Development of dual casein kinase 1δ/1ε (CK1δ/ε) inhibitors for treatment of breast cancer

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Abstract

Casein kinase 1δ/ε have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1δ/ε inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers.

Introduction

The casein kinase 1 (CK1) family consists of six monomeric serine/threonine-specific protein kinases (α, δ, ε, γ1, γ2 and γ3).1 All six human CK1 isoforms are highly homologous in their active site, with the delta (CK1δ) and epsilon (CK1ε) isoforms sharing 98% sequence identity in their protein kinase domains. Elsewhere in the enzyme, such as in the C-terminal, and non-catalytic domains, significant variances exist between isoforms.2 CK1 kinases play crucial roles in regulating a variety of cellular growth and survival processes including circadian rhythm,3 membrane trafficking,4 DNA damage repair,5 cytoskeleton maintenance,6 and notably Wnt signaling.7 A recent study showed that CK1δ/CK1ε might be involved in the etiology of addictive behavior and that their inhibition prevents relapse-like alcohol drinking.8 Importantly, abnormal regulation of these two CK1 family members is implicated in human cancers and several known CK1δ and/or CK1ε substrates control tumor cell growth, apoptosis, metabolism and differentiation.9 For instance, both isoforms are overexpressed in pancreatic ductal adenocarcinoma,10 ovarian cancer11 and chronic lymphocytic leukemia.12 Interestingly, expression of constitutively active, myristoylated CK1ε in mammary epithelial cells is sufficient to drive cell transformation in vitro via stabilization of β-catenin and the activation of Wnt transcription targets.13 At the same time, forced expression of kinase defective CK1δ mutants blocks SV40-driven cellular transformation in vitro and mammary carcinogenesis in vivo.14

The Wnt/β-catenin pathway has known roles in breast cancer, where: (i) Wnt signaling can contribute to triple negative breast cancer; (ii) nuclear β-catenin connotes metastasis and poor outcome; and (iii) β-catenin contributes to mutant ErbB2-driven breast cancer.15 Importantly, gain-of-function (e.g., in β-catenin) and loss-of-function (e.g., in CK1α APC and AXIN) mutations prevalent in other cancers are not found in breast cancer. Recent discoveries from our laboratories establish casein kinase 1 delta (CK1δ) as an essential regulator of β-catenin activity that is overexpressed and amplified in human breast cancer.16

CK1δ and CK1ε are eminently tractable for small molecule targeted drug discovery.17 Among the small molecules reported to inhibit CK1δ/CK1ε are CKI-7,18 (R)-CR8 and (R)-DRF053,19 IC261,20 D4476,21 LH846,22 benzimidazole 5,23 PF480056724 and PF670462.3(a), 24 Unfortunately, most of these compounds are not specific inhibitors of CK1δ/CK1ε and in some cases their pharmacological effects are now known to be (or are suspected to be) due to non-selective target engagement.20(b), 25 At the same time, the contribution of CK1δ and CK1ε to human cancer is still not fully understood and the non-selective nature of previously reported CK1δ/ε inhibitors has impeded pharmacological validation of these kinases as anti-cancer targets.3(a), 20(b) Nonetheless, available data clearly shows that targeted inhibition of CK1δ/CK1ε is a viable, highly attractive anticancer strategy yet to be fully developed.26

In research targeting the development of novel therapeutics for treatment of metastatic and resistant forms of cancer, the Roush laboratory at Scripps Florida identified highly potent and selective purine-derived dual inhibitors of CK1δ and CK1ε under the aegis of the NIH’s MLPCN program (Fig. 1).25 These agents induce proliferative arrest and rapid apoptosis of CK1δ/CK1ε-expressing human luminal B, HER2+ and triple negative cancer cells ex vivo and SR-3029 induces tumor regression in orthotopic xenograft models in vivo.26 Specifically, genetic knockdown or pharmacological inhibition of CK1δ using SR-3029 blocks β-catenin nuclear localization and TCF transcriptional activity, induces rapid apoptosis and provokes tumor regression in patient derived orthotopic models of triple negative breast cancer.25, 26 Mechanistic studies are consistent with the premise that CK1δ controls breast cancer tumorigenesis via its effect on β-catenin, which plays known roles as an effector of aberrant Wnt signaling in human breast cancer.26

Noteworthy, profiling of 442 kinases with SR-3029 (and three close analogs) confirmed their high selectivity vs. the plethora of kinases whose activity is impaired by the purportedly specific Pfizer CK1δ inhibitor (PF670462).25 The four off-target kinases that are inhibited by SR-3029 largely have no known function. High selectivity of N9-arylsubstituted purine scaffold could be explained by the unique structural features of the CK1δ/ε active site. Specifically, relatively small size of the gatekeeper residue Met82 in case of both CK1δ/ε creates large hydrophobic pocket which is occupied by N9 aryl residue of the inhibitors. This structural feature of CK1δ/ε was also encountered by Shokat and colleagues when developing analog-sensitive kinase technology.27

Herein we report structure-activity and structure-property relationship (SAR and SPR) studies that led to identification of dual selective CK1δ/ε inhibitors (including SR-3029) with physicochemical properties and in vitro and in vivo pharmacokinetic parameters suitable for use in murine xenograft studies against breast cancer.26

Section snippets

Synthetic chemistry

The general procedure for the synthesis of purine-based CK1δ/ε inhibitors is based on a previously reported sequence25, 28 which we improved by incorporating a more efficient method for arylation of the N9 nitrogen of dichloropurine (Scheme 1). Thus, N9 aryl purine intermediates (permutations of structure 6) were generated by copper (I) mediated coupling of commercially available dichloropurine 4 with a variety of symmetrical or unsymmetrical diaryliodonium salts 5.29 This reaction provides 6

Conclusions

We have developed a series of potent and selective purine-based CK1δ/ε inhibitors with excellent antiproliferative activities. A set of the most active compounds was also subjected to extensive physicochemical testing for solubility, permeability, and microsomal stability in an effort to predict their in vivo profile. These efforts led to the identification of 17 and 28 that has physical, in vitro and in vivo PK properties suitable for use in xenograph studies of human cancer. Such studies in

Material and methods

All reagents were purchased from commercial suppliers and were used without further purification. Dichloromethane, diethyl ether, N,N-dimethylformamide and tetrahydrofuran were dried by being passed through a column of desiccant (activated A-1 alumina). Triethylamine and diisopropyl amine was purified by distillation from calcium hydride. Reactions were either monitored by thin layer chromatography or analytical LC-MS. Thin layer chromatography was performed on Kieselgel 60 F254 glass plates

Acknowledgments

We thank Benjamin Huffman and Alexander Burns for contributions to the synthesis of several CK1δ/ε inhibitors in this work. We also thank the Scripps Florida NMR facility and Xiangming Kong for assistance. This work was supported in part by NIH NCI grant R01CA175094 (W.R.R., and D.R.D.) and NIH NCI NRSA postdoctoral fellowship F32CA200105 (A.M.).

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