KR20130046282A - Peptides comprising mdm2-binding motif and use thereof - Google Patents

Abstract

PURPOSE: A peptide containing an mdm2-binding motif and a use thereof are provided to suppress the function of mdm2, to activate p53 pathway, and to promote cancer cell apoptosis by suppressing binding of p53 and mdm2. CONSTITUTION: A peptide or a pharmaceutically acceptable salt thereof contains an mdm2(mouse double minute 2)-binding motif amino acid sequence of sequence number 1. The peptide contains the mdm2-binding motif with an amino acid sequence of sequence number 1 or 2. A pharmaceutical composition for preventing or treating cancer contains the peptide or a pharmaceutically acceptable salt thereof as an active ingredient. Cancer is selected from the group consisting of leukemia, lymphoma, esophageal carcinoma, neuroblastoma, soft tissue cancer, breast cancer, colon carcinoma, lung cancer, and stomach cancer.

Description

Peptides containing mdm2-binding motifs and uses thereof

The present invention relates to a peptide comprising an mdm2-binding motif and its use. More specifically, the present invention relates to an mdm2-binding motif sequence of SEQ ID NO: 1 capable of binding to mdm2 (mouse double minute 2) and inhibiting its function. Mdm2 function inhibitory peptide or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for the treatment or prevention of cancer diseases comprising the peptide as an active ingredient, and the therapeutic agent for cancer diseases using the mdm2-binding motif It is about how to.

The p53 protein is a protein involved in cell growth and apoptosis, and acts as a tumor suppressor gene. Inactivation by p53 mutation causes cancer. One such p53 inactivation mechanism is overexpression of the mdm2 protein, a cellular oncogene. p53 is automatically regulated by a feedback loop involving the mdm2 protein, which binds to the transcriptional activation domain (TAD) of p53 and inhibits p53 function, and mdm2 binds to p53 to promote p53 degradation in a ubiquitin-dependent pathway. To promote cancer [Oliner, JD et al . (1993) Nature 362, 857; Vassilev, LT meat al . (2004) Science 303, 844-848; and Maki et al . (1999) J. Biol. Chem. 274, 16531.

As it is found that mdm2 protein is amplified or overexpressed in many malignant tumors of humans, thereby inhibiting the function of p53, activation of the p53 pathway through the inhibition of mdm2 is a new target of cancer drug development.

One field of research is to develop a small molecular weight formulation that can inhibit the formation of a conjugate between p53 and mdm2, and to treat cancer by inhibiting the interaction of mdm2 with p53 in WO00 / 15657, US Pat. Useful piperazine derivatives are described, and International Application Publication No. WO03 / 051360 describes cis-imidazoline, which is useful for the treatment of breast cancer, colorectal cancer, lung cancer and the like as an mdm2 protein inhibitor. International application WO03 / 095625 describes 1,4-benzodiazepines that inhibit the interaction of mdm2 and p53. International Application WO03 / 106384A2 discloses boronic chalcone derivatives that inhibit the expression of mdm2 or the formation of mdm2 conjugates, see Stoll, Stoll, R. et. al . (2001) Biochemistry 40, 336-344, describe chalcone derivatives that inhibit the binding of mdm2 to p53.

Another field relates to the development of mdm2-specific antisense oligonucleotides that inhibit mdm2 protein expression. International applications WO99 / 49065, WO99 / 10486, US Patent US6013786 and US6238921, and European Patent EP1007658 and the like disclose such antisense oligonucleotides. Providing.

There have also been studies of peptides that specifically bind to mdm2 and inhibit the binding of p53 and mdm2. As part of this study, the present inventors have applied for a patent, confirming that the fragment of p53 can bind to mdm2 to inhibit mdm2 function and activate p53 (Korean Patent No. 10-0788928). These p53-derived peptides have the same recognition site for binding to mdm2 and compete with p53, leading to stabilization of p53. However, p53-derived peptides have problems of limited mdm2 inhibition and penetration into cells, and their practical use is very limited.

Therefore, the present inventors have newly conducted a new study to develop a peptide that can bind mdm2 and inhibit the function of mdm2 competitively with p53, and newly identified that a specific site of the protein named SUSP4 acts as an mdm2-binding motif. In addition, peptides containing the motif were confirmed to promote cancer cell death by activating p53 by binding to mdm2 competitively at binding sites such as p53, and completed the present invention.

It is an object of the present invention to provide a SUSP4 protein derived peptide or a pharmaceutically acceptable salt thereof that binds to mdm2 and inhibits mdm2 function.

Another object of the present invention to provide a pharmaceutical composition for the treatment or prevention of cancer diseases comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide a method for screening a therapeutic agent for cancer diseases using the peptide or a pharmaceutically acceptable salt thereof.

As one aspect for achieving the above object, the present invention provides a peptide for inhibiting mdm2 function derived from SUSP4 protein or a pharmaceutically acceptable salt thereof comprising the mdm2-binding motif sequence of SEQ ID NO: 1.

The term "peptide" of the present invention means a polymer consisting of amino acids linked by amide bonds or peptide bonds. For the purposes of the present invention, "mdm2-binding motif" refers to a peptide of conservative sequence capable of specifically binding to mdm2 and inhibiting the function of mdm2. The peptide is a peptide having an activity capable of binding to a p53 binding site on mdm2, and acts as a competitive substrate for p53, thereby inhibiting the binding of p53 and mdm2. The mdm2-binding motif according to the present invention has the amino acid sequences of SEQ ID NOs: 1 and 2.

In the present invention, "peptide including an mmd2-binding motif" is a peptide comprising mdm2-binding motifs of SEQ ID NO: 1 and 2 as long as the peptide can bind to the p53 binding site on mdm2 and inhibit the function of mdm2 without limitation It is included in the scope of the invention.

In a preferred embodiment of the invention, the peptides comprising the mdm2-binding motif of the invention are peptides P4S19 and P4L29 having the amino acid sequence of SEQ ID NO: 1 or 2 below.

P4S19: YQILVITEDIEKEIENALG (SEQ ID NO: 1)

P4L29: LTDQEKGREMYQILVITEDIEKEIENALG (SEQ ID NO: 2)

In the present invention, nuclear magnetic resonance spectroscopy and spin paramagnetic resonance spectroscopy have found that sites containing 273-291 residues and 263-291 residues in SUSP4 (201-300) are involved in mdm2 binding. The fragment portion is the same in mdm2 (3-109) to which p53-derived α-helix fragment peptide known to show anticancer function by binding to the hydrophobic binding site present in mdm2 (3-109) and p53 TAD (1-73) binds. Since it binds to a hydrophobic binding site, it was confirmed that the fragment peptide could inhibit the function of mdm2.

When a small protein called SUMO is bound to Mdm2, Mdm2 is stabilized to decrease the amount of p53. SUSP4 (SUMO-1 specific protease 4, Accession No. NP_694733) (201-300) removes SUMO bound to Mdm2. Action has been reported to promote the degradation of Mdm2 and consequently to increase the amount of p53, a carcin suppressor [Lee, MH et al. (2006) Nat. Cell Biol. 8, 1424-1431. The present invention has been found by the NMR technique and SPR technique that the essential motif is involved in the binding mechanism with mdm2, the inventors found that the fragment corresponding to 201-300 residues of SUSP4 is an amorphous protein without tertiary structure, but with mdm2 It was confirmed that it had a partial secondary structure before binding (see FIG. 5). In this regard, the present inventors first identified P4S19 and P4L29 as motif sequences necessary for binding to mdm2 in the SUSP4 (201-300) fragment.

According to an embodiment of the present invention, peptides P4S19 and P4L29 comprising the mdm2-binding motif of the present invention in chemical shift shift experiments and X-ray crystal structure bind the same as p53 TAD (1-73) and p53 α-helix fragment peptides on mdm2. Competitive binding to the site was confirmed (see FIGS. 9 and 10).

According to another embodiment of the present invention, the BIAcore experiments confirmed that the peptides P4S19 and P4L29 including the mdm2-binding motif of the present invention can specifically bind to the mdm2 (3-109) protein (see FIGS. 11 and 12). ).

Further, according to another embodiment of the present invention, the peptides P4S19 and P4L29 comprising the mdm2-binding motif of the present invention competitively bind to the same binding site as p53 TAD (1-73) and p53 α-helix fragment peptide on mdm2. It was confirmed that the cancer cell killing effect by inhibiting the function of mdm2 and activating p53 binding thereto (see FIGS. 13 and 14).

The term "pharmaceutically acceptable salts" of the present invention means all salts that retain the desired biological and / or physiological activity of the peptide, and exhibit minimal unwanted toxicological effects. For example, the peptide may contain inorganic acids (e.g. hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (e.g. acetic acid, halo acetic acid such as trifluoroacetic acid, propionic acid, maleic acid, succinic acid). , Malic acid, citric acid, tartaric acid, salicylic acid), and acidic sugars (e.g. glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides (e.g. hyaluronic acid, chondroitin sulfate, arginine acid), chondroitin sulfate It may be a salt formed by adding sulfonic acid such as pate, organic sulfonic acid including sugar esters (eg, methane sulfonic acid, p-toluene sulfonic acid), and the like, but is not particularly limited thereto.

In another aspect of the invention, the present invention provides a pharmaceutical composition for the treatment or prevention of cancer diseases comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a method for preventing or treating cancer diseases, comprising administering the pharmaceutical composition to a subject in need thereof.

In many cancers, amplification and overexpression of mdm2 is observed independent of p53 mutations. For example, increased levels of mdm2 transcription are observed in leukemia, lymphomas, and amplification of mdm2 in esophageal carcinomas, neuroblastoma, soft tissue tumors, and the like. do. Soft tissue cancers showed amplification of mdm2 in Ewing's sarcoma, leiomysarcoma, lipomas, liposarcoma, malignant fibrous histiocytomas, and maliginat schwannomas. (Jamil Momand et al, (1998) Nucleic Acids Research, 26, 3453-3459).

Therefore, the pharmaceutical composition of the present invention comprises a peptide containing a mdm2-binding motif capable of binding to mdm2 and inhibiting its function instead of p53 or a pharmaceutically acceptable salt thereof as an active ingredient, thereby amplifying and overexpressing mdm2. Prevent or develop various cancer diseases caused by soft tissue cancer such as leukemia, lymphoma, esophageal carcinoma, neuroblastoma, and Ewing's sarcoma, leiomyosarcoma, lipoma, liposarcoma, fibrous histiocytoma, malignant schwannoma It can be usefully used to treat the cancers.

The pharmaceutical composition of the present invention may be formulated into a suitable formulation with a pharmaceutically acceptable carrier and administered by various routes. The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. The carrier can be used without limitation so long as it is known in the art such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants. Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Typically available surfactants that facilitate migration across the membrane are those derived from steroids, but N- [1- (2,3-dioleoyl) propyl-N, N, N-trimethylammonium chloride] (DOTMA Cationic lipids, or cholesterol hemisuccinate.

In the present invention, the term "cancer disease" refers to a disease associated with regulation of cell death and is caused by overproliferation of cells when a normal apoptotic balance is broken. These abnormal hyperproliferative cells sometimes invade surrounding tissues and organs to form a mass, which destroys or transforms normal structures in the body. Such a condition is called cancer. Preferably, "cancer disease" in the present invention means a disease caused by p53 inactivation or overexpression or amplification of mdm2 due to the binding of p53 and mdm2.

In the present invention, the term "prevention" means any action that inhibits metastasis, growth, etc. or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention, and "treatment" means the administration of the pharmaceutical composition by administration of the pharmaceutical composition. Means any behavior that improves or beneficially changes the symptoms caused by cancer.

In the present invention, the term "individual" means all animals, including humans, who may or may have a cancer, and can effectively prevent or treat the cancer by administering the pharmaceutical composition of the present invention to an individual. The pharmaceutical composition of the present invention can be administered in parallel with existing anticancer agents.

The term "administration" of the present invention means introducing a predetermined substance into a patient in an appropriate manner, and the route of administration of the composition may be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may include at least one excipient such as starch, calcium carbonate, sucrose, or lactose, Prepare by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, flavoring agents, preservatives, etc. Can be. However, upon oral administration, since the peptides are easy to digest, it is desirable to formulate oral compositions to coat the active agent or protect it from degradation in the stomach. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. The suppository base includes Uittepsol, Macrogol, and Twin 61. Cacao butter, laurin butter, glycerogelatin and the like can be used. Carbohydrates such as glucose, sucrose, dextran, antioxidants such as ascorbic acid, glutathione, chelating substances, small molecule proteins or other stabilizers may be used to increase the stability or absorption of the peptide.

In addition, the pharmaceutical compositions of the present invention may be administered by any device in which the active substance may migrate to the target cell. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial growth Preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

On the other hand, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. Generally, the active substance can be administered at a dose of about 0.01 mg / kg / day to 1000 mg / kg / day. In the case of oral administration, a range of 50 to 500 mg / kg may be suitable and may be administered at least once a day.

According to another aspect of the present invention, the present invention provides a method for screening a therapeutic agent for cancer diseases using mdm2-binding motif.

The screening method according to the present invention,

1) synthesizing a peptide comprising an mdm2-binding motif;

2) analyzing whether the synthesized peptide can bind to mdm2 by competitively acting on the p53 binding site in mdm2; And

3) When the synthesized peptide binds to mdm2, the peptide may be determined as a therapeutic agent for cancer diseases.

Step 1) is a step of synthesizing any peptide comprising the sequence using the mdm2-binding motif having the amino acid sequence of SEQ ID NO: 1 according to the present invention. In this step, the peptide can be obtained by chemical or recombinant methods using a known peptide synthesizer.

Step 2) is a step of checking whether the peptide containing the synthesized mdm2-binding motif has binding activity with respect to mdm2 in the presence of p53. The binding of mdm2 to a peptide comprising an mdm2-binding motif at this step can be confirmed by any method capable of analyzing peptide-peptide binding known in the art, for example, the chemical shift described herein. shift) can be performed through variation analysis.

Step 3) is a step of determining the peptide as a therapeutic agent for cancer diseases when it is determined that the synthesized peptide binds to mdm2 as a result of the analysis in step 2).

The peptide containing the mdm2-binding motif of the present invention competitively binds to mdm2 at the same site as the p53 binding site on mdm2, thereby inhibiting the binding of p53 and mdm2, thereby inhibiting the function of mdm2 and activating the p53 pathway to promote cancer cell death. can do. Therefore, the peptide containing the mdm2-binding motif of the present invention can be usefully used in the design and development of drugs for the treatment or prevention of cancer diseases through mdm2 function inhibition and p53 pathway activation.

1 is a two-dimensional nuclear magnetic resonance spectroscopy of the P4L29 and P4S19 peptide according to an embodiment of the present invention.
2 is a 15N-1H HSQC spectrum of ¹⁵ N-SUSP4 (201-300) alone according to an embodiment of the present invention. This spectrum is measured at 5 ° C. with a 0.4 mM ¹⁵ N-SUSP4 (201-300) sample. The x- and y-axes of the spectrum show chemical shifts (in ppm) of 1H and 15N, respectively.
3 is a 15N-1H HSQC spectrum of a binding state of ¹⁵ N-SUSP 201-300 collected by titrating ¹⁵ N-SUSP4 (201-300) with mdm2 (3-109) according to an embodiment of the present invention. . They were collected at the molar concentrations of ¹⁵ N-SUSP4 (201-300): mdm2 (3-109) at 1: 0.5, 1: 1, 1: 2, and 1: 3 during the titration process.
4 is a diagram illustrating nuclear magnetic resonance signals of ¹⁵ N-SUSP4 (201-300) disappeared during the mdm2 (3-109) titration process mentioned in FIG. 2 according to an embodiment of the present invention. The missing nuclear magnetic resonance signals and weakened signals are represented by triangles, with signals slightly shifted to black and white circles, respectively.
5 is a NOE bar plot showing the structural characteristics of the SUSP4 201-300 of the present invention.
FIG. 6 is a 15N-1H HSQC spectrum of ¹⁵ N-mdm 2 (3-109) alone in accordance with an embodiment of the present invention. FIG. The spectrum is measured at 5 ° C. with a 0.05 mM ¹⁵ N-mdm 2 (3-109) sample. Peaks assigned chemical shifts indicated the corresponding residue names.
FIG. 7 is a diagram illustrating 15N-1H HSQC spectra of ¹⁵ N-mdm 2 (3-109) alone and SUSP4 (201-300) combined states according to an embodiment of the present invention. Both spectra were measured at 5 ° C using 0.05 mM ¹⁵ N-mdm2 (3-109) samples. Stand-alone state spectrum of mdm2 (3-109) is shown in black ^{15 N-mdm2 (3-109):} SUSP4 (201-300) the molar concentration ratio of 1: 3, the combined state measured at point ¹⁵ N-mdm2 ( The spectrum of 3-109) is shown in red.
FIG. 8 shows three 15N-1H HSQC spectra of a binding state of ¹⁵ N-mdm2 (3-109) collected by titration of ¹⁵ N-mdm2 (3-109) with P4L29 according to an embodiment of the present invention. Is nested. This spectrum was measured at 25 ° C. using 0.05 mM ¹⁵ N-mdm 2 (3-109) sample. The spectra of single state ¹⁵ N-mdm2 (3-109) are shown in black, and the appropriate, bound spectra are molar ratios of ¹⁵ N-mdm2 (3-109): P4L29 1: 0.5, 1: 1, and Collected at 1: 2 points and shown in blue, yellow, and red, respectively.
Figure 9 is a graph showing the variation range calculated from the difference in the chemical shift of ¹⁵ N-mdm2 (3-109) in a single state and P4L29 binding state according to an embodiment of the present invention. According to the formula published in the existing paper [Stoll, R. et al . (2001) Biochemistry 40, 336-344] The fluctuation range was calculated from the difference in the corresponding chemical shifts. The x-axis of the graph represents amino acid residues of the mdm2 protein, and the y-axis is the variation in chemical shift. The chemical shift of binding state ¹⁵ N-mdm2 (3-109) was measured at a molar ratio of ¹⁵ N-mdm2 (3-109): P4L29 at 1: 2.
Figure 10 is a graph showing the variation range calculated from the difference in the chemical shift of ¹⁵ N-mdm2 (3-109) in the single state and P4S19 binding state according to an embodiment of the present invention. According to the formula published in the existing paper [Stoll, R. et al . (2001) Biochemistry 40, 336-344] The fluctuation range was calculated from the difference in the corresponding chemical shifts. The x-axis of the graph represents amino acid residues of the mdm2 protein, and the y-axis is the variation in chemical shift. The chemical shift of binding state ¹⁵ N-mdm2 (3-109) was measured at a molar ratio of ¹⁵ N-mdm2 (3-109): P4S19 at 1: 2.
FIG. 11 is a sensogram of a BIAcore experiment measuring the binding and dissociation process between mdm2 (3-109) protein and P4L29 peptide according to an embodiment of the present invention.
FIG. 12 is a sensogram of a BIAcore experiment measuring the binding and dissociation process between mdm2 (3-109) protein and P4S19 peptide according to an embodiment of the present invention.
Figure 13 shows the results of cancer cell killing experiments by the P4L29 site fragment peptide according to an embodiment of the present invention.
Figure 14 shows the results of cancer cell death test by P4S19 site fragment peptide according to an embodiment of the present invention.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One: SUSP4 Wow mdm2  Production of protein

SUSP4 is known to promote mdm2 self-ubiquitination to actively regulate p53. As a result of analyzing the binding relationship between SUSP4 and mdm2, the present inventors found that 263-301 and 273-301 sites in SUSP4 are involved in binding to mdm2 using nuclear magnetic resonance spectroscopy and spin paramagnetic resonance spectroscopy. Accordingly, the SUSP4 fragment and the mdm2 protein fragment including the two sites were prepared and used in the experiment.

<1-1> SUSP4 Preparation of (201-300) Sections

First, residues 201-300 of SUSP4 were cloned. After synthesizing oligonucleotides corresponding to the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, a polymerase chain reaction (PCR) was performed. At this time, PCR was repeated for 30 minutes of stretching for 1 minute at 94 ° C, 1 minute at 60 ° C and 1 minute at 72 ° C after initial denaturation at 94 ° C for 10 minutes, and finally annealed at 72 ° C for 10 minutes. The amplified PCR product was inserted into the expression vector pET15b (Novagen) using NdeI and BamHI restriction sites to construct an expression vector capable of expressing residues 201-300 of SUSP4.

Forward primer: 5'-GGG AAT TCC ATA TGG AGG AGA GAG AGA AGT GGT GTT CTG-3 '(SEQ ID NO: 3)

Reverse primer: 3'-GCG GGA TCC TCA ACT TAG GAT TTC CTC TTG GGG-5 '(SEQ ID NO: 4)

Using the constructed expression vector, SUSP4 (210-300) is a His-tag-SUSP4 wherein glycine, serine, histidine and methionine derived from the vector are further included at the N-terminus and Histag is connected to the N-terminus. 201-300) in the form of a fusion protein.

Specifically, BL21 (DE3) E. coli transformed to overexpress His · tag-SUSP4 (201-300) Cells were incubated at 37 ° C. in M9 minimal medium. When the OD ₆₀₀ value was 0.6, IPTG (isopropyl thio-β-D-thiogalactopyranoside) was added to the medium to a final concentration of 0.5 mM, and the cells were further incubated for 7 hours. M9 minimal media used for ¹⁵ N isotope labeling included 1 mg / ml ¹⁵ NH ₄ Cl, 0.4% glucose, 2 mg / l biotin, 2 mg / l thiamine, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 0.05 mg / ml ampicillin was added. Then, the cultured cells were collected by centrifugation, buffer (50 mM sodium phosphate (pH 7.8), 1 mM PMSF, 10 mM β-mercaptoethanol) was added, and the cells were eluted by ultrasonic disruption. His-tag-SUSP4 (201-300) fusion protein was purified by nickel affinity resin (Ni-Sepharose affinity resin, GE Healthcare) and then cleaved with thrombin (Sigma). The cleaved SUSP4 (201-300) was purified using a SOURCE 15Q FPLC ion column and a Hyprep 26/60 Sephacryl S-200 FPLC column (GE Healthcare).

<1-2> mdm2 (3-109) Preparation of Section

The human mdm2 protein N-terminal domain corresponding to residue 3-109 in the pLM1 vector was converted to BL21 (DE3) E. coli. Overexpressed in cells [Uesugi, M. et. al . (1999) Proc. Natl. Acad. Sci. USA 96 (26), 14801-14806. Transformed E. coli Cells were incubated at 37 ° C. in LB medium and IPTG was added to the medium to a final concentration of 0.4 mM when the OD ₆₀₀ value was 0.6. Cells were then further incubated at 30 ° C. for 4 hours. The cultured cells were collected by centrifugation, and then buffered (50 mM TrisHCl (pH 7.5), 0.4 M NaCl, 1 mM PMSF, 10 mM β-mercaptoethanol) was eluted by sonication, and the cell eluate was centrifuged to separate proteins. Precipitated. Precipitated protein was dissolved in buffer (50 mM TrisHCl, pH 7.5), 0.4 M NaCl, 1 mM PMSF, 10 mM β-mercaptoethanol, followed by SP-Sepharose and Q-Sepharose column chromatography. Graphics were purified using a Hireprep 26/60 Sephacryl S-200 FPLC column (Pharmacia Biotech). ¹⁵ N and ¹⁵ N, ¹³ C isotopically labeled ¹⁵ N-mdm2 (3-109) and ¹⁵ N, ¹³ C-mdm2 (3-109) proteins were minimal in M9 with ¹³ C-glucose and ¹⁵ NH ₄ Cl. Cell culture was performed in the medium and further cultured at 20 ° C. for 16 hours after the addition of IPTG. The purification method after incubation is the same as described above.

Example  2: SUSP4  Intercept Peptides  synthesis

Peptides comprising the mdm2-binding motif according to the present invention is a peptide P4S19 corresponding to residues 273-291 of SUSP4 and peptide P4L29 corresponding to residues 263-291 of SUSP4 using APEX 348W (Advanced Chemtech) peptide synthesizer And organic synthesis by a solid phase method. The sequence of the peptide comprising the synthesized mdm2-binding motif is as follows.

P4S19: YQILVITEDIEKEIENALG (SEQ ID NO: 1)

P4L29: LTDQEKGREMYQILVITEDIEKEIENALG (SEQ ID NO: 2)

The C terminus of the peptide containing the synthesized mdm2-binding motif was all amidated and purified after synthesis using a Vydac C18 HPLC column. The molecular weight of the peptides containing the final purified mdm2-binding motif was confirmed by MALDI-TOF mass spectrometry.

Example  3: Nuclear magnetic resonance spectroscopy

All nuclear magnetic resonance data were measured at 5 ° C and 25 ° C with a Varian Unity INOVA 600 MHz nuclear magnetic resonance spectrometer. When collecting 15N-1H heteronuclear single quantum coherence spectroscopy (HSQC) spectra, the spectral width was 8000 Hz for the ¹ H axis and 1800 Hz for the ¹⁵ N axis. The number of data is 1024 points in the t2 domain and 256 points in the t1 domain.

Three-dimensional HNCA and HNCOCA experiments of ¹⁵ N, ¹³ C-mdm2 (3-109) prepared in Example <1-2> and 15N-edited TOCSY, 15N-edited NOESY of ¹⁵ N-mdm2 (3-109) (mixing time = 150 ms) The experiment was performed at 25 ° C. to perform chemical shift assignment of the mdm2 (3-109) protein in single and bound states. ¹⁵ N-mdm2 (3-109) and ¹⁵ dissolved in a buffer of 25 mM TrisHCl (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.1 mM PMSF, 0.1 mM EDTA, 0.1 mM benzamidine, 0.02% NaN ₃ N, ¹³ C-mdm 2 (3-109) was subjected to three-dimensional nuclear magnetic resonance spectroscopy in a single state at 0.4 mM mdm 2 (3-109).

Two-dimensional nuclear magnetic resonance experiment of P4L29 dissolved in 50 mM sodium acetate-d3 solution at 15 ° C. temperature, 1.6 mM concentration and pH 6.37 for the structural analysis of the P4L29 peptide as a peptide containing the mdm2-binding motif of the present invention Was performed.

TOCSY [Griesinger, C. et al . (1988) J. Am. Chem. Soc. 110, 7870-7872], sequence-specific resonance assignment was performed through NOESY (mixing time = 200 ms) and ROESY experiments, and DQF-COSY experiments [Rance, M. et. al . (1987) Biochem. Biophys. Res. Commun. 117, 479-485], the ³ J _{HNH α} coupling constant was measured. When the two-dimensional nuclear magnetic resonance spectra were collected, the width of the spectrum was 8000 Hz on both axes. The number of data is 2048 points in the t2 domain and 256 points in the t1 domain. All nuclear magnetic resonance data is stored on the Varian Vnmr and nmrPipe / nmrDraw software on Sun SPARCstation workstations [Delaglio, F. et. al . (1995) J. Biomol. NMR. 6, 277-293].

Two-dimensional nuclear magnetic resonance spectra of TOCSY, NOESY and ROESY measured in 50 mM sodium acetate-d3 solution were analyzed and signal designation of nuclear magnetic resonance peaks of P4L29 and P4S19 peptides was performed, and the observed NOE signals were analyzed to analyze the hydrogen source. Tracking distance information was derived. The results are shown in FIG. The hydrogen interatomic distance information, ³ J _HNHα coupling constant and chemical shift index (CSI) shown in FIG. 1 demonstrated that the P4L29 and P4S19 peptides form a partial secondary structure.

Example  4: chemical shift variation ( chemical shift perturbation ) Experiment

The ¹⁵ N isotopically labeled ¹⁵ N-SUSP4 (201-300) protein prepared in Example <1-1> was purified by 20 mM sodium acetate (pH 6.5), 50 mM NaCl (90% H ₂ O / 10% D). _It was dissolved in ₂ O) buffer and collected two-dimensional 15N-1H HSQC spectra at a concentration of 0.4 mM ¹⁵ N-SUSP4 (201-300) and a temperature of 5 ℃. After ¹⁵ N-SUSP4 (201-300) in mdm2 (3-109) sikimyeo then added to the protein ^N-15 SUSP4 (201-300) protein: mdm2 molar ratio of (3-109): 1, 1: 0.5 15N-1H HSQC spectra of the same ¹⁵ N-SUSP4 (201-300) were collected at points 1: 1, 1: 2, and 1: 3.

In contrast, ¹⁵ N isotopically labeled ¹⁵ N-mdm2 (3-109) protein was purified by 20 mM sodium acetate (pH 6.5), 100 mM NaCl, 1 mM DTT, 0.1 mM PMSF, 10 μM EDTA, 0.1 mM benzamidine, Dissolved in 0.02% NaN ₃ buffer and collected 15N-1H HSQC spectra at a concentration of 0.05 mM ¹⁵ N-mdm2 (3-109) and 5 ° C. Then ¹⁵ N-mdm2 (3-109) protein was added to the peptide containing the SPSP (201-300) or mdm2-binding motif prepared in Example 2 and bound state ¹⁵ N-mdm2 (3-109) during the titration process 15N-1H HSQC spectra of were collected. The molarity ratios of ¹⁵ N-mdm2 (3-109): binding peptide during the titration process were 1: 0, 1: 0.5, 1: 1, 1: 2, and 1: 3 for the SUSP4 (201-300) fragments. (FIGS. 2-4), 1: 2 for peptides with mdm2-binding motifs (P4S19 and P4L29) (FIGS. 9 and 10).

The chemical shift variation test results performed as described above are as follows.

<4-1> SUSP Chemical Shift Variation Test of (201-300)

FIG. 2 shows the results of collecting two-dimensional 15N-1H HSQC spectra of purified ¹⁵ N-SUSP4 (201-300) alone by labeling ¹⁵ N isotopes. MDM2 (3-109) was added to the ¹⁵ N-SUSP4 (201-300) to titrate and 15N-1H HSQC spectra of the bonded state SUSP4 (201-300) were collected and shown in FIG. 3. 4 shows a nuclear magnetic resonance peak corresponding to about 20 residues out of 100 residues lost due to binding with mdm2 (3-109) during the titration process. This is due to an increase in the linewidth of the nuclear magnetic resonance peak by chemical exchange between the single state signal and the binding state signal during the titration process.

<4-2> mdm2 Chemical shift variation experiment of (3-109)

During titration the process of adding the above <4-1> and is then purified as opposed to the ¹⁵ N isotope ¹⁵ N-mdm2 (3-109) to the cover in order to identify the binding site of mdm2, SUSP4 (201-300) Two-dimensional 15N-1H HSQC spectra were collected. First, the HSQC spectrum of ¹⁵ N-mdm 2 (3-109) in a single state was collected and shown in FIG. 6. Then, as shown in FIG. 7, the peaks corresponding to the SUSP4-binding site amino acid residues were shifted in the HSQC spectrum of the binding state ¹⁵ N-mdm2 (3-109) collected by adding SUSP4 (201-300). Can be. Three-dimensional nuclear magnetic resonance experiments, HNCA, HNCOCA, 15N-edited TOCSY, and 15N-edited NOESY spectra were used for chemical shift assignment of the single and bound states mdm2 (3-109). As shown in FIG. 8, the fluctuation range calculated from the chemical shift was almost similar to the chemical shift variation of the previously published p53 α-helix [Stoll, R. et. al . (2001) Biochemistry 40, 336-344. From the above results, it was confirmed that SUSP4 (201-300) binds to the same hydrophobic binding site in mdm2 (3-109) that binds p53 α-helix fragment peptide and p53 TAD (1-73) (transcriptional activation domain). (FIG. 7).

<4-3> P4L29  Secondary Structure Formation Section Peptide  Chemical Shift Variation Experiment

Two-dimensional 15N-1H HSQC spectra of purified ⁵ N-mdm2 (3-109) alone were collected by labeling ¹⁵ N isotopes, followed by titration by addition of peptide P4L29 containing an mdm2-binding motif according to the present invention. 15N-1H HSQC spectra of binding state ⁵ N-mdm 2 (3-109) were collected. Because P4L29 and ⁵ N-mdm2 (3-109) bind with low affinity, the rapid chemical exchanges mean that the nuclear magnetic resonance signals in single and bound states are averaged and observed as single peaks. As shown in FIG. 8, which overlaps a plurality of 15N-1H HSQC spectra obtained during the titration process, the average peak is continuously shifted during the titration process. FIG. 9 is a graph illustrating the variation in chemical shifts obtained from such chemical shifts.

<4-4> P4S19  Secondary Structure Formation Section Peptide  Chemical Shift Variation Experiment

Two-dimensional 15N-1H HSQC spectra of purified ⁵ N-mdm2 (3-109) alone were collected by labeling ¹⁵ N isotopes, followed by titration by addition of peptide P4S19 containing the mdm2-binding motif according to the present invention. 15N-1H HSQC spectra of binding state ⁵ N-mdm 2 (3-109) were collected. Since P4S19 and ⁵ N-mdm2 (3-109) bind with low affinity, the fast magnetic exchange results in averaging single and bound nuclear magnetic resonance signals, which were observed as single peaks. As shown in FIG. 7, which overlaps a plurality of 15N-1H HSQC spectra obtained during the titration process, the average peak is continuously shifted during the titration process. FIG. 10 is a graph illustrating the variation of chemical shifts obtained from such chemical shifts.

Example  5: BIAcore  Experiment

BIAcore experiments were performed on a BIAcore 3000 instrument. A CM5 sensor chip (Pharmacia Biosensor, Sweden) was mounted on the instrument, washed with HBS (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.005% Tween20) buffer, and 35 μl injection of EDC / NHS mixture. To activate the carboxymethyl dextran surface. 120 μl of mdm2 (3-109) dissolved in a buffer of 25 mM sodium acetate (pH 4.5) and 150 mM NaCl at a concentration of 1 mg / ml was injected and the mdm2 (3-109) protein was transferred to the CM5 sensor chip by amine coupling. Immobilized. 10 mM glycine (pH 2.0) to flush 1 M ethanolamine to inactivate activated carboxyl groups that remain unreacted with the mdm2 (3-109) protein and to wash proteins that are not bound to the sensor chip. ) Was injected. Then, 150 μl of peptides containing the mdm2-binding motif according to the present invention dissolved in HBS buffer by concentration (80 μM, 40 μM, 20 μM, 10 μM) were injected and the sensogram during the binding and dissociation process according to time. ) Data was collected. After each peptide injection, 10 μl of a 0.25 M sodium chloride and 0.025 M sodium hydroxide mixture was injected to dissociate the bound peptides. The flow rates were 5 μl / min for sensor chip activation, deactivation and immobilization and 30 μl / min for binding and sodium chloride washing. The equilibrium dissociation constant (K _d ) was calculated by plotting and fitting the sensorgram data at equilibrium according to the concentration [van Holde, KE et al . (1998) Principles of Physical Biochemistry (FIGS. 11 and 12).

FIG. 11 is a sensogram of a BIAcore experiment in which the binding and dissociation process between mdm2 (3-109) protein and P4L29 peptide is measured by time according to an embodiment of the present invention, and FIG. 12 is an embodiment of the present invention. This is a sensogram of the BIAcore experiment that measures the binding and dissociation process between mdm2 (3-109) protein and P4S19 peptide according to time.

As shown in Figures 11 and 12, the peptides including the mdm2-binding motif of the present invention P4L29 and P4S19 can specifically bind to mdm2 (3-109) protein, their equilibrium dissociation constant, K _d ) was found to be 58 μM and 264 μM, respectively.

Example  6: mdm2 Containing binding motifs Peptide  Cancer cell death effect

U2OS, SJSA-1, SAOS-2, and HCT116 cancer cells in a wet 5% CO ₂ atmosphere (Gibco-BRL, UK) and 100 U / ml with 10% fetal bovine serum (FBS) It was incubated at 37 ° C in Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium to which penicillin / streptomycin was added.

P4L29 and P4S19 peptides were treated with cultured cancer cells using palmitoyl acid or HIV-tat peptides for 24 hours and 48 hours, and cell survival rate was measured using MTS solution (Roche) to measure apoptosis. A cell survival assay was performed.

The cancer cell killing effects of P4L29 and P4S19, which are peptides containing the mdm2-binding motif according to an embodiment of the present invention, are shown in FIGS. 13 and 14, respectively.

As shown in Figures 13 and 14, the control group, the present invention cell death, while little, pal-P4L29 is exhibited an IC ₅₀ value of about 14 μM, HIV-tat P4S19 exhibits an IC ₅₀ value of 20 to 60 μM It was confirmed that the peptide containing the mdm2-binding motif according to the excellent cell death effect on cancer cells.

<110> Korea Research Institute of Biocience and Biotechnology <120> Peptides comprises mdm2-binding motif and use <130> PA110599 / KR <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> mdm2-binding motif P4S19 <400> 1 Tyr Gln Ile Leu Val Ile Thr Glu Asp Ile Glu Lys Glu Ile Glu Asn   1 5 10 15 Ala leu gly             <210> 2 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> mdm2-binding motif P4L29 <400> 2 Leu Thr Asp Gln Glu Lys Gly Arg Glu Met Tyr Gln Ile Leu Val Ile   1 5 10 15 Thr Glu Asp Ile Glu Lys Glu Ile Glu Asn Ala Leu Gly              20 25 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 gggaattcca tatggaggag agagagaagt ggtgttctg 39 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 gcgggatcct caacttagga tttcctcttg ggg 33

Claims (7)

A peptide or pharmaceutically acceptable salt thereof comprising a mouse double minute 2 (mdm2) -binding motif amino acid sequence of SEQ ID NO: 1.
The peptide of claim 1, or a pharmaceutically acceptable salt thereof, comprising an mdm2-binding motif, wherein the peptide has an amino acid sequence of SEQ ID NO: 1 or 2.
A pharmaceutical composition for the treatment or prophylaxis of cancer diseases comprising a peptide comprising the mdm2-binding motif of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
The pharmaceutical composition of claim 3, wherein the cancer disease is any one selected from the group consisting of leukemia, lymphoma, esophageal carcinoma, neuroblastoma, soft tissue cancer, breast cancer, colon cancer, lung cancer, and stomach cancer.
The pharmaceutical composition of claim 3, further comprising a pharmaceutically acceptable carrier.
A method for screening a therapeutic agent for cancer diseases using an mdm2-binding motif having the amino acid sequence of SEQ ID NO: 1, comprising the following steps.
1) synthesizing a peptide comprising an mdm2-binding motif;
2) analyzing whether the synthesized peptide can bind to mdm2 by competitively acting on the p53 binding site in mdm2; And
3) when the synthesized peptide binds to mdm2, determining the peptide as a therapeutic agent for cancer diseases.
A method for screening a therapeutic agent for cancer diseases using an mdm2-binding motif having the amino acid sequence of SEQ ID NO: 2, comprising the following steps.
1) synthesizing a peptide comprising an mdm2-binding motif;
2) analyzing whether the synthesized peptide can bind to mdm2 by competitively acting on the p53 binding site in mdm2; And
3) when the synthesized peptide binds to mdm2, determining the peptide as a therapeutic agent for cancer diseases.

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