KR101449623B1 - Multivalent peptide inhibitors against protein-protein interaction and a method for preparing thereof - Google Patents

Abstract

The present invention relates to a novel multivalent peptide inhibitor capable of inhibiting the binding of HIF-1α to a VBC protein and the binding of HIF-1α and a p300 protein at a concentration of several nM, a method for producing the same, will be.

Description

TECHNICAL FIELD The present invention relates to a multivalent peptide inhibitor and a method for preparing the same,

The present invention relates to a novel multivalent peptide inhibitor capable of inhibiting the binding of HIF-1α to a VBC protein and the binding of HIF-1α and a p300 protein at a concentration of several nM, a method for producing the same, will be.

The ability of animal cells to detect and respond to changes in oxygen levels is essential for survival and is essential for survival as well as developmental processes as well as physiological processes such as angiogenesis, myocardial ischemia, cerebral ischemia, pulmonary hypertension, It is known to be involved. One of the proteins that plays an important role in many diseases related to these oxygen levels is HIF-1 (hypoxia inducible factor-1: hypoxia inducer).

HIF-1 is a transcription factor that is induced in a hypoxic state and plays an important role in energy metabolism, vasomotor control, neovascularization, regulation of gene expression associated with apoptosis, and cell response in hypoxic state. HIF-1 is a heterodimer composed of α and β subunits. Β subunit is always expressed stably. However, HIF-1α is stabilized in hypoxia and affects the expression of various genes. In normoxia, it is characterized by proteosome degradation.

HIF-1α, stabilized in hypoxia, forms a dimer with HIF-1β, which is always present in the cell, and migrates to the nucleus, forming a complex by binding to p300 protein, a transcription aid protein in the nucleus. (Freedman, SJ, et al., PNAS, 99: 5367-5372, 2002) were found to form complexes between the 331-418 amino acid region of the protein and the 792-824 th amino acid peptide of HIF-1α. The complex of HIF-1α and p300 binds to the hypoxia-response element (HRE) present on the promoter of the target gene to activate the transcription of the target genes. Among the genes regulated by HIF-1, there are VEGF, Tie-2, Flt-1, Flk-1, PAI-1, EPO and NOS genes involved in angiogenesis. IGF-II, IGFBP-1, p21, NIP3, ADM, NOS2, and TGFA are genes that are involved in apoptosis resistance. Among the genes involved in invasion and metastasis are SDF-1, CXCR4, beta 2 integrin, prolyl-4-hydroxylase-alpha 1 and the like. By overexpression of these genes, cancer cells acquire traits such as resistance to cell death, increase in angiogenesis, increase in cell proliferation, increase in invasion ability, and resistance to chemotherapy, and eventually become malignant and transfer to other tissues . In addition, angiogenic factors such as VEGF induced by HIF that are activated in a hypoxic state are not only cancer but also diseases such as macular degeneration, diabetic retinopathy, arthritis and psoriasis And progression ( Pathology International , 2005, 55: 603-610; Ann. Rheum . Dis . , 2003, 62: ii60-ii67). Thus, inhibiting the interaction between HIF-1α and p300 may be useful in the treatment of diseases associated with worsening of disease, such as cancer, arthritis, psoriasis, diabetic retinopathy and macular degeneration, by activation of angiogenesis.

On the other hand, HIF-1α is degraded by proteasomes under normal oxygen conditions. This mechanism is caused by the interaction of pVHL (von Hippel-Lindau tumor suppressor) protein with HIF-1α. Specifically, the proline group located at position 402 or 564 of the amino acid sequence of the HIF-1α protein in the normal oxygen state is hydrolyzed by an enzyme called prolyl hydroxylase-2 (PHD2) to become a hydroxyproline group, and the pVHL protein is longin C protein and Elongin B protein to form a VBC protein complex. The 115th Histidine (His) group of the pVHL protein and the 111th serine (Ser) group and the 564th hydroxyproline group of HIF- (Min, JH, et al., Science, 296, 1886-1889, 2002), whereby HIF-1α is ubiquitinated and degraded by proteosomes. Therefore, inhibition of the interaction between HIF-1α and VBC increases the activity of HIF-1α in normal oxygen to promote cell cycle progression or enhance angiogenesis or cell survival, (U.S. Patent No. 6,787,326), which is useful for the treatment of ischemic diseases such as hypoplasia and angioplasty.

 In order to analyze the interaction between HIF-1α and p300 or VBC protein and to develop a therapeutic agent for various diseases associated therewith, it is necessary to use a substance directly controlling the binding of HIF-1α and p300 or VBC protein, ie, HIF-1α and p300 protein It is important to develop an inhibitor that inhibits binding, or an inhibitor that inhibits the binding of a proline group-hydroxylated HIF-1 alpha peptide to a VBC protein.

To this end, the present inventors have previously provided a method for quantitatively analyzing the formation of a complex between a fluorescence-labeled HIF-1α peptide and a VBC protein or a p300 protein through a change in fluorescence polarization value Korean Patent Application Nos. 2005-0066881 and 2006-0099389, which are incorporated herein by reference), a compound that inhibits the binding of HIF-1α with a hydroxylated proline group to a VBC protein using such a method baicalein and ciclopirox have been selected (Korean Patent Application No. 2007-0007036). On the basis of this, an inhibitor peptide which inhibits the binding of the proline group to the hydroxylated HIF-1.alpha. Peptide and the VBC protein has been provided (Korean Patent Application No. 2007-0013934, incorporated herein by reference). However, their inhibition constants (Ki) are only about several micromolar concentrations, which is too low to be used in clinical application technology in the future.

Therefore, for more practical applications, it is necessary to develop a peptide inhibitor that not only inhibits the binding between HIF-1α and its interacting protein but also exhibits higher inhibition.

Accordingly, the present inventors have devised a multivalent peptide inhibitor capable of eliciting a strong interaction with a weakly interacting unit, and have found that the binding of proline to hydroxylated HIF-1α and VBC proteins and the binding of HIF-1α and p300 proteins We have developed a novel multivalent peptide inhibitor which can be inhibited at a concentration level of several tens nM.

It is an object of the present invention to provide a multivalent peptide inhibitor that inhibits the binding of a proline group to a hydroxylated HIF-1 alpha and a VBC protein, a method of producing the inhibitor, and a pharmaceutical composition for prevention or treatment of ischemic diseases including the inhibitor .

Another object of the present invention is to provide a multivalent peptide inhibitor for inhibiting the binding of HIF-1 alpha and p300 protein, a method for producing the inhibitor, and a pharmaceutical composition for the prevention or treatment of angiogenic diseases including the inhibitor will be.

In order to achieve the above object, in one aspect, the present invention relates to a polyprotein inhibitor which inhibits the binding of a proline group to a hydroxylated HIF-1α and a VBC protein.

In the present invention, the term "HIF-1" is a heterozygous complex consisting of HIF-1 [alpha] subunit and HIF-1 [beta] subunit that are constantly expressed in an oxygen dependent manner as a hypoxia-induced transcription factor. The gene sequence of HIF-1 alpha is listed in GenBank accession number U22431, and the amino acid sequence of the protein is listed in GenBank accession number AAC50152. The amino acid sequence of HIF-1? Is shown in SEQ ID NO: 1 in the present specification.

The term "VBC protein" in the present invention means a protein complex in which the Elongin C protein and the Elongin B protein are sequentially linked to the beta region of the VHL protein, and the VHL, Elongin C and Elongin B genes are GenBank accession numbers NM000551 and NM007108 And NM005648.

Preferably, the peptide inhibitor of the present invention comprises (1) a core, and (2) a peptide having an amino acid sequence of SEQ ID NO: 2, wherein the peptide is formed by a peptide structure in which two or more of the peptides are bound to the surface of the core .

In the present specification, the peptide having the 556th to 575th amino acid sequences of the amino acid sequence of HIF-1α and the proline substituted with the hydroxyproline as the 564th amino acid is shown in SEQ ID NO: 2. In the present specification, a peptide structure comprising the above (1) core and (2) the peptide having the amino acid sequence of SEQ ID NO: 2 and having two or more of the peptides bound to the surface of the core is referred to as "556-575 Quot; sex peptide inhibitor ". Since the 556-575 polyvalent peptide inhibitor can bind to the 115th histidine and 111th serine of the pVHL protein forming the VBC protein complex by hydrogen bonding, the VBC protein competitively interacts with HIF-1α present in the body against the VBC protein To specifically inhibit the binding between HIF-1 alpha and VBC proteins.

The term "core" in the present invention means a metal or non-metal particle having a plurality of peptides directly bonded to its surface, which is a structure existing at the center so that a plurality of peptides can form aggregates. Examples of the core include inorganic nanoparticles, macromolecules, macromolecular micelles, proteins, and nucleic acids. More specifically, gold nanoparticles, silver nanoparticles, biodegradable polymers, polymer chains, protein self- assembled structures protein structures, DNA nanostructures, DNA self-assemblies, or streptavidin.

In order for the peptide having the amino acid sequence of SEQ ID NO: 2 to bind to the core surface, a functional group capable of binding to the core may be introduced at either end of the peptide. The functional group preferably includes a functional group having high affinity with the material forming the core, and may be variously selected depending on the material forming the core. For _{instance, -COOH, -NH 2, -SH,} -CONH 2, -PO 3 H, -PO 4 H, -SO 3 H, -SO 4 H, -CHO, comprise a -NCO, -OH, or . In the case where the core is streptavidin, biotin can be used as a functional group, but the scope of the present invention is not limited thereto.

Also, the linker may or may not be included between the functional group for binding to the core surface and the peptide of SEQ ID NO: 2. The linker is designed to secure a proper space between the core and the peptide to prevent unnecessary interaction between the core and the peptide and to impart flexibility to the peptide. The linker has the above effect and has a substantial effect on the binding between the peptide and the VBC Any linker that is not yet available can be used as a linker. For example, aminocaproic acid (ACA) or ethylene glycol may be used as the linker, but the present invention is not limited thereto.

The peptide structure of the present invention is characterized in that at least two peptides having the amino acid sequence of SEQ ID NO: 2 bind to the core surface to form a multivalent peptide. Accordingly, the weak interaction between the peptide unit of SEQ ID NO: 2 and the VBC protein can be amplified with strong interaction by the multivalent peptide construct. Depending on the type of core, the range of the peptide of SEQ ID NO: 2 may be varied on the surface of the core, preferably 2 or more, more preferably 2 to 100, of the peptide.

In a specific embodiment of the present invention, the polyvalent peptide inhibitor based on the 556th to 575th amino acids of HIF-1α competes with the monovalent HIF-1α peptide, F-HyP564, against the VBC protein to produce F-HyP564 And specifically inhibited complex formation between VBC proteins (Fig. 2). This suggests that HIF-1.alpha. Protein hydrolyzed under normal oxygen conditions can be accumulated in cells without degradation, and thus can be useful for the prevention or treatment of ischemic diseases such as coronary artery insufficiency, cerebral insufficiency, and angiotension.

Accordingly, in another aspect, the present invention relates to a pharmaceutical composition for preventing or treating ischemic diseases comprising 556-575 polyvalent peptide inhibitor as an active ingredient.

The peptide structure of the present invention is useful as an ischemic cardiovascular disease including heart failure, heart disease such as heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction and angina, stroke and peripheral vascular diseases, And is useful for the prophylaxis or treatment of at least one ischemic disease selected from the group consisting of insulin resistance, insufficiency, and angiotension.

In another aspect, the present invention relates to a method for producing the above-mentioned 556-575 polyvalent peptide inhibitor. Such a preparation method is not particularly limited, and in order to synthesize the novel multivalent peptide inhibitor devised in the present invention, a core capable of attaching peptides constituting a part of HIF-1?

1) synthesizing a peptide (peptide of SEQ ID NO: 2) having a 556th to 575th amino acid sequence in the amino acid sequence of HIF-1α, wherein proline, which is the 564th amino acid, is substituted with hydroxyproline , And 2) introducing a functional group capable of binding to the core at one end of the peptide to bind to the core, thereby producing a H556-575 polyvalent peptide inhibitor. At this time, the peptide synthesis can be carried out using a commonly used solid support phase method, and more specifically, the amino group of the substituted peptide can be carried out using amino acids protected with a protecting group (Fmoc or Boc) and a resin .

In another aspect, the present invention relates to a multivalent peptide inhibitor which inhibits the binding of HIF-1 alpha to the p300 protein.

In the present invention, the term "p300 protein" is a transcription-assisting protein in the nucleus, which binds to HIF-1α stabilized in a hypoxic state to form a complex, and then binds to an HRE present on the promoter of the target gene, . p300 protein has Zn ^{2 +} binding factor comprising a cysteine-/ histidine-rich has the (cysteine- / histidine-rich) CH1 domain, and is known to the CH1 domain is combined with the C- terminal region of HIF-1α. The gene sequence of p300 is listed in GenBank accession number U01877.

 Preferably, the peptide inhibitor comprises (1) a core, and (2) a peptide having an amino acid sequence of SEQ ID NO: 3, wherein the peptide has two or more peptides bound to the surface of the core.

In the present specification, the peptide having amino acid sequence 786 to 826 of the amino acid sequence of HIF-1? Is shown in SEQ ID NO: 3. In the present specification, the peptide structure comprising the above (1) core and (2) the peptide having the amino acid sequence of SEQ ID NO: 3 and having two or more of the peptides bound to the surface of the core is referred to as "786-826 Quot; sex peptide inhibitor ". Since the 786-826 polyvalent peptide inhibitor can bind to the amino acid region of the p300 protein, it acts competitively with the HIF-1α existing in the body against the p300 protein and specifically inhibits the binding between HIF-1α and p300 proteins .

The definition of the core is as described above.

In order for the peptide having the amino acid sequence of SEQ ID NO: 3 to bind to the core surface, a functional group capable of binding to the core may be introduced at either end of the peptide, which is described above. The linker may or may not be included between the functional group for binding to the core surface and the peptide of SEQ ID NO: 3, and the linker is as described above.

The peptide structure of the present invention is characterized in that at least two peptides having the amino acid sequence of SEQ ID NO: 3 bind to the core surface to form a multivalent peptide. Accordingly, the weak interaction between the peptide unit of SEQ ID NO: 3 and the p300 protein can be amplified by strong interaction with the multivalent peptide construct.

In a specific embodiment of the present invention, the multivalent peptide inhibitor based on the 786th to 826th amino acids of HIF-1α competes with the monovalent HIF-1α peptide, F-786-826, against the p300 protein, 786-826 and p300 protein (Fig. 3). This inhibits the binding of HIF-1α to p300, a transcriptional helper present in the nucleus, by activating HIF-1α in the hypoxic state, thereby inhibiting the transcription of various genes involved in angiogenesis by HIF-1α, It can be useful for the prevention and treatment of angiogenesis related diseases such as arthritis, psoriasis, diabetic retinopathy and macular degeneration.

Accordingly, in another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of angiogenesis diseases comprising 786-826 polyvalent peptide inhibitor as an active ingredient. The peptide structure of the present invention may be useful for the prevention or treatment of angiogenic diseases such as cancer, arthritis, psoriasis, diabetic retinopathy and macular degeneration.

The cancer is preferably a solid tumor and is preferably a solid tumor and is preferably a solid tumor, and is preferably a solid tumor. The cancer is preferably a solid cancer, and the cancer is preferably a solid tumor, such as bladder cancer, breast cancer, colon cancer, uterine cancer, head and neck cancer, leukemia, lung cancer, lymphoma, melanoma, non- Neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, retinoblastoma, glioma, Wilm's tumor, esophageal cancer, oral cancer, kidney cancer, multiple myeloma, pancreatic cancer, skin cancer and small cell lung cancer Lung cancer, < / RTI >

In another aspect, the present invention relates to a method for producing the above-mentioned 786-826 polyvalent peptide inhibitor. Such a preparation method is not particularly limited, and in order to synthesize the novel multivalent peptide inhibitor devised in the present invention, a core capable of attaching peptides constituting a part of HIF-1?

In a preferred embodiment, the present invention provides a method for producing a peptide comprising the steps of: 1) synthesizing a peptide having a 786th to 826th amino acid sequence (the peptide of SEQ ID NO: 3) in the amino acid sequence of HIF-1α, and 2) Lt; RTI ID = 0.0 > 786-826 < / RTI > polyvalent peptide inhibitor. At this time, the peptide synthesis can be carried out using a commonly used solid support phase method, and more specifically, the amino group of the substituted peptide can be carried out using amino acids protected with a protecting group (Fmoc or Boc) and a resin .

The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable additives in addition to the active ingredient, and the kind of the additive may include at least one of an excipient, a disintegrant, a binder and a lubricant, It is not. In addition, the composition can be used for oral use, and can be prepared, for example, as a warning agent, granule, powder, tablet, or capsule. At this time, the dose of the composition is suitably adjusted according to the purpose of use, the purpose of use, the condition of the patient, age, sex, weight, prescription drug and disease, and is usually 0.01 to 100 mg / kg, The dose may be 0.5 to 10 mg / kg, and may be administered once to several times a day, more preferably three times a day.

The 556-575 polyvalent peptide inhibitor according to the present invention specifically inhibits the binding of the proline to HIF-1α and VBC proteins, so that the hydroxylated HIF-1α protein is not degraded in the normal oxygen state, Can be useful not only for the prevention or treatment of ischemic diseases such as hypoxia and angiopathy, but also for studying intracellular physiological phenomena that may be associated with hydroxylated HIF-1α. In addition, the 786-826 polyvalent peptide inhibitor specifically inhibits the binding of HIF-1α and p300 proteins, and thus can inhibit the activity of HIF-1α, which is activated by hypoxia in most cancer cells and interacts with p300 Therefore, it can be used not only for the prevention or treatment of cancer, but also for studying intracellular physiological phenomena that may be associated with activated HIF-1α.

1 is a schematic diagram for explaining inhibition of protein interaction by a polyvalent peptide inhibitor,
FIG. 2 shows the result of fluorescence polarization measurement of inhibition of interaction between HIF-1α and VBC protein by 556-575 multivalent peptide inhibitor,
FIG. 3 shows the result of fluorescence polarization measurement of the degree of inhibition of interaction between HIF-1α and p300 protein by the concentration of the 786-826 multivalent peptide inhibitor.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. Versatile Peptides  Manufacture of inhibitors

The peptide of SEQ ID NO: 2 or the peptide of SEQ ID NO: 3 (80 [mu] M) and streptavidin (10 [mu] M) were mixed in PBS buffer (100 [mu] L) Shake it and mix it. Subsequently, streptavidin-based 556-575 polyvalent peptide inhibitors and 786-826 polyvalent peptide inhibitors were prepared by removing peptides not bound to streptavidin using G75 Sepharose resin. The sequences of SEQ ID NO: 2 and SEQ ID NO: 3 used are shown in Table 1 below.

SEQ ID NO: synthesis Peptides 2 Biotin-ACA-DLDLEALA-HyP- YIPADDDFQLR-NH 2 3 Biotin-ACA-SMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN-NH ₂

Example 2. Multivalency to binding of HIF- 1? And VBC proteins Inhibitory Effect Analysis of Peptide Inhibitors

The inventors of the present invention previously conducted a simple quantitative analysis on the formation of a complex between a fluorescently labeled HIF-1α peptide, a VBC protein and a p300 protein through the change of fluorescence polarization value (Korean Patent Application No. 2005-0066881 No. 2006-0099389, which is incorporated herein by reference). In an embodiment of the present invention, in order to measure whether or not the polyvalent peptide inhibitors produced can inhibit the formation of a complex between the HIF-1 alpha peptide and the related protein, the conventional method of quantitatively analyzing through the change of the fluorescence polarization value Was used.

NP-40 (nonidet P-40) was added so that the polyvalent peptide inhibitor was not added and the final concentration was 0.25% in order to examine the degree of inhibition of the polyvalent peptide inhibitor on the complex formation of HIF-1α and VBC protein a buffer solution (50 mM Tris, 120 mM NaCl , pH 8.0) to (named F-Hyp564 peptide, FITC-AHA-DLDLEALA-HyP -YIPADDDFQLR-NH 2) price peptide of 100 nM a, and 150 nM of VBC protein To form a complex, and the fluorescence polarization value was measured and used as an initial value. Then, 556-575 polyvalent peptide inhibitor prepared in Example 1 was added to the complex solution at a concentration of 0-200 nM at 25 ° C, and the fluorescence polarization value was measured according to the inhibitor peptide concentration. At this time, a fluorescence analyzer (Appliskan, Thermo Scientific) was used, and a filter corresponding to 488 nm was used for the exitation and emission. The results are shown in Fig.

In order to investigate the degree of inhibition of the polyvalent peptide inhibitor on the complex formation of HIF-1α and p300 protein, NP-40 (nonidet P-40) was added so that the polyvalent peptide inhibitor was not added and the final concentration was 0.25% buffer solution (50 mM Tris, 120 mM NaCl , pH 8.0) in the unit price of 100 nM peptide (786-826 F-labeled peptide, FITC-AHA-SMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN-NH 2) and 150 nM of the complex by the addition of p300 proteins And the fluorescence polarization value was measured and used as an initial value. Then, the complex solution was mixed at 25 ° C while increasing the concentration of the 786-826 polyvalent peptide inhibitor prepared in Example 1 to a concentration of 0-200 nM, and the fluorescence polarization value according to the concentration of the inhibitor peptide was measured. At this time, a fluorescence analyzer (Appliskan, Thermo Scientific) was used, and a filter corresponding to 520 nm was used for the exitation and emission. The results are shown in Fig.

As shown in Figs. 2 and 3, when the polyvalent peptide inhibitor was added, the fluorescence polarization value was decreased in a concentration-dependent manner as compared with the case where it was not added. The IC ₅₀ value for the F-786-826 and p300 protein binding of the IC ₅₀ value and 786-826 multivalent peptide inhibitors for the F-HyP564 and VBC protein binding of such polyhydric 556-575 peptide inhibitors sphere through a curve fitting Respectively. The dissociation constants (Kd = 134 nM) between the previously known F-HyP564 and VBC proteins and the dissociation constant (Kd = 198 nM) between F-786-826 and p300 proteins and the IC ₅₀ values obtained above were assigned to the following equations (Ki) values of the 556-575 polyvalent peptide inhibitor and the 786-826 polyvalent peptide inhibitor, respectively, and these values are shown in Table 2. Compared with the inhibition constant of the monovalent inhibitor, the inhibition of the multivalent peptide inhibitors was about 100 times and 15 times better, respectively.

[Equation] Ki = IC ₅₀ / (S / Kd + 1)

Here, S refers to the concentration of the fluorescence-labeled peptide.

Inhibitor Peptides Monovalent peptide inhibitor
(F-Hyp564 peptide) 556-575 Multivalent Peptide Inhibitor Monovalent peptide inhibitor
(F-786-826 peptide) 786-826 Multivalent Peptide Inhibitor Ki
( nM ) 3700 39 1627 108

From these results, it can be seen that the 556-575 polyvalent peptide inhibitor and the 786-826 polyvalent peptide inhibitor of the present invention inhibit VBC / F-HyP564 complex formation and p300 / F-786-826 complex formation, respectively. In addition, their inhibition showed about 100 times improvement in inhibitory effect when compared with the respective monovalent peptide inhibitors. That is, the multivalent peptide inhibitors act as inhibitors that inhibit the interaction between HIF-1.alpha. And VBC proteins and the interaction between HIF-1.alpha. And p300 proteins, and act as inhibitors that are far superior to monovalent peptide inhibitors have.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Multivalent peptide inhibitors against protein-protein          interaction and a method for preparing it <130> DPP20120318KR <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 826 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE &Lt; 222 > (1) .. (826) &Lt; 223 > HIF-1a (GenBank accession No. AAC50152) <400> 1 Met Glu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu   1 5 10 15 Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys              20 25 30 Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu Pro His          35 40 45 Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg Leu Thr Ile      50 55 60 Ser Tyr Leu Arg Val Lys Leu Leu Asp Ala Gly Asp Leu Asp Ile  65 70 75 80 Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys Ala Leu                  85 90 95 Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile             100 105 110 Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr         115 120 125 Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Met     130 135 140 Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys Gly Lys Glu 145 150 155 160 Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr                 165 170 175 Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr Trp Lys Val Leu             180 185 190 His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn Ser Asn Gln Pro         195 200 205 Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile Cys     210 215 220 Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys 225 230 235 240 Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp                 245 250 255 Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly             260 265 270 Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr         275 280 285 Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr Thr Gly Gln     290 295 300 Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp Val Glu Thr Gln 305 310 315 320 Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro Gln Cys Ile Val                 325 330 335 Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His Asp Leu Ile Phe             340 345 350 Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp         355 360 365 Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser     370 375 380 Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu 385 390 395 400 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn                 405 410 415 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn             420 425 430 Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu         435 440 445 Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser     450 455 460 Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro 465 470 475 480 Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp                 485 490 495 Gln Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser G             500 505 510 Pro Asn Ser Ser Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val         515 520 525 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr     530 535 540 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser                 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser             580 585 590 Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln         595 600 605 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp Glu Leu     610 615 620 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile Leu Ile Ala 625 630 635 640 Ser Pro Ser Pro Thr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser                 645 650 655 Ser Pro Tyr Arg Asp Thr Gln Ser Arg Thr Ala Ser Pro Asn Arg Ala             660 665 670 Gly Lys Gly Val Ile Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro         675 680 685 Asn Val Leu Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu     690 695 700 Glu Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg 705 710 715 720 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly Thr                 725 730 735 Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser Leu Ser Trp             740 745 750 Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln Asn Gly Met Glu Gln         755 760 765 Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly     770 775 780 Gln Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys 785 790 795 800 Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu                 805 810 815 Glu Leu Leu Arg Ala Leu Asp Gln Val Asn             820 825 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 556-575 amino acids of HIF-1a <220> <221> PEPTIDE <222> (1) <223> Xaa is hydroxyproline <400> 2 Asp Leu Asp Leu Glu Ala Leu Ala Xaa Tyr Ile Pro Ala Asp Asp Asp   1 5 10 15 Phe Gln Leu Arg              20 <210> 3 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> 786-826 amino acids of HIF-1a <400> 3 Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys Glu   1 5 10 15 Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu Glu              20 25 30 Leu Leu Arg Ala Leu Asp          35 40

Claims (14)

(1) a core comprising streptavidin, and (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 2,
Biotin is introduced at one end of the peptide,
Wherein two or more of said peptides are bound to one surface of said core on the surface of said core.
delete delete delete A pharmaceutical composition for preventing or treating an ischemic disease comprising the peptide structure of claim 1.
6. The method of claim 5, wherein the ischemic disease is selected from the group consisting of heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, stroke, peripheral vascular disease, angina pectoris, coronary artery insufficiency, Composition.
6. The composition of claim 5, wherein the composition further comprises a pharmaceutically acceptable additive.
(1) a core comprising streptavidin, and (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 3,
Biotin is introduced at one end of the peptide,
Wherein two or more of said peptides are bound to one surface of said core on the surface of said core.
delete delete delete A pharmaceutical composition for preventing or treating angiogenic diseases selected from the group consisting of cancer, arthritis, psoriasis, diabetic retinopathy, and macular degeneration comprising the peptide structure of claim 8.
13. The method of claim 12, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, colon cancer, uterine cancer, head and neck cancer, leukemia, lung cancer, lymphoma, melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer, Neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, retinoblastoma, glioma, Wilm's tumor, esophageal cancer, oral cancer, kidney cancer, multiple myeloma, pancreatic cancer, skin cancer and small cell lung cancer Lung cancer. &Lt; / RTI &gt;
13. The composition of claim 12, wherein the composition further comprises a pharmaceutically acceptable additive.

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