WO2015014329A1 - Pharmaceutical composition comprising monensin for treating of diseases associated with deregulated wnt signaling pathway - Google Patents

Abstract

The invention relates to novel biological activities of monensin, an antibiotic isolated from Streptomyces cinnamonensis. Monensin was identified, as potent Inhibitor of the canonical Wnt signalling pathway and its activity in various in vitro and in vivo assays was demonstrated. Particularly, the invention relates to pharmaceutical composition comprising monensin or its pharmaceutically acceptable salt for treating diseases associated with the deregulated Wnt signalling pathway, preferably intestinal diseases, more preferably familial adenomatous polyposis, colon cancer, rectal cancer and colorectal carcinoma.

Description

Pharmaceutical composition comprising monensin for treating diseases associated with the deregulated Wnt signalling pathway

Field of the invention The invention relates to novel biological activities of monensin. an antibiotic isolated from Streptomyces cinnamonensis. Monensin was identified as potent inhibitor of the canonical Wnt signalling pathway and its activity in various in vitro and in vivo assays was demonstrated. Particularly, the invention relates to pharmaceutical composition comprising monensin or its pharmaceutically acceptable salt for treating diseases associated with the deregulated Wnt signalling pathway.

Background of the invention

The Wnt signalling pathway is evolutionary conserved in nematodes, insects and all vertebrates. It plays an essential role during embryonic development as well as in homeostasis maintenance and tissue renewal of stem cells in adult organism. Aberrant activation of this crucial pathway leads to various type of human diseases including cancer (1).

A key component of the canonical branch of Wnt signalling, β-catenin, has several cellular functions including participation in adherent junctions through association with E-cadherin at the cell membrane (2). In the cytoplasm, β-catenin is phosphorylated and targeted for degradation by the complex, which is composed of the scaffolding protein Axin, the tumour suppressor adenomatous polyposis coli (APC) protein, casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). This continual β-catenin depletion impedes its translocation into the nucleus in the absence of the Wnt signal (3). Binding of the extracellular Wnt ligand to a transmembrane Frizzled (Fz) receptor and its co-receptor, low-density lipoprotein receptor related protein 5 and 6 (LRP5/6), leads to the phosphorylation of the L P5/6 intracellular domain by CKly. This event activates LRP5/6 and promotes membrane recruitment of the degradation complex. The recruitment of the degradation complex to the Wnt-Fz-LRP5/6 complex induces stabilization of β-catenin. Thus, β-catenin accumulates and enters the nucleus where it associates with the HMG box transcription factors from the LEF/TCF family. The constitution of these complexes, in which β-catenin provides a strong trans-activation domain, results in the activated transcription of the Wnt target genes (4).

In humans, the β-catenin protein consists of 781 amino acids (aa) residues. Its structure is formed by sequentially conserved central region (aa 141-664) composed of 12 Armadillo repeats. The central region is flanked by N- and C-terminal domains with potentially flexible structure. Proximally to the C-terminal domain there is a specific rigid helix structure, next to the last Armadillo repeat (Helix-C). The central region of the protein represents a stable scaffold providing an interaction surface for β-catenin binding partners (5). The regions involved in the stabilization and transcriptional activity have been localized in the N- and C-terminal parts of β-catenin. In the degradation complex, β-catenin is phosphorylated on N-teiminal serines and a threonine (S33, S37, T41 and S45), leading to its targeting to polyubiquitination and degradation by the proteasome (6). Conversely, phosphorylation on the C-terminal residues (S552 and S675) promotes transcriptional activity of β-catenin. The presumptive mechanism is though the binding of chromatin remodelling histon acetylases such as CBP/p300. Moreover, the C-terminal domain of β-catenin affects the binding of LEF/TCF transcription factors (7). Although the mechanism underlying this activation is not known in detail, the Helix-C within the C-terminal part was shown to be essential for the signalling activity of β-catenin (5).

Deregulation of the canonical Wnt signalling is a hallmark of many types of human cancers, including colorectal carcinoma, mammary gland tumours or melanomas (8). The term "deregulation" means especially the activation of the Wnt signalling pathway in the cell type or tissue, where this pathway is not active under normal conditions. Another possibility is an increase of the signal above its normal physiological level. Such deregulation may be related to the overproduction of Wnt ligands or to the mutations that cause a loss of internal control mechanisms of the Wnt signalling pathway. Among many components of the pathway, the APC tumour suppressor is the most frequently mutated gene in human cancers. Specific mutations in the APC gene are the cause of familial adenomatous polyposis (FAP), a heritable disease in which patients develop large number of polyps in the colon, prograding to colorectal cancer. APC is also mutated in the majority of sporadic colorectal tumours. The loss of function (frameshift or nonsense) mutations occur in both alleles of this gene and consequentially truncated APC protein is not able to regulate β-catenin stability. In colorectal tumours with wild-type APC protein, point mutations in the gene for β-catenin (CTNNB1) were found. These mutations "protect" β-catenin from the degradation. Similar mutations were found also in other cancer types such as melanomas and hepatocellular carcinomas (9). Initial mutations in APC or CTNNB1 are usually followed by changes in other genes like KRAS or TRP53 (10). Taken together, those facts make the Wnt signalling pathway an attractive object for a targeted anti-cancer therapy.

Monensin (4-[2-[5-ethyl-5-[5-[6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-oxan-2-yl]-3- methyl-oxolan-2-yl]oxolan-2-yl]-9-hydroxy-2₅8-dimethyl-l,6-dioxaspiro[4.5]dec-7-yl]-3- methoxy-2-methyl-pentanoic acid) is a polyether antibiotic isolated from Streptom ces cinnamonensis having the following chemical formula:

Monensin has been known since 80' s of the last century. Monensin is ranked among natural carboxylic polyether ionophores; such substances are subjects of great attention due to their antibacterial, antifungal and antiparasitic biological activity (11). Interestingly, monensin has been used in veterinary medicine and as a food additive in animal husbandry for several years. It is known to promote muscle growth and to increase milk production of dairy cattle whereas no significant effect on the reproduction and health of animals was proved. Some studies also show the relationship between monensin diet and reduced milk fat (12). Nevertheless, little is known about its molecular mechanism f action. It was published recently, that monensin inhibits androgen signalling and induces apoptosis in prostate cancer cells (13). In these studies, monensin is shown to decrease the amount of androgen receptors (A ) and therefore inhibit prostate cancer cells growth. It is not surprising taken that β-catenin directly interacts with AR and enhances their transcriptional activity (14). In respect to other physiological activity, monensin was reported as Signal transduction and activation of transcription 3 (STAT3; CN 102552300 A). Monensin was proposed also as an agent for depression treatment (EP 1034782 Al). Monensin was disclosed as a component of the composition that elongates neurites of motor nerve cells (JP 2006052192 A). Monensin was also suggested as an agent against corona-virus infection (US 7 544 721 Bl).

Description of the invention

In the study which forms a background of the present invention, the inventors performed high throughput screen aimed to find novel chemicals with the ability to modulate Wnt signalling. Monensin, an antibiotic isolated from Streptomyces cinnamonensis, was identified as a potent inhibitor of the canonical Wnt signalling pathway. The inventors demonstrated the activity of monensin in various in vitro assays including reporter STF (pSuperTOPFLASH HE 293) cells and four cancer cell lines derived from human colorectal tumours. Furthermore, it was shown that monensin also displayed its efficiency in vivo by reducing secondary body axis formation in Xenopus embryos, preventing regeneration of injured tail fin of zebra fish (D nio rerio) and decreasing the size of continually developing tumours in APC^Mm mice.

The inventors used STF cells harboring the genome-integrated Wnt-responsive luciferase reporter SuperTOPFLASH (15) to search for novel inhibitors of Wnt/p-catenin signalling. The screen included 2448 different compounds obtained from three commercially available collections (see Examples for details). The primary screen identified seven compounds displaying a profound inhibitory effect on the pSuperTOPFLASH activity. These "small molecules" included the previously identified Wnt pathway inhibitors indometacin (16), thapsigargin (17), and harmine (18). Additionally, four compounds without any relation to Wnt signalling were discovered. The putative novel Wnt pathway modulators were examined for their effective concentration range, cell toxicity and direct repressive effect on the luciferase reaction. A polyether antibiotic monensin that suppressed Wnt signalling (without affecting cell viability) at concentrations 0.2 to 5 μΜ was selected for subsequent studies. In variety of in vitro and in vivo experiments (closely described in Examples) it was found that monensin

- inhibits canonical Wnt signalling in reporter STF cells,

- inhibits canonical Wnt signalling during regeneration of tail fin of Danio rerio,

- inhibits canonical Wnt signalling in Xenopus embryos,

- acts at the level of the β-catenin degradation complex, wherein it decreases the amount of β-catenin in cells,

- downregulates expression of the Wnt signalling target genes in SW480, COLO320 and LS174T colorectal cancer cell (CRC) lines,

- inhibits proliferation in above mentioned CRC lines and affects cell cycle progression in SW480 cells,

- inhibits Wnt signalling stimulated with wild-type and mutant β-catenin mRNA (that is constitutively active due to point mutations), and

reduces tumour development in APC^Mm mice.

Given that monensin is able to decrease Wnt signalling at the level of the β-catenin degradation complex or downstream, the inventors suggests that the compound could be applicable in the medical treatment of colorectal tumour cells, which have the main mutations mostly at this level of the cascade. Although the exact mechanism of action is not known, the inventors present here the convincing results obtained by many proven methods to demonstrate the ability of monensin to inhibit canonical Wnt signalling and colon cancer cell growth. Since the compound is widely used in veterinary practice, future trouble-free pharmaceutical applications can be anticipated.

Therefore, one aspect of the invention relates to monensin or its pharmaceutically acceptable salt for use in a treatment of the diseases associated with the deregulated Wnt signalling pathway. Among the typical diseases associated with the deregulated Wnt signalling pathway belong intestinal diseases.

Another aspect of the present invention relates to monensin or its pharmaceutically acceptable salt for use in a treatment of the intestinal diseases associated with deregulated Wnt signalling pathway. Familial adenomatous polyposis, colon cancer, rectal cancer and colorectal carcinoma belong among intestinal diseases associated with deregulated Wnt signalling. Consequently, preferred aspect of the present invention relates to monensin or its pharmaceutically acceptable salt for use in a treatment of familial adenomatous polyposis, colon cancer, rectal cancer and colorectal carcinoma.

Further aspect of the present invention relates to the use of monensin or its pharmaceutically acceptable salt for manufacturing of pharmaceutical composition for treating diseases associated with the deregulated Wnt signalling pathway. Preferred aspect relates to the use of monensin or its pharmaceutically acceptable salt for manufacturing of pharmaceutical composition for treating intestinal diseases associated with the deregulated Wnt signalling pathway. Familial adenomatous polyposis, colon cancer, rectal cancer and colorectal carcinoma belong among such intestinal diseases. Consequently, more preferred aspect of the present invention relates to the use of monensin or its pharmaceutically acceptable salt for manufacturing of pharmaceutical composition for treating familial adenomatous polyposis, colon cancer, rectal cancer and colorectal carcinoma.

The pharmaceutical compositions according to the present invention are useful mainly for the treatment of humans, but can be also used in veterinary medicine. Medical treatment involves prophylactic as well as curative treatment.

In the pharmaceutical composition according to the invention, monensin may also be present in the form of pharmaceutically acceptable salts (non-toxic, physiologically acceptable), of inorganic or organic nature. A person skilled in the art is capable to prepare routinely appropriate salts.

The pharmaceutical compositions according to the invention comprise monensin or its pharmaceutically acceptable salt in a pharmaceutically effective amount as the active substance. The method for detemiining the pharmaceutically effective amount is the routine procedure well known to the person skilled in the art.

Typically, the active substance is present in the pharmaceutical composition together with excipients, such as fillers, disintegrators, diluents, solvents, binders, emulsifying agents, buffers, stabilizing agents, preservatives and colouring agents. The excipients and their use are well known to those skilled in the art.

The pharmaceutical compositions comprising monensin or its pharmaceutically acceptable salt can be formulated for systemic administration, e.g. enteral administration, such as oral administration, e.g. in the form of tablets or capsules, for rectal administration, e.g. in the form of suppositories, for nasal administration or for inhalation, e.g. in the form of spray or drops. The compositions according to the invention can be formulated for parenteral administration, such as via injection (i.v., i.m., s.c), infusion or implanted reservoir system. It is obvious for a person skilled in the art that this specification is not exhaustive, and other appropriated methods of adn inistration will be known to a skilled person. Monensin can be comprised in the pharmaceutical composition in combination with other active substances, for example with a compound exhibiting synergistic effects.

The determination of the dosage of the monensin^' as an active substance in unit dosage form, e.g. in the capsule, or e.g. suitable concentration in solution for injection or infusion is also a routine procedure known to a skilled person. The relevant guidelines pertaining to the pharmacological compositions, dosage forms, excipients etc., are summarized in the specialized literature (Gennaro, A.R. et al. Remington: The Science and Practice in Pharmacy. 20. Edition. Lippincot Williams & Wilkins, Baltimore, MD, 2000, Kibbe, A. H. Handbook of Pharmaceutical Excipients. Pharmaceutical Press, London, 2000, Chalabala, M. et al.: Technologie Leku. Galen, Praha, 2001) readily available to a skilled person, and also in the Czech Pharmacopoeia (CL 2009), in the European Pharmacopoeia (Ph. Eur.) and/or in the U.S. Pharmacopoeia (USP).

Description of the drawings

Fig. 1. The primary screen identified potential small molecule inhibitors of Wnt signalling

A. High throughput screening of three commercially available "small molecule" libraries aimed to find new canonical Wnt signalling inhibitors was preformed by luciferase assay using Wnt/p-catenin responsive STF cells; the Wnt signalling pathway was activated by cell treatment with recombinant Wnt3a ligand. Three primary hits are shown. Harmine and thapsigargin are known Wnt pathway inhibitors in contrast to monensin, a carboxylic polyether antibiotic. B. Simultaneously, the effect of compounds on the cell viability at the same concentrations was measured using CellTiter-Blue Cell Viability assay. C. Left: Quantification of regeneration of Danio rerio tail fin. The last third of the tail fin was resected and the fishes were kept for one week in a medium supplemented with 2 μΜ monensin or the solvent (ethanol) alone. After that the percentage of regeneration of tail fin tissue was evaluated. The data represent 3 independent experiments, n indicates the number of individuals in the group. Right: Images representing the recovered part of the fin missing pigmentation. ** P < 0.01 (t-test).

Fig. 2. Monensin specifically reduces the level of canonical Wnt signalling in reference HEK293 cells and also in Xenopus embryos

A. Luciferase activity in HEK293 cells, transfected with the Wnt P-catenin reporter pSuperTOPFLASH and Renilla plasmid ensuring control of transfection efficiency. The Wnt signalling pathway was stimulated by adding recombinant Wnt3a ligand into the medium and the cells were further cultured with monensin at concentrations of 1 and 5 μΜ, or with vehicle (DMSO) alone. The effect of either concentration on decrease in the luciferase activity is clearly visible. On the other hand, monensin at the same concentrations had no effect on the luciferase activity in HEK293 cells transfected with a NF-κΒ reporter pNF-icB-Luc and Renilla plasmid and stimulated with 10 ng/mL lymphotoxin a (LT ). B. Quantitative RT-PCR (qRT-PCR) analysis of Wnt target genes in HEK293 cells stimulated with Wnt3a ligand and incubated with monensin at concentrations of 1 and 5 μΜ (20 hours). Control cells were cultured with DMSO. C. Cellular distribution and stability of β-catenin in mouse LT - cells. Cells were stimulated with Wnt3a ligand and incubated overnight with 5 μΜ of monensin or with DMSO. After the incubation, cells were stained with antibody against β-catenin and DAPI nuclear stain. Magnification: 63 Ox. D. Western blot analysis of lysates from the parental STF cell line and Wntl producing STF cell line incubated with monensin at concentrations of 0.5; 1 μΜ and 5 μΜ or DMSO, respectively (20 hours). Antibodies against Axin2, total amount of β-catenin, active forms of β-catenin (β-catenin phosphorylated on S675, marked P-S675^-catenin, and β-catenin non-phosphorylated on S33/37/T41) and TCF4 were used, a-tubulin documented equal protein loading. ** P < 0.01 (t-test).

Fig. 3. Monensin operates at the level of β-catenin stability A. Luciferase assay in STF cells stimulated either with Wnt3a Hgand, 1 μ of BIO or 3 μΜ of CHIR99021 and cultivated with 1 and 5 μΜ of monensin (final concentration in culture medium) or DMSO, respectively. Monensin decreased Wnt^-catenin signalling induced by both BIO and CHIR99021 as well as Wn a. A percentage of luciferase count is presented. B. Monensin decreases the amount of β-catenin in mouse L_TK-, stimulated with 1 μΜ of BIO. Cells were grown overnight with 5 μΜ of monensin or with DMSO. After the incubation, cells were stained with antibody against β-catenin and DAPI nuclear stain. Magnification: 630x. C. Western blot analysis of lysates from STF cell line stimulated with 1 μΜ of BIO and incubated with monensin at concentrations of 0.5; 1 and 5 μΜ or DMSO, respectively (20 hours). Antibodies against total amount of β-catenin and active forms of β-catenin (P-S675^-catenin and ηοη-Ρ-β-catenin, see legend to Fig. 2) and TCF4 were used, a-tubulin documented equal protein loading. D. Left: Quantification of body axis duplication in Xenopus laevis embryos induced by injection of 20 pg XWntS mRNA or 800 pg β-catenin mRNA into the marginal zone of the ventral blastomeres of 4- cell stage embryo. In both instances was injected monensin, together with RNA, in the total amount 0.04 pmol per injection, or DMSO in final concentration 0.4 %, respectively. After 36 h incubation the number of embryos with a secondary body axis was evaluated. The results involve 4 or 3 independent experiments, respectively, n is number of embryos evaluated. Right: Representative images of embryos with duplicated or single body axis, respectively, after 36 h incubation. E. Luciferase test with STF cells transiently transfected with constructs containing normal form of β-catenin (wt β-catenin) and constitutively active forms of β-catenin: S33Y, S37A, S45A, AS45 and ΔΝ. Treatment with 1 μΜ and 5 μΜ monensin results in reduction in luciferase activity for all β-catenin constructs, however, no significant effect of monensin was observed in cells transfected with a construct encoding Lefl fused to the VP 16 activating domain from HSV. Renilla plasmid was used as control of transfection efficiency. ** P < 0.01; * P < 0.05 (t-test). Fig. 4. Monensin inhibits canonical Wnt signalling in cells of SW480 line but not in HCT116 cells

A. Luciferase activity in SW480 and HCT116 cell lines transfected with Wnt p-catenin reporter plasmid TOPFLASH or non-reactive FOPFLASh and Renilla, respectively. Cells were cultured overnight with monensin at concentrations of 1 μΜ and 5 μΜ or with vehicle (DMSO) alone. * P < 0.05 (t-test). B. Quantitative RT-PCR analysis demonstrated decreased expression of the Wnt target genes in SW480 cell line after incubation with monensin at concentrations of 1 and 5 μΜ (42 hours). In HCT116 cells the effect of the compound was not statistically significant. Control cells were cultured with DMSO. C. Beta-catenin cell distribution and quantity in SW480, and HCT116 cell lines. Cells were cultured overnight with 5 μΜ monensin or DMSO. After incubation the cells were stained with anti-p-catenin antibody or DAPI nuclear-specific stain. Magnification: 400x. D. Western blot analysis of lysates from SW480 and HCT116 cells incubated with monensin at concentrations of 0.5; 1 and 5 μΜ or DMSO, respectively (42 hours). Antibodies against total β-catenin and active forms of β-catenin (P-S675-p-catenin and ηοη-Ρ-β-catenin) were used; a-tubulin documented equal protein loading. E. Relative ³H-thymidine incorporation in colorectal cancer cell lines SW480, HCT116 (left) and control Wnt-independent HeLa and HE 293 cells (right). Cells were incubated overnight with 1 μθί of ³H-thymidine and with monensin at concentrations of 0.5; 1 and 5 μΜ or DMSO, respectively. The counts of control cells cultivated in DMSO were defined as 100 %. The graphs depict the mean values from 4 independent experiments.

Fig. 5. Monensin reduces the size of intestinal tumour in APC¹™*" mice

A. Representative microphotographs (a,b) and more detailed microscopy images (a', b') showing the different size of adenomas (marked by black arrows) in the jejunum of APC¹™¹¹ mice treated daily with DMSO (a, a') or monensin (b, b') for 6 weeks. The sections were stained with β-catenin antibody and hematoxylin nuclear stain. B. Left: The average tumour area in the small intestine was significantly decreased in monensin treated group. Middle: The total number of tumours in the small intestine remains similar in both groups. Right: Statistical box chart with the quantification of the tumour area in the small intestines of APC™¹ mice showing statistically significant decrease of total tumour area in monensin treated animals, *, P < 0.05 (t-test). C. Representative images showing that rumours in monensin-treated mice have unchanged amounts of cells positive in proliferation marker i67 (c, c', d, d'). By contrast, after monensin treatment there is a higher number of cells producing cell cycle inhibitor p21 (e, e', f, f") and undergoing apoptosis (TACS, g, g h, h'). D. Representative images of non-adenomatous epithelial tissue of the small intestine demonstrate that the architecture of the epithelium was not damaged by monensin treatment Sections were stained with antibodies against the proliferation marker Ki67 (i,k) and cytokeratin 20 ( rt20, j,l), a marker of terminally differentiated cells. Tissues were counterstained with haematoxylin nuclear stain. The black bars at the bottom corners of the images represent 0.6 mm (a,b), 0.2 mm (a', b', c, d, e, f, g, h) and 0.08 mm (c', d'₅ e', f , g', h').

Examples of the invention Example 1

This example briefly describes screening wherein monensin was identified as an inhibitor of the Wnt signalling pathway. Furthermore, the example describes also the methods used in the following examples demonstrating in vitro and in vivo anti-tumour activity of monensin.

High throughput screen for inhibitors of the canonical Wnt signalling pathway STF cells harbouring the genome-integrated Wnt-responsive luciferase reporter SuperTOPFLASH (15) were used to search for novel inhibitors of Wnt/p-catenin signalling. The screen included 2448 different compounds obtained from three commercially available collections (see Materials and Methods for details). STF cells were stimulated with recombinant Wnt3a ligand and, simultaneously, a specific compound was added to culture medium to 1 μΜ concentration. The luciferase activity was quantified 18 hours later using bioluminescent signal detection. The primary screen identified seven compounds displaying a profound inhibitory effect on the pSuperTOPFLASH activity. These "small molecules" included the previously identified Wnt pathway inhibitors indometacin (16), thapsigargin (17), and harmine (18). Additionally, four compounds without any known relation to Wnt signalling were discovered. The putative novel Wnt pathway modulators were examined for their effective concentration range, cell toxicity and direct repressive effect on the luciferase reaction. A polyether antibiotic monensin that suppressed Wnt signalling without affecting cell viability at concentrations 0.2 to 5 μΜ was selected for subsequent studies (Fig. 1A, B).

Specific effect of monensin was verified in in vivo experiment wherein the ability of monensin to prevent regeneration of zebra fish tail fin tissue after injury. This fish, similarly to other lower vertebrates, exerts the ability to regenerate injured tissues and organs with the help of stem cells. The process called epimorphic regeneration is controlled by Wnt/p-catenin signalling cascade (19). Due to a simple structure, speed of regeneration and absence of pigment in regenerating tissue such experiment is well-suited for primary confirmation of Wnt signalling inhibition. After surgical resection of the terminal third of tail fin the fishes were kept in E3 medium supplemented with 2 μΜ monensin or adequate amount of solvent (ethanol). Evaluation of tissue regeneration one week after resection shows 50% loss of regeneration in animals kept in medium with monensin in comparison to the control group (Fig. 1C).

M terials and methods

Recombinant Wnt3a purification Mouse Wnt3a ligand was isolated from the culture medium of Wnt3a~producing L cells without the heparin purification step according to a detailed protocol of Willert and colleagues (20).

Cell lines and luciferase assays

Human HEK293, SW480, Colo320, LS174T, HCT116 and HeLa cells, mouse L_TK- cells and Wnt3a-producing L cells were purchased from American Type Culture Collection. STF cells containing the genome-integrated Wnt/ -catenin-responsive luciferase reporter, pTOPFLASH were obtained from Q. Xu and J. Nathans (15). Construct encoding mouse wild-type Wntl (kindly provided by O. Machon) protein was generated in the mammalian lentiviral vector, pCDHl (System Biosciences). Wntl producing STF cells were generated by the transduction of STF cells with pLHCX retrovirus (BD Clontech) containing the mouse Wntl gene. Plasmids NF-KB-LUC and pRL-TK (Renilla) were purchased from Promega. The luciferase assays were performed using ONE-Glo Luciferase Assay System (Promega) for primary high throughput screening and Dual-Glo Luciferase Assay System (Promega) for subsequent assays with Renilla and measured in EnVision Multilabel Reader (PerkinElmer).

Primary screening

STF cells were plated in 384- well plates (Corning) at a density of 2500 cells/25 μΐ/well using a Multidrop Combi dispenser (Thermo Scientific) and cultured overnight Then, Wnt3a was added and, immediately, library compounds were added using pintool (V&P Scientific) coupled to a JANUS Automated Workstation (PerkinElmer) to a final concentration of 1 uM. The compound library included the Library of Pharmacologically Active Compounds (LOPAC1280, Sigma- Aldrich), the Prestwick Chemical Library (Illkirch, France) and the NIH Clinical Trial Collection (NIH, USA). The cells were cultured for 24 hours and the luciferase activity was determined. Cell viability was determined after 48 hours incubation using the CellTiter-Blue Cell Viability Assay (Promega).

Chemical compounds

Except of the above presented libraries, following commercially available compounds were used: monensin sodium salt (M5273, Sigma- Aldrich), BIO (B1686, Sigma- Aldrich), CHIR99021 (S1263, Selleckchem).

Plasmids and transfections

Constructs encoding wilt-type β-catenin, β-catenin harbouring mutation in serine 45 (S45A, AS45) and murine Lefl fused with the VP 16 viral activating domain (Lefl-VP16) were generated by PCR and cloned into mammalian expression vectors pClNEO (Promega) or pEGFP-C3 (Clontech), respectively. Constructs encoding β-catenin with mutation in serine 33 and serine 37 (S33Y, S37A) and β-catenin lacking the N-terminal domain (ΔΝ, aal32-781) (kindly provided by T.Valenta) were generated in mammalian expression vectors pCS2 and pcDNA3.1 (Invitrogen). Transfections were performed using Lipofectamine 2000 reagent (Invitrogen). Regeneration of Danio rerio tail fin

Zebra fishes younger than 6 months were kept in E3 medium (5 mM NaCl, 0.17 mM KC1, 0.33 mM CaCl₂ and 0.33 MgS0₄ in distilled water) at 28 °C. Fishes of the size about 2.5 cm were narcotized by tricaine (ethyl-3-aminobezoate methanesulphonate, Sigma) and approximately 1/3 of the tail fin was resected. Then the fishes were randomly divided into groups and kept in E3 medium supplemented with 2 μΜ monensin or adequate amount of ethanol alone for 1 week. After this week the fishes were photographed and the size of regenerated tissue, clearly visible due to the absence of pigment, was evaluated as percentage using Image! software. Xenopus double axis formation assay

Capped Xenopus laevis Wnt8 mRNA (XWnt8) was synthesized from linearized plasmid template using the mMESSAGE mMACHINE kit (Ambion). XWntS mRNA (20 pg) or a wild-type β-catenin mRNA (800 pg) with either 0.04 pmol monensin or corresponding volume of DMSO (final concentration 0.4%) was injected (total injected volume 4 nl) into the marginal zone of the ventral blastomeres of 4-cell stage Xenopus laevis embryos. The embryos were incubated at 20 °C and axis duplication was scored after 36 hours.

RNA Purification and RT-qPCR

Total RNAs were isolated from cells using RNA Blue reagent (Top-Bio) and then reversely transcribed. Real-time PCR (SYBR Green PCR Master mix, Roche) was performed in LightCycler 480 System (Roche). Two different genes were used as an endogenous control for every qRT-PCR experiment.

Statistical analysis

Data obtained in the analyses were evaluated by Student's t-test. Statistically significant data (*, P = < 0.05) and very significant data (**, P = < 0.01) were distinguished. Immunocytochemistry and immunohistochemistry

Both techniques were performed according to standard protocols. The following commercially available antibodies were used: mouse monoclonal anti-p-catenin, (sc-7963; Santa Cruz), mouse monoclonal anti- i67, (Mob 237; Diagnostic BioSystems), mouse monoclonal anti-Krt20, (M7019; Dako), mouse monoclonal anti-p21 (556431 , BD Pharmingen). For the detection of apoptotic cells TumourTACS In Situ Apoptosis Detection Kit (4815-30-K, D Systems) was used.

Immunoblotting Cell lysates were immunoblotted using the following primary antibodies: mouse monoclonal anti^-catenin (610153; BD Transduction Laboratories™), rabbit polyclonal anti-non- phospho-p-catenin/S33/S37/T41 (#4270; Cell Signalling), rabbit polyclonal anti-phospho-β- catenin/Ser675 (#4176; Cell Signalling), rabbit monoclonal anti-Axin2 (#2151; Cell Signalling), rabbit monoclonal anti-TCF4 ((#2569; Cell Signalling), mouse anti-a-tubulin (TU-01 ; Exbio CZ).

Cell proliferation assay

Metabolic incorporation of [³H]-thymidine (M.G.P., final concentration 0.1 mCi/μΙ) was measured in MicroBeta2 Microplate Counter (PerkinElmer) after overnight incubation at 37 °C. Cell cycle analysis

Cells were fixed by 70% ethanol and stained by propidium iodide (Sigma, final concentration 20 μg ml). Cell fluorescence was measured in a flow cytometer (BD LSR II). Cells were stained with Hoechst 33342 (Invitrogen) to discriminate between G2 cells and so called cell doublets. Tumour treatment in APC^M,n mice

Related 4 weeks old pups were weaned, genotyped and randomized. Twelve APC^Mm mice were divided into two groups and treated with either monensin (10 mg kg) or vehicle (DMSO) alone. Daily per oral applications started at the same day and continued for 6 weeks. The mice were sacrificed and the intestines were dissected, washed in PBS and fixed in 4% formaldehyde for 3 days. Fixed intestines were embedded in paraffin, sectioned and immunohistochemically stained. The number and size of the intestinal lesions were quantified by using the Ellipse software (ViDiTo). Example 2

Monensin inhibits canonical Wnt signalling in reporter cells HE 293

To confirm the specificity of the monensin action reporter genes assays in HE 293 cells were performed using the Wnt^-catenin-responsive reporter plasmid pTOPFLASH, negative- control reporter pFOPFLASH, and NF- Β pathway luciferase reporter plasmid pNF- B-Luc. Cells transiently transfected with the reporters were stimulated with recombinant Wnt3a or lymphotoxin-a (LTa) and incubated overnight with increasing concentrations of monensin; all transfection mixtures contained Renilla expressing plasmid to monitor transfection efficiency. In agreement with the results obtained in STF cells, 1 and 5 μ monensin reduced the TOPFLASH activity to 34% and 32%, respectively. In contrast, monensin had no effect on the transcription from the pNF-KB-Luc and pFOPFLASH reporters (Fig. 2A, data for FOPFLASH not shown).

Next, qRT-PCR analysis of HEK293 cells cultured with Wnt3a and DMSO or 1 or 5 μΜ of monensin, respectively, was preformed. The downregulation of the known Wnt target genes (Axin2, CYCLIN Dl, LGR5, NKDl, SP5) was recorded after 20 hours treatment with monensin in comparison with DMSO treated cells (Fig. 2B). Because the expression of the Wnt target genes is regulated by degradation of β-catenin in the cytoplasm, we examined the cellular localization and amount of β-catenin by immunofluorescent staining in mouse LiK-cells. The pathway was stimulated with Wnt3a and cells were cultured either with 5 μΜ of monensin or DMSO, respectively. A visible reduction of total β-catenin was recorded after overnight incubation with monensin (Fig.2C).

Additionally, western blot analysis in parental STF cell line and STF cell line producing exogenous Wntl was performed. After 20-hour incubation with various monensin concentrations, decreasing amount of total β-catenin as well as two transcriptionally active forms of β-catenin (non-phosphorylated at S33/37AT41 and phosphorylated at S675) was recorded. This result indicates ongoing destabilization and degradation of cellular β-catenin. Moreover, Axin2 protein, the product of the Wnt target gene, was also decreased after monensin treatment. Interestingly, Tcf4 transcriptional factor remained unchanged (Fig. 2D). Example 3

Monensin functions at the level of β-catenin destabilization and degradation

Next, the experiments were aimed to explore at which level of the Wnt signalling cascade monensin functions. First, the pathway was stimulated using two specific GSK3 inhibitors with similar mechanism of action: BIO ((2'Z,3'E)-6-Bromoindirubin-3'-oxime) and CHIR99021 (21). If the effect of monensin was at the level of Wnt Hgand or membrane receptors, it would not be able to inhibit the Wnt pathway activity triggered by substances, activating the cascade at the level of the β-catenin degradation complex. Luciferase assay in STF cells revealed potent inhibition of both Wnt agonists BIO and CHIR99021 in presence of monensin (Fig. 3 A). Considering this, monensin may function at the level of the degradation complex or downstream. To revise changes in the amount and distribution of β-catenin when stimulated with BIO and treated with monensin or DMSO, immunofluorescent staining in mouse L_TJ - cells was preformed. A reduction of both cytoplasmatic and nuclear β-catenin confirmed previous results (Fig. 3B). Also western blot analysis in STF cells cultured for 20 hours with BIO and three monensin concentrations displayed decreasing amount of all used forms of β-catenin (Fig. 3C).

In order to validate the efficiency of monensin in the live organism, the secondary body axis formation assay in Xenopus laevis embryos was carried out. The injection of capped ectopic Xenopus Wnt8 (XWnt8) mRNA into the marginal zone of two ventral blastomeres of four-cell stage Xenopus embryo stimulates canonical Wnt signalling at the ventral side of the embryo and induces a secondary body axis formation (22). Alternatively, β-catenin mRNA was used to achieve the same effect. By using a specific Wnt inhibitor, the axis duplication can be blocked and the normal phenotype is thus rescued. A co-injection of 20 pg of XWnt8 with 0.04 pmol of monensin resulted in 30.7% of Xenopus embryos with double axis (n = 209) whereas a co-injection of 20 pg of XWnt8 with DMSO displayed 65.9% of embryos with double axis (n = 205; Student's t-test: 0.002). When used 800 pg of wild-type β-catenin mRNA co-injected with monensin, the secondary axis formation decreased again to 40.7 % (n = 130) when compared with 78.1% in co-injections with DMSO (n = 118; Student's t-test: 0.0407; Fig. 3D). To uncover the mechanism of function of monensin, the modulatory effect of the compound in STF cells transiently transfected with constructs containing various forms of the gene for β-catenin and Renilla plasmid as a transfection control was examined. Wild-type β-catenin and β-catenin with mutations causing constitutive activation of the protein (S33Y, S37A, S45A, AS45) including protein lacking the N-terminal domain (ΔΝ) were used. Luciferase analysis displayed statistically significant inhibition of all of these protein variants using two different monensin concentrations. By contrast, we recorded no significant effect of monensin in cells transfected with fusion protein containing the Lefl transcription factors the with VP 16 activating domain from herpes simplex virus (Lefl -VP 16) indicating that monensin does not influence the activity of LEF/TCF transcription factors per se. This result indicated the β-catenin-dependent effect of monensin (Fig. 3E). Further, the inventors hypothesized that there could be inhibition of some kinase (or a group of kinases) family as the result of monensin treatment. Thus, the mammalian "kinome" inhibition analysis was done with monensin at concentrations of 0.1 and 1 μΜ. However, even though there were slight changes in various kinases activities (the kinases represented different kinase families), no significant inhibition was proved (data not shown).

Example 4

Monensin downregulates expression of the Wnt target genes and inhibits proliferation of S W480, COLO320 and LS 174T human colorectal cancer cell (CRC) lines

Given that monensin is able to decrease Wnt signalling at the level of the β-catenin degradation complex (or downstream), the inventors assumed that the compound could be applicable in the medical treatment of colorectal tumour cells, which harbour mutations triggering the pathway at this specific level. The effect of monensin was investigated in four CRCs: SW480 and COLO320 harbouring mutation in the APC gene; LS174T and HCT116 with a mutation in the CTNNB1 gene. Primary luciferase experiment with SW480 and HCT116 lines transfected with plasmids TOPFLASH, FOPFLASH and Renilla revealed statistically significant monensin reactivity of SW480 cells. HCT116 cells showed very low level of Wnt signalling activation and culture with monensin had no significant inhibitory effect on aberrant Wnt signalling (Fig. 4A). Expression analysis of all 4 cell lines showed downregulation of the Wnt target genes (Axin2, CYCLI Dl, NKD1, SP5) in SW480, COLO320 and LS174T cells incubated 42 hours with monensin at two different concentrations. Surprisingly, no specific effect in HCTl 16 was observed (Fig. 4B, and data not shown). Following visualisation of β-catenin cell distribution and quantity confirmed that the protein level significantly decreased in SW480, COLO320 and LS174T cells. All 3 cell lines exhibited high amounts of cytoplasmic β-catenin which is typical for active canonical Wnt signalling. By contrast, HCTl 16 cells exhibited almost exclusive membrane localization of β-catenin that is maintained after culture with monensin, despite the fact that the cells changed partially their shape (such phenomenon was observed also in other cell lines) (Fig. 4C, and data not shown). Western blot analysis in all CRCs cultured with monensin showed surprisingly less visible decay of total and active forms of β-catenin in SW480, COLO320 and LS174T cells. The reason could be the presence of membrane-bound β-catenin in the lysate, the amount of which is supposedly not affected by monensin. There was no perceivable effect in the protein level in HCTl 16 cells (Fig. 4D, and data not shown).

Upon inhibition of the canonical Wnt signalling pathway, the proliferation of human colorectal cancer cells was reduced. The effect of monensin on cell proliferation was tested in the panel of CRCs, wherein the cells were incubated overnight with various concentration of monensin and with [³H]-thymidine. During the cell-cycle progression, [³H]-thymidine is incorporated in the newly synthesized DNA and thus, the radioactivity of the whole DNA from cells can be measured. Monensin-treated SW480 and COLO320 cells displayed more than 50% decreased proliferation when compared to control cells treated with DMSO whereas LS174T cells proliferated about 25% less then control cells. As expected, results showed no difference in proliferation of HCTl 16 cells as well as control cells HEK293 and HeLa (Fig. 4E and data not shown). Moreover, cell cycle analysis revealed that monensin decreased the proportion of cells in the S phase (from 42.3% in control cells to 33.8% in cells treated with 5μΜ of monensin) and in G /M phase (from 11.3% to 5.2%) and increased the cell fraction in the G_t phase (from 44.4% to 59.2%) in SW480 cells. In HCT 116 cells incubated with 1 μΜ of monensin we recorded no significant change in cell cycle phases. Interestingly, when incubated with 5μΜ of monensin, HCTl 16 cells displayed moderate rise of cells in the S phase (from 50.8% to 58.4%) and decline of cells in the G₂/M phase (from 18.5% to 1.9%) whereas a rate of cells in the Gj phase remained unchanged (not shown).

Example 5 Monensin reduces tumour development in APC"™ mice

APC^Mm mice (Min - multiple intestinal neoplasia) harbours a mutation in one allele of the APC gene. The mutated allele produces truncated APC protein. Because of frequent spontaneous mutation in the second allele, APC""¹ mice develop multiple polyps, most frequently in the small intestine (29). Therefore APC^Mm mice provide a valuable animal model for human intestinal cancer including familial adenomatous polyposis and sporadic tumours. The APC^Mm model is useful for testing chemical agents targeted against early stage adenomas, because the earliest polyps appear during the third week after birth and consequently rise in numbers and size during the life (23). The effect of monensin in APC^Mm mice was examined during 6 weeks of daily treatment. Two equivalent groups of 6 mice (3 males and 3 females) at the same age were exposed to daily per oral application of monensin (10 mg kg) or vehicle (DMSO), respectively. After 6 weeks, mice were sacrificed; the dissected intestines were embedded in paraffin and sectioned. Immunohistochemical staining visualized elevated expression of β-catenin, the hallmark of canonical Wnt signalling, in all lesions. Stained tumours contrasted with the healthy mucosa, enabling quantitative analysis of tumour size and number in the small intestine using image analysis program Ellipse. In the small intestine, the significant reduction of the total tumour area was observed in the monensin-treated group (average: 10.16 mm²) when compared with the control group (average: 16.46 mm²; Student's t-test: P=0.0125; Fig.6A). Moreover, the average area of one tumour in the small intestine was decreased in monensin-treated animals (average: 0.199 mm ) when compared with control animals (average: 0.299 mm ; Student's t-test P=0.0144). Both groups displayed very similar numbers of tumours in the small intestine (Fig. 5A, B).

Furthermore, the intestine was itrrmunohistochemically stained using antibody against Ki67, proliferating cell marker, and p21, cell cycle inhibitor. Moreover, TumourTACS In Situ Kit for detection of apoptotic cells via double strand DNA breaks marking was used. Monensin-treated tumours displayed almost identical amounts of proliferating cells, however, stronger staining for p21 and for apoptotic cells was observed compared with control tumours from the same part of the gut. The staining appeared preferentially at the surface of lesions observed (Fig. 5C).

Further, healthy mucosa in animals from both groups was examined with antibodies against epithelial cell populations, such as Ki67, marking stem and progenitor cells in intestinal crypt, or Krt20, marking protein produced in terminally differentiated epithelial cell on intestinal villi. No changes in proliferation and differentiation of cells or in the architecture of the intestinal epithelium were observed (Fig. 5D). Thus it can be summarized that APC^Mm mice treated 6 weeks with monensin developed smaller tumours in comparison to the control group as a result of inhibition of the canonical Wnt signalling pathway without negative side effects on homeostasis of untreated intestinal epithelium.

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Claims

L Monensin or its p¾ rmace«ticaUy acceptable salt for se in a tre tment of diseases associated with the deregulated Writ signalling pathway,

2» Moneosm or its pharmaceutically acceptable salt Ibr use in a treatment of intestinal diseases associated with the deregulated Wnt signalling pathway.

3. Monensin or its pharmaceutically acceptable salt ibr use in a treatment of familial adenomatous polyposis, colon cancer, rectal cancer and colorectal carcinoma.

4. A pharmaceutical composition comprising monensin or its pharmaceutically acceptable salt tor use i a treatment of diseases associated with the deregulated Wnt signalling pathway,

5. The pharmaceutical composition according to claim 4, wherein the disease-associated with the deregulated Wat signalling pathway is an intestinal disease associated with the deregulated Wnt signalling pathway. o. The pharmaceutical composition according to claim 5, wherein the intestinal disease associated with the- deregulated Wnt signalling pathway is familial adenomatous polyposis, colon cancer, rectal cancer and colorectal carcinoma.

7, A use of monensin or its pharmaceutically acceptable salt tor manthseturing of pharaiaeeutieal -composition for treating diseases associated with the deregulated Wnt signallin pathwa .

8, The use according to claim 7, wherein the disease associated with the deregulated Wnt signallin pathway is an intestinal disease associated, with the deregulated W t signalling pathway. The use according to ekkn 8, wherein the intestinal disease associated with deregulated Wnt signalling pathwa is iami!ia! adenomatous polyposis, colon cancer, rectal cancer aad colorectal carcinoma.