Patent Application
20220016080
The present invention relates to a novel class of compounds and to compositions comprising the same. The compounds and compositions (such as pharmaceutical compositions) of the present invention can be used as medicaments in the treatment of cancer.
Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In other works, the authors of the present invention have employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Their results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, these findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT, such as carcinomas, tumors with mesenchymal phenotype or tumors where enhanced Hakai expression is detected respect to normal tissues. However, until now, compounds capable of effectively inhibiting Hakai-mediated ubiquitination that are especially suitable as therapeutic tools for the treatment of carcinomas have not been disclosed.
The present invention provides for such class of compounds, which includes enantiomers and pharmaceutically acceptable salts thereof, that selectively and effectively inhibit Hakai-mediated ubiquitination, preferably without affecting Hakai protein levels, and that at the same time represent excellent anti-cancer drugs useful in the treatment of a variety of cancers, such as carcinomas
The present invention provides a class of compounds, which includes enantiomers and pharmaceutically acceptable salts thereof, that selectively and effectively inhibit Hakai-mediated ubiquitination, preferably without affecting Hakai protein levels, thereby representing excellent anti-cancer drugs useful in the treatment of a variety of cancers, such as carcinomas, in particular, tumors arising from the epithelial layers of the gastrointestinal track including month (oral cancer), esophagus, stomach, and small and large intestines (such as rectal or colon cancer). It also includes skin cancer, mammary gland (breast cancer), pancreas cancer, lung cancer, head and neck cancer, liver cancer, ovary cancer, cervix cancer, uterus cancer, gallbladde cancer, penile cancer, and urinary bladder cancer (such as renal, prostate or bladder cancer). The compounds of the present invention also show lower toxicity which renders the present compounds very attractive. Such compounds are represented by formula (I) below.
Therefore, a first aspect of the present invention, refers to a compound of formula (I)
and pharmaceutically acceptable salts thereof;
for use in the treatment of cancer, in particular for use in the treatment of carcinoma, more particularly for use in the treatment of carcinomas, which include tumors arising from the epithelial layers of the gastrointestinal track including month (oral cancer), esophagus, stomach, and small and large intestines (such as rectal or colon cancer). It also includes skin cancer, mammary gland (breast cancer), pancreas cancer, lung cancer, head and neck cancer, liver cancer, ovary cancer, cervix cancer, uterus cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (such as renal, prostate or bladder cancer).
It is herein noted, that in the context of the present invention, the term “carcinoma” is understood as a type of cancer arising in the epithelial tissues. These cover the outside of the body, as the skin and also cover and line all the organs inside the body, such as the organs of the digestive system. Furthermore, they line the body cavities, such as the inside of the chest cavity and the abdominal cavity. Carcinomas are the most common type of cancer. These tumors are responsible for more than 80% of the cancer-related deaths in the Western world.
In a preferred embodiment of the first aspect of the invention, each R2 and R3 independently represents a group selected from cyclopropyl or linear or branched C1-C6 alkyl.
In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, R2 and R3 form a 3-or 4-membered spiro ring together with the carbon atom to which they are both attached.
In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, z is an integer selected from 1, 2 or 3; wherein R4 represents a group selected from —CN, cyclopropyl or linear or branched C1-C6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 halogen atoms; and wherein group R4 replaces the hydrogen atom of one of the groups CH present in the phenyl ring to which R4 is attached.
In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, one or both of the integers x and y equal 1 and A represents a group selected from aryl, heteroaryl and cyclic amides optionally substituted by 1 or 2 groups that are independently selected from halogen atom, —CN, —N(Ra)Rb, —ORa, —C(═O)Ra, —C(═O)ORa, —C(═O)N(Ra)Rb, —OC(═O)—Ra, —N(Rc)C(═O)Rb, —NRcSO2Ra, —SO2N(Ra)Rb, —SRa, —S(O)Ra, —S(O)2Ra, linear or branched C1-C6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 halogen atoms; C3-C6 cycloalkyl which optionally contains 1 or 2 heteroatoms selected from O, S and N, and which ring is optionally substituted by C1-C3 alkyl; phenyl or C5-C6 heteroaryl each optionally substituted by halogen atom, C1-C3 alkyl or cyclopropyl.
In yet another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the compound is selected from the list consisting of any of the following compounds:
The compounds identified above are further indicated herein below:
More preferably, the compound is
4-(5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid
Still more preferably, both of the integers x and y equal 0 and A represents a benzyl substituted by 1 group, preferably at the para-position, selected from halogen from a halogen atom, —ORa, —OC(═O)—Ra, —N(Rc)C(═O)Rb, —NRcSO2Ra, —SO2N(Ra)Rb, —SRa, —S(O)Ra, or —S(O)2Ra. Preferably, said group is selected from a halogen atom, or -ORa. Preferably said compounds are selected from the group consisting of hits 5 to 9 above. More preferably, said compound is hit 7.
In yet another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the compound is a ketoheteroaryl, preferably selected from the list consisting of any of the following compounds:
The compounds identified above are further indicated herein below:
More preferably, a still more preferred embodiment of the present invention refers to any of the ketoheteroaryls compounds useful to practice the present invention as illustrated through-out the present specification. In particular, preferably, both of the integers x and y equal 0 and A represents a heteroaryl optionally substituted by a halogen atom or —ORa. Preferably, said compound is selected from the group consisting of hit 23 or hit 25 above.
In yet another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the compound is a cyclic amide, preferably selected from the list consisting of any of the following compounds:
The compounds identified above are further indicated herein below:
Preferably, the compound is the substituted indoline (and indole) analog illustrated below:
Wherein R represents a group selected from hydrogen, cyclopropyl or linear or branched C1-C6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 halogen atoms. More preferably, R is a methyl group. Preferably, said compound is selected from the group consisting of hit 10 or hit 16 above.
In yet another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the compound is benzylamide, preferably selected from the list consisting of any of the following compounds:
Further compounds useful in the present invention are illustrated through-out the present specification.
The present invention identifies 4-Tetrazolylbenzoic acids Hakin-1, Hakin-2 and Hakin-6 (from hereinafter compounds #1, 2 and 6)
as inhibitors of Hakai capable of competing for the HYB binding site that is only present in the Hakai dimer. Hakai has been reported to be involved in tumor progression; therefore inhibitors of the interaction between Hakai and E-cadherin might be useful for the treatment of cancer. In this sense and as shown in the examples, inhibition of tumor progression has been herein demonstrated in vitro and in vivo utilizing compound #1. Analogs #2 and 6 have not been available for testing, but they are structurally closely related to compound #1 and the present description makes it plausible that these compounds also inhibit tumor progression.
Therefore, the present invention solves the technical problem of providing compounds having excellent anti-oncogenic effects and low toxicity. Thus, the compounds of the present invention can advantageously be used as a medicament and, particularly, in the treatment of a variety of cancers, such as carcinomas, in particular the gastrointestinal track cancer including month (oral cancer), esophagus, stomach, and small and large intestines (such as rectal or colon cancer). It also includes skin cancer, mammary gland (breast cancer), pancreas cancer, lung cancer, head and neck cancer, liver cancer, ovary cancer, cervix cancer, uterus cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (such as renal, prostate or bladder cancer. Indeed, collectively, our data, as illustrated in the examples, show that Hakin-1 is a specific inhibitor for Hakai-mediated ubiquitination, without affecting Hakai protein levels (Example 1). In addition, Hakin-1 was able to suppress proliferation in Hakai-MDCK cell while no effect was detected in MDCK cells (
Compound #1 (as already stated, also referred to as Hakin-1) is a 1,5-disubstituted tetrazole, a chemically and metabolically stable pharmacophore fragment frequently used in drug development (Tetrazole Derivatives as Promising Anticancer Agents, E. A. Popova et al., Anticancer Agents Med Chem. 2017 Mar 27. doi: 10.2174/1871520617666170327143148, Epub ahead of print). The tetrazole ring is substituted with a 4-carboxyphenyl group in position 1. In position 5, it is connected via a mercaptomethylcarbonyl linker with another phenyl ring.
Important physico-chemical parameters like molecular weight, calculated lipophilicity logP and polar surface area are in the desired range for orally absorbed drugs that are unlikely to pass the blood-brain barrier. According to a snapshot from a docking study with compound 1 in the Hakai HYB site, the carboxyphenyl moiety makes 3 important hydrogen bond interactions with the protein and is likely to mimick the binding mode of the phosphotyrosine subtrates. Further hydrogen bonds are formed by two of the tetrazole nitrogen atoms which provide additional stability and hold the tetrazole-carboxyphenyl unit on both ends in a defined binding mode.
Therefore, the following subunit seems to be essential and shall be considered as the core structure for identifying derivatives of compound #1:
Common structural feature of compounds #1, 2 and 6.
Departing from such common structural core, a number of modifications in order to optimize the activity (and other properties) of these compounds and provide analogs of these compounds, can be made. In this sense, there are several opportunities of introducing modifications in different parts of the molecule that represent an important objective in the optimization process and in the provision of analogs of compounds #1, 2 and 6. In this sense, a medicinal chemistry program would start focusing on the chemical space around compound #1 and with less priority around compounds #2 and #6. With this in mind we herein provide a group of different analogs of compounds #1, #2 and #6, useful in the present invention. Notably, useful as medicaments and, particularly, in the treatment of a variety of cancers, such as carcinomas, in particular tumors arising from the epithelial layers of the gastrointestinal track including month (oral cancer), esophagus, stomach, and small and large intestines (such as rectal or colon cancer). It also includes skin cancer, mammary gland (breast cancer), pancreas cancer, lung cancer, head and neck cancer, liver cancer, ovary cancer, cervix cancer, uterus cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (such as renal, prostate or bladder cancer).
A first class of analogs is provided by connecting the core structure to a ring structure (Cy=any cycle) in order to find those analogs that are similar to compounds #1 and #2.
These types of compounds are grouped below according to the following structural subclasses a) to c).
a) Ketophenyls (including compound #1):
It is noted that these structures can comprise a substituent on the phenyl ring or in another part of the molecule, e.g. on the carboxyphenyl ring or on the carbon atom between the sulfur and the carbonyl group. The latter case has been exemplified as follows:
Moreover, the following examples, illustrate the introduction of modifications such as a cyclopropyl bridge or the addition of substituents on the carboxyphenyl ring:
Particular examples of Ketophenyl compounds useful to practice the present invention are illustrated below:
b) Ketoheteroaryls:
Further substructures provided by connecting the core structure to a ring structure (Cy=any cycle) are herein listed as Ketoheteroaryls. In this sense, a typical exploration in medicinal chemistry would be to replace the phenyl group present in compound #1 by a heteroaryl group which represents a similar aromatic ring with additional heteroatoms. We herein provide 6 examples of analogs that fall into this category:
6-membered heteroaryls (like pyridines, pyrimidines, pyridazines) that are structurally closer to phenyl as illustrated below, also form part of the present invention:
Moreover, particular examples of ketoheteroaryls compounds useful to practice the present invention are illustrated below:
c) Cyclic amides (including Compound #2):
Still further substructures provided by connecting the core structure to a ring structure (Cy=any cycle) are herein listed as Cyclic amides, as depicted below:
In particular, substituted indoline (and indole) analogs as illustrated below:
More particularly, any of the compounds substituted on the phenyl ring shown herein below:
With modifications in other parts of the molecule:
In addition, the different substructures a) to c) above provided by connecting the core structure to a ring structure (Cy=any cycle) may be further substituted as follows:
1. Examples of Structures Having a Substitution on the Mercaptoacetyl Linker
2. Examples of Structures Having a Substitution on the Carboxyphenyl Moiety
On the other hand, an entirely new group or class of compounds that derives from extending the core structure with a nitrogen atom, a carbon atom and a cyclic group to provide compounds resembling compound #6, is herein provided under the subclass benzylamides:
Compounds falling within this category are herein indicated below:
Other compounds pertaining to this class of compounds are:
With modifications in other parts of the molecule:
All of the above compounds, #1, #2 and #6, as well as those grouped as structures provided by connecting the core structure to a ring structure (Cy=any cycle) to provide compounds resembling compound #1 or #2, or as a class of compounds that derives from extending the core structure with a nitrogen atom, a carbon atom and a cyclic group to provide compounds resembling compound #6, are herein named “compounds of the invention”.
It is herein noted that all compounds of the invention are encompass by the following general formula:
as well as any pharmaceutically acceptable salts thereof.
Preferably, each R2 and R3 independently represents a group selected from cyclopropyl or linear or branched C1-C6 alkyl.
More preferably, R2 and R3 form a 3-or 4-membered spiro ring together with the carbon atom to which they are both attached.
More preferably, z is an integer selected from 1, 2 or 3; R4 represents a group selected from —CN, cyclopropyl or linear or branched C1-C6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 halogen atoms; and wherein the group R4 replaces the hydrogen atom of one of the groups CH present in the phenyl ring to which R4 is attached.
More preferably, one or both of the integers x and y equal 1 and A represents a group selected the list consisting of aryl, heteroaryl and cyclic amides substituted by 1 or 2 groups that are independently selected from halogen atom, —CN, —N(Ra)Rb, —ORa, —C(═O)Ra, —C(═O)ORa, —C(═O)N(Ra)Rb, —OC(═O)—Ra, —N(Rc)C(═O)Rb, —NRcSO2Ra, —SO2N(Ra)Rb, —SRa, —S(O)Ra, —S(O)2Ra, linear or branched C1-C6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 halogen atoms; C3-C6 cycloalkyl which optionally contains 1 or 2 heteroatoms selected from O, S and N, and which ring is optionally substituted by C1-C3 alkyl; phenyl or C5-C6 heteroaryl each optionally substituted by halogen atom, C1-C3 alkyl or cyclopropyl.
Still more preferably, the compounds of the invention are any of compounds #1, #2 or #6, or any of those identified above and grouped as structures provided by connecting the core structure to a ring structure (Cy=any cycle) to provide compounds resembling compound #1 or #2, or any of those identified above and grouped as a class of compounds that derives from extending the core structure with a nitrogen atom, a carbon atom and a cyclic group to provide compounds resembling compound #6.
In a preferred embodiment, the “compounds of the invention” useful to work the present invention are selected from any of the following list:
The compounds of the present invention can be in a free form or in the form of a pharmaceutically acceptable salt.
Examples of pharmaceutically acceptable salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate or hydrobromide, etc., organic acid salt such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate or maleate, etc. Also, when the compound has a substituent such as carboxyl group, there may be mentioned a salt with a base (for example, alkali metal salt such as sodium salt, potassium salt, etc. or alkaline earth metal salt such as calcium salt, etc.).
The compounds of the present invention or their enantiomers or pharmaceutically acceptable salts can be in any of its intramolecular salt or adduct, or its solvate or hydrate.
When the compounds of the present invention or a pharmaceutically acceptable salt thereof of the present invention is used as an effective ingredient for medical use, it can be used with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is an inert carrier suitable for each administration method, and can be formulated into conventional pharmaceutical preparation (tablets, granules, capsules, powder, solution, suspension, emulsion, injection, infusion, etc.). As such a carrier, there may be mentioned, for example, a binder (such as gum arabic, gelatin, sorbitol and polyvinylpyrrolidone), an excipient (such as lactose, sugar, corn starch and sorbitol), a lubricant (such as magnesium stearate, talc and polyethylene glycol), a disintegrator (such as potato starch) and the like, which are pharmaceutically acceptable. When they are used as an injection solution or an infusion solution, they can be formulated by using distilled water for injection, physiological saline, an aqueous glucose solution.
The administration method of the compounds of the present invention and/or a pharmaceutically acceptable salts thereof of the present invention is not particularly limited, and a usual oral or parenteral administration method (intravenous, intramuscular, subcutaneous, percutaneous, intranasal, and as others, transmucosal, enteral, etc.) can be applied.
The dosage of the compounds of the present invention or a pharmaceutically acceptable salts thereof of the present invention may be optionally set in a range of an effective amount sufficient for showing a pharmacological effect, in accordance with the potency or characteristics of the compound to be used as an effective ingredient. The dosage may vary depending on administration method, age, body weight or conditions of a patient.
The following examples are merely illustrative of the present invention and do not limit the same.
The compounds listed through-out the present specification can be prepared following the general synthetic route below:
2.1. Materials and Methods
Protein and ligands models. The X-ray crystal structure of the phosphotyrosine-binding domain of Hakai (PDB 3VK6) was downloaded from the Protein Data Bank and the dimer modelled using the proper symmetry operations. Amino acid protonation was carried out using the pdb2pqr server at a pH of 7.2. 3D models for the ligands were built using the Virtual Screening and Data Management Integrated Platform (VSDMIP), as described elsewhere. Briefly, the initial 3D coordinates for each ligand were generated with CORINA [Sadowski, J.; Gasteiger, J.; Klebe, G. Comparison of Automatic Three-Dimensional Model Builders Using 639 X-Ray Structures. J. Chem. Inf. Comput. Sci. 1994, 34, 1000-1008 (DOI: 10.1021/ci00020a039)]. Thereafter, ALFA [4] was used to generate a large variety of conformers for each of which MOPAC-calculated atomic partial charges were assigned by employing the AM1 semiempirical model and the ESP method. All ligand models were stored in the VSDMIP database to be used in the different virtual screening campaigns.
Virtual Screening. Ligands in the eMolecules catalogue [https://www.emolecules.com/info/products-screening-compounds.html] were downloaded and processed as described in the preceding section. Only molecules presenting a carboxylic acid moiety and/or a phosphate group capable of mimicking a phosphotyrosine residue were considered. Next, CRDOCK was used to lodge the selected molecules inside the binding pocket of Hakai by using the CRScore scoring function and the BFGS energy minimizer. The ligands were then ranked according to the predicted score and the top 350 molecules were re-evaluated by using an in-house implementation of the HYDE scoring function. Finally, the best 20 molecules were visually inspected to select a final set of 6 molecules.
Binding pocket analysis. To better analyse the results of the virtual screening campaign, we used our in-house cGRILL software [6] to produce affinity maps within the binding pocket of Hakai's phosphotyrosine-binding domain based on the van der Waals, Coulombic and hydrogen bonding interactions of hypothetical atomic probes. The negatively charged acceptor probe (═O) was used to map possible locations for the molecular recognition of the phosphotyrosine residue to help filtering the docking solutions during the visual inspection of the poses.
Plasmids, Inhibitors and Antibodies
pcDNA-Flag-Hakai, pBSSR-HA-ubiquitin, pSG-v-Src and pcDNA-myc-E-Cadherin plasmids were previously described. Compounds Hakin-1 [4-(5-{[2-(4-nitrophenyl)-2-oxoethyl]thiol}-1H-tetrazol-1-yl)benzoic acid] and Hakin-5 [(2E,4E,8E)-7,13-Dihydroxy-4,8,12-trimethyl-2,4,8-tetradecatrienoic acid] were obtained from ChemBridge Corporation and TimTec or Analyticon
Discovery, respectively. The rest of the analogues tested (Ketophenyls A-1, A-7, A-8, A-9; ketoheteroaryls: A-23, A-25; Cyclic amides: A-10, A-16 and Bencylamide: A-6.1) were obtained from Vitasmlab. Compounds were re-suspended in DMSO (Sigma) at 100 mM for in vitro assays, and Hakin-1 was at 100 mM for in vivo assays. The highest concentration of DMSO was used as the vehicle control for the experiments. Note that Hakin-5 chemical structure is in
Cell Culture
MDCK, HEK293T, HepG2, MCF7 and ACHN cells were cultured in Dulbecco's Modified Eagles Medium (DMEM). MDCK stably expressing Hakai cells (Hakai-MDCK) were previously reported and were growth in DMEM with G418 (800 μg/ml). Different clones of Hakai-MDCK cells shown comparable phenotypes and characteristics as demonstrated previously. LoVo and PC-3 cells were cultured in F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) and HT-29 cells in McCoy's 5a Medium Modified. 5637 cells were cultured in RPMI medium. All culture media were supplemented with 1% penicillin/streptomycin and 10% of heat-inactivated fetal bovine serum (FBS) at 37° C. in a humidified incubator with 5% CO2. Cells were monthly tested for mycoplasma contamination and used only for 1-3 months after defrosted. LoVo and HT29 cells were authenticated with the StemElite ID system (Promega). For phase-contrast images, culture cells were photographed with a Nikon Eclipse-TI microscope.
Ubiquitination Assays
For ubiquitination assays, 750.000 HEK293T cells were seeded in 6-well cell culture plates and after 24 h were transfected with 0.25 μg Src, 0.75 μg Flag-Hakai, and 0.5 μg HA-ubiquitin with Lipofectamin 2000 (Invitrogen, UK). Six hours after transfection cells were treated with indicated concentrations of Hakin-1, Hakin-5 or the rest of the analogues tested for 36 h. Whole cell extracts were obtained in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1% Triton X-100) containing 10 μg/ml leupeptin, 10 μg/ml aprotinin and 1 mM phenylmethanesulphonyl fluoride (PMSF), supplemented with 10 mM N-ethylmaleimide. Cells were harvested and subjected to western blotting using anti-HA antibody to detect ubiquitination.
Immunoprecipitation
For immunoprecipitation experiments, 293 cells were transfected with 3 μg Src, 4 μg Flag-Hakai, and 2 μg HA-ubiquitin and 3 μg E-cadherin with Lipofectamin 2000 (Invitrogen, UK). 24 h after transfection, cells were lysed for 20 min in 1 ml of 1% Triton X-100 lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1% Triton X-100) containing 10 μg/ml leupeptin, 10 μg/ml aprotinin and 1 mM phenylmethanesulphonyl fluoride (PMSF), supplemented with 10 mM N-ethylmaleimide and 2.5 mM sodium orthovanadate. After centrifugation at 18.000 g for 10 min, the supernatants were immunoprecipitated for 2 h with 2 μg of anti-E-cadherin antibody bound to 60 μl of protein G PLUS-Agarose beads, followed by SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting with the indicated antibodies as previously reported.
Viability Assays
For cytotoxicity assays, 1×104 cells were seeded per well into a 96-well plate. After 24 h cells were treated with the indicated inhibitors for 72 h and a MTT colorimetric cell viability assay was performed following manufacturer's instructions (Sigma Aldrich, St Louis, Mo.). Absorbance was measured at 570 and 630 nm using a Multiskan Plus Reader (Nanoquant Infinite M200 Tecan Trading AG, Switzerland). Dose-response curves were designed with Graph Pad Prism Software and the half-maximal inhibitory concentration (IC50) values were calculated. Represented data are the mean±SEM of at least three independent experiments with six replicates per condition.
Western Blotting and Immunofuorescence
For western blot analysis, cells were treated with the indicated inhibitors for 48 h and the whole cell extracts were obtained as described previously. Twenty micrograms of lysates were resolved on a 10% polyacrilamide SDS-PAGE followed by western blot analysis performed as previously described. For immunofluorescence assays cells were grown for 24 h on glass coverslips and treated with the indicated inhibitors for 48 h. Cells were fixed with 4% PFA for 15 min, permeabilized with 0.5% Triton X-10 and incubated with E-cadherin antibody for 2 h. Coverslips were incubated with fluorescein-tagged secondary antibody (Dakopatts, Sweden) for 1. Finally coverslips were mounted with ProLong Gold antifade reagent (LifeTech, UK) and images were taken in epifluorescence microscope (Olympus) using 40× objective.
Proliferation Assays
For BrdU assays, 1×104 of indicated cells were plated per well into a 96-well plate. After 24 h, cells were treated with the indicated inhibitors for 48 h. Three independent experiments were plated with six replicates per each condition. Cells were treated with 10 mM BrdU for 2 h. BrdU incorporation into newly synthesized DNA was measured using a cell proliferation colorimetric immunoassay kit according to the manufacturer's instructions (Roche, Switzerland). Results are expressed as mean±S.D. Results are represented as percentage of positive cells (mean±S.D) of three independent experiments.
Soft Agar-Colony Formation Assay
Soft agar-colony formation assay was performed on 12-well plates in triplicates at a density of 5×103 MDCK and MDCK-Hakai cells/well, or 12×103 HT29 cells/well. Cells were seeded in medium with 0.5% low-melting agarose over a layer with 0.75% low-melting agarose (Lonza Rockland, Me., USA). Cells were treated with the indicated inhibitors and DMSO was used as vehicle. Treatment was refreshed every 3 days and, after 21 days for MDCK and MDCK-Hakai cells or 28 days for HT29 cells, number of colonies were quantified. Quantification of five randomly-selected fields of each condition was photographed with a Nikon Eclipse-TI microscope (objective 4×). Experiments were conducted with three triplicates and were repeated three times. Data are represented as mean±SD.
Migration and Invasion Assay
For invasion assays, cells were treated with Hakin-1 or DMSO as vehicle for 48 h using 1% FBS during the last 24 hours. 3×105 MDCK, MDCK-Hakai or LoVo cells were seeded in a cell invasion chamber (Cell invasion assaykit, Chemicon International) containing medium with 2% FBS. After 72 hours for MDCK and MDCK-Hakai Invasive and 16 h for LoVo cells, invasive cells invaded that reach the lower chamber containing 30% FBS were fixed and stained with crystal violet (Sigma Aldrich, St Louis, Mo.) following the manufacturer's specifications. For migration assays, HT29 cells were cultured with Hakin-1 or DMSO as vehicle for 48 h, using medium without serum the last 24 h. In the cell migration chamber were seeded 3×105 HT29 cells (Cell migration kit, Millipore, Bedford, Mass.) containing medium without serum. After 16 h, migrated cells in the lower chamber containing serum with 30% FBS were stained with crystal violet and counted following the manufacturer's specifications. For both invasion and migration assays cells were counted in five fields photographed with an Olympus microscope using a 20× objective, experiments were performed in triplicates for each condition and the assays were repeated at least three times. Results are expressed as mean±SD.
Tumour Xenograft Model
Xenografts experiments were performed in Experimental Surgery Unit—Technological Training Center from INIBIC in compliance with the European Community Law (86/609/EEC) and the Spanish law (R.D. 53/2013). The experiment was approved by the Ethics Committee for Animal Experimentation of Xerencia de Xestion Integrada da Coruna (XXIAC). Mice were in a 12/12 hours light/dark cycle with water and food available ad libitum. Six weeks old athymic nu/nu mice were randomly distributed in groups. One million of MDCK cells, resuspended in DMEM without serum and antibiotic, were subcutaneously inoculated in both flanks in two groups of 3 animals. The same number of Hakai-MDCK cells were injected in two groups of 4 animals. Twenty days after inoculation tumours in Hakai-MDCK were palpable. Then, half of the animals were treated with Hakin-1 (5 mg/kg) and the other half with the same concentration of DMSO every 3 days. Tumour outgrowth was monitored twice a week taking measurements of tumour length (L) and width (W) with an electronic calipter. Tumour volume was calculated as pLW2/6. Forty days after inoculation, animals were sacrificed. Tumours, lungs, kidneys and livers were collected and fixed in 4% PFA and embedded in paraffin blocks for histology and/or immunohistochemistry (IHC) analyses.
Histology and Immunohistochemistry
Tumours and tissues were deparaffinised, rehydrated and stained with haematoxylin and eosin (H&E) as previously described. Tumour sections (4 μm) were also deparaffinised and hydrated for immunohistochemistry. Antigen retrieval was carried by heating the samples (2100 Retriever; PickCell Laboratories) in citrate buffer (Dako REAL, Denmark) or in EDTA buffer. Then, endogenous peroxidase activity was blocked with peroxidase blocking (DakoCytomation, Denmark). Samples were blocked and permeabilized with 0.2% BSA and 0.1% Tx-100 for 1 hour and incubated with the indicated primary antibodies overnight at 4° C. in a wet chamber. Slides were incubated for 1 hour at room temperature with the secondary antibody and detection was carried our using DAB (DakoReal Envision kit) according to manufacturer instructions. Finally, nuclei were counterstained with Gill's Hematoxylin and mounted with DePeX. Pictures were taken with an Olympus microscope. Quantification of images was performed taken 5 photographs of each animal with Image J programme and the represented results are shown as mean±SEM. The number of mitosis was counted in sections stained with H&E. In this case, ten pictures of each tumour were taken with an Olympus BX50 microscope (objective 40×) and the number of mitosis was counted manually. Results are represented as mean±SEM and a representative photograph is shown for each condition.
Quantification of Lung Metastasis from In Vivo Mouse Model
Real-time PCR was used to study the presence of metastasis in the lung mice. Primers for HA epitope and Hakai present in ectopic HA-tagged Hakai expressed in MDCK-Hakai cells (5′-TCTGGGACGTCGTATGGGTA-3′; 5′-TTCTTCATCACCTTGCGGG-3′) were used for the quantification. Primers for mouse apolipoprotein B (apob) (5′-CGTGGGCTCCAGCATTCTA-3′; 5′-TCACCAGTCATTTCTGCCTTTG-3′) were used as endogenous control. MDCK cell line was used as negative control. Lung DNA was extracted from 10-15 sections of paraffin blocks (4 μm) using with QIAamp DNA Mini Kit (Qiagen). The amplification and quantification of DNA was carried by quantitative PCR in technical triplicates by using a LightCycler 480 real-time lightcycler (Roche). Relative DNA levels were calculated by 2−ΔΔCt method.
Statistical Analysis
Shapiro-Wilk test was used to check a normal distribution and Levene test to assess the equality of variances. Statistical significance of data was determined with ANOVA with Bonferroni test or Kruskal-Wallis with Tukey correction test. Significance among the experimental groups indicated in the figures is shown as * p<0.05, **p<0.01 and ***p<0.001. Results obtained are expressed as mean±SD or mean±SEM as indicated. Survival graphic in xenograft assay was analysed with GraphPad Prism software and the test of Breslow was used to calculate p values. Results are represented as fold induction of treated cells over the values obtained in the untreated cells.
List of antibodies used to carry out the present invention.
In Vivo TUNEL Assay
Tissue sections from tumours were deparaffinised and rehydrated using standard protocols. The slides were rinsed twice with PBS and treated with citrate buffer buffer (Dako REAL, Denmark) in microwave at 350 W for 5 min. The tissue sections were then analysed with an in situ Cell Death Detection Kit, Fluorescein (Roche) following the manufacturer's instructions. Then, slides were incubated with Hoechst for 5 min in darkness. The reaction was visualized under an epifluorescence Olympus microscope using 20× objective. Five representative pictures of each section were taken. The percentage of positive cells was calculated and results are represented as mean±SEM.
2.2. Results
Identification of Putative Selective Hakai Inhibitors
With the aim of finding candidate molecules with the required potential to inhibit Hakai, we designed a virtual screening workflow based on the structural information available and the nature of the phosphotyrosine-binding pocket, which was explored with the aid of affinity probes. As a first step, we considered only molecules in our chemical library that display a negatively charged carboxylate or phosphate group that would be complementary to the highly positive molecular electrostatic potential of the binding pocket. The selected molecules were then docked into the Hakai dimer to evaluate all possible binding poses and then ranked using the HYDE postprocessing scoring function to estimate the interaction energy of the hypothetical Hakai-inhibitor complexes. The first 20 top-ranking molecules were visually inspected and two of them were selected for subsequent experimental validation, namely Hakin-1 and Hakin-5 (
Effect of Hakin-1 Inhibitor on Hakai-Induced Ubiquitination
We first investigated the effect of Hakin-1 inhibitor on the ubiquitination induced by the E3 ubiquitin-ligase Hakai by using culture tumour cells. 293T cells were transfected with Src, Hakai and ubiquitin in presence of Hakin-1 inhibitor or DMSO as control. Hakin-1 strongly reduces the ubiquitination mediated by Hakai in a doses dependent-manner (
Hakai Inhibition by Hakin-1 Activates Epithelial Differentiation on Tumour Cells
Next, we studied the effect of Hakai inhibition on the cell viability of cancer cells. For this objective, we generated a dose-response curve by using Hakin inhibitors in several epithelial cells as we previously reported. First, we analysed the cytotoxicity effect on Hakin-1 on HT-29 and LoVo colon tumour cell lines showing an important inhibitory response (
Hakin-1 Inhibits Proliferation, Oncogenic Potential and Invasion in Tumour Culture Cells
We next characterized the effect of Hakin-1 in tumour cell lines using standard proliferation and soft agar colony-forming assays. Given that Hakai affects not only cell-cell contacts but also proliferation in fibroblast and epithelial cells, we decided to determine the possible effect of Hakin-1 in cell proliferation. Hakin-1 reduced cell proliferation in HT29 and LoVo cell lines (
In Vivo Antitumor Effect of Hakin-1 in Tumour Xenografts
The acquisition of migratory and invasive abilities during EMT are crucial events in the formation of distant metastasis, therefore targeting these events is therefore an ideal approach for cancer treatment. Since we have shown that Hakin-1 effectively inhibits cell proliferation, oncogenic potential and cellular invasion and motility in cell cultures, we decided to study the efficacy of Hakin-1 on this suppression of pre-existing tumours in vivo. For this purpose, MDCK and Hakai-MDCK cell were subcutaneously injected into the flank of nude mice. As previously reported, Hakai-MDCK cells formed primary tumours whereas parental MDCK cells were unable to do so. Hakin-1 displayed a potent effect on inhibiting xenograft tumour growth in vivo (
Hakin-1 Treatment Reduces N-Cadherin Mesenchymal Markers of Tumours Xenograft and Micrometastasis Formation in Lung In Vivo
We further evaluated the in vivo effect of Hakin-1 on the reversion of the EMT, as crucial process in tumour progression and cell invasion. First, we confirmed that Hakai protein expression levels were not affected by Hakin-1 action in xenograft tumours of nude mice (
Effect of Hakin-1 Analogues on Cytotoxicity and on Hakai-Induced Ubiquitination
Next, we studied the effect of the selected analogues on HT29 colon cancer cells. First, we analysed cytotoxicity effect of the following analogues: Ketophenyls A-1, A-7, A-8 and A-9; ketoheteroaryls: A-23 and A-25; Cyclic amides: A-10 and A-16 and Bencylamide A-6.1 on HT-29 colon tumour cell lines. It is shown an important inhibitory response of the Ketophenyls A-7, A-8, A-9 and ketoheteroaryls: A-23, A-25, however no cytotoxic effect was detected by the action of Ketophenyls A-1, Cyclic amides: A-10 and A-16 and Bencylamide A-6.1 (
| Number | Date | Country | Kind |
|---|---|---|---|
| 18382809.4 | Nov 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/081522 | 11/15/2019 | WO | 00 |
