KR102261371B1 - Anticancer peptide for targeting CP2c - Google Patents

Abstract

The present invention relates to a CP2c targeting peptide-based anticancer agent.

Description

CP2c targeting peptide-based anticancer drug {Anticancer peptide for targeting CP2c}

The present invention relates to a CP2c targeting peptide-based anticancer agent.

To date, many protein/peptidic anticancer drugs and low-molecular compound-based anticancer drugs have been developed and used, but they do not selectively act on cancer cells in vivo, but cause serious side effects that affect normal cells as well. The effect is insignificant. In addition, many protein/peptidic anticancer drugs under development have a short half-life in vivo, and various studies are underway to overcome this. As such, although efforts are being made to develop anticancer drugs worldwide, anticancer drugs that are free from problems with in vivo stability, safety and drug resistance have not yet been developed. Therefore, if it is possible to secure long-term stability in vivo, to selectively remove only various types of cancer cells, and to develop an anticancer drug that also acts on drug-resistant cancer cells, it will bring high added value worldwide.

Grant et al., Antiproliferative small-molecule inhibitors of transcription factor LSF reveal oncogene addiction to LSF in hepatocellular carcinoma, Proc. Natl. Acad. Sci. 2012; 109(12): 4503-4508 Kang et al., Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2, FEBS Journal 2005; 272:1265-1277

An object of the present invention is to provide a CP2c target peptide-based anticancer agent that can ensure long-term stability in vivo, can selectively remove only various types of cancer cells, and also act on drug-resistant cancer cells.

The CP2c target peptide according to the present invention refers to a peptide that binds to the transcription factor CP2c and inhibits the formation of a transcription factor complex including CP2c (CP2c homotetramer and CP2c/CP2b/PIAS1 heterohexamer), thereby inducing cancer cell-specific cell death. The CP2c target peptide corresponds to a peptide of the smallest size in which Tyr-Pro-Gln-Arg (SEQ ID NO: 1) consists only of amino acids essential for an anticancer effect. Therefore, a peptide that essentially contains the above four amino acids and exhibits an anticancer effect by interacting with the CP2c protein can be used as the CP2c target peptide according to the present invention.

The CP2c target peptide according to the present invention may be a peptide consisting of 4 to 20 amino acids including the amino acid sequence of SEQ ID NO: 1. Preferably, it may be a peptide (ACP52) comprising 6 amino acids (6 aa) 'NYPQRP (Asn-Try-Pro-Gln-Arg-Pro, SEQ ID NO: 2)'.

The CP2c target peptide according to the present invention may be used by binding a cell-penetrating peptide (CPP) to enhance cell-penetrating activity.

For example, the CP2c target peptide according to the present invention is a peptide (ACP52) consisting of 'NYPQRP (Asn-Try-Pro-Gln-Arg-Pro) and a cell-penetrating peptide (CPP: cell-penetrating peptide) of 9 amino acids ( 9aa) 'CRGDKGPDC (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, SEQ ID NO: 3)' may be a peptide including iRGD (internalizing RGD). 'ACP52C', which is an example of the CP2c target peptide according to the present invention, has Ac (acetyl group, Acetyl group) at the N-terminus of ACP52 and iRGD at the C-terminus Pro, and NH ₂ (amide at the C-terminus of iRGD) group, amide group) to which the peptide is bound (see FIG. 7 ).

In addition, in the present invention, a CP2c target peptide-fatty acid conjugate coupled with a fatty acid was prepared for the purpose of securing the in vivo stability of the previously developed CP2c target peptide. Binding of fatty acids to the CP2c target peptide increases in vivo stability by binding to albumin in the blood, thereby providing a high therapeutic effect.

Although not limited thereto, the fatty acid may be a C ₁₂ to C ₂₀ fatty acid. In one embodiment of the present invention, the fatty acid may be a C ₁₆ fatty acid, for example, palmitoyl acid (also expressed as 'pal').

The CP2c targeting peptide-fatty acid conjugate according to the present invention may include a CP2c targeting peptide providing CP2c targeting (eg ACP52) and CPP (eg iRGD) and/or a linker peptide linking them between fatty acids. As the linker peptide, a peptide comprising amino acids or a combination thereof known in the art may be used without limitation. Specifically, the linker peptide may include glycine (Gly, G), such as Gn (wherein n is an integer of 1 to 6). In another embodiment of the present invention, the linker peptide may _{consist of an amino acid sequence represented by G n} KG _m (wherein n and m are each independently an integer of 0 to 6). For example, when n or m is 0, lysine (Lys, K) may be located at the N-terminus or C-terminus of the linker peptide, and when n and m are not 0, between glycine can be located Lysine (Lys, K) included in the linker peptide is included for conjugation of the fatty acid with the peptide. Specifically, the terminal functional group of lysine (-HN ₂ ) may enable the binding of additional amino acids. Accordingly, the linker peptide of the present invention may further include an amino acid sequence represented by glutamic acid (Glu, E) or EGLFG, which is the target sequence of the proteolytic enzyme Catepsin B, that binds to the functional group of lysine.

In another embodiment of the present invention, the amino acid sequence represented by the glutamic acid (Glu, E) or EGLFG, which is the target sequence of the proteolytic enzyme Catepsin B, is a part of the linker peptide binding to the fatty acid, and the CP2c target peptide according to the present invention- In the fatty acid conjugate, the lysine of the linker peptide represented by _{the G n} KG _{m is linked with a peptide bond between a functional group of lysine (NH 2} ) and an alpha or gamma carboxyl group of glutamic acid, and a carboxyl group of a fatty acid and a peptide can be linked by a peptide bond between the amino groups of

When synthesizing the CP2c target peptide-fatty acid conjugate according to the present invention, the amino acid sequence represented by the glutamic acid (Glu, E) or EGLFG, which is the target sequence of the proteolytic enzyme Catepsin B, is first bound to the linker peptide represented by _{G n} KG _m Then, it may be linked to a fatty acid, but is not limited to the above synthesis sequence.

For example, when the amino acid sequence represented by the glutamic acid (Glu, E) or EGLFG, which is the target sequence of the proteolytic enzyme Catepsin B, is first combined with a fatty acid, palmitoyl acid combined with glutamic acid in the present specification with respect to such a fatty acid is As “E-pal”, palmitoyl acid combined with an amino acid sequence represented by EGLFG may be referred to as “EGLFG-pal”.

On the other hand, the CP2c target peptide-fatty acid conjugate used in the present invention has improved stability of the peptide, enhanced pharmacological properties (half-life, absorption, potency, potency, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced To obtain antigenicity, the N- and/or C-terminus of the peptide may be modified. Preferably, the formula is an acetyl group, a fluorenyl methoxy carbonyl group, an amide group, a formyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG) is bonded to the N- and/or C-terminus of the peptide. It may be in the form, but if it is a component capable of improving the modification of the peptide, particularly the stability of the peptide, it may be included without limitation. As used herein, the term "stability" refers to storage stability (eg, room temperature storage stability) as well as in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo. In the CP2c target peptide-fatty acid conjugate according to the present invention, the N-terminus and C-terminus of the peptide may be modified with an acetyl group and an amide group, respectively.

The structure of the CP2c target peptide-fatty acid conjugate according to an embodiment of the present invention will be described as follows:

1) Ac-K(E-pal)-GG- was attached to the N-terminus of ACP52C without the N-terminal Ac (C16-ACP52Cn, ACP52CG);

2) Ac-K(EGLFG-pal)-GG- was attached to the N-terminus of ACP52C without the N-terminal Ac (A16-GFLG-ACP52Cn, ACP52CK);

3) Among the GG-K-GG linking sites located between ACP52 and iRGD, the functional group of K was attached to the carboxyl group of glutamic acid alpha carbon of E-Pal (C-16-ACP52Cm, ACP52GK)

4) Among the GG-K-GG linking sites located between ACP52 and iRGD, the functional group of K was attached to the gamma carbon carboxyl group of glutamic acid of E-Pal (γC16-ACP52Cm, ACP52CGK).

For example, the structures of ACP52GK and ACP52CGK in the CP2c target peptide-fatty acid conjugate according to the present invention are specifically as follows:

In the drawings, the dotted line only indicates the bonding structure, and does not indicate the bonding order.

In an example of the present invention, in vivo stability and anticancer effect in liver cancer cell xenograft (Hep3B) and breast cancer cell xenograft (MDA-MB-231) mouse models using four synthesized CP2c target peptide-fatty acid conjugates. , tumor metastasis inhibitory effect and physiological toxicity were analyzed, and as a result, it was confirmed that in vivo stability, tumor suppression and tumor metastasis inhibitory effect were excellent, and it was confirmed that it was a safe substance without toxicity to the living body.

In addition, in one embodiment of the present invention, in the process of confirming the anticancer effect by treating the synthesized four types of CP2c target peptide-fatty acid conjugates, cells with resistance to the CP2c target peptide-fatty acid conjugate are generated. Confirmed. Accordingly, it was confirmed that the mechanism and cause of the resistance was due to the decrease of MDM2p90 in the cells treated with the CP2c target peptide-fatty acid conjugate. Accordingly, as a result of treatment with a CP2c target peptide-fatty acid conjugate with a caspase2 inhibitor capable of maintaining or increasing the number of MDM2p90 by inhibiting the degradation of MDM2p90 in resistant cells, it was confirmed that the tumor suppressive effect was maintained even in resistant cancer cells.

Accordingly, the present invention also provides a pharmaceutical composition for preventing, improving and treating cancer and a health functional food composition comprising the CP2c target peptide-fatty acid conjugate according to the present invention as an active ingredient.

In the present invention, the cancer may include a resistant cancer that exhibits resistance to an anticancer agent, in particular, a CP2c target peptide-fatty acid conjugate-based anticancer agent according to the present invention.

In the present invention, the term “treatment” refers to any action in which the symptoms of cancer are improved or beneficially changed by administration of the peptide according to the present invention or a pharmaceutical composition comprising the same.

In the present invention, the term "administration" refers to introducing a predetermined substance, ie, a peptide derivative according to the present invention or a pharmaceutical composition comprising the same, to a subject by any suitable method, and the route of administration thereof is such that the drug reaches the target tissue. As long as possible, it may be administered through any general route. For example, the route of administration may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc., but is not limited thereto. . However, since peptides are digestible upon oral administration, oral compositions are preferably formulated to coat the active agent or to protect it from degradation in the stomach. Preferably, it may be administered in the form of an injection. In addition, the pharmaceutical composition according to the present invention can be administered by any device capable of transporting the active substance to a target cell.

In the present invention, the term "contained as an active ingredient" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type, severity, and drug of the patient. Activity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The peptide according to the present invention or a pharmaceutical composition comprising the same may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, or may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. The dosage and frequency of administration of the pharmaceutical composition of the present invention is determined according to the type of drug as the active ingredient, together with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

Accordingly, the pharmaceutical composition according to the present invention may include various pharmaceutically acceptable carriers as long as the peptide according to the present invention is contained as an active ingredient. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc., for oral administration, and in the case of injections, buffers and preservatives , an analgesic agent, a solubilizer, an isotonic agent, a stabilizer, etc. can be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. can be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it may be prepared in the form of unit dose ampoules or multiple doses. . Other solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc. may be formulated.

Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, it may further include a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, a preservative, and the like.

The peptide according to the present invention can also be contained as an active ingredient in a health functional food composition, and the food composition according to the present invention can be prepared in the form of a composition by mixing with a known active ingredient known to have an anticancer function. It may further include a scientifically acceptable food supplement additive. The health functional food composition of the present invention includes a composition for all types of food, such as functional food, nutritional supplement, health food, and food additives.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, health food may be prepared in various forms such as tablets, pills, powders, capsules, gums, vitamin complexes, juices and drinks, and granulating food compositions containing the peptide according to the present invention as an active ingredient, It can be ingested by encapsulation or powder. The food composition of the present invention may include ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared as a drink, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, licorice extract, etc. may be additionally included in addition to the peptide of the present invention. In addition, as a food supplement additive, food additives conventional in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like may be included. In addition, as in conventional beverages, various flavoring agents or natural carbohydrates may be contained as additional ingredients. Specific examples of natural carbohydrates include, for example, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and common sugars such as polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol, sugar alcohols such as erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be included. have.

Furthermore, the food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components may be used independently or in combination.

Hereinafter, the present invention will be described in more detail by way of examples, but the following examples are only provided to help the understanding of the present invention, and do not limit the scope of the present invention.

In the present invention, in order to identify final candidates that improved the in vivo stability of the transcription factor CP2c target peptide ACP52C, which showed an effect as a general-purpose anticancer agent, C ₁₆ fatty acids were linked to the CP2c target peptide lead material ACP52C to demonstrate in vivo stability and anticancer effect. It was confirmed in the cancer cell xenograft mouse model. In addition, the cancer cell-specific anticancer effect of the final anticancer candidate was confirmed in various cancer cells and normal cells. The anticancer effect of these final candidates was shown to be caspase2-dependent, and the therapeutic effect on cancer cells with anticancer drug resistance was confirmed by the combined treatment of the caspase2 inhibitor and the final candidates.

1 is a graph showing _{GI 50} values according to ACP52c treatment in various cancer cell lines.
2 shows the analysis results of G2/M cell cycle arrest and cell death induction (subG1 cells) according to ACP52c treatment using FACS.
3 is a diagram showing that ACP52C treatment causes an increase in the expression of p53, which regulates cell cycle regulation and apoptosis.
4 is a diagram showing that apoptosis is induced by ACP52C treatment.
5 shows the results of analysis of the migration pathway, intracellular location, and intracellular residence time of ACP52C over time in the MDA-MB-231 cell line.
6 shows the in vivo half-life analysis results for the ACP52C peptide.
7 shows the construction of a C16 fatty acid binding peptide for improving in vivo stability.
8 shows the results of cell growth analysis according to C ₁₆ fatty acid binding peptide (ACP52C; ACP52CG; ACP52CK) treatment in various cancer cell lines.
9a and 9b show the results of cell growth analysis according to treatment with _{C 16} fatty acid binding peptide (ACP52C; ACP52CG; ACP52CK) in various p53 mutant or null cancer cell lines.
10A to 10B show cells treated with ACP52CGK in normal cell lines (BEAS2B, hMSC) and cancer cell lines (Hep3B, Hs746T, Caov-3, MDA-MB-231, U343, HCT116, PANC1, PC3, A549, THP-1). Show the results of the growth analysis.
11a and 11b show the results of analysis of the anticancer effect of ACP52CG and ACP52CK in the Hep3B cell line xenograft mouse model.
12a and 12b show the results of analysis of the anticancer effect of ACP52CK in a mouse model of liver cancer induced by DEN treatment.
13a and 13b show the results of analysis of the anticancer effect of ACP52CG and ACP52CK in a mouse model of liver cancer induced by DEN treatment.
14a and 14b show the results of analysis of the anticancer effect of ACP52CG and ACP52CK in the MDA-MB-231 (LM1) cell line xenograft mouse model.
15A to 15D show the results of analysis of the anticancer effect (tumor size, weight, body weight and hematological analysis) and metastasis inhibition effect when ACP52CGK was treated with three different doses at 3-day intervals in the Hep3B cell line xenograft mouse model.
16A to 16D show the results of analysis of the anticancer effect (tumor size, weight, body weight and hematological analysis) and metastasis inhibition effect when ACP52CGK was treated with three different doses at 5-day intervals in the Hep3B cell line xenograft mouse model.
17A to 17D show the anticancer effect (tumor size, weight, body weight and hematological analysis) and metastasis when ACP52CGK was treated with three different doses at 3-day intervals in the MDA-MB-231 (LM1) cell line xenograft mouse model. Shows the results of the inhibitory effect analysis.
18A to 18D show the anticancer effect (tumor size, weight, body weight and hematological analysis) and metastasis when ACP52CGK was treated with three different doses at 5-day intervals in the MDA-MB-231 (LM1) cell line xenograft mouse model. Shows the results of the inhibitory effect analysis.
19 shows the results of in vivo half-life analysis using the fluorescent label ACP52CGK.
20 shows the results of analysis of the migration pathway, intracellular location and intracellular residence time of ACP52CGK over time in the MDA-MB-231 cell line.
Figure 21 shows the results of analysis of whether antibodies to ACP52C, ACP52CG, ACP52CK and ACP52CGK through ELISA.
22 shows the results of an in vivo emergency and repeated toxicity test for ACP52C.
23 shows the results of a repeated toxicity test for ACP52CGK.
24 shows the results of histological analysis of major organs during repeated toxicity testing of ACP52CGK.
25 shows the results of analysis of the potency of ACP52CGK in cells cultured from tumor tissue of a breast cancer patient.
26 shows the results of analysis of the potency of ACP52CGK in cells cultured from cryopreserved breast cancer patient cancer tissues.
27 shows the results of comparative analysis of the potency of ACP52CGK in cells cultured from PDX tumor tissues by generation.
28 shows the results of analysis of potency and resistance to ACP52CGK in cells derived from patient tumor tissue.
29 shows the expression analysis results of CP2c transcription-activity-independent pathway proteins in lung cancer cell lines showing resistance to ACP52C.
30 shows the expression analysis results of CP2c transcription-activity-independent pathway proteins according to ACP52C treatment in lung cancer cell lines (A549, PC9, KCL22).
Figure 31 shows the expression analysis results of CP2c transcription-activity-independent pathway proteins according to MDM2 overexpression in lung cancer cell lines (A549, PC9).
Figures 32a and 32b show the results of cell growth analysis according to ACP52C treatment in the A549 cell line induced long-term MDM2 overexpression.
33 shows the results of MDM2p60 expression model and lung cancer cell line-specific alternative splicing and SNP analysis.
34 shows the results of analysis of potency according to the Caspase2 inhibitor and ACP52C complex treatment in ACP52C-resistant cells.
35 shows cell morphological changes according to the Caspase2 inhibitor and ACP52C complex treatment in ACP52C-resistant cells.

Hereinafter, the present invention will be described in more detail by way of Examples, but the following Examples are only provided to aid the understanding of the present invention, and do not limit the scope of the present invention.

[Preparation Example 1] Preparation of CP2c target peptide bound to cell penetrating peptide

The transcription factor CP2c is known to be overexpressed in various cancers. According to a study by a US research team, inhibition of CP2c expression in liver cancer cell lines inhibits growth, and overexpression of CP2c has been reported to cause cancer malignancy and metastasis. [Grant et al., Antiproliferative small-molecule inhibitors of transcription factor LSF reveal oncogene addiction to LSF in hepatocellular carcinoma, Proc . Natl . Acad. Sci. 2012; 109(12): 4503-4508].

The present inventors identified peptides binding to the transcription factor CP2c (also known as Tfcp2, LSF, LBP1, UBP1, etc.) by phage display method [Kang et al., Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2, FEBS Journal 2005; 272:1265-1277], one peptide (CP2c target peptide, SEQ ID NO: 2) that interferes with the DNA binding of CP2c was selected, and in order to improve cell permeability, nine peptides as cell-penetrating peptides (CPPs) were selected. Amino acid (9aa) 'CRGDKGPDC (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, SEQ ID NO: 3)', iRGD (internalizing RGD), a peptide in the form of binding to the CP2c target peptide ACP52C A lead material was synthesized (FIG. 7).

[Example 1] Confirmation of anti-cancer effect of ACP52C

The selected peptide binds to CP2c and inhibits the formation of a transcription factor complex including CP2c (CP2c homotetramer and CP2c/CP2b/PIAS1 heterohexamer), thereby indirectly interfering with DNA binding of CP2c. As a result of analyzing the anticancer effect in various carcinomas targeting the synthesized ACP52C, it was confirmed that it exhibits cancer cell-specific growth inhibition and cell death efficacy (FIG. 1).

The growth inhibition and cell death efficacy of ACP52C was confirmed by FACS analysis of the cell cycle change by treating ACP52C after synchronizing the cell cycle in the G1/S phase through the double thymidine block method. As a result, it stops in the G2/M phase and forms a polyploid. Confirmed. On the other hand, it was confirmed that when ACP52C was treated in the cell line synchronized to the G2/M phase according to the nocodazole treatment, cell death was induced by stopping the subG1 phase (FIG. 2).

ACP52C-induced G2/M phase arrest was indicated by increased expression of CHK1/2 protein and decreased expression of Cdc25c, CDK1 and cyclin B proteins, whereas induction of apoptosis resulted in decreased expression of anti-apoptotic marker protein and expression of pro-apoptotic protein. It was confirmed by apoptosis leading to increase and activation of caspase ( FIGS. 3 and 4 ).

The migration pathway, distribution, and stability of ACP52C entering the cell were analyzed by confocal microscopy after treating the Cy5-labeled peptide (Cy5-ACP52C) in the cell line culture medium for 30 minutes and then analyzing the migration pathway of the peptide over time. The peptide passed through the cytoplasm and was mostly located in the nucleus from 4 hours, and came out from the cytoplasm after 8 hours and was degraded at 16 hours. The peptide was located in the nucleus from 1 hour after treatment and CP2c and CP2c tended to be distributed in the cytoplasm after 8 hours ( FIG. 5 ).

ACP52C half life in serum was analyzed by HPLC (Model: UHPLC DIONEX Ultimate 3000; Flow = 1.000ml/min). After incubation with serum for various times, serum proteins were removed with Centricon (Mw10,000) before HPLC analysis. As a result of the experiment, ACP52C was not degraded at all until 24 hours. Meanwhile, after ACP52C was injected into the tail vein, blood was extracted over time and the degree of degradation of ACP52C was analyzed by HPLC. As a result, EC50 was about 2 hours. Additionally, after injecting the Cy5-labeled peptide (Cy5-ACP52C) into the tail vein of the mouse, the fluorescence intensity remaining in the mouse body was measured for each hour, and as a result, it showed a half-life of about 7.95 hours. However, even after 5 days after injection, ACP52C was observed in cancer tissues (FIG. 6).

[Preparation Example 2] Synthesis of CP2c target peptide-fatty acid conjugate

Combining the results of Examples confirming the anticancer effect on ACP52C, it was determined that the stability of ACP52C was low, and to overcome this, four kinds of albumin-affinity C ₁₆ fatty acid (palmitoyl acid, palmitoyl acid) binding peptides were synthesized ( Fig. 7). First, each peptide modified at the N-terminus and C-terminus was synthesized, and finally, four types of fatty acid-binding peptides were synthesized by binding _{C 16 fatty acids.}

[Example 2] Confirmation of in vitro anticancer effect of CP2c target peptide-fatty acid conjugate

The specific cell killing effect in various cancer cells was analyzed for the CP2c target peptide-fatty acid conjugate synthesized in Preparation Example 2 above. As a result, the three C ₁₆ fatty acid binding peptides ACP52CG, ACP52CK and ACP52CGK all induced cancer cell-specific cell death similar to the control group ACP52C, and the GI ₅₀ value confirmed at 48 hours after treatment was similar or more efficient (Fig. 8). , Fig. 10). In addition, even cancer cell lines with various p53 mutations were treated with ACP52CG and ACP52CK to obtain a cell survival curve through MTT analysis _{and measure the GI 50} value. As a result, the GI ₅₀ value for each cell line showed a deviation, but was less than 10 mM, It was clearly distinguished from the result of approaching 1,000 mM in normal cell lines. (Fig. 9). In conclusion, all of ACP52CG, ACP52CK and ACP52CGK had anticancer effects similar to those of ACP52C, and the fatty acids bound to this complex did not show any negative effects on cancer cells and normal cells.

[ Example 3] In a mouse model transplanted with various cancer cell lines CP2c Target peptide-fatty acid conjugate Confirmation of tumor growth inhibitory effect and general physiological toxicity according to treatment

[3-1] Gammam and in a mouse model transplanted with a breast cancer cell line. ACP52CG , ACP52CK and ACP52GK Confirmation of tumor growth inhibitory effect and general physiological toxicity

To analyze the anticancer efficacy of the CP2c target peptide-fatty acid conjugate synthesized in Preparation Example 2 in an animal model, ACP52CG and ACP52CK were injected into the tail vein 5 times at 3-day intervals in Hep3B xenograft mice. As a result, both ACP52CG and ACP52CK showed similar efficacy to sorafenib, but the efficacy was lower than that of ACP52C. However, no specific abnormalities were found in normal tissues and blood levels, and no toxicity was observed (FIG. 11).

In addition, for mice 22 weeks after DEN treatment, as a control group, only vehicle treatment (mock), sorafenib and ACP52CG-GFLGE (ACP52CK), the only approved liver cancer treatment, 3 groups (7~8) two/group), and the drug was injected through the tail vein at a concentration of 5 mg/Kg at 3-day intervals for a total of 12 times. As a result of the analysis, the sorafenib treatment group also showed a significant anticancer effect (p = 0.04), whereas the ACP52CG-GFLGE (ACP52CK) treatment group showed a similar anticancer effect (p = 0.001) to the sorafenib treatment group (Fig. 12). In addition, the results of analyzing the effects of ACP52CG and ACP52CK in mice 15 weeks after DEN treatment were also superior to the FQI used as a control, but did not show superior efficacy compared to ACP52C (FIG. 13).

In MDA-MB-231 (LM1) xenograft mice, when ACP52CG and ACP52CK were injected into the tail vein 5 times at 3-day intervals, both ACP52CG and ACP52CK showed tumor regression efficacy, but the efficacy was not significantly superior. As no specific abnormality was found in blood levels, toxicity was not observed (FIG. 14).

Hep3B xenograft mice were injected with ACP52GK 5 times at 3-day intervals into the tail vein, and then the efficacy was analyzed. As a result, the efficacy was lower than that of ACP52C, but it was confirmed that the effect was superior to that of the control group, FQI.

[3-2] Gammam and in a mouse model transplanted with a breast cancer cell line. ACP52CGK Confirmation of tumor growth inhibitory effect, metastasis inhibitory effect and general physiological toxicity

In Hep3B xenograft mice, ACP52CGK was injected into the tail vein 5 times at 3 concentrations (1.39, 2.77 or 5.54 mg/kg) at 3-day intervals to measure the tumor volume, and the mice were sacrificed on the 24th day after tumor cell injection. to extract the tumor and major tissues. The collected blood was subjected to basic CBC analysis using a Coulter LH 750 hematology analyzer. The tumor was weighed and stained with 4% formaldehyde along with the major organs. After making tissue slices through paraffin sections, hematoxylin /eosin staining was performed. The various acquired data were statisticalized using an Excel program.

As a result, ACP52CGK exhibited a tumor suppressive effect similar to that of ACP52C, and no abnormalities were found in normal tissues and blood levels ( FIGS. 15a , 15c and 15d ). In addition, according to FIG. 15b showing the average and standard deviation of the tumor area metastasized to the lung, it was confirmed that ACP52CGK had a superior effect on inhibiting tumor metastasis than ACP52C.

In Hep3B xenograft mice, ACP52CGK was injected 5 times at 3 concentrations (1.39, 2.77, 5.54 mg/kg) at intervals of 5 days to evaluate the anticancer effect in the same manner as described above. As a result, in all three concentrations of ACP52CGK It exhibited superior tumor suppression and metastasis inhibitory effects than the sorafenib and ACP52C treatment groups, and the inhibitory effect was concentration-dependent ( FIGS. 16a and 16b ). In addition, when ACP52CGK was injected at intervals of 3 days, it showed more effective tumor suppression and metastasis suppression effect when injected at intervals of 5 days (compare Figs. 15 and 16). /kg was the optimal concentration. In addition, no particular abnormality was found in blood levels, so toxicity was not observed ( FIGS. 16c and 16d ).

As a result of analyzing the anticancer effect of ACP52CGK on MDA-MB-231 (LM1) xenograft mice in the same manner as in the Hep3B xenograft mouse model experiment, in the same way, in all three concentrations (1.39, 2.77, 5.54 mg/kg) of ACP52CGK It exhibited superior tumor suppression and metastasis inhibitory effects than the phenib and ACP52C treatment groups (Fig. 17a, Fig. 17b, Figs. 18a and 18b), and showed a better inhibitory effect when injected at 5-day intervals rather than 3-day intervals, and 2.77 mg/kg was the optimal concentration. In addition, no particular abnormality was found in blood levels, so toxicity was not observed ( FIGS. 17c , 17d , 18c and 18d ).

[Example 4] Stability and safety evaluation of CP2c target peptide-fatty acid conjugate

To analyze the in vivo half-life of ACP52CGK, after injecting Cy5-labeled ACP52CGK into the tail vein of the mouse, the fluorescence intensity remaining in the mouse body was measured for each hour, and as a result, the half-life of about 20.2 hours was shown (FIG. 19). These results indicate that the in vivo stability was significantly improved compared to the lead material ACP52C, which showed a half-life of 7.95 hours by conjugation of fatty acids.

In order to analyze the migration pathway, distribution and stability of ACP52CGK entering the cell, Cy5-labeled ACP52CGK was treated in cell line culture medium for 30 minutes and then the peptide migration pathway was traced over time, and also into mitochondria (Hsp60) and lysosomes (LC3). Movement was analyzed by confocal microscopy. As a result, ACP52CGK passed through the cytoplasm and was located almost in the nucleus from 4 hours, and came out from the cytoplasm after 8 hours, and some were located in the mitochondria, but moved to the lysosome and degraded at 16 hours. The intracellular pathway, distribution and stability of ACP52CGK were similar to those of ACP52C (FIG. 20).

On the other hand, it was assumed that the ACP52C peptide would not exhibit immunogenicity as 15 amino acids. To verify this, immunogenicity was confirmed. Since the final candidate material will bind C-16 palmitoyl acid to ACP52C, it was decided to analyze whether antibodies were formed on ACP52CG and ACP52CK in the process of deriving the final new drug candidate. As a result of performing ELISA analysis on sera isolated from animals injected with ACP52CG and ACP52CK three times, both rabbit sera did not induce an immune response in ACP52CG, ACP52CK, and ACP52CGK, including ACP52C. Therefore, it was determined that the final candidate material (ACP52CGK) would not exhibit immunogenicity even in the human body (FIG. 21).

A repeated toxicity test for the in vivo administration of ACP52CGK was conducted using female and male mice. As a preliminary experiment, 100 mg/kg and 1000 mg/kg of ACP52C were administered twice as anti-venom, both survived, did not show liver toxicity, and did not affect major organs. As this experiment, after intravenous infusion of ACP52CGK into solvent group and high-dose group (100 mg/Kg) consisting of 6 females and males at 3-day intervals for 28 days, the weight change, drinking water intake, and main Histological analysis of organs, organ weight, hematological tests and blood biochemical tests were analyzed. As a result, no abnormal findings were found (Fig. 22, Fig. 23, Fig. 24, Table 1, Table 2, Table 3, Table 4).

In order to find out whether the efficacy of ACP52CGK tested on cancer cell lines and xenograft mouse models can be equally applied in actual clinical practice, the efficacy of ACP52CGK was evaluated in cells cultured from the tumor tissue of breast cancer patients in a fresh state. analyzed. As a result, it was confirmed that ACP52CGK induces cell death at GI50: ~ 2 uM, similar to that in the cancer cell line experiment (FIG. 25). In addition, the primary culture was performed by thawing the cancer tissues of breast cancer patients in cryopreservation, and as a result of analyzing the effect of ACP52CGK on the cells obtained therefrom, it was confirmed that cell death was also induced at GI50: ~ 2 uM (Fig. 26). ).

[ Example 5] CP2c Target peptide-fatty acid to the conjugate showing resistance to Causes of resistance to cells analysis

As a result of analyzing the efficacy of ACP52CGK in cells primary cultured from patient derived xenograft (PDX) tumor tissues for each generation, it was confirmed that the sensitivity to ACP52CGK was not significantly different in cells derived from the same origin (FIG. 27). However, there was a case that showed resistance to ACP52CGK in cells primary cultured from some PDX tumor tissues (FIG. 28).

Cells exhibiting resistance to these ACP52CGK (mainly lung cancer cell lines) showed a tendency to have a low MDM2p90 expression level and a relatively high MDM2p60 expression level. In fact, three types of lung cancer cell lines (A549, PC9, KCL22) were treated with ACP52C and then protein expression was analyzed by immunoblot. As a result, the expression of YY1 was decreased in all cell lines, but the expression of p53, p63 and p73 did not show any change. didn't In particular, there was no decrease in the expression of MDM2 (p90 and p60) in these cell lines. MDM2p60 is known to be a protein in which the C-terminal region of MDM2p90 including the sites phosphorylated by ATM (S386, S395, S407) is deleted, so it was determined that the degradation would not be regulated by ACP52C treatment (Fig. 29, Fig. 30). .

In accordance with this, it was confirmed that MDM2p90 was continuously overexpressed by treatment with doxycycline for 3 weeks, and the CP2c transcriptional activity-dependent and independent marker proteins were confirmed by week by week by immunoblot. Interestingly, it was confirmed that the expression of p53 protein and YY1 was decreased. did. Meanwhile, as a result of analyzing the induction of cell death according to ACP52C treatment by MTT assay, it was confirmed that cell death was induced from the second week. Therefore, it was demonstrated that the low expression of MDM2p90 in lung cancer cell lines is the cause of ACP52C resistance ( FIGS. 31 and 32 ).

Two models have been proposed that the generation of MDM2p60 is due to caspase2 cleavage of MDM2p90 or alternative splicing. First, as a result of performing RT-PCR using a primer capable of detecting an alternative splicing form of MDM2 in various cancer cell lines, a lung cancer cell-specific splice form was not found. In addition, as a result of analyzing the nucleotide sequence after PCR cloning of genomic DNA to determine whether MDM2p60 is generated according to the generation of truncated mRNA due to the presence of SNP in the MDM2 gene specifically for lung cancer cell lines, lung cancer cell-specific SNPs were found not (Fig. 33).

On the other hand, in order to identify the phenomenon that occurs due to the high activity of caspase2 specifically in lung cancer cells, the Caspase2 inhibitor (AC-VDVAD-CHO) was treated and the MDM2 protein was confirmed by immunoblot. As a result, the protein amount of MDM2p90 by the caspase2 inhibitor treatment was While increasing, it was confirmed that the protein amount of MDM2p60 decreased. Therefore, the MDM2p60 form, which is overexpressed in lung cancer cells, is the result of cleavage of MDM2p90 by caspase2. Based on these results, the cell death-inducing effect was analyzed by complex treatment of caspase 2 inhibitor and ACP52C in cells (A549, PDX cell; Breast F0-JOS) that showed resistance to ACP52C. It was confirmed that the effect was shown to be around GI50 ~ 1 uM in the complex-treated group (Fig. 34, Fig. 35).

Therefore, the ACP52CGK peptide improved in vivo stability compared to the existing ACP52C peptide and showed growth inhibition/cell death in all carcinoma cells as in ACP52C, but had no significant effect on normal cells. In addition, it was confirmed that cancer cells exhibiting drug resistance to ACP52C exhibited anticancer effects on all carcinomas when combined with a caspase 2 inhibitor.

<110> Industry-University Cooperation Foundation Hanyang University <120> Anticancer peptide for targeting CP2c <130> P19U10C0757 <150> KR 10-2019-0038023 <151> 2019-04-01 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide for targeting CP2c <400> 1 Tyr Pro Gln Arg One <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptide for targeting CP2c <400> 2 Asn Tyr Pro Gln Arg Pro 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> cell-penetrating peptide <400> 3 Cys Arg Gly Asp Lys Gly Pro Asp Cys 1 5

Claims (12)

Transcription factor CP2c target peptide consisting of the amino acid sequence shown in SEQ ID NO: 1;
a linker peptide bound to the C-terminus of the CP2c target peptide and _{consisting of an amino acid sequence represented by G n} KG _m , wherein n and m are each independently an integer of 1 to 6;
a cell membrane penetrating peptide (CPP) bound to the C-terminus of the linker peptide; and
Including; palmitoyl acid conjugated with the linker peptide;
In the conjugate of the linker peptide and palmitoyl acid, the gamma carbon or alpha carbon carboxyl group of glutamic acid (Glu, E) is bonded to the functional group of lysine (Lys, K) of the linker peptide, and the carboxyl group of the palmitoyl acid is the carboxyl group of the glutamic acid The CP2c target peptide-fatty acid conjugate, which is bound to an amino group. Transcription factor CP2c target peptide consisting of the amino acid sequence shown in SEQ ID NO: 2;
a linker peptide bound to the C-terminus of the CP2c target peptide and _{consisting of an amino acid sequence represented by G n} KG _m , wherein n and m are each independently an integer of 1 to 6;
a cell membrane penetrating peptide (CPP) bound to the C-terminus of the linker peptide bound to the C-terminus of the CP2c target peptide; and
Including; palmitoyl acid conjugated with the linker peptide;
In the conjugate of the linker peptide and palmitoyl acid, the gamma carbon or alpha carbon carboxyl group of glutamic acid (Glu, E) is bonded to the functional group of lysine (Lys, K) of the linker peptide, and the carboxyl group of the palmitoyl acid is the carboxyl group of the glutamic acid The CP2c target peptide-fatty acid conjugate, which is bound to an amino group. 3. The method of claim 1 or 2,
The peptide N-terminus and/or C-terminus of the CP2c target peptide-fatty acid conjugate is modified to increase stability, CP2c target peptide-fatty acid conjugate. 4. The method of claim 3,
The CP2c target peptide-fatty acid conjugate, wherein the peptide N-terminus of the CP2c target peptide-fatty acid conjugate is modified with an acetyl group and the C-terminus of the peptide is modified with an amide group. delete delete delete delete A pharmaceutical composition for preventing or treating cancer comprising the CP2c target peptide-fatty acid conjugate of claim 1 or 2 as an active ingredient. A health functional food composition for preventing or improving cancer comprising the CP2c target peptide-fatty acid conjugate of claim 1 or 2 as an active ingredient. A pharmaceutical composition for preventing or treating cancer, comprising the CP2c target peptide-fatty acid conjugate of claim 1 or 2 and a Caspase 　 2 　 inhibitor as active ingredients. A pharmaceutical composition for the prevention or treatment of cancer resistant to the CP2c target peptide-fatty acid conjugate of claim 1 or 2, comprising the CP2c target peptide-fatty acid conjugate of claim 1 or 2 and a Caspase 2　 inhibitor as active ingredients .

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