KR102397817B1 - Composition for prevention or treatment of triple-negative breast cancer through blocking microtubule acetylation - Google Patents

Abstract

The present invention relates to a triple-negative breast cancer inhibitor through inhibition of microtubule acetylation, and specifically, triple-negative breast cancer prevention or therapeutic pharmaceutical compositions; Health functional food composition for preventing or improving triple negative breast cancer; And it relates to a method for preventing or treating triple-negative breast cancer comprising administering the pharmaceutical composition to a subject other than a human.
The compound of the present invention has confirmed its excellent inhibitory ability to target triple-negative breast cancer rather than normal cells or receptor-existing breast cancer through 2D and 3D cell culture and xenograft tumor experiments, and confirms that there is no side effect such as weight loss Therefore, it may be effectively used to inhibit, prevent, or treat triple-negative breast cancer, which has been difficult to target due to the absence of a receptor.

Description

A composition for preventing or treating triple-negative breast cancer through inhibition of microtubule acetylation {Composition for prevention or treatment of triple-negative breast cancer through blocking microtubule acetylation}

The present invention relates to a triple-negative breast cancer inhibitor through inhibition of microtubule acetylation, and specifically, triple-negative breast cancer prevention or therapeutic pharmaceutical compositions; Health functional food composition for preventing or improving triple negative breast cancer; And it relates to a method for preventing or treating triple-negative breast cancer comprising administering the pharmaceutical composition to a subject other than a human.

Worldwide, more than one million women are diagnosed with breast cancer each year. Breast cancer is a highly heterogeneous disease composed of many different types that are distinguished using a histological classification system. Many subtypes and most of the patients are histologically identified as luminal type A or lumen B, which can be clearly characterized as exhibiting estrogen receptor (ER) expression with low or high grade histological structures, respectively (Santana-Davila). and Perez, 2010). Immunohistochemical methods are used to measure the expression of the progesterone receptor (PR), which when coupled with an ER-positive status allows for the classification of tumors as being hormone responsive. In addition, overexpression or amplification of human epidermal growth factor receptor 2 (HER2) can be observed by immunohistochemistry or fluorescence in situ (FISH). In general, expression of these three markers in breast tumors is associated with better clinical outcomes, including tamoxifen, Arimidex™ (anastrozole), Aromasin™ (exemestane), the protein comprising Femara™ (letrozole), Faslodex™ (fulvestrant), Herceptin™ (trastuzumab), or Tykerb™ (lapatinib) This is because there are several treatment options available for targeted patients (de Ruijter et al., 2011). Another histological subtype of breast cancer consists of basal cancers, particularly associated with high histological grade, increased mitotic index and high Ki67 expression (Santana-Davila and Perez, 2010). The majority of basal cancers include patients with triple-negative breast cancer (TNBC), which constitute 15-20% of all diagnosed breast cancer patients (Ismail-Khan and Bui, 2010).

Triple-negative breast cancer is defined as a lack of protein expression in ER, PR and an absence of HER2 protein overexpression. The relationship between basal-positive and triple-negative breast cancers is not easily explained, as not all triple-negative breast cancers are basal-positive, and not all basal-positive cancers are triple-negative breast cancer, but about 75% of patients in this class have both. because they share the characteristics of TNBC is associated with a poor prognosis consisting of a low 5-year survival rate and high recurrence.

Patients with triple-negative breast cancer develop their disease earlier in life compared to other breast cancer subtypes, and are often diagnosed in the premenopausal stage (Carey et al., 2006). Triple-negative breast cancer shows an increased tendency to relapse after treatment and appears to be more aggressive than other breast carcinoma subtypes similar to those of the basal-positive breast cancer subtype (Nofech-Mozes et al., 2009). As a result, the overall 5-year survival of patients with triple-negative breast cancer is significantly lower than that of patients diagnosed with breast cancer of other subtypes.

According to a report published in Cancer Research in 2015, microtubule acetylation is increased in triple-negative breast cancer cell lines compared to normal cells and other subtypes of breast cancer cell lines (AE Boggs et al). In addition, this research team found that cancer cell mobility was significantly reduced when microtubule acetylation was inhibited by overexpressing the mutant gene.

On the other hand, anticancer drugs that control microtubules, which are cytoskeletal proteins that play a very important role in cell division and migration, have already been developed and are being applied to cancer patients. These drugs are divided into a group that inhibits microtubule polymerization, such as vinca alkaloid and cholhicine, and a group that stabilizes microtubules, such as Docetaxel and Paclitaxel. Both of these drugs act to suppress the role of microtubules during cell division and can effectively control cancer cells that undergo rapid cell division.

On the other hand, side effects such as vomiting and hair loss appeared because these drugs also act on normal cell groups showing rapid cell division, such as gastric mucosal cells and hair follicle cells. In addition, according to a paper published in 2015, Taxol, a microtubule-stabilizing drug well known as an anticancer drug for breast cancer, shows its drug effect only in Her2-positive breast cancer, which is a part of the subtype of breast cancer patients, gradually revealing its limitations. Therefore, there is an increasing need for the development of excellent new triple-negative breast cancer therapeutics with excellent triple-negative breast cancer-specific microtubule inhibitory effects and fewer side effects for the prevention, improvement or treatment of triple-negative breast cancer.

Under this background, the present inventors continued their efforts to develop novel triple-negative breast cancer therapeutics. As a result, the compounds of the present invention act particularly sensitively on triple-negative breast cancer, thereby inhibiting the growth of cancer cells through inhibition of microtubule acetylation. And, by confirming that there is no side effect such as weight loss, the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating triple-negative breast cancer comprising a compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or improving triple-negative breast cancer comprising a compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Another object of the present invention is to prevent triple-negative breast cancer comprising administering to an individual other than a human, a pharmaceutical composition comprising a compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient Or to provide a treatment method.

One embodiment of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating triple-negative breast cancer comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient. :

Specifically, the composition aims to inhibit acetylation of microtubules, and more specifically, the inhibition of microtubule acetylation is one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 1 to 3 Inhibition of binding to microtubule may be achieved by binding to the microtubule binding site of alpha-tubulin acetyltransferase 1 (αTAT1), and the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent. and the composition may further include an anticancer agent, but is not limited thereto.

In the present invention, to check that microtubule acetylation is actively occurring in triple-negative breast cancer, and to suppress it, 30,000 compounds from the Cambridge small chemical library are screened to select and synthesize a compound with excellent microtubule acetylation inhibitory activity did

The synthesis method thereof is shown in FIGS. 9 to 10 .

The term, “microtubule” is also called microtubule, and as a member of the cytoskeleton, it refers to a framework that maintains the cell shape of a thin tube that is widely spread throughout the cytoplasm. Polymerization of a protein called tubulin produces long tethered microtubules, some exceeding 50 micrometers in length. It assembles and disassembles very dynamically. The tube has an outer diameter of 24 nanometers and an inner diameter of about 12 nanometers, and is found in all eukaryotic cells. is known to do

As used herein, the term “acetylation” refers to a reaction for substituting an acetyl functional group in a compound.

The term, “inhibition” means that the activity is lowered due to interference or interference with enzyme activity, and the inhibition can be used in the same sense as “inhibition”.

As used herein, the term “pharmaceutically acceptable salt” refers to a salt in a form that can be used pharmaceutically among salts, which are substances in which cations and anions are combined by electrostatic attraction, and are usually metal salts, organic bases It may be a salt with, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine salts with, etc.; salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid, or the like. More specifically, a preferred acceptable salt in the present invention may be HCl, and may be represented by the following formula (3), but is not limited thereto:

As used herein, the term “active ingredient” refers to a component capable of exhibiting the desired activity alone or in combination with a carrier having no activity by itself.

In one embodiment of the present invention, microtubule acetylation expression was analyzed according to the tissues of breast cancer patients having ER, PR, or HER2 receptors, triple-negative breast cancer patients, and breast cancer cell lines of various subtypes ( FIGS. 1 to 2 ), Microtubule acetylation expression was analyzed in aTAT1-knock-out MDA-MB-231 cell line (Fig. 3), and tumorigenesis was induced by xenografting MDA-MB-231 in NOD/SCID mice, followed by in vivo tumorigenesis. and SPIN90 Knockout mouse embryonic fibroblast microtubule acetylation expression was compared (Figs. 4 to 5), and 30,000 compounds from the Cambridge library were treated to confirm the microtubule acetylation inhibitory activity (Figs. 6 to 8). ), a compound having excellent efficacy in inhibiting microtubule acetylation was selected and synthesized ( FIGS. 9 to 10 ), and its microtubule acetylation inhibitory activity was confirmed ( FIG. 11 ).

As used herein, the term “triple-negative breast cancer” is a type of breast cancer lacking three hormone receptors, estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (Human epidermal growth factor receptor 2, HER2) is not expressed, so it does not respond well to anti-hormonal agents or targeted therapies, making it difficult to treat and has a poor prognosis.

In the present invention, as a result of diligent efforts to discover a compound specific for the treatment of triple-negative breast cancer, it was confirmed that microtubule acetylation actively occurs in triple-negative breast cancer, and a compound having the effect of inhibiting microtubule acetylation was discovered, and this was By treating breast cancer, it was confirmed that there was an anticancer effect, and the triple-negative breast cancer specific therapeutic effect was confirmed through the compound of the present invention.

As used herein, the term “prevention” refers to any action that inhibits or delays the onset of triple-negative breast cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of triple-negative breast cancer are improved or beneficially changed by the administration of the pharmaceutical composition.

In another embodiment of the present invention, the microtubule acetylation inhibitory efficacy of the compounds GM-90257, GM-90568, and GM-90631 of the present invention was confirmed through cell experiments (FIG. 12), GM-90257, GM- When 90568, and GM-90631 were removed again after treatment, a change in microtubule acetylation was confirmed (FIG. 13), and colony formation of the triple-negative breast cancer cell line MDA-MB-231 was inhibited in a concentration-dependent manner by the compound. After confirming (FIG. 14), normal cell line (MCF-10A), receptor-present breast cancer cell line (MCF-7), and triple-negative breast cancer cell line (MDA-MB-231) were cultured in a 2D environment, MTT assay The specific inhibitory efficacy of the compound of the present invention on breast cancer cells was confirmed through (FIG. 15), the apoptosis pattern was confirmed through Annexin V/PI staining (FIG. 16), and the triple-negative breast cancer cell line MDA- in NOD/SCID mice After xenocrafting MB-231 to induce tumorigenesis, GM-90257 or GM-90631 was injected intraperitoneally to confirm tumorigenesis inhibitory efficacy ( FIGS. 17 to 18 ), and the mechanism of inhibiting microtubule acetylation of the compound was confirmed (FIG. 19).

Through this, the in vitro microtubule acetylation inhibitory efficacy of the compound of the present invention was confirmed, and excellent triple-negative breast cancer-specific therapeutic efficacy through microtubule acetylation inhibition in an animal model was confirmed.

As used herein, the term “pharmaceutical composition” may further include a pharmaceutically acceptable carrier, excipient or diluent, and the carrier may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid), poly-L-lactic acid (poly-L-lactic acid), mineral oil, and the like.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The content of the compound of the present invention contained in the pharmaceutical composition of the present invention is not particularly limited.

As used herein, the term “anticancer agent” is a chemotherapy agent used to inhibit the proliferation of cancer cells, and specifically, mechloethamine, chlorambucil, phenylalanine, mustard ), cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa ( thiotepa), cisplatin, carboplatin, dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin, idarubicin, mitoxantrone (mitoxantrone), plicamycin, mitomycin, C bleomycin; From the group consisting of vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan and iridotecan It may be any one or more selected, but is not limited thereto.

Another embodiment of the present invention for achieving the above object provides a health functional food composition for preventing or improving triple-negative breast cancer comprising a compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient do.

In this case, the terms, “pharmaceutically acceptable salt”, “active ingredient”, “triple-negative breast cancer”, and “prevention” are the same as described above.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated by administration of the composition according to the present invention, for example, the degree of symptoms.

As used herein, the term “health functional food” refers to a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the It refers to obtaining useful effects for health purposes such as regulating nutrients with respect to structure and function or physiological actions. On the other hand, health food means food that has an active health maintenance or promotion effect compared to general food, and health supplement means food for the purpose of health supplementation. In some cases, the terms health functional food, health food and health supplement food can be mixed.

The composition of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, disinfectants, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, emulsifying agents, thickeners, filming agents, gum base agents, foam inhibitors, solvents, and improving agents. may include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of drugs using food as a raw material, and is excellent in portability, so the present invention Foods of can be taken as an adjuvant to enhance the effect of prevention or improvement of triple-negative breast cancer.

The composition of the present invention may be included in various weight % in the food composition if it can exhibit the effect of preventing or improving triple-negative breast cancer. Specifically, it may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. In the case of long-term ingestion for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another embodiment of the present invention for achieving the above object is to administer a pharmaceutical composition comprising a compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient, to an individual other than a human. It provides a method for preventing or treating triple-negative breast cancer comprising the steps of.

In this case, the terms, “pharmaceutically acceptable salt”, “active ingredient”, “pharmaceutical composition”, “triple-negative breast cancer”, “prevention” and “treatment” are as described above.

As used herein, the term "administration" means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two to three times. It can also be used alone or in combination with other drug treatments. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, that have or can develop triple-negative breast cancer.

Another embodiment of the present invention for achieving the above object is a triple-negative breast cancer prevention or treatment use of a composition comprising a compound represented by Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient. to provide.

Another embodiment of the present invention for achieving the above object provides a triple-negative breast cancer prevention or treatment use of a composition comprising the compound represented by Formula 3, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Another embodiment of the present invention for achieving the above object provides a compound represented by Formula 2, or a pharmaceutically acceptable salt thereof.

The composition for inhibiting microtubule acetylation according to the present invention exhibits excellent microtubule acetylation inhibition and growth inhibition ability targeting triple-negative breast cancer other than normal cells or receptor-existing breast cancer, thus preventing or treating triple-negative breast cancer. It may be included in a target pharmaceutical composition, a food composition for prevention or improvement, and since the food composition can be consumed on a daily basis, a high effect can be expected for the prevention or improvement of triple-negative breast cancer.

The compound of the present invention has confirmed its excellent inhibitory ability to target triple-negative breast cancer, not normal cells or receptor-existing breast cancer, through 2D and 3D cell culture and xenograft tumor experiments, and it is confirmed that there is no side effect such as weight loss Therefore, it may be effectively used to inhibit, prevent, or treat triple-negative breast cancer, which has been difficult to target due to the absence of a receptor.

1 is a photograph and graph showing the expression of microtubule acetylation through immunohistochemical staining of tissues of breast cancer patients having ER, PR, or HER2 receptors and tissue slides of triple-negative breast cancer patients.
2 is a photograph showing the microtubule acetylation expression pattern according to various subtypes of breast cancer cell lines.
3A is an electrophoretic photograph showing the expression of microtubule acetylation in the MDA-MB-231 cell line in which αTAT1 was knocked out.
3B is a photograph and graph showing the expression of microtubule acetylation in the MDA-MB-231 cell line in which αTAT1 was knocked out.
4 is a graph and photograph comparing changes in tumorigenesis in vivo after xenotransplantation of MDA-MB-231 to NOD/SCID mice to induce tumorigenesis.
5 is an electrophoresis photograph showing the expression of microtubule acetylation of SPIN90 Knockout mouse embryonic fibroblasts.
6 is a photograph confirming the inhibition of acetylation of microtubules through fluorescence staining by treating the candidate compound.
7 is a photograph showing the microtubule acetylation inhibitory activity of 20 candidate compounds.
8 is a photograph showing the microtubule acetylation inhibitory activity of a candidate compound.
9 is a schematic diagram showing a method for synthesizing compound GM-90257.
10 is a schematic diagram showing a method for synthesizing the derivatives GM-90631 and GM-90568 of compounds GM-90257.
11 is a photograph showing the microtubule acetylation inhibitory activity by concentration of the candidate compounds GM-90257 and GM-90568.
12A is a photograph showing the microtubule acetylation inhibitory efficacy of GM-90257, GM-90568, and GM-90631.
Figure 12B is a photograph showing the microtubule acetylation inhibitory efficacy of each concentration of GM-90257, GM-90568, and GM-90631.
13 is a photograph showing the change in the aspect of microtubule acetylation when removed again after treatment with GM-90257, GM-90568, and GM-90631.
14 is a photograph and graph showing that the compound of the present invention inhibits colony formation in the triple-negative breast cancer cell line MDA-MB-231 in a concentration-dependent manner.
15 is a normal cell line (MCF-10A), a receptor-present breast cancer cell line (MCF-7), and a triple-negative breast cancer cell line (MDA-MB-231) after culturing in a 2D environment, the present invention through MTT assay It is a graph showing the breast cancer cell inhibitory effect of the compound.
Figure 16 shows Annexin V/PI staining after treatment with the compound of the present invention in a normal cell line (MCF-10A), a receptor-present breast cancer cell line (MCF-7), and a triple-negative breast cancer cell line (MDA-MB-231). It is a graph showing the pattern of apoptosis through
17 is a graph and photograph showing the tumorigenesis inhibitory efficacy results by intraperitoneal injection of GM-90257 after xenotransplantation of the triple-negative breast cancer cell line MDA-MB-231 into NOD/SCID mice to induce tumorigenesis.
18 is a graph and photograph showing the results of tumorigenesis inhibition efficacy by concentration by intraperitoneal injection of GM-90631 after xenotransplantation of the triple-negative breast cancer cell line MDA-MB-231 into NOD/SCID mice to induce tumorigenesis.
19A is a photograph showing the structure at the binding site of the compound of the present invention.
19B is a photograph showing the result of co-localization of αTAT1 with microtubules through fluorescence staining by treating αTAT1 overexpressing cells with the compound of the present invention.
19C is a photograph showing the microtubule polymerization change through the OD value.
19D is a photograph showing the results of an in vitro experiment in which the compound of the present invention directly inhibits microtubule acetylation due to the interaction between αTAT1 and microtubule.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Experimental Example 1: Preparation of patient tissue

Paraffin-sampled slides for breast cancer tissue analysis and patient pathology data were purchased from US Biomax (BR10011), and were reviewed by the Gwangju Institute of Science and Technology (GIST) in order to obtain and analyze human tissue slides and patient information. study was approved. (IRB number: 20191128-BR-49-05-02).

Experimental Example 2: Cell Culture

MDA-MB-231 was cultured in Dulbecco's Modified Eagle's Medium (high glucose, Gibco) containing 10% FBS, 50 μg/ml of Streptomicyn, and 50 units/ml of penicillin. ZR-75-1, T47D, HCC-1428, HCC-1419 and Hs578T were purchased from the Korea Cell Line Bank and cultured according to the method specified in the data sheet provided by the institution.

Experimental Example 3: Western blot analysis

RIPA containing the collected cells 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 1% SDS, 0.25% sodium deoxycholate, 10mM NaF, 1mM Na ₃ VO ₄ and protease inhibitor cocktail (Roche) dissolved in buffer. Primary antibodies were performed using Acetyl-α-tubulin (Sigma, USA), α-tubulin (Sigma, USA), and acetyl-histone H3 sampler kit (Cell signaling technology, USA).

Experimental Example 4: Immunohistochemistry analysis

Tumors collected from xenograft mice were fixed in 10% formalin and embedded in paraffin blocks. Thereafter, paraffin was removed, re-hydration with ethanol, and antigen exposure by heating in Retrieval solution (IHC World, USA) for 30 minutes, acetyl-α-tubulin (Abcam, UK), Ki67 (Sigma, USA), and Mayer hematoxylin (Dako, USA) for cell nucleus staining. Then, the stained slide samples were scanned and analyzed by Aperio Image Scope (Leica biosystems, Germany).

Experimental Example 5: αTAT1 KO cell generation method through CRISPR-Cas9

αTAT1 knock-out using the CRISPR-Cas9 system was performed in the same manner as in the following paper (Oh, You, Ko, Jeong, Keum & Rhee, 2017). Specifically, after cloning the anealed sgRNA into the pLenti-CRISPR v2 plasmid, αtat1-targting cloning vector, psPAX2 and pMD2.G and polyethyenimine were co-transfected into the HEK293T cell line for lentivirus production. After 48 hours of incubation, lentivirus secreted from HEK293T was collected from conditioned media (CM). Lentivirus was injected into MDA-MB-231 with polybrene (8 μg/ml) for 48 hours and screened with puromycin (1 μg/ml) for 2 weeks.

Experimental Example 6: Analysis of soft agar colony formation

To confirm anchorage-independent cancer cell growth, a 1% agarose solution and a 2X DMEM culture mixture were mixed in a 1:1 ratio, and then 1.5 ml of each was distributed to a 6 well plate and then solidified at room temperature for 30 minutes. made it Next, after mixing with MDA-MB-231 suspended in the culture medium (5 x 10 ³ cells per well) and 0.6% agarose solution maintained at 40 °C, a 6-well plate in which the previously prepared 1% agarose solution is hardened was added to After the plate was hardened at room temperature for 30 minutes, 300 μl of the complete culture solution was added to each well along with the candidate compound and incubated at 37°C for 3 weeks.

Experimental Example 7: Xenograft tumor injection experiment

Female NOD/SCID mice (8-12 weeks old) were used in a breast cancer xenograft model. MDA-MB-231 (1 x 10 ⁶ cells) mixed with Matri-gel was injected into the left groin mammary fat pad, and the developed tumor was quantified by the following formula: [V = (L x W2) / 2, L; length, W; width]. When the tumor reached 100 mm 3 , each compound was injected intraperitoneally for 15 days once every 2 days. All experimental procedures on animals were performed with the approval of GIST's Animal Care and Ethics Committee (GIST-2017-003).

Experimental Example 8: Compound screening

As a 10 mM stock solution, a Cambridge chemical library (NM1117-1) containing 30,000 small compounds dissolved in DMSO (dimethyl sulfoxide) was used. Specifically, Spin90-KO MEFs (You et al., 2017) with consistently high expression of acetyl-α-tubulin were cultured in a 96 well culture plate and each compound was treated at a concentration of 10 μM. After primary detection with Acetyl-α-tubulin (Sigma, USA), it was detected and stained with Alexa fluorescence secondary antibody to confirm under a microscope. Finally, individual analysis was performed using an IX81 fluorescence microscope, and 20 selected candidates were quantitatively analyzed through Western blot.

Experimental Example 9: Microtubule acetylation analysis

The tubulin protein was purified from αTAT1 KO MDA-MB-231 that was not acetylated in microtubules. The MTS buffer contained 80 mM PIPES, 1 mM EGTA, 1 mM MgCl ₂ , 40% glycerol, PMSF 1 mM, Na3VO4 1 mM and NaF 10 mM, and this solution was added to the cells in an incubator at 37° C. for 20 minutes. After resuspension of the cells in MTS buffer, paclitaxel was added to a final concentration of 20 μM. Then, the solution was homogenized for about 3 times with a 25G syringe needle, and then incubated for 10 minutes at room temperature. Using a Ti-70 rotor (Beckman Coulter, USA), centrifugation was performed at 31100 rpm for 30 minutes at room temperature to separate the precipitate and the supernatant. Thereafter, the Pellet was resuspended in MTS buffer containing 20 μM paclitaxel, and 1 mg/ml of His-αTAT1, 1 uM acetyl-coA and each microtubule acetylation inhibitory compound (1 mM) were added thereto, followed by 30 °C at 37 °C. incubated for min. The enzyme activity of αTAT1 was stopped by addition of 4X SDS sample solution and analyzed by SDS-PAGE followed by Western blot.

Experimental Example 10: Annexin V/PI staining analysis

After using the compound, annexin V-PI apoptosis kit (BD bioscience, USA) was used to analyze cancer cell apoptosis and dead cells. After the compound was treated in the cancer cell line and incubated for 24 hours, the cells were analyzed using flow cytometry (flow cytometry, BD bioscience, USA) after PI-annexin V staining according to the manufacturer's protocol.

Experimental Example 11: Immunofluorescence staining analysis

To detect acetyl-α-tubulin expression after compound treatment, MDA-MB-231 was fixed with PBS containing 4% paraformaldehyde, permeabilized with 0.1% triton-X 100, and 0.1% BSA solution After blocking, the cells were stained with acetyl-α-tubulin (Sigma, USA), which is a primary antibody. In addition, to analyze the co-localization of αTAT1 and α-tubulin, MDA-MB-231 transfected with Dsred2-αTAT1 was sampled in the same manner and stained with a primary antibody, α-tubulin (Sigma, USA). All fluorescence images were acquired using a confocal microscope (FV1000, Olympus).

Experimental Example 12: Microtubule polymerization (polymerization) analysis

Tubulin protein (Cytoskeleton, USA) purified from pig brain was used for in vitro microtubule polymerization analysis. Tubulin protein was adjusted to 3 mg/ml and individually mixed with each compound containing paclitaxel, nocodazole, GM-90257, GM-90631 and DMSO as controls. Then, each OD value at 340 nm was checked every 30 seconds while polymerization was carried out at 37° C. for 1 hour.

Experimental Example 13: Statistical Analysis

Data requiring quantification were analyzed using two-tailed Student's t-test to prove a statistically significant difference between the two groups. All results represent the mean (n=3), bars and mean ± standard deviation (SD) were included. All P values are two-sided. Data were considered statistically significant at P values less than 0.05; *, P ≤0.05; **, P ≤ 0.01; ***, P ≤0.001.

Example 1: Comparison of microtubule acetylation expression patterns according to breast cancer cell types

Breast cancer is divided into various subtypes based on receptors on the surface of cancer cells. These receptors are sometimes used for targeted therapy, and specific anticancer effects can be expected. However, in the case of triple-negative breast cancer where none of the three receptors exist, targeted therapy is difficult and cancer cell migration and invasiveness are higher than other breast cancers. Therefore, the mortality rate due to metastasis is high. Therefore, for the inhibition and treatment of triple-negative breast cancer, the expression pattern of microtubule acetylation, one of the characteristics of triple-negative breast cancer, was compared with other breast cancer cells.

Specifically, the microtubule acetylation pattern was compared by performing immunohistochemistry of the method of Experimental Example 4 on tissue slides of breast cancer patients.

As a result, as shown in FIG. 1 , it was confirmed that microtubule acetylation was significantly increased in triple-negative breast cancer, unlike the tissues of breast cancer patients having ER, PR, or HER2 receptors.

In addition, the expression pattern of microtubule acetylation according to various subtypes of breast cancer cell lines based on the receptor was confirmed.

cell line surface receptor Subtype MCF-10A - Normal MEF-7 ER+, PR+, Her2- Luminal A ZR-75-1 ER+, PR+, Her2- Luminal A T47D ER+, PR+, Her2- Luminal A HCC-1428 ER+, PR+, Her2- Luminal A HCC-1419 ER+, PR-, Her2+ Luminal B Hs578T ER-, PR-, Her2- TNBC MDA-MB-231 ER-, PR-, Her2- TNBC

As a result, as can be seen in Tables 1 and 2, it was confirmed that microtubule acetylation was significantly increased in the triple-negative breast cancer cell line.

Through this, it was confirmed that microtubule acetylation was more actively expressed in triple-negative breast cancer than in other breast cancer subtypes in both comparison of tissue slides of breast cancer patients and comparison of breast cancer cell lines.

Example 2: Growth comparison of triple-negative breast cancer according to microtubule acetylation expression

In Example 1, it was confirmed that microtubule acetylation occurred actively in triple-negative breast cancer cells. In order to confirm the effect of microtubule acetylation expression on the growth of triple-negative breast cancer, MDA- After knock-out (KO) of alpha-tubulin acetyltransferase 1 (αTAT1), a major acetylase, in MB-231 cell line, growth changes of the breast cancer cell line were analyzed. Specifically, αTAT1 KO cell generation was carried out through Experimental Example 5.

As a result, as shown in FIG. 3 , it was confirmed that the expression of microtubule acetylation was significantly reduced in the MDA-MB-231 cell line in which αTAT1, a major acetylase, was knocked out ( FIG. 3A ).

In addition, through the method of Experimental Example 6, it was confirmed that the number of colonies formed was significantly reduced in the colony formation experiment of MDA-MB-231 cells in a 3D environment using soft agar (FIG. 3B).

In addition, through the method of Experimental Example 7, after xenotransplantation of MDA-MB-231 into NOD/SCID mice to induce tumorigenesis, changes in tumorigenesis in vivo were compared.

As a result, as shown in FIG. 4 , the tumor size of the MDA-MB-231 cell line in which αTAT1 was knocked out was significantly reduced, and its weight was also significantly reduced compared to that in MDA-MB-231 without knock-out. confirmed that.

Through this, it was confirmed that inhibition of microtubule acetylation inhibited the growth of triple-negative breast cancer.

Example 3: Primary screening for selection of microtubule acetylation inhibitor candidate compounds

In Examples 1 and 2, it was confirmed that the expression of microtubule acetylation in triple-negative breast cancer was higher than that of other subtype cancer cell lines, and when the expression of microtubule acetylation was suppressed, it was confirmed that the growth of breast cancer was inhibited. Therefore, in order to discover compounds effective for inhibiting microtubule acetylation in triple-negative breast cancer, a large-scale screening of 30,000 samples was performed through the Cambridge library.

Specifically, the method of Experimental Example 8 was used, and SPIN90 Knock-out mouse embryonic fibroblast (SPIN90 KO MEF), which is known to continuously express microtubule acetylation during screening, and is known for rapid and stable cell growth, was used. was done (FIG. 5).

More specifically, through the method of Experimental Example 9, after the candidate material was treated, the acetylation of the microtubule was observed under a microscope through staining.

5342294

9080518

5375229

9083907

7945423

9084483

7945622

9090545

7947446

9093074

7968593

5934703

9130673

7906450

9076292

7905037

9078355

7916195

9079730

7804168

As a result, as can be seen in FIGS. 6 to 2, 20 compounds showing the ability to inhibit the acetylation of microtubules among about 30,000 candidate compounds were identified.

Example 4: Secondary screening for selection of microtubule acetylation inhibitor candidate compounds

Secondary screening was performed based on the 20 candidate compounds selected in Example 3, and the microtubule acetylation inhibitory efficacy was compared.

Specifically, in the secondary screening, microtubules were separated and purified from SPIN90 KO MEF, and acetylation was performed by treating candidate compounds, and the degree of acetylation of microtubules was confirmed through electrophoresis.

As a result, as shown in FIG. 7 , it was confirmed that the microtubule acetylation inhibitory activity of No. 5934703 was the most excellent among 20 candidate compounds.

Through this, compound 5934703 was selected as a candidate compound for the study of inhibition of microtubule acetylation of triple-negative breast cancer cells.

Example 5: Synthesis of derivatives of microtubule acetylation inhibitor candidate compounds

A derivative was synthesized based on the compound 5934703 selected in Example 4, and the efficacy thereof was compared.

Specifically, the group in which the salt was removed from compound 5934703 (none), the group in which the salt was substituted with HCl (HCl), the form of compound 5934703 as it is (HBr), and 5931471 (#1) and 5933489 derived through similarity analysis The microtubule acetylation inhibitory activity of (#2) was compared through the method of Example 4.

As a result, as shown in FIG. 8 , it was confirmed that the highest microtubule acetylation inhibitory activity was shown in the compound 5933489 (#2) derived through similarity analysis.

Through this, compound 5933489 (Formula 1) was selected as a candidate compound for the study of inhibition of microtubule acetylation of triple-negative breast cancer cells, and it was named GM-90257.

[Formula 1]

.

.

The synthesis method of GM-90257 is shown in FIG. 9 .

Example 6: Structure optimization of derivatives of microtubule acetylation inhibitor candidate compounds

After the structure optimization of GM-90257 selected and named in Example 5, its efficacy was measured.

Specifically, its efficacy was confirmed in the same manner as in the experimental method of Example 4.

No. Chemical structure Effect -

+++++ One

++++ 2

- 3

- 4

+ 5

+ 6

- 7

- 8

- 9

- 10

++ 11

+ 12

+

As a result, as can be seen in Table 3, it was confirmed that the microtubule acetylation inhibitory effect of the compound of GM-90257 represented by Formula 1 was superior to 5934703 selected in Example 4 through the structure optimization experiment.

Through this, the excellent microtubule acetylation inhibitory efficacy of the compound GM-90257 represented by Formula 1 was confirmed.

Example 7. Synthesis of a derivative of compound GM-90257 and analysis of its efficacy in inhibiting microtubule acetylation

Derivatives were synthesized based on the compound GM-90257 selected in Example 6. Specifically, GM-90631 (Formula 3) containing GM-90568 (Formula 2) and its salt (HCl), a derivative of GM-90257, was synthesized:

[Formula 2]

[Formula 3]

.

.

The synthesis method of the derivative is shown in FIG. 10 .

Accordingly, the inhibition of microtubule acetylation of compounds GM-90257 and GM-90568 was compared.

As a result, as shown in FIG. 11 , it was confirmed that the microtubule acetylation inhibitory activity of the derivative GM-90568 synthesized based thereon was superior to the microtubule acetylation inhibitory activity of the compound GM-90257.

Through this, the excellent effect of inhibiting microtubule acetylation of compound GM-90568 was confirmed.

Example 8: Analysis of the efficacy of inhibiting microtubule acetylation in triple-negative breast cancer cell lines

The microtubule acetylation inhibition efficacy of GM-90257, GM-90568, and GM-90631 in triple-negative breast cancer confirmed above was confirmed through the triple-negative breast cancer cell line MDA-MB-231.

Specifically, after separation and purification of microtubules from the MDA-MB-231 cell line in which αTAT1 was knocked out, acetylation was performed by treating the compound, and the degree of microtubule acetylation was measured by the method of Experimental Example 9. Confirmed.

As a result, as shown in FIG. 12 , it was confirmed that all of the above GM-90257, GM-90568, and GM-90631 had the effect of inhibiting microtubule acetylation in the triple-negative breast cancer cell line MDA-MB-231, and also GM It was confirmed that the inhibitory ability was more excellent in GM-90568 and GM-90631 compared to -90257 ( FIGS. 12A to 12B ).

Through this, it was confirmed that the compound of the present invention exhibits excellent microtubule acetylation inhibitory efficacy even in triple-negative breast cancer cells, and in GM-90568 and GM-90631 including structure optimization and salt (HCl), structure optimization Compared to the previous compound GM-90257, it was confirmed that there was a better inhibitory effect on microtubule acetylation.

In addition, it was confirmed whether the expression of microtubule acetylation was restored by removing it again after treatment with the compound.

As a result, as can be seen in FIG. 13 , it was confirmed that the expression of microtubule acetylation was resumed when the compound treatment was removed. In addition,

Through this, it was confirmed that the expression of microtubule acetylation in triple-negative breast cancer cells was directly inhibited by treatment with the compound of the present invention.

Example 9: Analysis of triple-negative breast cancer cell line growth inhibitory efficacy

In Example 8, GM-90257, GM-90568, and GM-90631 confirmed the efficacy of inhibiting microtubule acetylation in the triple-negative breast cancer cell line MDA-MB-231, and whether the compounds actually inhibit the growth of triple-negative breast cancer. To confirm, its efficacy was confirmed through MDA-MB-231.

Specifically, as in the method of Experimental Example 6, after culturing MDA-MB-231 cells in a 3D environment using soft agar, the change in the degree of colony formation by the compound was compared.

As a result, as shown in FIG. 14 , it was confirmed that colony formation of the triple-negative breast cancer cell line MDA-MB-231 was decreased in a concentration-dependent manner by the compound.

Through this, it was confirmed that the compound of the present invention has the effect of inhibiting the growth of triple-negative breast cancer cells.

In addition, the normal cell line (MCF-10A), the receptor-present breast cancer cell line (MCF-7), and the triple-negative breast cancer cell line (MDA-MB-231) were cultured in a 2D environment, The efficacy of inhibiting breast cancer cells was confirmed.

As a result, as shown in FIG. 15 , it was confirmed that the cancer cell growth rate was significantly reduced in MDA-MB-231 compared to MCF-10A and MCF-7 in which normal cell lines and receptors were present.

Through this, it was confirmed that the compound of the present invention has excellent inhibitory efficacy targeting triple-negative breast cancer.

In addition, after the compound was treated with a normal cell line (MCF-10A), a receptor-present breast cancer cell line (MCF-7), and a triple negative breast cancer cell line (MDA-MB-231), through the method of Experimental Example 10, Annexin V/PI staining was performed to confirm the apoptosis pattern.

As a result, as shown in FIG. 16 , it was confirmed that the apoptosis pattern was significantly increased in MDA-MB-231 compared to MCF-10A and MCF-7 in which normal cell lines and receptors were present, and the compound in MDA-MB-231 It was confirmed that the increased apoptosis pattern decreased again upon removal of the .

Through this, it was confirmed that the compound of the present invention effectively induced apoptosis in triple-negative breast cancer, and when the compound was removed, apoptosis was reduced again. confirmed that.

Example 10: triple-negative breast cancer inhibition efficacy analysis through xenocraft

In Example 9, GM-90257, GM-90568, and GM-90631 confirmed the growth inhibitory efficacy of triple-negative breast cancer cell lines, and their efficacy was confirmed through xenotransplantation.

Specifically, through the method of Experimental Example 7, the triple-negative breast cancer cell line MDA-MB-231 in NOD/SCID mice was induced to form tumors through xenograft tumor inoculation, and then GM-90257 and GM-90631 were injected intraperitoneally. Thus, the effect of inhibiting tumor formation was confirmed.

As a result, as shown in FIG. 17 , when GM-90257 was injected, it was confirmed that the size and weight of the tumor were significantly reduced compared to the control group, and there was no side effect such as weight loss.

In addition, as shown in FIG. 17, when GM-90631 was injected, it was confirmed that the size and weight of the tumor were significantly reduced in a concentration-dependent manner, and there was no side effect such as weight loss.

Through this, it was confirmed that the compound of the present invention inhibited tumor formation without side effects such as weight loss, thereby effectively inhibiting triple-negative breast cancer.

Example 11: Mechanistic analysis of compounds

In order to confirm the mechanism of the compounds of the present invention, their structures were analyzed

Specifically, this was confirmed through the method of Experimental Example 11.

As a result, as shown in FIG. 19A, it was confirmed that the compound was attached to the αTAT1 binding site of alpha-tubulin, a subunit of microtubule, GM-90257 had a binding affinity of -16.452, whereas GM-90568 had -21.217 was confirmed to be.

Through this, it was confirmed that the compound of the present invention was attached to the αTAT1 binding site and inhibited the binding of αTAT1 and microtubulin. Through the binding affinity value, it was confirmed that the inhibitory ability of GM-90568 was superior to that of GM-90257. did

In addition, after overexpressing αTAT1, co-localization between αTAT1 and microtubule was confirmed through fluorescence staining.

As a result, as can be seen in FIG. 19B, in the control group, it was confirmed that co-localization occurred well by matching the pictures of αTAT1 and microtubule, but when the compound was treated, it was confirmed that co-localization was inhibited. .

Through this, it was confirmed that the compound of the present invention inhibited the binding of αTAT1 and microtubule.

In addition, through the method of Experimental Example 12, taxol, which controls the conventional microtubule polymerization, and nocodazole, and the compounds GM-90257 and GM-90568 of the present invention were treated, and the change in microtubule polymerization was confirmed through the OD value. .

As a result, as shown in FIG. 19C , it was confirmed that the compound was not involved in the polymerization of microtubules itself, unlike taxol and nocodazole.

Through this, it was confirmed that the compound of the present invention has the ability to inhibit microtubule acetylation by adhering to the αTAT1 binding site and inhibiting the binding of αTAT1 to the microtubule without being involved in the polymerization of the microtubule itself.

In other words, for the effective treatment of triple-negative breast cancer, which is dangerous because of the absence of ER, PR, and HER2 receptors, as well as difficult to target treatment, the compound of the present invention has excellent microtubule acetylation inhibitory efficacy, which has a strong metastatic history. Or, the excellent growth inhibition ability targeting triple-negative breast cancer rather than receptor-existing breast cancer was confirmed through 2D and 3D cell culture and xenograft tumor experiments. It was confirmed that it can be effectively used to suppress, prevent, or treat triple-negative breast cancer.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (12)

A pharmaceutical composition for preventing or treating triple-negative breast cancer comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient:
[Formula 1]

[Formula 2]

.
The pharmaceutical composition for preventing or treating triple-negative breast cancer according to claim 1, wherein the pharmaceutically acceptable salt of the compound represented by Formula 2 is represented by Formula 3 below:
[Formula 3]

.
The pharmaceutical composition according to claim 1 or 2, wherein the triple-negative breast cancer prevention or treatment is achieved through inhibition of microtubule acetylation.
[Claim 4] The microtubule binding site of alpha-tubulin acetyltransferase 1 (αTAT1) according to claim 3, wherein at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 3 inhibits microtubule acetylation. By binding to the microtubule binding inhibition is achieved, the pharmaceutical composition.
The pharmaceutical composition according to claim 1 or 2, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
delete A health functional food composition for preventing or improving triple-negative breast cancer comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient:
[Formula 1]

[Formula 2]

.
The health functional food composition according to claim 7, wherein the composition comprising a pharmaceutically acceptable salt of the compound represented by Formula 2 as an active ingredient is represented by Formula 3 below:
[Formula 3]

.
A method for preventing or treating triple-negative breast cancer, comprising administering to a subject other than a human, a pharmaceutical composition comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2, or a pharmaceutically acceptable salt thereof, as an active ingredient:
[Formula 1]

[Formula 2]

.
[Claim 10] The method according to claim 9, wherein the method for preventing or treating triple-negative breast cancer comprising the pharmaceutically acceptable salt of the compound represented by Formula 2 as an active ingredient is represented by Formula 3 below:
[Formula 3]

.
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