and the mixture was stirred at room temperature (or at 80 • C) for 4-16 h. The solvent was removed and the crude products were purified by column flash-chromatography on silica gel (gradient EE/PE).

Cell Lines and Transfection
Parental HeLa cells, as well as HeLa CSNK1D knock-out cells (CK1δ −/− ) (generated by CRISP/Cas9 technology and provided by EDIGENE, Beijing, China), were cultured in Dulbecco's Modified Eagle medium (DMEM). HCT-116 and HT-29 colon cancer cells were cultured in McCoy's 5A medium whereas SW480 and SW620 colon cancer cells were cultured in Leibovitz's L-15 medium. All media were supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. DMEM-based medium for the culture of HeLa CSNK1D knock-out cells was additionally supplemented with 1 µg/mL puromycin. Cell cultures were maintained at 37 • C in a humidified atmosphere containing 5% CO 2 . Cells were transfected using Effectene transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For selection of stably transfected clones, transfected cells were grown in selective medium, containing 0.6 mg/mL of G-418 (Calbiochem-Merck Millipore, Darmstadt, Germany).

MTT Viability Assay
For performing MTT viability assays, cells were seeded in a 96-well plate (5 × 10 4 cells/well for HeLa cells (parental, CK1δ −/− , or CK1δ −/− cells stably transfected with wild type or mutant CK1δ), HCT-116, and HT-29 cells or 7.5 × 10 4 cell/well for SW480 and SW620 cells). After 24 h, old medium was replaced with fresh medium containing either the small molecule of interest or DMSO as control. After 48 h of treatment, 10 µL MTT (5 mg/mL) was added and incubated at 37 • C for 4 h. The medium was then gently removed and 100 µL of acidic isopropanol was added (90% (v/v) isopropanol, 10% (v/v) 1 N HCl). Cell culture plates were then shaken for 30 min at room temperature. Absorbance was measured at 570 nm with TriStar 2 LB942 Microplate Reader (Berthold Technologies, Bad Wildbad, Germany) and data analysis was performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA).

Statistical Methods
Results were presented as the mean of experiments at least performed in triplicate ± SD. In the case of IC 50 data, the standard error was calculated based on values obtained from three independent experiments. Enzyme kinetic data as well as IC 50 and EC 50 data was calculated using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Other data calculation and presentation were performed using either GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) or Microsoft Excel 2016 (Microsoft, Redmond, Washington, USA). Where indicated, statistical analysis was done by performing two-way ANOVA. P-values smaller than 0.05 were considered to be statistically significant. Levels of significance were indicated by asterisks as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns, not significant.

Conclusions
In order to develop treatment strategies for application in personalized medicine, profound understanding of the effects of mutations of CK1δ, being associated with increased oncogenic potential, is crucial. In the present study, we characterized several mutations of CK1δ, which resulted in an increase of kinase activity. Furthermore, we demonstrated conclusively that CK1-specific small molecule inhibitors also have the potential to inhibit the tested CK1δ mutants. By selectively targeting tumorigenesis-associated CK1δ mutants instead of wild type CK1δ, tumor-specific effects could be obtained, offering a much wider therapeutic window than for targeting CK1δ in general. In this regard, new CK1 isoform-and mutant-specific inhibitors like the ones described in the present study represent interesting tools for future therapeutic applications in personalized medicine.