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Development of IWP-related compounds as selective wt/mutCK1d/e inhibitors, characterization in vitro and in vivo in animal models for their use in new anticancer concepts

Subject Area Pharmacy
Term from 2019 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 420839669
 
Final Report Year 2023

Final Report Abstract

CK1 family members phosphorylate a broad range of substrate proteins involved in various physiological processes including metabolism, transcription, cell cycle progression, apoptosis, and differentiation, thereby being key players in many cellular functions. Consequently, aberrant expression and activity levels of CK1 isoforms contribute to the pathogenesis of neurodegenerative, inflammatory, and proliferative diseases. The importance of CK1 isoforms in the pathogenesis of malignant tumors is leading to approaches for pharmacological intervention and has already resulted in the development of several CK1-specific SMIs, including D4476 and PF-670462. In this context, the groups of Peifer and Knippschild could already characterize highly potent and specific CK1δ/ε SMIs with IC50 values in the low nanomolar range. The antineoplastic efficacy of some of these compounds has already been shown in cell culture models as well as in animal models. However, there is still considerable interest in further optimizing CK1 isoformspecific SMIs concerning their affinity, isoform and mutant selectivity, efficacy, and their biological availability. The application of such compounds might be of special interest in the treatment of aggressive tumor entities with poor prognosis, and to improve the lives of cancer patients. In the first part of the project, we characterized the kinetic properties of CK1δ mutants, which were detected in different tumor entities. Subsequently, we developed and synthesized two new sets 2 of novel selective and effective wt/mutCK1δ/ε small molecule inhibitors, that were developed on the basis of the previously characterized IWP scaffold (Scheme 1). DBU MeOH, 0 °C, 5 h Pyridine, 110 °C, 24 h 80 °C, over night, N2-Atm. N2-Atm. TEA, DCM, RT, 24 h TEA, DMF, 80 °C, 2 h N2-Atm. IWP-CK1 scaffold Scheme 1. General synthesis towards CK1 inhibitors showing the IWP-derived scaffold We next designed a set of benzyl-substituted compounds and performed docking experiments with the reported CK1δ ligand-protein-structure (pdb 5OKT) suggesting suitable aromatic substitution patterns to increase ligand affinity. By the optimized preparation method, we synthesized targeted derivatives for subsequent SAR determination (Table 1). Table 1. Set of synthesized IWP derivatives showing systematic variations of aromatic substituents. 3 Furthermore, we attached chiral scaffolds to a CK1 inhibitor core structure to investigate SAR in terms of stereochemistry. The core of these compounds is binding to the hinge region, whereas the chiral scaffolds are situated in the hydrophobic region II of CK1. Noteworthy, this strategy has been a part of ligand design in this project. Unfortunately, we could not determine a significant impact on both, affinity and specificity of these compounds. These results are published in Lit. 2 of the DFG grant output (Thorsten von Drathen, Elizabeth M. Ure, Stefan Kirschner, Aileen Roth, Laura Meier, Anthony D. Woolhouse, Scott A. Cameron, Uwe Knippschild, Christian Peifer, Andreas Luxenburger. C5-Iminosugar modification of casein kinase 1δ lead 4-[3-(4-fluorophenyl)- 5-isopropylisoxazol-4-yl]pyridine promotes enhanced inhibitor affinity and selectivity. Arch. Pharm. 2022; e2100497. https://doi.org/10.1002/ardp.202100497). Additionnally, we used amino acid moieties attached to the IWP core. However, comparable to the above mentioned chiral scaffolds, these compounds also showed no suitable biological activities against CK1 isoforms. Hence, we focused on the IWP-derived derivatives given in Table 1. Prior to a biological characterization of the novel compounds, we developed a systematic workflow for complementing and validating results from CK1 high-throughput screening as well as increasing the comparability of CK1 isoform-specific inhibitor parameters for radiometric as well as non-radiometric assays. Following determination of CK1 isoform selectivity of the most potent inhibitor compounds, compound 18 (= SK-006) demonstrated highest CK1 isoform selectivity and inhibition of hyperactive CK1δ mutants (also see Table 4). With the aim to develop and identify more potent CK1δ-specific inhibitors, a second set of IWP-derived compounds were designed showing significantly improved inhibition for compounds 20 to 22. Compound 20 (SK- 001) was identified to affect metastatic colon tumor cell viability thereby representing an attractive agent for systemic treatment of metastatic disease. Our results showed that the most effective compounds represent a promising base for the development of novel colon and rectal cancer therapy concepts. Based on the compound set, especially compounds SK-003 and SK-004 demonstrated increased selectivity towards CK1δ compared to compound 20. Unfortunately, only SK-005 had an inhibitory effect on pancreatic and bladder cancer cell viability. Since promising highly specific and potent compounds are normally assessed in vivo in specific disease-associated or developmental animal models regarding their pharmacokinetics and toxicology, such in vivo models are inevitable. While mice still are the most widely used but strictly regulated animal model, the search towards meaningful alternatives is in full swing. In line with this notion, the early zebrafish model shows a rising popularity also in drug discovery research. To evaluate the eligibility of the zebrafish animal model for CK1 inhibition assays using novel 4 compounds, we determined the kinetic parameters of zebrafish and human CK1 isoforms and tested different selected human CK1-specific compounds on the inhibition of human and zebrafish CK1 isoforms showing high similarities between human and zebrafish CK1 isoforms. Since all CK1 isoforms are continuously expressed during zebrafish embryogenesis, we analyzed the effects of CK1-inhibition on embryonic development of zebrafish. Overall, the phenotype induced by CK1-specific compound treatment corresponds to the developmental impairments after CK1δdownregulation using morpholinos. Therefore, we concluded that the early zebrafish model can be considered to be a suitable in vivo vertebrate animal model for initial studies of CK1δ inhibition in vivo. Taken together, we identified highly effective as well as mutant and isoform-selective CK1 inhibitors with significant therapeutic potential for novel therapeutic approaches against aggressive tumor entities. In this project, we have laid the groundwork for testing these novel compounds in a vertebrate animal model (zebrafish embryo model) as a meaningful alternative to complex in vivo animal models.

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