|Home | About | Journals | Submit | Contact Us | Français|
The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and β-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts.
The serine/threonine protein kinase CK2 is a constitutively active heterotetrameric complex composed of two catalytic (α or α′) and two regulatory (β) subunits,1 which has emerged as an attractive drug discovery target in oncology.2 Researchers from Cylene have recently advanced CX-4945, a selective, orally available inhibitor of CK2 into the clinic for treatment of patients with solid tumors and hematological malignancies.3
Among its diverse functions, CK2 interacts with and regulates multiple components of the Wnt pathway, an evolutionarily conserved signaling network that regulates embryonic development and the regeneration of intestinal epithelial cells.4 Certain cancers, including colorectal carcinoma (CRC), arise due to gene mutations among constituents of the Wnt pathway, including the CK2 substrates dishevelled (Dvl), APC, and β-catenin.5 Inhibition of CK2, either by RNA knockdown or with small molecules, decreases β-catenin–Tcf-mediated transcription of Wnt target genes such as survivin and leads to cell death and apoptosis in a range of CRC lines.6,7 In addition, elevated levels of CK2 activity have been reported in CRC tissue samples and expression levels correlate with poor prognosis in CRC patients.8,9 Taken together, these data illustrate the potential utility of CK2 inhibitors in CRC and other cancers characterized by aberrant Wnt pathway activity.
We sought to identify a potent, selective inhibitor of CK2 kinase for hypothesis testing in vivo using preclinical models of CRC. An early probe from our previously described series of ATP-competitive pyrazolo[1,5-a]pyrimidine-derived inhibitors of CK2 (1, 2; Figure Figure11) was used to assess the link between CK2 inhibition and Wnt signaling.10
Treatment of DLD-1(APC mutant) cells with 2 inhibits β-catenin phosphorylation and decreases Wnt-mediated gene transcription as shown in a Luciferase reporter assay in APC mutant DLD-1 cells (IC50 = 0.06 μM).11 In an acute dose pharmacokinetic/pharmacodynamic (PK/PD) study, treatment of DLD-1/AKT1 overexpressing murine xenografts with 2 (10 mg/kg, PO) resulted in the 20% inhibition of Wnt-mediated luciferase gene transcription at 8 h, and coincided with an unbound drug concentration at the level of the Wnt reporter IC50. When tested in disease model studies using a murine DLD-1(APCmut) xenograft, compound 2 showed limited tumor growth inhibition.10
Our medicinal chemistry strategy subsequently focused on achieving potent inhibition of the Wnt pathway, as demonstrated using a Wnt luciferase reporter assay in DLD-1 cells,11 while seeking to improve physicochemical properties, enhance target coverage, and potentially deliver increased in vivo efficacy in preclinical models characterized by aberrant Wnt signaling.
To aid our design efforts we obtained the X-ray crystallograhic structure of human CK2α at 2.55 Å resolution in complex with compound 2 (Figure Figure22).12 The inhibitor occupies the ATP-binding cleft and is anchored to the hinge region via a pair of hydrogen bonds. The C7 aminocyclopropane group is directed toward solvent and forms a hydrogen bond with the main-chain carbonyl oxygen of V116, while the N1 position of the pyrazolopyrimidine core interacts with the amide NH of the same residue. An additional interaction is observed between an ordered water molecule and the C5 NH of the inhibitor. The ortho-methyl substituent enforces an energetically disfavored cisoid configuration of the acetamide that permits coordination of the carbonyl group with a nearby water molecule. This bound water, adjacent to the gatekeeper residue F113, also interacts with the cyano group of the pyrazolo[1,5-a]pyrimidine core and the main-chain amide of D175. In addition, the solved structure suggested that substitution of the ortho-position (R1 in Figure Figure11, Scheme 1, and Table 1) with polar functionality could enable additional energetically favorable interactions with the protein.
Analogues of 2 were synthesized by adapting the convergent approach described in our earlier work (Scheme 1).10 Treatment of N-(2-fluoro-5-nitro-phenyl)acetamide (3) with secondary amines, followed by reduction of the nitro group in the resulting products (4), afforded anilines of general structure 5. Palladium-catalyzed or KF-promoted coupling of these intermediates (5) with 5-chloro-7-(cyclopropylamino) pyrazolo[1,5-a]pyrimidine-3-carbonitrile (6) provided the desired analogues (Table 1) either directly (7a–c, f, g, j, k), via the methansulfonyl derivative of 7f or, as in the case of 7h–i, l, and q, following TFA deprotection of the corresponding Boc derivatives (Scheme 1)
Introduction of an N-methylpiperazinyl substituent (7a) enhanced solubility, but reduced enzymatic and cellular potency relative to 2. However, the acyclic 1,2-diaminoethyl substituent of 7b imparted high solubility in addition to potent enzymatic (CK2 IC50 < 3 nM) and cellular activity (Wnt DLD-1 Luciferase IC50 = 75 nM). Further substitution of the terminal amino group, as in N,N-diethyl analogue 7c, reduced the biochemical and cellular potency. Weakly basic or nonbasic groups at the terminal position (7d–7f) contributed to a reduction in Wnt reporter activity. Extension of the linker segment in 7b by an additional methylene unit, producing analogue 7g, results in a greater than 10-fold reduction in enzyme activity and suggested that optimal positioning of the charged dimethylamino group was achieved with a three atom linker. Pharmacokinetics for 7b in the rat following oral administration of 10 mg/kg dose are characterized by low exposure (AUC = 0.36 μM·h) and high clearance (CL = 65 mL/min/kg). In vitro metabolite identification studies using rat hepatocytes identified a series of products derived from 7b that likely arise via oxidative demethylation of the side chain nitrogen atoms. Incubations in the presence of ABT in rat and human microsomes give less extensive metabolite formation and suggest a predominantly oxidative mechanism of clearance for 7b. We subsequently synthesized the didemethylated analogue 7h, which exhibits improved cellular activity (Wnt DLD-1 Luciferase IC50 = 50 nM), high solubility, and reduced intrinsic clearance in rat hepatocytes and human microsomes relative to 7b. In addition, 7h showed greatly reduced activity (IC50 > 100 μM) compared to 7b (IC50 = 4 μM) in our hERG ion channel assay. Interestingly, rigidification of the linker segment through the use of 3-aminopyrrolidino- (7l, m) and 3-aminopiperidino- substituents (7n, o), despite contributing to an overall increase in lipophilicity, afforded compounds with reduced turnover in rat hepatocyte and human microsome preparations but led to a reduction in Wnt reporter assay potency (Table 1). Substitution adjacent to the primary amine, as in 7p and 7q, led to further improvement in the in vitro metabolic stability while preserving cellular activity.
To confirm our initial design hypothesis, we determined the X-ray crystallographic structures of CK2α with 7b and 7h (Figure Figure33).12 Both inhibitors are bound in the ATP-binding site in a manner analogous to that of 2. However, while the side chain of 7b positions the terminal dimethylamino group for an electrostatic interaction with D175, the unsubstituted amine of 7h is able to directly coordinate an ordered water molecule and the side-chain carbonyl group of N161.
A number of compounds in this series possess half-maximum inhibitory potency below the lower limit of detection (IC50 < 3 nM) in our enzymatic assay, which measures the inhibition of recombinant human full-length CK2α mediated phosphorylation of a synthetic peptide substrate at Km ATP concentration. To better understand the contribution of the side-chain interactions on ligand binding we developed a surface plasmon resonance (SPR) assay to determine the binding affinities of 2, 7b, and 7h.14 The results (Table 2) indicate that 2 and 7h possess similar affinity and are approximately 10-fold more potent than 7b. Comparison of 7b and 7h indicate that the dimethylamino side-chain of 7b is disfavored compared to the primary amine of 7h.
Kinase selectivity profiling of 7h at a concentration of 0.1 μM against a panel of 402 kinases revealed a high degree of selectivity (Figure Figure44).15 The limited off-target activity (12 kinases with >50% inhibition) was restricted to members of the CMGC family, including isoforms of the dual-specificity tyrosine-regulated kinases (Dyrk) and CAMK kinases such as the death-associated protein kinases (Dapk) and homeodomain interacting protein kinases (Hipk). Moderate inhibition (74%) of bone morphogenetic protein receptor type-1B kinase (BMPR1b) was also observed. Biochemical IC50 determinations ([ATP] = Km) revealed that 7h exhibits moderate-to-weak activity against the Hipk and Dyrk isoforms (IC50s = 0.04–1.3 μM) and is active in the 10–20 nM range against Dapk2 and Dapk3.14
The ability of our compounds to inhibit the Wnt pathway, as measured using the DLD-1 Topflash reporter assay, correlates well with inhibition of a direct CK2 substrate, pAKTS129, in cells.16 These data are consistent with the finding that CK2α-dependent up-regulation of β-catenin driven transcriptional activity requires phosphorylation of AKT.17 In addition, Wnt pathway inhibition correlates with antiproliferative effects in DLD-1 (APC mutant) cells (Figure Figure55). Similar levels of activity are observed in other CRC cell lines with constitutively activated Wnt signaling that is driven either by β-catenin (HCT-116) or APC mutations (SW620) (Table 3).
To further strengthen the mechanistic data, we subsequently demonstrated that 7h induced a concentration-dependent decrease in the active form of β-catenin in Wnt3a expressing mouse fibroblast L-cells,14 an in vitro system in which pathway up-regulation is triggered by constitutive Wnt3a expression (Figure S1). In addition, the mouse L S/L line contains a TCF4 driven luciferase construct and the degree of active β-catenin inhibition seen with 7h correlates with the potency of the compound in the corresponding Wnt3a L S/L reporter assay (IC50 = 0.05 μM).14
Based on the in vitro biomarker, pathway and growth inhibition data of 7h, characterization of this compound in vivo was undertaken. Oral dosing of 7h resulted in low bioavailability and limited the unbound drug concentration to levels expected to be subtherapeutic. However, intraperitoneal (IP) or intravenous (IV) dosing regimens delivered sustained free drug concentrations above efficacious levels.
The ability of compound 7h to inhibit substrate and downstream marker phosphorylation in an APC mutant CRC model was evaluated in SW620 tumor-bearing murine xenografts. Administration of 7h induced dose-dependent modulation of the downstream markers pAKTS129 and β-catenin, as determined by Western blot analysis of tumor cell lysates (Figure Figure66A).14 Similarly, DLD-1 TOPflash luciferase (APC mutant) xenografts were utilized to assess the effect of the compound on Wnt-associated gene transcription. In this model, treatment with a single dose (10 mg/kg, IV) of 7h led to 40–50% inhibition of Tcf4-luciferase signal at the 8 h time point with suppression of this signal, and AKTS129 phosphorylation (not shown), still evident at 24 h, by which time no detectable drug remained in plasma (Figure Figure66B).
The durable substrate and pathway suppression observed with 7h following its clearance from plasma may be due, in part, to the high (pM) affinity of the compound (Table 2) and its associated slow dissociation rate (kd = 0.00025 s–1). These and other data18 suggested the potential for an intermittent dosing schedule as a means to achieve activity in disease model studies while minimizing tolerability issues.
Compound 7h showed a dose-dependent tumor growth inhibition, achieving 94% TGI in a HCT-116 (β-catenin mutant/model) and 74% TGI in a SW620 (APC mutant) model at a 30 mg/kg dose given weekly for 3 cycles. Reversible dose-proportional body weight loss was observed in both experiments; in the SW620 study, mean body weight changes observed on day 25 in treated animals ranged from −0.9 to −6.8%.
In conclusion, we have identified a series of potent and selective CK2 kinase inhibitors that decrease AKTS129 phosphorylation in cells and whose antiproliferative effects correlate with inhibition of Wnt luciferase reporter gene transcription. Using the in vivo probe 7h, we have demonstrated a reduction in the downstream biomarkers pAKTS129 and β-catenin and a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts.18 Further studies, using 7h and related analogues, are planned to more fully understand the dependence of CRC on CK2-mediated Wnt pathway inhibition and will be reported in future communications.
The authors wish to thank Mei Su and Helen Xiaomei Feng for compound synthesis, and Vicki Racicot and Zhong-Ying Liu for in vitro and in vivo biology support, respectively.
The authors declare no competing financial interest.