KR20180130364A - An anti-cancer peptide inhibiting Bcl-XL and a method for screening an anti-cancer agent - Google Patents

Abstract

The present invention relates to a peptide for preventing or treating cancer which can induce apoptosis in a transcription-independent manner, a pharmaceutical composition comprising the peptide, and a screening method using the same. The peptide of the present invention can induce apoptosis by forming a complex with Bcl-X_L, and thus can be used for treating cancer, wherein the structure of the complex can be used to screen anticancer agents.

Description

An anticancer peptide which inhibits Bcl-XL and an anti-cancer peptide inhibiting Bcl-XL and a method for screening an anti-cancer agent,

The present invention relates to a peptide for preventing or treating cancer, and a pharmaceutical composition comprising the peptide. Also disclosed is a method for inducing apoptosis and release of cytochrome c from mitochondria comprising the step of treating the peptide. Furthermore, the present invention relates to a screening method using the structure of the complex comprising the peptide.

p73 is one of the p53 family of transcription factors and is known to play an important role in tumor suppression. p73 induces apoptosis and cell cycle arrest through transactivation of the gene responsive to p53.

p73-mediated apoptosis is based on the nuclear transactivation of the nucleus of a cell death-inducing target gene (e.g., PUMA, Bax, death receptor CD95). However, p73 may also be located in the cytoplasm or mitochondria during apoptosis, suggesting that cytoplasmic p73 may have a transcription-independent apoptosis inducing activity. The tumor suppressor, Wwox, induced the redistribution of p73 from the nucleus to the cytoplasm and could induce apoptosis through mutual action. Thus, p73 has been a major research tool for anticancer activity.

For example, Korean Patent No. 10-1655649 discloses the use of a compound that targets the p53 gene in cancer prevention and treatment. US Patent Publication No. 2016-0008330 discloses a method of treating cancer by regulating the level of p73 .

Under these circumstances, the inventors of the present invention have made extensive efforts to identify peptides that induce apoptosis and to identify molecular structures capable of screening anticancer agents. As a result, p73 induces apoptosis in a transcription-independent manner through TAD, , Confirming the fact that the 16-mer p73-TAD peptide (p73-TAD16) mediates transcription-independent cell death through interaction with mitochondrial anti-apoptotic Bcl-X _L , / Bcl-X _L complex. Thus, it is deduced that the core region of p73 having the preventive or therapeutic effect of cancer can be excavated, and the anticancer agent can be screened from the structure of p73-TAD16 / Bcl-X _L complex, thereby completing the present invention.

It is an object of the present invention to provide a peptide for preventing or treating cancer.

It is another object of the present invention to provide a method for inducing apoptosis in a transcription-independent manner comprising the step of treating the peptide.

It is another object of the present invention to provide a method for inducing the release of cytochrome c from mitochondria comprising the step of treating said peptide.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer comprising the peptide for preventing or treating cancer.

It is yet another object of the present invention to provide a method for screening a substance that regulates the interaction between Bcl-X _L and p73-TAD16 using the structure of p73-TAD16 peptide and Bcl-X _L protein complex.

It is another object of the present invention to provide a method of screening anticancer agents using the structure of p73-TAD16 peptide and Bcl-X _L protein complex.

To achieve the above object, one aspect of the present invention provides a peptide for preventing or treating cancer comprising 13 to 70 consecutive amino acids including the amino acid sequence of SEQ ID NO: 2 in the amino acid sequence of SEQ ID NO: 1 .

Specifically, the peptide may be a peptide for preventing or treating cancer, which is composed of 16 to 67 amino acids, but is not limited thereto.

In the present invention, the amino acid sequence of SEQ ID NO: 1 is a full-length p73 protein (TAp73), which is known to induce apoptosis as one of the p53 protein family. Specific nucleotide sequences of the p73 protein and the gene encoding the p73 protein can be obtained from a known database such as GeneBank of NCBI. However, as far as having the activity of inducing apoptosis of the present invention, not only the above-mentioned known sequences but also sequences having 80% or more homology, specifically 85% or more, more specifically 90% or more, Sequences having 95% or more homology may also be included within the scope of the present invention.

In the present invention, " homology " is intended to indicate a similar degree to the amino acid sequence of a wild-type protein or a nucleotide sequence encoding the amino acid sequence, and the amino acid sequence or base sequence of the present invention and the above- Or more of the same sequence. This homology can be determined by comparing the two sequences visually, but can be determined using a bioinformatic algorithm that aligns the sequences to be compared and analyzes the degree of homology. The homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis. USA). The automated array algorithms in this module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithm and homology determination for other useful arrays is automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

In the present invention, even if it is described as 'a peptide consisting of a specific sequence number', if it has the same or equivalent activity as a peptide consisting of the amino acid sequence of the corresponding SEQ ID NO: Or a mutation that may occur naturally or a silent mutation thereof, and it is obvious that the sequence addition or mutation is within the scope of the present invention.

In the examples of the present invention, not only TAp73 protein of SEQ ID NO: 1 but also p73-TAD (SEQ ID NO: 8) containing the 1-67 residue thereof can induce apoptosis through cell survival experiment and colony formation assay (Fig. 1). When actinomycin A (ActD, transcription inhibitor) was treated (Fig. 3) to exclude transcription-dependent cell apoptosis of p73, it was confirmed that TAp73 and p73-TAD did not significantly affect apoptosis , And TAp73 and p73-TAD release cytochrome c from the mitochondria into the cytoplasm. These results indicate that TAp73 and p73-TAD can induce transcription-independent cell death through the mitochondrial pathway.

Furthermore, it was confirmed that the p73-TAD16 (SEQ ID NO: 2) peptide containing residues 10-25 of TAp73 induces apoptosis at a level similar to that of TAp73 (FIGS. 2 and 10).

From these experimental results, the inventors of the present invention found that the peptides of SEQ ID NO: 2 corresponding to residues 10 to 25 in the p73 protein or the peptides of SEQ ID NO: 2 alone or in the range of 13 to 70, particularly 16 to 67 It was confirmed that the peptide composed of amino acid can induce apoptosis and thus can be effective for the prevention and treatment of cancer.

In addition, the peptide of the present invention may be in any pharmaceutically acceptable form that can be used for prevention and treatment of cancer.

In the present invention, " pharmaceutically acceptable " means a substance which can be effectively used in a desired use without causing excessive toxicity, irritation, or allergic reaction within the scope of medical judgment.

Another aspect of the present invention provides a peptide for preventing or treating cancer comprising an amino acid sequence of the following general formula:

DGGTTX ₁ EHX ₂ X ₃ SSX ₄ EPD (general formula 1)

In the general formula 1,

X ₁ to X ₄ are phenylalanine, tryptophan, isoleucine, leucine, proline, methionine, or valine, respectively.

Specifically, the peptide may be composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The peptide represented by the general formula 1 of the present invention can induce apoptosis in a transcription-independent manner through a mitochondrial pathway, and thus can have a preventive and therapeutic effect on cancer.

In the example of the present invention, it was confirmed that the p73-TAD16 peptide composed of the amino acid sequence of SEQ ID NO: 2 (DGGTTFEHLWSSLEPD) was able to induce apoptosis by forming a complex with the Bcl-X _L protein (FIG. 10) , It was also confirmed that hydrophobic interaction by hydrophobic residues at specific positions (6, 9, 10, and 13) is important in the complex formation (FIG. 16).

From this, it can be concluded that the peptide represented by the general formula 1 can also form a complex with the Bcl-X _L protein and eventually induce apoptosis as long as the hydrophobic interaction by the hydrophobic moiety is maintained at the above position can do.

Accordingly, the amino acid residues 6, 9, 10, and 13 in the peptide represented by the general formula 1 may be any residue of a hydrophobic residue phenylalanine, tryptophan, isoleucine, leucine, proline, methionine, or valine, (F), the 9th amino acid residue is leucine (L), the 10th amino acid residue is tryptophan (W), the 13th amino acid residue is leucine (L), and the 6th amino acid residue is phenylalanine Specifically, it may be the amino acid sequence of SEQ ID NO: 2.

In addition, since the peptide of the present invention has a form in which an arbitrary amino acid is added to its N-terminal or C-terminal, it can exhibit the effect of the peptide provided in the present invention as it is. Therefore, . As an example, the peptide may be a form in which one or two amino acids are added to the N-terminal or C-terminal of the peptide.

Meanwhile, the cancer preventive or therapeutic peptide of the present invention may interact with Bcl-X _L to induce apoptosis in a transcription-independent manner, and may induce the release of cytochrome c from mitochondria Lt; / RTI >

In the examples of the present invention, it was confirmed that the peptide of the present invention induces the release of cytochrome c from mitochondria (Fig. 1). The peptide interacts with Bcl-X _L , _L , thereby inducing mitochondrial apoptosis in a transcription-independent manner (FIGS. 8 and 9).

In the present invention, the " Bcl-X _L " protein is one of the Bcl-2 family proteins, and the Bcl-2 protein family is an important factor of the mitochondrial apoptosis pathway by regulation of external mitochondrial membrane permeability and cytochrome c release. Anti-apoptotic Bcl-2 family protein performs anti-apoptotic function through sequestration of apoptosis-inducing BH3 protein.

Anti-apoptotic Bcl-2 family proteins can be produced by separating pro-apoptotic BH3-unique proteins such as PUMA, Bid, and Bim, which can activate Bak and Bax, apoptotic factors, Function. This modulation of apoptosis is carried out by the formation of a bond between the anti-apoptotic Bcl-2 family protein and the BH3 domain of the apoptosis-inducing BH3-unique protein. Anti-apoptosis and induction of apoptosis The inhibition of the interaction between Bcl-2 family proteins is an effective strategy for cancer treatment, so that the structure of the apoptosis-inducing BH3 peptide bound to the anti-apoptotic Bcl-2 family protein is anti- It is important in excavation.

Another embodiment of the present invention provides a method for inducing apoptosis in a transcription-independent manner by interacting with Bcl-X _L in vitro or ex vivo, comprising the step of treating the peptide.

Another aspect of the invention provides a method of inducing the release of cytochrome c from mitochondria in vitro or ex vivo, comprising the step of treating said peptide.

Hereinafter, the method will be described in detail. On the other hand, as described above, the definitions and the aspects of the terms described above are also applied to the following.

Since the peptide of the present invention interacts with Bcl-X _L to form a complex, Bid that induces apoptosis is released from Bcl-X _L , thereby inducing apoptosis in a transcription-independent manner. Therefore, By treating the peptide in vitro or ex vivo, it is possible to induce apoptosis in a transcription-independent manner.

Embodiment of the present invention, p73-TAD is anti-apoptotic (anti-apoptotic) Bcl-2 family proteins _{(Bcl-X L, Bcl-} 2, Bcl-w) and air down directly cross GST pool to function And confirmed by pull-down assay (Fig. 5).

In addition, immunoprecipitation experiments confirmed the formation of Bcl-X _L / TAp73 and Bcl-X _L / p73-TAD complexes, and overexpressed TAp73 interacted with endogenous Bcl-X _L or Bcl-2 .

Confocal microscopy (confocal microscopy) immunofluorescence (immunofluorescence) analyzed by p73 and Bcl-X _L are mutually determine the location, and ETO blood seed (etoposide) mitochondria in endogenous p73 and Bcl-X _L and after the treatment that acts with the And coexistence of transfected TAp73 and p73-TAD with Bcl-X _L was also confirmed.

From this, we confirmed that p73-TAD directly interacts with anti-apoptotic Bcl-2 family proteins and coexists in mitochondria.

On the other hand, evidence of mitochondrial apoptosis induced by TAp73 and p73-TAD (decreased cell survival, increased sub-G1 population, and cytochrome c release from mitochondria) was confirmed by transduced Bcl-X _L It was observed that the above phenomenon was suppressed (increased cell survival, sub-G1 group reduction, and decreased cytochrome c release from mitochondria) (Fig. 8).

These results confirm that the transcription-independent apoptotic activity of TAp73 and p73-TAD is inhibited by Bcl-X _L.

Further, evidence that the p73-TAD16 peptide of the present invention induces apoptosis including cytokine c release from mitochondria in a transcription-independent manner (cell survival reduction, sub-G1 population increase, etc.) was observed and transduced Bcl- It was confirmed that X _L inhibited cell death (increased cell survival, sub-G1 population reduction) (FIGS. 1 and 8).

In addition, direct interaction of p73-TAD16 peptide with Bcl-X _L was confirmed by immunoprecipitation (FIG. 2) and immunofluorescence analysis revealed that GFP-tagged p73-TAD16 peptide binds to Bcl-X _L and mitochondria (Fig. 7).

From these results, it was confirmed that the peptide of the present invention can induce the release of cytochrome c from mitochondria, has a binding activity to Bcl-X _L , and can induce apoptosis through such interaction.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the peptide for preventing or treating cancer.

The pharmaceutical composition for preventing or treating cancer according to the present invention may contain a peptide for preventing or treating cancer that can induce apoptosis as an active ingredient and may exhibit the preventive or therapeutic effect of cancer.

In the present invention, " cancer " means a tumor that is abnormally grown by autonomous overgrowth of body tissues or a tumor that forms a tumor, and is particularly caused by loss or lack of normal cell killing activity, Cancer can be treated by controlling cell death.

Specifically, the cancer is not particularly limited as long as the symptom can be alleviated, alleviated, ameliorated or treated by the peptide of the present invention, and includes all cancers known to be induced by Bcl-X _L overexpression, It does not. Specific examples thereof include gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, small bowel cancer, rectum cancer, , Endometrial cancer, cervical cancer, vaginal cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, (CNS) tumors, spinal cord tumors, brainstem glioma, or pituitary adenoma, and more specifically, lung cancer cells may be used for the treatment of lung cancer, But is not limited thereto.

As described above, the peptide of the present invention has an effect of inducing apoptosis, and is not limited to a specific type of cancer, which is a cancer that can be treated by inducing apoptosis. In the pharmaceutical composition of the present invention, ≪ / RTI >

In the examples of the present invention, lung cancer cells were selected as a representative example of inducing apoptosis through inhibition of Bcl-X _L to confirm the apoptosis-inducing effect and the anti-cancer effect by the peptide of the present invention.

In the present invention, " prevention " means any action that inhibits or delays cancer by administration of a composition comprising the peptide.

In the present invention, the term " treatment " means any action that improves or alters the symptoms of cancer by administration of a composition containing the peptide.

The pharmaceutical composition of the present invention can be used as a single preparation and can be used as a combination preparation containing a drug known to have an approved cancer treatment effect and can be formulated using a pharmaceutically acceptable carrier or excipient to provide a unit dose ≪ / RTI > or by intrusion into a multi-dose container.

In addition, if necessary, other conventional additives such as an antioxidant, a buffer and / or a bacteriostatic agent can be added and used. A diluent, a dispersant, a surfactant, a binder, a lubricant, Pills, capsules, granules or tablets, and the like.

In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of a peptide. The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and is generally in the range of 0.001 to 1000 mg / kg, preferably 0.05 To 200 mg / kg, more preferably 0.1 to 100 mg / kg, may be administered once a day to several times per day. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer an amount that can achieve the maximum effect in a minimal amount without causing side effects, and can be readily determined by those skilled in the art.

The mode of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be conventionally used in the art. As a non-limiting example of such a mode of administration, the compositions may be administered orally or parenterally. The pharmaceutical composition according to the present invention can be manufactured into various formulations according to the intended administration mode.

The administration frequency of the composition according to the present invention is not particularly limited, but may be administered once a day or divided into several doses.

The pharmaceutical compositions comprising the peptides of the invention may comprise a pharmaceutically acceptable carrier. Such a carrier may be non-naturally occurring.

The term " pharmaceutically acceptable " in the present invention means that the compound does not cause a sufficient amount and side effects sufficient to exhibit the therapeutic effect, and the kind of the disease, the age, body weight, health, sex, , The route of administration, the manner of administration, the frequency of administration, the duration of treatment, the formulations, or the medicaments used concurrently, well known in the medical arts.

The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter and a perfume in the case of oral administration, A solubilizer, an isotonic agent, and a stabilizer may be used in combination. In the case of topical administration, a base, an excipient, a lubricant and a preservative may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, unit dosage ampoules or multiple dosage forms may be prepared. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules and sustained release formulations.

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, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the present invention, " administration " means introducing a predetermined substance into a patient by any suitable method, and the administration route of the conjugate can be administered through any ordinary route so long as the drug can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary and rectal administration. However, since the peptide is extinguished upon oral administration, it is preferable that the oral composition is formulated so as to coat the active agent or protect it from decomposition at the top. Specifically, it can be administered in the form of an injection. In addition, the pharmaceutical composition may be administered by any device capable of transferring the active ingredient to the target cell.

In addition, the pharmaceutical composition of the present invention is determined depending on the kind of drug, which is an active ingredient, together with various 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.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Specifically, the preferred total dose of the peptides of the invention may be from about 0.0001 mg to 500 mg per kg of patient body weight per day. However, the dose of the peptide depends on various factors such as age, body weight, health condition, sex, severity of disease, diet and excretion rate as well as administration route and frequency of treatment of the pharmaceutical composition, It will be understood by those of ordinary skill in the art that appropriate effective dosages may be determined according to the particular use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

Another aspect of the present invention provides a method of preventing or treating cancer comprising administering the peptide or a composition comprising the peptide to a subject.

In the present invention, an " individual " may mean all animals, including humans, who have developed or are likely to develop cancer. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The preventive or therapeutic method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject who has developed or is at risk of developing cancer.

According to another aspect of the present invention,

(a) interaction between the p73-TAD16 peptide and the Bcl-X _L protein using the tertiary structure of the p73-TAD16 peptide consisting of the amino acid sequence of SEQ ID NO: 2 and the Bcl-X _L protein complex consisting of the amino acid sequence of SEQ ID NO: Generating or screening a regulatory candidate material; And

(b) confirming whether or not the interaction between Bcl-X _L and p73-TAD16 of the candidate substance generated or selected in the step (a)

A method for screening for a substance that modulates the interaction between Bcl-X _L and p73-TAD16 is provided.

The tertiary structure may be defined by the atomic coordinates described in any one of Tables 2 to 22.

In addition, the screening method of the present invention may further include a step of designing a tertiary structure of a protein complex using a tertiary structure defined by the atomic coordinates described in any one of Tables 2 to 22, but the present invention is not limited thereto.

In the present invention, the " screening method " is a method for screening or searching for a target substance among various candidate substances. In the present invention, by using the tertiary structure of the Bcl-X _L / p73-TAD16 complex identified by the present inventors , A method for screening or searching for a substance having an activity of modulating the interactions between the proteins or inhibiting proteins in the complex. Also, in the present invention, a method of designing a desired substance or drug using the tertiary structure may be included in the screening method.

In the present invention, the " candidate substance " is a substance including, without limitation, a compound, DNA, RNA, antibody, compound, peptide or a combination thereof, and the tertiary structure of the Bcl-X _L / p73-TAD16 complex Synthesized, manufactured or modified so as to have a structure that is predicted to have an activity capable of regulating the interactions between the proteins or can be predicted to have a structure capable of binding to the protein complex, .

In the present invention, " a candidate substance for regulating the interaction between p73-TAD16 peptide and Bcl-X _L protein " has activity to regulate the interaction between p73-TAD16 peptide and Bcl-X _L protein according to a conventional selection method Or may be individual nucleic acids, proteins, other extracts or natural products, or compounds randomly selected.

In the present invention, " a method of screening a substance that regulates the interaction between Bcl-X _L and p73-TAD16 " is a method of screening a substance that controls Bcl-X _L / p73-TAD16 < / RTI > is inhibited or facilitated.

In one embodiment of the present invention, in order to confirm the interaction mechanism between the p73-TAD16 peptide of SEQ ID NO: 2 and Bcl-X _L , NMR titration and NMR analysis of the Bcl-X _L / p73-TAD16 peptide complex structure (Fig. 11 and 12). The determined complex structure is defined by the atomic coordinates of Tables 2 to 22. The atomic coordinates in Tables 2 to 22 are the ensemble of the complex structure obtained through NMR analysis, and each atomic coordinate represents a complex structure, which can be used for screening a desired substance. The atomic coordinates in Table 2 are representative examples of the Bcl-X _L / p73-TAD16 peptide complex structure identified in the present invention.

^{When the} p73-TAD16 peptide was titrated to Bcl-X _L labeled with ¹⁵ N isotope, the chemical shift perturbations corresponding to amino acids E92, E129, N136 and F146 of Bcl-X _L were confirmed, The altered amino acids are located in the BH1, BH2, and BH3 domains of Bcl-X _L , indicating that the site is involved in binding of the p73-TAD16 peptide (FIG. 12).

Specifically, in the Bcl-X _L / p73-TAD16 peptide complex structure, the p73-TAD16 peptide binds to the hydrophobic groove composed of the BH1, BH2, and BH3 domains of Bcl-X _{L and} the α3 helix, The disordered protein p73-TAD16 peptide changes to a structure that forms an amphipathic α-helix.

The hydrophobic interactions between the F15, L18, W19 and L22 amino acids of the p73-TAD16 peptide and the A93, F97, V141, A142, F191, L194 and Y195 amino acids of Bcl-X _L and the hydrophobic interaction between the p73-TAD16 peptide E23, through the electrostatic interaction between Bcl-X _L of R100, R139 is formed in Bcl-X _L / p73-TAD16 peptide complex structure, in particular, F97, in Bcl-X _L Y195 is as W19, F15 of the p73-TAD16 peptide Intermolecular π-stacking formation contributes to the stability of the complex.

In order to determine the position of the p73-TAD16 peptide, which plays a crucial role in binding to Bcl-X _L , the inventors of the present invention found that the p73-TAD16 peptide involved in the hydrophobic interaction in the Bcl-X _L / p73- F15, L18, W19, and L22 amino acids were replaced with alanine. As a result, it was found that a chemical shift perturbation observed by the binding of wild-type p73-TAD16 peptide to Bcl-X _L , When this p73-TAD16 peptide mutant was titrated to Bcl-X _L , it was not confirmed. This means that hydrophobic residues on p73-TAD16 peptide play an important role in the interaction of Bcl-X _L with p73-TAD16 peptide (Fig. 16).

The Bcl-X _L / p73-TAD16 peptide complex of the present invention exhibits a distinct difference in binding mode with Bcl-X _L when compared to the previously known Bcl-X _L / BH3 peptide complex. Specifically, the p73-TAD16 peptide is a non-canonical region consisting of the BH1, BH2, and BH3 domains that are not typical canonical groove regions formed by BH1 and BH3 of Bcl-X _L to which the known BH3 peptide binds site) (Fig. 16).

Furthermore, it was confirmed that the p73-TAD16 peptide binds in the opposite direction to that of the BH3 peptide bound to Bcl-X _L.

The structures of the Bcl-X _L / p73-TAD16 peptide complexes identified by the present inventors are listed in Protein Data Bank (PDB) under the number 5H3Y and are shown in Tables 2 to 22. The atomic coordinates of the peptide complex can be stored in a medium for subsequent use with a computing device such as a computer. Typically, the coordinates may be stored in a medium useful for storing large amounts of data, such as magnetic or optical media (i.e., floppy disks, hard disks, compact disks, magneto-optical media or electronic media, etc.). The selection of computers, storage media, networking and other devices or technologies in this regard will be familiar to those skilled in the art of structural / computational chemistry.

Based on the three-dimensional structure of the Bcl-X _L / p73-TAD16 peptide complex identified in the present invention, a computer-readable medium containing data on atomic coordinates and / or three- Information on various protein sites can be provided. Through this process, it is possible to predict the reaction pattern without conducting a substantial experiment on a number of new drug candidate substances, and it is possible to increase the economical efficiency of new drug development by carrying out an experiment only on the selected substances according to the result.

The screening method of the present invention comprises (a ') a p73-TAD16 peptide consisting of the amino acid sequence of SEQ ID NO: 2 and represented by the atomic coordinates of any one of Tables 2 to 22, and Bcl-X _L Protein complexes may be further utilized to design the tertiary structure of the complexes,

Wherein said step comprises the steps of: inputting data on atomic coordinates and the like to the tertiary structure of said proteins on a computer together with an appropriate software program; And a three-dimensional protein structure capable of visualization and further computer manipulation. When the tertiary structure of the Bcl-X _L / p73-TAD16 peptide complex is designed using the atomic coordinates shown in the above table, the amino acid modified for the wild-type protein is substituted for the wild-type amino acid, But it is not limited thereto.

(A) generating or selecting the interaction between p73-TAD16 adjustment candidate peptide and Bcl-X _L protein using the three-dimensional structure of the present invention,

When all or a part of the tertiary structure of the Bcl-X _L / p73-TAD16 peptide complex identified in the present invention is used, a candidate substance capable of controlling the interaction of the Bcl-X _L and p73-TAD16 peptide complexes is specifically Can be selected or generated. In other words, it is possible to design a substance that controls the interaction of Bcl-X _L and p73-TAD16 peptides based on the tertiary structure. It is also possible to determine whether the known substances can control the interaction, It can also be selected. The generation or selection of the candidate substance can be performed by a method known in the art.

(B) confirming whether or not the interaction between Bcl-X _L and p73-TAD16 of the candidate substance generated or selected in the step (a)

The present invention is not limited to the above-mentioned method, but it is not limited thereto, and it may be confirmed whether or not the candidate substance generated or selected in the step (a) inhibits or promotes the complex formation of Bcl-X _L and p73-TAD16. Inhibition or promotion of complex formation may be accomplished by any method known in the art and may be performed by any method known in the art including but not limited to pulldown assay, NMR, immunoprecipitation, isothermal titration calorimetry, gel permeation chromatography permeation chromatography, and the like, but the present invention is not limited thereto.

The method of screening for a substance that modulates the interaction between Bcl-X _L and p73-TAD16 of the present invention is characterized in that certain candidate substances are selected from the three-dimensional structures of the Bcl-X _L / p73-TAD16 complexes (Tables 2-22) by detecting whether or not the forming, may be screened for a substance that controls the interaction between Bcl-X _L and p73-TAD16 and, by the screening material is controlling the interaction Bcl-X _L and p73-TAD16, various fields . Particularly, in the present invention, it is confirmed that the above-mentioned interaction is involved in induction of apoptosis, and thus it can be used for screening substances inducing apoptosis.

According to another aspect of the present invention,

(a) using a three-dimensional structure of Bcl-X _L protein complex consisting of the amino acid sequence of SEQ ID NO: 2 the amino acid sequence consisting of p73-TAD16 peptide and SEQ ID NO: 9 generates a candidate to inhibit the Bcl- X _L or Selecting step

(b) confirming whether the candidate substance generated or selected in the step (a) inhibits Bcl-X _L ,

A method for screening a substance that inhibits Bcl-X _L is provided.

Here, the tertiary structure may be defined by the atomic coordinates described in any one of Tables 2 to 22.

In addition, the screening method of the present invention may further include the step of designing a tertiary structure of a candidate substance inhibiting Bcl-X _L using a tertiary structure defined by the atomic coordinates described in any one of Tables 2 to 22 But is not limited thereto.

In the present invention, the " candidate substance inhibiting Bcl-X _L " is a substance that is presumed to have an activity of inhibiting Bcl-X _L according to a conventional selection method, or may be a randomly selected individual nucleic acid, protein, , Or a compound, but is not limited thereto. In the present invention, the candidate substance that inhibits Bcl-X _L has an activity of interfering with p73-TAD16 peptide and Bcl-X _L protein, or an activity of inhibiting Bcl-X _L that forms a conjugate, , But is not limited thereto.

In the present invention, " a method of screening for a substance that inhibits Bcl-X _L " means that the test group treated with the selected or generated candidate substance has an activity of inhibiting Bcl-X _L It was designed in such a way as to confirm whether or not.

The substance that inhibits Bcl-X _L screened by the screening method of the present invention may be one that mimics the structure of p73-TAD16 peptide, but is not limited thereto.

The screening method of the present invention comprises (a ') a p73-TAD16 peptide consisting of the amino acid sequence of SEQ ID NO: 2 and represented by any one of the atomic coordinates described in Tables 2 to 22, and Bcl-X _L protein complex of the present invention may be used to further include a step of designing a tertiary structure of a candidate substance inhibiting Bcl-X _L ,

Wherein the step comprises: inputting data on atomic coordinates and the like to a tertiary structure for the proteins on a computer together with an appropriate software program; And a three-dimensional protein structure capable of visualization and further computer manipulation. In addition, when designing the tertiary structure of Bcl-X _L inhibitory substance using the atomic coordinates shown in the above table, the modified amino acid of the wild-type protein is substituted with the amino acid of the wild type, Amino acids and the like can be appropriately modified within a range not bringing the amino acid derivative of the present invention, but the present invention is not limited thereto.

The step of (a) generating or screening the inhibitory substance of Bcl-X _L using the tertiary structure,

When all or a part of the tertiary structure of the Bcl-X _L / p73-TAD16 peptide complex identified in the present invention is used, by controlling the interaction of Bcl-X _L or Bcl-X _L and the p73-TAD16 peptide complex, A substance capable of inhibiting Bcl-X _L that can induce apoptosis can be specifically selected or produced. That is, a candidate substance having a structure similar to that of p73-TAD16, which regulates the interaction of Bcl-X _L and p73-TAD16 peptides based on the tertiary structure of the protein complex or complexed with Bcl-X _L Or may be screened for candidate Bcl-X _L inhibitors by determining whether previously known substances can modulate the interaction. The generation or selection of the candidate substance can be performed by a method known in the art.

The step (b) of confirming whether or not the candidate substance generated or selected in the step (a) of the present invention inhibits Bcl-X _L ,

Wherein (a) X Bcl- _L generated or selected candidate in the step form a complex - by acting on or Bcl-X _L does not form the complex, to determine whether has the activity to inhibit the Bcl- X _L But is not limited thereto. The inhibitory activity against Bcl-X _L can be confirmed by any method known in the art and specifically includes a cell death activity assay, Caspase-3/7 activation assay, cytochrome c release assay, etc. But are not limited thereto.

The Bcl-X _L inhibitor screened by the screening method of the present invention can induce apoptosis, and thus can be used as an anticancer agent.

According to another aspect of the present invention,

(a) producing or selecting a candidate compound for chemotherapy using a tertiary structure of a p73-TAD16 peptide consisting of the amino acid sequence of SEQ ID NO: 2 and a Bcl-X _L protein complex consisting of the amino acid sequence of SEQ ID NO: 9; And

(b) confirming the anticancer effect of the candidate substance generated or selected in the step (a).

A method for screening an anticancer agent is provided.

Here, the tertiary structure may be defined by the atomic coordinates described in any one of Tables 2 to 22.

In addition, the screening method of the present invention may further include a step of designing a tertiary structure of a protein complex using a tertiary structure defined by the atomic coordinates described in any one of Tables 2 to 22, but the present invention is not limited thereto.

In the present invention, the " anticancer therapeutic agent candidate " is a substance that is presumed to have an anticancer activity, specifically, a cell killing activity according to a conventional selection method, or a randomly selected individual nucleic acid, protein, other extract or natural product, But is not limited thereto. In the present invention, the anticancer therapeutic agent has an activity of regulating the interaction between the p73-TAD16 peptide and the Bcl-X _L protein, thereby inducing apoptosis.

In the present invention, the " method for screening anticancer agent " is designed to confirm whether or not the test group treated with the selected or generated candidate substance has anticancer effect as compared with the control group not treated.

(A ') a p73-TAD16 peptide consisting of the amino acid sequence of SEQ ID NO: 2, defined by any one of the atomic coordinates set forth in Tables 2 to 22, and a Bcl-X _L protein complex consisting of the amino acid sequence of SEQ ID NO: The step of designing the tertiary structure of the composite may further include using a differential structure,

Wherein said step comprises the steps of: inputting data on atomic coordinates and the like to the tertiary structure of said proteins on a computer together with an appropriate software program; And a three-dimensional protein structure capable of visualization and further computer manipulation. When the tertiary structure of the Bcl-X _L / p73-TAD16 peptide complex is designed using the atomic coordinates shown in the above table, the amino acid modified for the wild-type protein is substituted for the wild-type amino acid, But it is not limited thereto.

The step (a) of generating or selecting a candidate substance for an anticancer therapeutic agent using the tertiary structure of the present invention comprises:

When all or a part of the tertiary structure of the Bcl-X _L / p73-TAD16 peptide complex identified in the present invention is used, the interaction of Bcl-X _L and p73-TAD16 peptide complex is controlled to induce apoptosis The candidate cancer agent can be specifically selected or generated. Specifically, in the present invention, it was confirmed that the p73-TAD16 peptide can induce apoptosis in a transcription-independent manner by forming a Bcl-X _L / p73-TAD16 peptide complex. As a result, To induce the formation of the complex or to select or produce candidate substances that can sustain the complex. That is, it is possible to design an anticancer therapeutic agent that controls the interaction of Bcl-X _L and p73-TAD16 peptides based on the tertiary structure, and judge whether known substances can control the interaction, Material may be selected. The generation or selection of the candidate substance can be performed by a method known in the art.

(B) confirming the anticancer effect of the candidate substance generated or selected in the step (a)

The present invention is not limited thereto, but may be to confirm whether the candidate substance generated or selected in the step (a) promotes the complex formation of Bcl-X _L and p73-TAD16 or can maintain the formed complex. The formation or maintenance of the complex can be confirmed by any method known in the art and specifically includes pulldown assay, NMR, immunoprecipitation, Isothermal titration calorimetry, Gel permeation chromatography ), But the present invention is not limited thereto.

The present inventors confirmed that when the Bcl-X _L / p73-TAD16 peptide complex is formed, the p73-TAD16 peptide can induce apoptosis in a transcription-independent manner. Therefore, compared with the control group in which the candidate substance was not treated, -X _L and p73-TAD 16 peptides can be antitumor agents for cancer and the like and can be considered as a cancer treatment agent.

In the present invention, the cancer may be included in the present invention without limitation as long as it is a cancer that is prevented or treated by the screened substance, and specific examples thereof include gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, Hodgkin's disease, esophageal cancer, endometrial cancer, endometrial cancer, uterine cancer, vulvar cancer, ovarian cancer, colon cancer, small intestine cancer, rectum cancer, Lymphoma, renal cancer, ureteral cancer, renal pelvic cancer, hematologic cancer, brain cancer, central nervous system, lymph node metastasis, lymph node metastasis, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, A central nervous system (CNS) tumor, a spinal cord tumor, a brainstem glioma, or a pituitary adenoma, and more specifically may be, but is not limited to, lung cancer.

Since the peptide for cancer prevention or treatment of the present invention is a peptide capable of inducing apoptosis in a transcription-independent manner, the pharmaceutical composition containing the same has the use for prevention or treatment of cancer. In addition, a substance or an anticancer agent capable of controlling the interaction between the proteins can be screened using the structure of the Bcl-X _L / p73-TAD16 peptide complex.

Figure 1 shows that p73-TAD can induce apoptosis in a transcription-independent pathway. (a) shows the results of cell survival experiments using TAp73 and p73-TAD. H1299 cells were transiently transfected with peGFP-C3-TAp73, peGFP-C3-p73-TAD, or a control vector, and viable cells were measured using a WST-1 assay. n = 6; Compared to the control, P <0.0001 (b) is the result of colony formation assay using TAp73 and p73-TAD. TAp73 and p73-TAD inhibited colony formation of H1299 cells transfected with the indicated plasmid. n = 3; P <0.001 compared with the control group. (c) is the result of annexin-PE cell death assay of TAp73 and p73-TAD. Cell death of H1299 cells transfected with the indicated plasmids was assessed by Annexin V-PE and 7-AAD staining. Then, flow cytometry was performed. (d) is a result of caspase-3/7 activity measurement. Caspase-3/7 activity was measured in H1299 cells transfected with peGFP-C3-TAp73, peGFP-C3-p73-TAD, or a control vector. n = 3, P < 0.0005 compared to the control group. (e) is the result of cytochrome c release assay of TAp73 and p73-TAD. H1299 cells were transfected with the indicated plasmids and cytosol and mitochondrial fractions from cell lysates were immunoblotted with anti-cytochrome c or anti-GFP antibodies. GAPDH and VDAC1 were used as cytosol and mitochondrial fraction markers, respectively. The graph on the panel below shows the relative band strength. In the same blot, each band intensity was normalized for GAPDH or VDAC1 band intensities. n = 4; P <0.05 compared to the control group. (a) - (e), the data are presented as mean ± sd and the statistics were performed using one-way ANOVA test.
Figure 2 shows DNA fragmentation assay results with TAp73, p73-TAD, and p73-TAD16. (a) shows the DNA fragmentation assay results of TAp73 and p73-TAD. H1299 cells transfected with the indicated plasmids were stained with DAPI solution and flow cell analysis of DNA content was performed for cell death determinations. n = 3; (B), pGFP-C3-p73-TAD16 (c), or a control vector as analyzed by a DNA fragmentation assay. _Lt ; RTI ID = 0.0 &gt; Bcl-X < / RTI &gt; _L to the death of infected H1299 cells. n = 3; P < 0.05 (b), P < 0.0001 (c). (ac), the data were expressed as mean ± sd and the statistics were performed using one-way ANOVA test.
Figure 3 shows the transcription-independent cell death of TAp73 and p73-TAD. (a) shows H1299 cells transfected with peGFP-C3-TAp73 or peGFP-C3-p73-TAD treated with DMSO (dimethyl sulfoxide) or 0.5 nM ActD and measured for cell survival with WST-1 assay . n = 5; P <0.005 compared to the control group. (b) shows the cell survival assay results of TAp73 (A156V) transcription-inactive mutants. peGFP-C3-TAp73, peGFP-C3-TAp73 (A156V), or H1299 cells as a control vector, and viable cells were measured with WST-1 assay. n = 3; P <0.005 compared to the control group. (ab), data were presented as mean ± sd and the statistics were performed using one-way ANOVA test.
4 is a graph showing the results of measurement of caspase-3/7 activity in Bax / Bak DKO HCT116 cells. caspase-3/7 activity was measured in wild type (WT) and Bax / Bak DKO HCT116 cells after transfection of peGFP-C3-TAp73, peGFP-C3-p73-TAD or control vector. n = 3; P <0.0005 compared to the control group. Data were presented as mean ± sd and the statistics were performed using a one-way ANOVA test.
Figure 5 shows that p73-TAD binds to the anti-apoptotic Bcl-2 family protein. (a) is a p73-TAD and Bcl-X _L - is a diagram showing the interaction of, Bcl-2, and Bcl-w to the GST pull-down assays. GST-p73-TAD pull down with GST-tagged lysates of HEK293T cells transfected with p73-TAD and labeled Flag-tagged plasmids were performed and immunoprecipitated with anti-FLAG antibody. (b) shows the interaction of TAp73 or p73-TAD with Bcl-X _L - in vivo. H1299 cells transfected with the indicated plasmid were immunoprecipitated with anti-Bcl-X _{L <} - &gt; or anti-Flag antibodies and detected with anti-GFP antibody. (c) shows the interaction between overexpressed TAp73 or p73-TAD and endogenous Bcl-X _L -. The lysates of H1299 cells overexpressing GFP-p73 were immunoprecipitated with anti-Bcl-X _L -antibody and detected with anti-GFP antibody. (d) shows the interaction between endogenous TAp73 and Bcl-X _L - or Bcl-2. H1299 cells were treated with 50 μM ethopocide, and the cell lysates were immunoprecipitated with anti-Bcl-X _L - or anti-Bcl-2 antibodies and detected as anti-p73 antibodies. (e) shows confocal analysis of TAp73 and endogenous Bcl-X _{L <} - &gt;. HeLa cells were irradiated (X-irradiated) for 3 hours at 5 Gy and not irradiated (control group). And immunostained with TAp73 (violet) or Bcl-X _L - (red). Cell nuclei and mitochondria were stained with DAPI and MitoTracker Deep Red, respectively. The scale bar is 10 μm. The white box represents the enlarged area in each panel. The illustrations and arrows show examples of co-localization of TAp73, Bcl-X _L- , and mitochondria (bottom panel), or Bcl-X _L- and mitochondria (top panel).
Fig. 6 is a graph showing that endogenous TAp73 is not immunoprecipitated with apoptosis-inducing BH3-only proteins Bad, Puma, Bik, Bid, and Bim. The lysates of H1299 cells were immunoprecipitated with antibodies against BH3-only protein and immunoblotted with anti-p73 antibody.
FIG. 7 shows the coexistence of TAp73 and p73-TAD in the mitochondria. peGFP-C3-TAp73, peGFP-C3-p73-TAD or H1299 cells as a control vector. After subcellular fractionation, the cytosol and mitochondrial fractions of cell lysates were immunoblotted with anti-GFP antibody. GAPDH and VDAC1 were used as cytosol and mitochondrial fraction markers, respectively.
Figure 8 shows that the transcription-independent cell death induced by p73-TAD can be inhibited by Bcl-X _L -. (a) shows the results of cell survival assay of TAp73 and p73-TAD in the presence or absence of Bcl-X _{L &lt;} - &gt;. the H1299 cells with pCMV2-Flag-Bcl-X _L with, or peGFP-C3-p73, peGFP- C3-p73-TAD, or without peGFP-C3 were transfected. peGFP-C3 and pCMV2 were used as controls. After transfection, the cells were treated with DMSO or ActD for 24 hours at < RTI ID = 0.0 &gt; 37 C. &lt; / RTI &gt; Cell viability was then measured with the WST-1 assay. Data were expressed as fold change to vector-transfected cells. n = 3; P <0.0001 compared to the control group. (b) shows the effect of Bcl-X _L - on the apoptosis of peGFP-C3-p73, peGFP-C3-p73-TAD or H1299 cells transfected with the control vector through Annexin V-PE cell death assay It is a degree. n = 3; P <0.0001 compared to the control group. (c) cytochrome c release assays of TAp73 and p73-TAD in the presence or absence of Bcl-X _L &lt;&quot;&gt;. pCMV2-Flag-Bcl-X _L with, or without, a co-transfected peGFP-C3-p73 or H1299 cells peGFP-C3-p73-infected TAD (contransfected). After subcellular fractionation, the cytosol and mitochondrial fractions from cell lysates were immunoblotted with anti-cytochrome c antibody. GAPDH and VDAC1 were used as cytosol and mitochondrial fraction markers. The graph on the lower panel shows relative strength. Each band intensity was normalized to GAPDH or VDAC1 band intensities. The total amount of cytochrome c was also shown, and the experiment time of cytochrome c release assay was 16 hours after transfection. n = 4; P <0.05 compared to the control group. In (a) to (c), the data were expressed as mean ± sd and the statistics were performed using a one-way ANOVA test.
FIG. 9 is a graph showing that Bid, a pro-apoptotic BH3-only protein, is released from Bcl-X _L - by interaction with p73-TAD. Immunoprecipitation experiments were performed to confirm competition for TAp73 (a) or p73-TAD (b). HEK293T cells were transfected with pCMV-Myc-Bid and peGFP-C3-TAp73 (a) or peGFP-C3-p73-TAD (b). Cell lysates were immunoprecipitated with anti-Bcl-X _L -antibodies and detected with the indicated antibodies.
FIG. 10 shows that p73-TAD16 mediates transcription-independent cell death by interaction with Bcl-X _L -. (a) The physical relationship between p73-TAD16 and Bcl-X _L - was determined by immunoprecipitation. with _{pCMV2-Flag-Bcl-X L} , or without, were plated peGFP-C3-p73-TAD16 lysate antibody immunoprecipitation and immunoblotting as shown in the use of H1299 cells transfected with. (b) cell survival assay with p73-TAD16. (c) Caspase-3/7 activity measured by p73-TAD16. n = 3; P <0.001 compared with the control group. (de) pCMV2-Flag-Bcl -X L with, or without, peGFP-C3-p73-TAD , peGFP-C3-p73-TAD16, WST- or cell survival and cell death of H1299 cells transfected with control vector 1 (d) and Annexin-PE cell death assay (e). (f) is the cytochrome c release assay result of H1299 cells transfected with peGFP-C3-p73-TAD16. n = 3; Compared to the control group, P &lt; 0.005. (g) Caspase-3/7 activity was measured by peGFP-C3-TAp73 (Δ1-25), peGFP-C3-p73-TAD (Δ1-25), peGFP-C3-TAp73, or peGFP-C3-p73-TAD Were measured in transfected H1299 cells. n = 3; P <0.0005 (b, d, e, g) compared to the control group. Data were expressed as mean ± sd and statistical analysis was performed using one-way ANOVA test (be, g) or Student's t-test (f).
11 shows the structure of the Bcl-X _L - / p73-TAD16 peptide complex. (a) is a diagram showing the presence (black) or without under (red), ¹⁵ N- overlay the 2D ¹ H- ¹⁵ N HSQC spectrum of the labeled (labeled) Bcl-X _L in the p73-TAD16 peptide (left) . Bcl-X _L residues exhibiting chemical shifts of ΔCS> 0.15 ppm and 0.1 ppm <ΔCS <0.15 ppm are shown in red and yellow respectively (right). The α-helical and BH domains are shown graphically below and above, respectively. (b) shows the ensemble of the final 20 lowest-energy NMR structures of the Bcl-X _L / p73-TAD16 peptide complex (left) and the representative structure (right). The Bcl-X _L- and p73-TAD16 peptides were designated purple and light green, respectively. (c) shows the spiral wheel diagram (left) of p73-TAD16 peptide bound to Bcl-X _L -, and the corresponding region in the structure (right). N-terminal. and a spiral region containing F15-E23 of the p73-TAD16 peptide. In the p73-TAD16 peptide, the bulky hydrophobic residues (F15, L18, W19, L22, orange) face the hydrophobic blemishes (groove, purple) of Bcl-X _L- while the remaining hydrophilic residues (cyan) . (d) shows the binding surface between Bcl-X _L - and p73-TAD16 peptides. Bcl-X _L - p73-TAD16 residues involved in binding to the green, Bcl-X _L involved in the p73-TAD16 peptides and hydrophobic and electrostatic interactions - moiety of each are shown in purple, and blue. (e) shows two intermolecular π-stacking interactions between F97 and Y195 of Bcl-X _L - and W19 and F15 of the p73-TAD16 peptide.
Figure 12 shows the results of NMR and ITC analyzes on the interaction between Bcl-X _L &lt;&quot;&gt; and p73-TAD16 peptides. (a) shows the 2D ¹ H- ¹⁵ N HSQC spectrum of ¹⁵ N-labeled Bcl-X _L bound to unlabeled p73-TAD16 peptide. (b) shows the chemical shift perturbation of several Bcl-X _L - residues according to p73-TAD16 peptide titration not labeled with ¹⁵ N-labeled Bcl-X _L. Unlabeled p73-TAD16 peptide was added at a molar ratio of 1: 0 (black), 1: 1 (green), 1: 2 (red), 1: 3 (blue), and 1: ¹⁵ N-Bcl-X _L was observed. (c, d) shows the ITC profile following binding of p73-TAD13 (c) and p73-TAD16 peptide (d) of Bcl-X _L.
Figure 13 summarizes the secondary structure and NMR restraint of p73-TAD16 peptide. (a) was calculated from the difference in the experimental peak shift of p73-TAD16 combined with three other arbitrary coil reference values for the second Hα peak shift of Bcl-X _L - bound to the p73-TAD16 peptide. An arbitrary coil value can be determined by the method of Schwarzinger et al., Sequence-specific random coil NMR chemical shifts, J Am Chem Soc 123, 2970-8, 2001, Tamiola et al. was obtained from proteins. J Am Chem Soc 132, 18000-3, 2010), and Zhang et al (a database of uniformly referenced protein chemical shifts. J Biomol NMR 25, 173-95, 2003), respectively, blue, yellow, and Green. The combined structure information was obtained from the NOESY of the p73-TAD16 peptide in the presence of Bcl-X _L &lt; &quot;&gt;. (b) shows the NOE connectivity of p73-TAD16 in the presence of Bcl-X _L &lt; &quot;&gt;. (c) shows the number of intermolecular (green) and intramolecular (light green) NOEs of the Bcl-X _L -coupled p73-TAD16 peptide used in the structure calculation.
Figure 14 is a bond p73-TAD16 peptides of Bcl-X _L - the Bak (a), Bad (b ), Bim (c), and PUMA (d) the Bcl-X _L binding to cell death induced BH3 peptide of - and Fig. The PDB codes for each structure are 1BXL (a), 1G5J (b), 3FDL (c), and 2M04 (d). The? 1 helices not involved in binding and the apoptosis inducing BH3 peptide were not shown for clarity. The Bcl-X _L -protein bound to the p73-TAD16 peptide was shown in purple, and the Bcl-X _L -protein bound to the apoptosis-inducing BH3 peptide was light blue to dark blue.
15 is a graph showing an overlaid 2D ¹ H- ¹⁵ N HSQC spectrum of ¹⁵ N-labeled Bcl-X _L under the absence (blue) and presence (red) of mutant p73 peptide containing F15A, L18A, W19A or L22A to be.
16 is a diagram showing a new mode of recognition of Bcl-X _L by p73-TAD. (a) shows the structure of p73-TAD16 peptide bound to Bcl-X _{L &lt;} - &gt;. The p73-TAD16 peptide, light green, is located at the center of the BH1, BH2, and BH3 domains and can interact with the residues of the three BH domains. (b) shows that the p73-TAD16 peptide is equally involved in the interaction with Bcl-X _L - residues of the BH1, BH2, and BH3 domains. (cf) shows the complex structure of pro-apoptotic BH3 peptide and Bcl-X _L - in Bak (c), Bad (d), Bid (e), and PUMA (f) BH3 peptides of PUMA (PDB code: 1BXL), Bad (PDB code: 1G5J), Bid (PDB code: 830 4QVE), and PUMA (PDB code: 2M04) were light blue to dark blue. The? 1 helix, which does not participate in any peptide binding, is not shown for clarity.
17 compares Bcl-X _L - binding and MDM2 binding by p73-TAD16 peptide. (a) is a structural comparison between the Bcl-X _L- / p73-TAD16 peptide and (b) is a structural comparison between the MDM2 / p73-TAD16 peptide complex (PDB: 2MPS). The p73-TAD16 peptide was expressed in light green and indicated an important determinant of Bcl-X _L - binding.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example 1 Cell Culture and Construction of Plasmid Construct

p53-null human lung cancer cell line H1299 was cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS (fetal bovine serum). HEK293T cells were cultured in DME medium containing 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL). 5% CO ₂ in a humid cell 37 ℃ And maintained in the atmosphere.

All p73 constructs including TAp73, p73-TAD (1-67 residues) and p73-TAD16 (10-25 residues) were subcloned into the peGFP-C3 vector. Flags - a (flag-tagged) Bcl-X L, Bcl-2, and Bcl-w tag was prepared by inserting the respective structures in pCMV2- Flag vector (obtained from Dr. Kim Jung Hoon, Korea Research Institute of Bioscience and Biotechnology). GST-tagged p73-TAD and Myc-tagged Bid were inserted into pEBG and pCMV-Myc vectors, respectively. Bax / Bak DKO HCT116 cells were obtained from Dr. Richard J. Youle.

Example 2. Immunoblotting

The cell lysate was heated in SDS (sodium dodecyl sulfate) sample buffer and resolved by SDS-polyacrylamide gel electrophoresis (10 or 15% polyacrylamide) to give a polyvinylidene fluoride (PVDF) . After transfer, the membrane was blocked with 5% milk in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 60 minutes and incubated at room temperature for 2 hours Were incubated with primary antibodies in 5% milk in TBST.

The membrane was washed three times with TBST and cultured in TBST containing horseradish-peroxidase-linked anti-immunoglobulin G (IgG). After washing three times in TBST, the immunoreactivity products were detected by chemiluminescence using ECL (enhanced chemiluminescence system, Thermo Scientific).

Each primary antibody, Bcl-X _L specific antibody (1: 1000 dilution, Cell Signaling Technology, clone: 54H6), TAp73 (1: 500 dilution, Abcam, clone: 5B1288; 1: 1000 dilution, Bethyl Laboratories, Inc.) , Bid (1: 1000 dilution, Cell Signaling Technology, Cat. No. 2002S), Bad (1: 1000 dilution, Cell Signaling Technology, clone: D24A9), PUMA (1: 1000 dilution, Cat. No. A300-126A) Bim (1: 1000 dilution, Cell Signaling Technology, Cat. No. 4592), Bim (1: 1000 dilution, Cell Signaling Technology, clone: C34C5), VDAC1 (Voltage- (GAPDH) (1: 1000 dilution, Santa Cruz Biotechnology, clone: FL-335), cytochrome c (1: 1000 dilution, Thermo Scientific, Cat. No. 31460), GFP (1: 1000 dilution, Santa Cruz Biotechnology, clone: FL), normal rabbit IgG (1: 2000 dilution, BD Pharmingen, Cat. , And normal mouse IgG (1: 10000 dilution, Thermo Scientific, Cat. No. 314 30) were purchased. The same applies to the antibodies used in the following examples.

Example 3 Immunoprecipitation

For the endogenous protein complex, the lysates of HEK293T cells were immunoprecipitated with 1 [mu] g of antibody and immunoblotted. Specifically, whole cell lysates were obtained using RIPA (radioimmunoprecipitation assay), and the obtained lysates were immunoprecipitated using rabbit IgG and anti-Bcl-X _L antibody.

The cells were then incubated with 20 μL of antibody and protein Sepharose beads (GE Healthcare) for 2 hours. The beads were washed three times with buffer (150 mM NaCl, 20 mM Tris.HCl, pH 7.5, 0.5% NP-40). To demonstrate that an equivalent amount of cell lysate was used in the immunoprecipitate, a sample representing 0.01% of total lysate was included.

Example 4. Cell survival assay

H1299 cells were seeded into 96-well plates, allowed to attach for 24 hours, and transfected with p73-expression plasmid. Twenty-four hours after transfection, the number of surviving cells was determined using a tetrazolium salt reduction assay (EZ-Cytox Cell Proliferation Assay, LPS solution) according to the manufacturer's protocol. Each treatment was repeated three times.

Example 5. Colony formation assay

Clonogenic cell viability was assessed in a colony forming assay. Ten million H1299 cells were seeded in a 6-well dish one day prior to transient transfection. The cells were transfected using Lipofectamine 2000 (Invitrogen) transfection reagent and grown to induce colony formation under G418 sulphate (200 μg / μL) selection. After 2 weeks of incubation, the cells were fixed with 3.7% formaldehyde and stained with crystal violet. Colonies larger than 0.6 mm in diameter were counted. Each treatment was repeated three times.

Example 6. DNA fragmentation assay [

2 X 10 ⁵ H1299 cells were seeded into 12-well plates and transfected with TAp73, p73-TAD, and p73-TAD16 plasmid constructs and incubated for 24 hours. The cells were then fixed with 70% ethanol and stored for at least 12 hours. Apoptotic cells were determined using a DNA fragmentation assay (Solution 3 [1 μg / mL 4'6-diamidino-2-phenyldole (DAPI), 0.1% Triton X-100 in PBS], Chemometech) according to the manufacturer's protocol .

Example 7. Cell death assay

Cell death was assessed using Annexin V-PE Cell Death Detection Kit (BD Biosciences) according to the manufacturer's instructions. Flow cytometry analysis was performed using 7-aminoactinomycin (7-AAD) staining to detect the percentage of apoptotic cells.

Flow cytometry results were collected using a FACS Calibur 4-color flow cell analyzer (BD Biosciences, San Jose, Calif., USA) and CellQuest ^TM software version 6.

Caspase-3/7 activity was measured using the Caspase-Glo ^® 3/7 Assay Kit (Promega) according to the manufacturer's instructions.

Example 8. Cytochrome c release assay

Using Lipofectamine 2000, pCMV2-Flag-Bcl -X L with or without the vector, were transfected co-infection of H1299 cells with peGFP-C3-TAp73, peGFP- C3-p73-TAD, or peGFP-C3 control vector . Cytochrome c release was detected 16 h after transfection using the cytochrome c cell death kits (Thermo Scientific) according to the manufacturer's instructions.

Example 9. GST pull-down assay (GST pull-down assay)

After 48 hours of co-transfection, HEK293T cells were harvested, washed in PBS, and resuspended in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and 1% glycerol, Roche). &Lt; / RTI &gt;

The resulting lysate was centrifuged at 15,000 x g for 15 min and GST pulldown assay was performed with supernatant.

For the GST pull-down assay, 500 μg of the lysate was mixed with 50 μL of glutathione agarose beads (GE Healthcare) while shaking gently at 4 ° C for 4 hours. Protein complexes were obtained by centrifugation, washed three times with lysis buffer and heated in SDS sample buffer for 5 minutes to elute protein conjugate from glutathione agarose beads.

After the reducing agent was added to the eluted sample, SDS-PAGE and immunoblotting were performed. For immunoblotting, the proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore) and immunoblotted with antibodies according to the manufacturer's instructions. Primary antibodies were detected with anti-rabbit or anti-mouse immunoglobulin coupled to horseradish peroxidase (Santa Cruz Bitoechnology) and visualized using electrochemiluminescence. Antibodies to α-Flag and α-GST were purchased from Sigma.

Example 10. Immunofluorescence

HeLa cells were grown in chamber slides and incubated in a 37 ° C CO ₂ incubator for 3 h after 5 Gy X-ray exposure. Thereafter, the cells were stained was fixed in 4% formaldehyde, and transmitted through the 0.5% Triton X-100, and wherein -TAp73 wherein -Bcl-X _L antibody.

Secondary anti-rabbit antibodies linked to Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) and secondary anti-mouse antibodies linked to Alexa Fluor 594 (Molecular Probes) were visualized with a laser scanning microscope (Zeiss LSM800 with Airyscan) Respectively. Cell nuclei were stained with DAPI according to the manufacturer's instructions, and mitochondria were stained with 50 nM Mito-Tracker Deep Red (Molecular Probes, Cat. No. M22426).

Example 11. X-ray irradiation of cells

Cells were placed in chamber slides with 80% confluence and exposed to X-rays using X-RAD 320 (Precision X-ay Inc., North Branford, USA).

Example 12. Protein expression and purification

To determine the structure of the protein, the Bcl-X _L construct and the C-terminal transmembrane domain (residues 212-233) without α1-α2 loop (45-84 residues) were expressed and purified .

For NMR experiments, ¹⁵ N- ¹⁵ of the cover 1 g for _{Bcl-X L N-NH 4} Cl or a ^{13 C, 15 N- 15 N-} NH of 1 g to a labeled Bcl-X _{L 4} Cl and The isotope-labeled Bcl-X _L protein was grown on M9 medium containing 2 g of ¹³ C-glucose.

Example 13. Peptide synthesis

Various versions of p73-TAD-derived peptides were purchased from Peptron, Inc. (Daejeon, Republic of Korea). The peptides were synthesized and stagnated at> 95% purity using high performance liquid chromatography (HPLC). The peptide sequence is as follows.

Wild-type p73-TAD16, DGGTTFEHLWSSLEPD (SEQ ID NO: 2);

p73-TAD13, TTFEHLWSSLEPD (SEQ ID NO: 3);

p73-TAD16 (F15A), DGGTTAEHLWSSLEPD (SEQ ID NO: 4);

p73-TAD16 (L18A), DGGTTFEHAWSSLEPD (SEQ ID NO: 5);

p73-TAD16 (W19A), DGGTTFEHLASSLEPD (SEQ ID NO: 6);

p73-TAD16 (L22A), DGGTTFEHLWSSAEPD (SEQ ID NO: 7)

Example 14. ITC measurement

The ITC measurement was carried out in a buffer containing 20 mM sodium phosphate at pH 6.5 at 25 ° C and 50 mM NaCl using an Auto-iTC200 Microcalorimeter (GE Healthcare) of Korea Basic Science Research Institute (Ochang, Korea) Respectively. The p73-TAD13 or p73-TAD16 peptide (3.6 mM each) was titrated with 0.12 mM Bcl-X _L in calorimeter cells. ITC data were analyzed using MicroCal Origin ^TM software.

Example 15. NMR Spectroscopy

All NMR data were collected at 25 ° C on a Bruker Avance II 800 spectrometer equipped with a cryogenic probe at the Korea Basic Science Institute (Ochang, Korea). All NMR samples were prepared in buffer containing the pH 6.5 20 mM sodium phosphate, 150 mM NaCl, 2 mM dithiothreitol (DTT), and 10% (v / v) D 2 O.

The interaction between Bcl-X _L- and p73-TAD16 peptides was carried out in the presence or absence of unlabeled p73-TAD16 peptide in the presence of 1 mM ¹⁵ N-labeled Bcl-X _L 2D ¹ H- ¹⁵ N HSQC spectra. A molar ratio of 1: the weighted average of the peak movement of the complex in 2 (weighted average chemical shift perturbation) is using the following equation ^{Δδ = {[Δδ (1 H} )] 2 + 0.2 * [Δδ (15 N)] 2} 1/2 Respectively. Sequence-specific backbone assignments were performed using 3D HNCACB and CBCA (CO) NH and side-chain assignments were performed with 1 mM ¹⁵ N, ¹³ C labeled Bcl-X _L- and 2 mM Was performed using the 3D HCCH-TOCSY and (H) CCH-TOCSY experiments with a complex of unlabeled p73-TAD16 peptide.

Distance constraints were collected from simultaneous 3D ¹³ C / ¹⁵ N-edited NOESY-HSQC spectra ⁴⁶ (τ _m = 250 ms). The distance restraint of p73-TAD16 peptide bound to Bcl-X _L- was collected using a 2D transferred NOESY ³¹ experiment at a molar ratio of 10: 1 (τm = 250 ms). Intermolecular NOEs between the Bcl-X _L and p73-TAD16 peptides were collected from ¹⁵ N / ¹³ C-edited, ¹⁵ N / ¹³ C-filtered 3D NOESY ⁴⁷ (τ _m = 250 ms). NMR data were analyzed with NMR Pipe ⁴⁸ and analyzed using CARA ⁴⁹ .

Example 16. Structural calculation

The complex structure of Bcl-X _L and p73-TAD16 peptides was calculated by the SA (simulated annealing) protocol using Xplor-NIH program ⁵⁰ . For coupled Bcl-X _L -, we used the TALOS program to predict dihedral angles for φ and ψ. The secondary structure of the bound Bcl-X _L - is derived from chemical shifts and medium-range NOE patterns in the TALOS + program to an artificial neural network (ANN) . The generic hydrogen bond restraint was applied to well-defined residues in the α-helical region. The proton distance was calculated from the NOE peak of the NOESY experiment based on the intensity. For the bound p73-TAD16 peptide, the secondary structure was predicted from secondary H _[alpha] chemical shifts using arbitrary coil values and NOE connectivity (Fig. 13).

Generic hydrogen bonds and dihedral angle restraints were used for well-defined residues in the α-helical region. Peak intensity was translated from the transferred NOESY spectrum to the inter-proton distance constraint. The original complex structure docked the p73-TAD16 peptide to Bcl-X _L - using the role of a rigid body / torsion angle and further refined the SA for all experimental limitations . The ensemble of 20 lowest-energy structures was used for verification with PROCHECK-NMR.

Example 17. Statistical analysis

Statistical analysis was performed using mean ± standard deviation (s.d.) and one way analysis of variance (ANOVA) or Student's t-test. Mean ± s.d at n ≥ 3. Are shown in the figure. A p-value of less than 0.05 was considered statistically significant.

Example 18. Use of data

The atomic coordinates of the Bcl-X _L / p73-TAD16 peptide complex were registered in the Protein Data Bank (PDB) under the number 5H3Y (Tables 2 to 22). NMR chemical shift assignments and restraints used in the structure calculations of the Bcl-X _L / p73-TAD16 peptide complexes were registered with the BMRB ID 25747 number in the Biological Magnetic Resonance Bank (BMRB) database.

The results of the experiments of the above examples were analyzed as in the following experimental examples.

Experimental Example 1. Identification of transcription-independent cell death via mitochondrial pathway of p73-TAD

The present inventors performed cell viability assays using H1299 lung cancer cells. TAp73 or p73-TAD was prepared against the cells transfected with the control vector when H1299 lung cancer cells were transfected with the full length p73 (TAp73, SEQ ID NO: 1) and p73-TAD (1-67 residues, SEQ ID NO: 8) About 50% of the expressing cells survived (Fig. 1 (a)).

In cell survival, the effect of TAp73 or p73-TAD was additionally confirmed through a colony-forming assay. Overexpression of p73-TAD in H1299 cells inhibited colony formation. However, it was suppressed to a lower extent than TAp73 (Fig. 1 (b)).

As shown in the DNA fragmentation assay data (FIG. 2 a), an analysis of the sub-G1 population of H1299 cells transfected with TAp73 or p73-TAD revealed that both constructs had approximately three times more apoptosis than the control .

In addition, the results of Annexin V-PE cell death and caspase-3/7 activation assays show that apoptosis induced by TAp73 and p73-TAD is mediated by caspase- It indicates death.

To exclude the effect of inducing transcription-dependent apoptosis via TAp73 transactivation, we performed cell survival assays in the presence of the transcription inhibitor actinomycin D (ActD). No significant cell viability changes were observed by ActD treatment (FIG. 3 a). In addition, it was confirmed that TAp73 (A156V), a transcription-inactivated TAp73 mutant, induces apoptosis similar to TAp73, suggesting that p73 can induce apoptosis in a transcription-independent manner (Fig. 3b) .

Analysis of cytochrome c release from mitochondria confirmed that transcription-independent apoptosis mediated by TAp73 and p73-TAD occurs via the mitochondrial pathway (FIG. 1e).

 To determine whether Bax and Bak, the apoptosis inducing proteins of the Bcl-2 family, are required for the induction of transcription-independent cell death, we used caspase-3 / 7-active assay was performed (Figure 4).

As a result, it was confirmed that TAp73- or p73-TAD-induced apoptosis was not observed in the cells due to the loss of Bax and Bak activity, and it was confirmed that the activation of Bax or Bak in the transcription-independent p73- Is required.

Taken together, these results indicate that p73-TAD mediates transcription-independent apoptosis through the intrinsic, mitochondrial pathway.

Experimental Example 2. Anti-apoptotic physicological relationship between Bcl-2 family protein and p73-TAD

To determine if p73-TAD is involved in the interaction with anti-apoptotic Bcl-2 family proteins, we performed glutathione S-transferase (GST) pool-down assay to determine the expression of p73-TAD and anti- And to detect complex formation between the apoptotic Bcl-2 family proteins (FIG. 5).

When HEK293T cells were co-transfected with GST-tagged p73-TAD with the anti-apoptotic Bcl-2 family proteins Bcl-X _L , Bcl-2, or Bcl-w, Glutathione agarose beads, confirming that p73-TAD complexed with the anti-apoptotic Bcl-2 family protein.

In addition, immunoprecipitation results showed that when H1299 cells were cotransfected with TAp73 or p73-TAD and Bcl-X _L -, Bcl-X _L - / TAp73 and Bcl-X _L - / p73-TAD complex (Fig. 2 (b)). The same cells overexpressing TAp73 were also found to bind to endogenous Bcl-X _L.

In order to confirm that such a protein complex is formed between endogenous proteins, the present inventors conducted immunoprecipitation experiments using the same cells treated with DNA-damaging reagent etoposide.

As a result, TAp73 formed a specific interaction with Bcl-X _L and Bcl-2. In contrast, endogenous TAp73 did not interact with the BH3-only proteins Bad, PUMA, Bik, Bid, and Bim, which induce apoptosis (Figure 6).

To understand the localization of TAp73 and Bcl-X _L- , the present inventors performed immunofluorescence using a confocal microscope in HeLa cells (FIG. 5e).

As a result, the endogenous TAp73 was observed that most, but present, X- ray irradiation after was much movement in the mitochondria and the cytoplasm, where with TAp73 and Bcl-X _L- are in the mitochondria and cytosol from the nucleus (Fig. 5 e, below panel and drawing). The mitochondrial location of TAp73 and p73-TAD was further confirmed by subcellular fractionation and Western blotting (Fig. 7).

Experimental Example 3. Bcl-X _L- -Independent cellular apoptosis regulation of p73-TAD by

To confirm the effect of the interaction with the transcription-independent p73-mediated p73-Bcl-X _L on cell death, we transfected Bcl-X _L with TAp73 or p73-TAD into H1299 cells And the cell survival experiment was carried out using the transfected cells (Fig. 8 (a)).

As a result, it was confirmed that transcription-independent cell death induced by TAp73 or p73-TAD was inhibited by overexpression of Bcl-X _L- . This was also confirmed by Annexin V-PE staining (Figure 8b) and DNA fragmentation assay results (Figure 2b). In addition, the release of mitochondrial cytochrome c induced by TAp73 or p73-TAD was also inhibited by overexpression of Bcl-X _L (FIG. 8 c).

These results indicate that the transcription-independent cell death induced by p73-TAD is regulated by Bcl-X _L.

Experimental Example 4 Bcl-X by p73-TAD _L Release of Bid from

The present inventors have found that cell death induced BH3 only protein Bid and Bcl-X _L is a direct interaction, and were expected to compete with p73 Bid for binding to Bcl-X _L. In order to confirm this, the present inventors conducted a competition experiment using immunoprecipitation with Bcl-X _L, Myc-Bid, and green fluorescent protein (GFP) -TAp73 or GFP-p73-TAD ).

Bid interacted with Bcl-X _L , but did not interact with TAp73 (Figure 6). In particular, the inventors have found that by combining and TAp73 and p73-TAD both Bcl-X _L and disassemble the Bcl-X _L / Bid composite, whereby, Bid the capacity from the Bcl-X _L - it was confirmed that the release dependent.

These results indicate that p73-TAD induces mitochondrial apoptosis in a transcription-independent manner by releasing Bid, a direct apoptotic activity factor, from Bcl-X _L.

Experimental Example 5: Bcl-X of p73-TAD16 _L  Binding-independent cell death

In order to confirm the motif of p73-TAD involved in Bcl-X _L binding, the inventors of the present invention performed immunization using GFP-tagged 16-mer p73-TAD peptide (10-25 residues, hereinafter referred to as p73-TAD16) Sedimentation experiments were carried out. As a result, it was confirmed that GFP-p73-TAD16 specifically binds to Bcl-X _L (Fig. 10A).

To confirm whether the motif binding to Bcl-X _L alone induces transcription-independent cell death, we performed cell survival and cell death assays with p73-TAD16.

As a result, the results of cell survival, caspase-3/7 activity, annexin V-PE cell death, and DNA fragmentation assay indicate that p73-TAD16 can induce transcription-independent cell death at a level similar to p73-TAD Respectively. In addition, it was also confirmed that such cell death was inhibited by overexpression of Bcl-X _L (FIG. 10 b to e; FIG. 2 c).

Furthermore, it has also been shown that p73-TAD16 induces the release of cytochrome c from the mitochondria, suggesting that p73-TAD16 induces transcription-independent cell death through the intrinsic mitochondrial pathway.

In addition, to analyze whether binding of p73-TAD16 with Bcl-X _L is necessary to mediate transcription-independent p73-induced cell death, we prepared p73-TAD mutants that do not contain TAp73 and p73-TAD16, The binding of p73-TAD16 and Bcl-X _L was degraded and caspase-3/7 activity assays were used to determine if the mutants could induce apoptosis.

As a result, it was confirmed that TAp73 (Δ1-25) and p73-TAD (Δ1-25) mutants lost a significant portion of apoptotic activity compared to the wild type (FIG. This indicates that the binding of p73-TAD16 and Bcl-X _L is important for induction of transcription-independent p73-mediated apoptosis.

That is, the above results suggest that p73-TAD16 mediates transcription-independent cell death through direct binding with Bcl-X _L.

Experimental Example 6: Bcl-X _L / p73-TAD16 complex structure

In order to analyze the characteristics of molecular interaction between Bcl-X _L and p73-TAD16 peptides, we used nuclear magnetic resonance spectroscopy (NMR) to measure the chemical shift perturbation of Bcl-X _L with p73-TAD16 peptide ) (Fig. 11 (a)).

As a result, it was confirmed from the result of 2D ¹ H- ¹⁵ N HSQC (Heteronuclear Single Quantum Coherence) that most of the cross-peptides of Bcl-X _L migrated significantly by binding with p73-TAD16 peptide b).

The peak changes induced by the p73-TAD16 peptide binding were mostly mapped to the? 2-a5 and? 8 helical structures corresponding to the BH1, BH2, and BH3 domains (FIG. 11 b). This suggests that the region is involved in binding to the p73-TAD16 peptide. From the results of the isothermal titration calorimetry (ITC) measurement, peptides p73-TAD16 and p73-TAD13 (13-25 residues, SEQ ID NO: 3) bind to Bcl-X _L at K _D values of 80 and 87 μM, respectively (Fig. 12 (c) and (d)).

In order to understand the structure of the molecular interaction between the Bcl-X _L and p73-TAD16 peptides, the present inventors confirmed the structure of the Bcl-X _L - / p73-TAD16 peptide complex using NMR (FIG. The structure of p73-TAD16 peptide bound to Bcl-X _L was calculated using a distance restraint derived from nuclear overhauser effect spectroscopy (NOESY) (Fig. 13).

NOE between molecules were obtained from ¹³ C, ¹⁵ N- filtered spectrum of binding to unlabeled p73-peptide TAD16, ¹³ C, ¹⁵ N--labeled Bcl-X _L. From the NOE, we calculated the structure of the Bcl-X _L / p73-TAD16 peptide complex. The statistics of the final 20 low-energy NMR structures are shown in Table 1.

As is consistent with the observed peak change (Fig. 11a), the p73-TAD16 peptide binds to the hydrophobic groove of Bcl-X _L in which BH1, BH2, BH3 domains and a3 helix are clustered together. The p73-TAD16 peptide is inherently disordered in the free state, but the F15-L22 residue has an amphiphilic a-helical structure due to its binding to Bcl-X _L (Fig. 13).

In the helical structure, the hydrophobic moiety faces the hydrophobic groove of Bcl-X _L , and the remaining hydrophilic moiety is exposed to the solvent (Fig. 11 (c)). A structural comparison between Bcl-X _L / p73-TAD16 peptide and Bcl-X _L / BH3 peptide (Bak, Bad, Bim and BH3 of PUMA) showed that the overall folding of Bcl-X _L bound to p73- -X _L / BH3 peptide complex (Fig. 14).

The complex structure of Bcl-X _L / p73-TAD16 identified by the present inventors is shown in Tables 2 to 22 below.

Experimental Example 7 Bcl-X _L  And &lt; RTI ID = 0.0 &gt; p73-TAD16 &lt;

As shown in the Bcl-X _L / Bad BH3 peptide and Bcl-X _L / Bim BH3 peptide complexes of the above examples, the Bcl-X _L / p73-TAD16 complex exhibits a broad hydrophobic action (F15, L18, W19, L22 of p73- ; and Bcl-X _L - liver function of A93, F97, V141, A142, F191, L194, Y195) and electrostatic action (E23, D25 of the p73-TAD16; and Bcl-X _L - liver function of R100, R139) (Fig. 11 (d)).

In particular, F97 and Y195 of Bcl-X _L are involved in? -Stacking binding of p73-TAD16 peptide with W19 and F15, respectively (FIG. 11 e). As is consistent with the intramolecular π-stacking interactions observed in the structure of the Bcl-X _L / PUMA BH3 peptide complex, such interactions can contribute significantly to the stability of the protein conjugate.

In order to confirm the main determinant factors involved in the binding of Bcl-X _L and p73-TAD16 peptides, the present inventors carried out NMR binding experiments using p73-TAD16 mutants containing alanine substitutions (F15A, L18A, W19A and L22A) Respectively.

As a result, ¹⁵ as shown in the spectrum of the 2D ¹ H- ¹⁵ N HSQC N--labeled Bcl-X _L and the mutant peptide (FIG. 16), due to the alanine mutant of the p73-TAD16, observed in the binding of the wild-type peptide NMR peak change completely disappears.

Results as described above, the p73-TAD16 bulky (bulky) hydrophobic residues in the motif indicates that play an important role in the p73-TAD16 and the interaction Bcl-X _L.

Experimental Example 8. Bcl-X by p73-TAD _L  New mode of recognition

Despite the overall similarities (Fig. 14), Bcl-X _L - structure / p73-peptide complexes are TAD16 Bcl-X _L - has the structure with the difference between / BH3 peptide complexes. The p73-TAD16 peptide binds to a unique position that differs from the typical binding groove of apoptosis-inducing BH3 peptide (Figure 16, a and b).

Bak, Bad, Bid, and PUMA are known to bind to a typical groove formed by the BH1 and BH3 domains in Bcl-X _L (Fig. 16 (c)).

On the other hand, the p73-TAD16 peptide binds at non-typical positions including the BH1, BH2, and BH3 domains of Bcl-X _L - (FIG. 16 a). At the binding junction, nine residues of Bcl-X _L are involved in forming interactions with the six residues of p73-TAD16 (F15, L18, W19, L22, E23, D25) and BH1 (R139, V141, A142 ), BH2 (F191, L194, Y195), and BH3 (A93, F97, R100) (Fig.

In addition, the p73-TAD16 peptide binds to Bcl-X _L in the opposite direction to the cell death-inducing BH3 peptide of Bak, Bad, Bid, PUMA (FIG. 16).

From the above experiments, the present inventors confirmed that the p73-TAD16 peptide can induce apoptosis in a transcription-independent manner through interaction with Bcl-X _L inhibiting apoptosis. In addition, it was confirmed that, p73-TAD16 / identified by the structure of Bcl-X _L complex, may, for screening an anticancer agent that induces apoptosis using the structure.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY An anti-cancer peptide inhibiting Bcl-XL and a method for          screening an anti-cancer agent <130> KPA170373-KR <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 636 <212> PRT <213> Homo sapiens <400> 1 Met Ala Gln Ser Thr Ala Thr Ser Pro Asp Gly Gly Thr Thr Phe Glu   1 5 10 15 His Leu Trp Ser Ser Leu Glu Pro Asp Ser Thr Tyr Phe Asp Leu Pro              20 25 30 Gln Ser Ser Arg Gly Asn Asn Glu Val Val Gly Gly Thr Asp Ser Ser          35 40 45 Met Asp Val Phe His Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln      50 55 60 Phe Asn Leu Leu Ser Ser Thr Met Asp Gln Met Ser Ser Ala Ala  65 70 75 80 Ser Ala Ser Pro Tyr Thr Pro Glu His Ala Ala Ser Val Pro Thr His                  85 90 95 Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Thr Met Ser Pro Ala             100 105 110 Pro Val Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro His His Phe Glu         115 120 125 Val Thr Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala Thr Trp Thr Tyr     130 135 140 Ser Pro Leu Leu Lys Lys Leu Tyr Cys Gln Ile Ala Lys Thr Cys Pro 145 150 155 160 Ile Gln Ile Lys Val Ser Thr Pro Pro Pro Gly Thr Ala Ile Arg                 165 170 175 Ala Met Pro Val Tyr Lys Lys Ala Glu His Val Thr Asp Val Val Lys             180 185 190 Arg Cys Pro Asn His Glu Leu Gly Arg Asp Phe Asn Glu Gly Gln Ser         195 200 205 Ala Pro Ala Ser His Leu Ile Arg Val Glu Gly Asn Asn Leu Ser Gln     210 215 220 Tyr Val Asp Asp Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Tyr 225 230 235 240 Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Ile Leu Tyr Asn Phe                 245 250 255 Met Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg Arg Pro Ile Leu             260 265 270 Ile Ile Ile Thr Leu Glu Met Arg Asp Gly Gln Val Leu Gly Arg Arg         275 280 285 Ser Phe Glu Gly Arg Ile Cys Ala Cys Pro Gly Arg Asp Arg Lys Ala     290 295 300 Asp Glu Asp His Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser Ser Ala 305 310 315 320 Lys Asn Gly Ala Ala Ser Lys Arg Ala Phe Lys Gln Ser Pro Ala                 325 330 335 Val Pro Ala Leu Gly Ala Gly Val Lys Lys Arg Arg His Gly Asp Glu             340 345 350 Asp Thr Tyr Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu         355 360 365 Met Lys Leu Lys Glu Ser Leu Glu Leu Met Glu Leu Val Pro Gln Pro     370 375 380 Leu Val Asp Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser 385 390 395 400 His Leu Gln Pro Pro Ser Tyr Gly Pro Val Leu Ser Pro Met Asn Lys                 405 410 415 Val His Gly Gly Met Asn Lys Leu Pro Ser Val Asn Gln Leu Val Gly             420 425 430 Gln Pro Pro Pro His Ser Ser Ala Ala Thr Pro Asn Leu Gly Pro Val         435 440 445 Gly Pro Gly Met Leu Asn Asn His Gly His Ala Val Pro Ala Asn Gly     450 455 460 Glu Met Ser Ser Ser His Ser Ala Gln Ser Met Val Ser Gly Ser His 465 470 475 480 Cys Thr Pro Pro Pro Pro Tyr His Ala Asp Pro Ser Leu Val Ser Phe                 485 490 495 Leu Thr Gly Leu Gly Cys Pro Asn Cys Ile Glu Tyr Phe Thr Ser Gln             500 505 510 Gly Leu Gln Ser Ile Tyr His Leu Gln Asn Leu Thr Ile Glu Asp Leu         515 520 525 Gly Ala Leu Lys Ile Pro Glu Gln Tyr Arg Met Thr Ile Trp Arg Gly     530 535 540 Leu Gln Asp Leu Lys Gln Gly His Asp Tyr Ser Thr Ala Gln Gln Leu 545 550 555 560 Leu Arg Ser Ser Asn Ala Thr Ile Ser Ile Gly Gly Ser Gly Glu                 565 570 575 Leu Gln Arg Gln Arg Val Met Glu Ala Val His Phe Arg Val Arg His             580 585 590 Thr Ile Thr Ile Pro Asn Arg Gly Gly Pro Gly Gly Gly Pro Asp Glu         595 600 605 Trp Ala Asp Phe Gly Phe Asp Leu Pro Asp Cys Lys Ala Arg Lys Gln     610 615 620 Pro Ile Lys Glu Glu Phe Thr Glu Ala Glu Ile His 625 630 635 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> p73-TAD16 <400> 2 Asp Gly Gly Thr Thr Phe Glu His Leu Trp Ser Ser Leu Glu Pro Asp   1 5 10 15 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> p73-TAD13 <400> 3 Thr Thr Phe Glu His Leu Trp Ser Ser Leu Glu Pro Asp   1 5 10 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> p73-TAD16 (F15A) <400> 4 Asp Gly Gly Thr Thr Ala Glu His Leu Trp Ser Ser Leu Glu Pro Asp   1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> p73-TAD16 (L18A) <400> 5 Asp Gly Gly Thr Thr Phe Glu His Ala Trp Ser Ser Leu Glu Pro Asp   1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> p73-TAD16 (W19A) <400> 6 Asp Gly Gly Thr Thr Phe Glu His Leu Ala Ser Ser Leu Glu Pro Asp   1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> p73-TAD16 (L22A) <400> 7 Asp Gly Gly Thr Thr Phe Glu His Leu Trp Ser Ser Ala Glu Pro Asp   1 5 10 15 <210> 8 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> p73-TAD <400> 8 Met Ala Gln Ser Thr Ala Thr Ser Pro Asp Gly Gly Thr Thr Phe Glu   1 5 10 15 His Leu Trp Ser Ser Leu Glu Pro Asp Ser Thr Tyr Phe Asp Leu Pro              20 25 30 Gln Ser Ser Arg Gly Asn Asn Glu Val Val Gly Gly Thr Asp Ser Ser          35 40 45 Met Asp Val Phe His Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln      50 55 60 Phe Asn Leu  65 <210> 9 <211> 233 <212> PRT <213> Homo sapiens <400> 9 Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys   1 5 10 15 Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser Asp Val Glu Glu              20 25 30 Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro          35 40 45 Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala      50 55 60 Val Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp Ala Arg Glu Val  65 70 75 80 Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu Ala Gly Asp Glu                  85 90 95 Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gln Leu             100 105 110 His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu Gln Val Val Asn         115 120 125 Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe     130 135 140 Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gln 145 150 155 160 Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp                 165 170 175 His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp Asp Thr Phe Val             180 185 190 Glu Leu Tyr Gly Asn Asn Ala Ala Glu Ser Arg Lys Gly Gln Glu         195 200 205 Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val     210 215 220 Leu Leu Gly Ser Leu Phe Ser Arg Lys 225 230

Claims (16)

A prophylactic or therapeutic cancer preventive or therapeutic peptide consisting of 13 to 70 consecutive amino acids comprising the amino acid sequence of SEQ ID NO: 2 in the amino acid sequence of SEQ ID NO: 1.
2. The peptide according to claim 1, wherein the peptide for preventing or treating cancer is composed of 16 to 67 amino acids.
A prophylactic or therapeutic peptide for cancer comprising an amino acid sequence of the following general formula:
DGGTTX ₁ EHX ₂ X ₃ SSX ₄ EPD (general formula 1)
In the general formula 1,
X ₁ to X ₄ are phenylalanine, tryptophan, isoleucine, leucine, proline, methionine, or valine, respectively.
2. The peptide for preventing or treating cancer according to claim 1, wherein the cancer preventive or therapeutic peptide comprises the amino acid sequence of SEQ ID NO: 2.
The cancer prevention or treatment peptide according to any one of claims 1 to 4, wherein the peptide for preventing or treating cancer induces apoptosis in a transcription-independent manner by interacting with Bcl-X _L .
5. The peptide according to any one of claims 1 to 4, wherein said cancer preventive or therapeutic peptide induces release of cytochrome c from mitochondria.
A method for inducing apoptosis in a transcription-independent manner by complexing Bcl-X _L in vitro or ex vivo, comprising the step of treating the peptide according to any one of claims 1 to 4.
A method for inducing the release of cytochrome c from mitochondria in vitro or ex vivo, comprising the step of treating a peptide according to any one of claims 1 to 4.
A pharmaceutical composition for preventing or treating cancer, which comprises the peptide for cancer prevention or treatment according to any one of claims 1 to 6.
10. The method of claim 9, wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, colon cancer, breast cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, Cancer of the prostate, cancer of the endometrium, cancer of the endometrium, cancer of the uterine cervix, vaginal cancer, Hodgkin's disease, esophageal cancer, lymphadenocarcinoma, bladder cancer, endometrioid cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, Or prevention or treatment of cancer which is acute leukemia, lymphocytic lymphoma, renal cancer, ureter cancer, renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma or pituitary adenoma A pharmaceutical composition.
The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
(a) interaction between the p73-TAD16 peptide and the Bcl-X _L protein using the tertiary structure of the p73-TAD16 peptide consisting of the amino acid sequence of SEQ ID NO: 2 and the Bcl-X _L protein complex consisting of the amino acid sequence of SEQ ID NO: Generating or screening a regulatory candidate material,
Wherein the tertiary structure is defined by atomic coordinates as set forth in any one of Tables 2 to 22; And
(b) confirming whether or not the interaction between Bcl-X _L and p73-TAD16 of the candidate substance generated or selected in the step (a)
A method of screening for a substance that modulates the interaction between Bcl-X _L and p73-TAD16.
(a) using a three-dimensional structure of Bcl-X _L protein complex consisting of the amino acid sequence of SEQ ID NO: 2 the amino acid sequence consisting of p73-TAD16 peptide and SEQ ID NO: 9 generates a candidate to inhibit the Bcl- X _L or A step of sorting,
Wherein the tertiary structure is defined by atomic coordinates as set forth in any one of Tables 2 to 22; And
(b) confirming whether the candidate substance generated or selected in the step (a) inhibits Bcl-X _L ,
A method for screening a substance that inhibits Bcl-X _L.
14. The method of claim 13, wherein the Bcl-X _L inhibiting substance mimics the structure of the p73-TAD16 peptide.
Methods for screening for substances that inhibit Bcl-X _L
(a) a step of generating or screening a candidate substance for an anticancer drug using the tertiary structure of the p73-TAD16 peptide consisting of the amino acid sequence of SEQ ID NO: 2 and the Bcl-X _L protein complex consisting of the amino acid sequence of SEQ ID NO: 9,
Wherein the tertiary structure is defined by atomic coordinates as set forth in any one of Tables 2 to 22; And
(b) confirming the anticancer effect of the candidate substance generated or selected in the step (a).
A method for screening an anticancer agent.
The method of claim 15, wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, colon cancer, breast cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, colon cancer, small bowel cancer, Cancer of the prostate, cancer of the endometrium, cancer of the endometrium, cancer of the uterine cervix, vaginal cancer, Hodgkin's disease, esophageal cancer, lymphadenocarcinoma, bladder cancer, endometrioid cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, Or an anticancer agent selected from the group consisting of acute leukemia, lymphocytic lymphoma, renal cancer, ureteral cancer, renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma or pituitary adenoma Way.

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