KR100847694B1 - Peptides inhibiting the binding of proline-hydroxylated hif-1a with vbc and a method for preparing thereof - Google Patents

Abstract

Peptides for inhibiting the binding of proline-hydroxylated HIF-1a(hypoxia inducible factor-1a) with VBC(a complex with elongin B and elongin C) are provided to inhibit degradation of HIF-1a under normoxia condition, so that the peptides are useful for treatment of ischemic diseases including coronary failure, brain insufficiency and vascular failure. A peptide for inhibiting the binding of proline-hydroxylated HIF-1a with VBC has the amino acid sequence comprising 556th-575th amino acids of HIF-1a(Genebank access number : u22431), wherein a 564th amino acid is substituted by an amino acid selected from an amino acid having acyclic hydrophobic group selected from valine, leucine and isoleucine, a hydrogen bond-forming amino acid selected from homoserine, threonine and asparagines, and an amino acid prepared by inserting a group other than hydroxyl group into a praline ring selected from 4-fluoroproline, 4-aminoproline and 4-thioproline.

Description

Peptides INHIBITING THE BINDING OF PROLINE-HYDROXYLATED HIF-1a WITH VBC AND A METHOD FOR PREPARING THEREOF}

1 is a diagram showing the structure of an inhibitor peptide in which hydroxyproline of HIF-1a is substituted with another amino acid,

Figure 2 shows the fluorescence polarization (FP) value of the binding of the fluorescent label peptide (F-HyP564, SEQ ID NO: 2) and the VBC protein having an amino acid sequence containing a proline group hydroxy position already at position 564 of HIF-1a Is a graph measured by

3A and 3B are graphs showing the inhibitory effect of various inhibitor peptides on the presence or absence of conjugate formation of F-HyP564 and VBC protein by the change in fluorescence polarization (FP) value, and Ki value is the F-HyP564 peptide that binds to VBC protein. It shows binding inhibition constant of the inhibitor peptide that competitively inhibits.

3A: 4FP (4-trans-fluoro-L-proline), □: leucine (Leu), Δ: valine (Val), ▼: asparagine (Asn),

In Figure 3b: Hse (L-homoserine), □: isoleucine (lle), ▲: aspartic acid (Asp), ▽: threonine (threonine, Thr).

The present invention relates to hydroxyproline of HIF-1a peptide that inhibits binding between HIF-1a and VBC protein having a proline group hydroxylated by HIF-1a spcific proryl hydroxylase 2 (PHD2). ) Relates to an inhibitor peptide substituted with another amino acid and a method for producing the same.

Hypoxia inducible factor-1 (HIF-1) plays an important role in regulating the expression of genes involved in energy metabolism, angiogenesis control, angiogenesis, apoptosis and various cellular responses in hypoxia It is a protein.

Among heterozygotes of HIF-1, HIF-1a is stabilized in hypoxia and affects the expression of various genes, but in normal oxygen state, it is degraded by proteasome in minutes. Has characteristics. In particular, the regulatory mechanism by proteosomes proceeds by decomposing HIF-1a with pVHL (von Hippel-Lindau tumour suppressor) protein, which is a substrate constitutive protein of the E3 ubiquitin-ligase complex. Therefore, inhibiting the binding between HIF-1a and pVHL may be useful in the treatment of ischemic diseases such as coronary insufficiency, brain insufficiency, and vascular insufficiency because normal cell cycle promotion, angiogenesis, or cell survival are enhanced in normal oxygen. Can be used (US 6,787,326 B1). On the other hand, facilitating interactions between them can inhibit the growth of cancer tissues through inhibition of angiogenesis in hypoxia, which can be very useful for cancer treatment research (Amato, G., et al., Nature Reviews, 2). , 1-9, 2003).

The binding of pVHL protein to human HIF-1a is dependent on the hydroxyproline group located at 402 or 564 of the amino acid sequence of HIF-1a, wherein the specificity of HIF-1a is determined by the prolyl hydroxylase-2 (PHD2) enzyme. The proline group is hydroxylated to regulate its interaction with the pVHL protein (Masson, N. & Ratcliffe, PJ, Journal of Cell Science, 116, 3041-3049, 2003). In particular, the prolyl hydroxylase-2 enzyme reacts in the presence of oxygen, iron, and the cofactor, 2-oxoglutarate, and the enzyme itself is oxidized to deactivate at a rapid rate. Ascorbate is required to prevent this (Ivan, M. et al., PNAS, 99 (21), 13459-13464, 2002).

The von Hippel-Lindau tumour suppressor (pVHL) protein consists of a β domain consisting of about 100 amino acids and a domain consisting of about 35 amino acids. The β region binds to the Elongin C protein to form a pVHL-ElonginC complex, and the Elongin B protein binds to the Elongin C portion of the complex to form a VBC protein complex. HIF-1a is bound to the a region of the VBC protein, where hydrogen bonding of the 115th histidine (His) group and the 111th serine (Ser) group and the 564th hydroxyproline group of HIF-1a is very important. (Min, JH, et al., Science, 296, 1886-1889, 2002).

Therefore, in order to analyze the interaction between the HIF-1a and the VBC protein and develop a therapeutic agent for various diseases related thereto, a substance that directly modulates the binding of the HIF-1a and the VBC protein, that is, a proline group-hydroxylated HIF-1a It is important to develop inhibitors that inhibit the binding of peptides to VBC proteins.

In the preceding invention, a method for simply and quantitatively analyzing the interaction between a probe having a fluorescent substance attached to a peptide derived from HIF-1a having a hydroxyproline group and a VBC protein complex using a change in fluorescence polarization has been developed. (Korean Patent Application No. 2005-0066881). Also, by using this method, compounds such as baicalein and cyclopirox, which inhibit the binding between HIF-1a having a hydroxylated proline group and VBC protein, are used. It has already been selected (Domestic Patent Application No. 2007-0007036). However, no biological inhibitors have been reported so far that can inhibit the binding between the two proteins additionally selected in addition to these compounds.

Accordingly, the present inventors have described a novel inhibitor peptide capable of inhibiting the binding of proline group-hydroxylated HIF-1a and VBC protein at a concentration of μM, that is, HIF-1a fragment peptide comprising the 556th to 575th amino acid sequence of HIF-1a. The present invention was completed by developing a synthetic peptide in which proline, which is the 564th amino acid of HIF-1a related to the hydroxylation reaction, is substituted with another amino acid.

Accordingly, it is an object of the present invention to provide an inhibitor peptide that inhibits the binding of HBC-1a and VBC protein to which a proline group is hydroxylated.

It is also an object of the present invention to provide a method for preparing the peptide.

In addition, another object of the present invention is to provide a composition for preventing or treating ischemic diseases such as coronary artery disease, brain failure, and vascular failure, comprising the peptide as an active ingredient.

In order to achieve the above object, the present invention provides a biological inhibitor, preferably an inhibitor peptide for inhibiting the binding of the proline group hydroxylated HIF-1a and VBC protein. These inhibitor peptides have a feature that specifically inhibits the formation of the F-HyP564-VBC protein complex because they act competitively with the F-HyP564 peptide against the VBC protein.

The present invention also provides a method for preparing the peptide. The preparation method is not particularly limited, and the novel peptides designed in the present invention may be prepared by a peptide synthesis method on a general solid support using amino acids protected with a protecting group (Fmoc or Boc). One such synthesis method is described by way of example as follows:

That is, the present invention

1) amino acid sequence having the 556th to 575th amino acid sequence of HIF-1a (GeneBank accession no. U22431) and proline, the 564th amino acid, having an acyclic hydrophobic functional group and an amino acid capable of hydrogen bonding; And synthesizing by substituting any one amino acid selected from the group consisting of amino acids formed by inserting a functional group other than a hydroxyl group into a proline ring; And

2) separating and purifying the synthesized peptide,

Provided are methods of making inhibitor peptides. In this case, the peptide synthesis may use a solid support phase method commonly used, and more specifically, may be performed using an amino acid and a resin in which the amino group of the substituted peptide is protected with a protecting group (Fmoc or Boc).

In an embodiment of the present invention, the peptide is a complex between the F-HyP564 and the VBC protein by competitively interacting with the proline-hydroxylated HIF-1a peptide (F-HyP564, SEQ ID NO: 2) located at position 564 relative to the VBC protein. Specifically inhibited formation. This suggests that the HIF-1a protein hydrated in the normal oxygen state is accumulated in the cell continuously without being degraded, and thus can be usefully used for the prevention or treatment of ischemic diseases such as coronary failure, brain failure, and vascular failure.

Therefore, in another aspect of the present invention, the peptide is an ischemic cardiovascular disease including heart disease such as heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction and angina and stroke and peripheral vascular disease, more preferably. May be usefully used for the prevention or treatment of one or more ischemic diseases selected from the group consisting of coronary artery failure, brain failure, and vascular failure.

Accordingly, the present invention provides a composition for preventing or treating at least one ischemic disease selected from the group consisting of coronary artery failure, brain failure, and vascular failure, comprising the peptide as an active ingredient.

Hereinafter, the present invention will be described in detail.

In the foregoing invention, the present inventors can easily quantitatively analyze whether or not a complex of a fluorescently labeled HIF-1a peptide and a VBC protein is formed through a change in fluorescence polarization value (Domestic Application No. 2005-0066881, the present invention). Was used as a reference to screening compounds such as baicalein and cyclopirox, which are inhibitor compounds that specifically inhibit the binding of proline groups to hydroxylated HIF-1a and VBC proteins (domestic) Patent Application No. 2007-0007036, incorporated by reference herein).

Accordingly, the present invention provides a synthetic peptide in which proline, the 564th amino acid of HIF-1a related to the hydroxylation reaction, is substituted with another amino acid, which is different from the inhibitor compound. The biological inhibitor is not particularly limited, and may be any antibody, peptide, and oligonucleotide that specifically inhibits the binding of the proline group to hydroxylated HIF-1a and the VBC protein, and more preferably, may be a synthetic peptide. .

More specifically, the synthetic peptide is a peptide derived from HIF-1a and includes the 556th to 575th amino acid sequence of HIF-1a, and the 564th of HIF-1a related to hydroxyproline, ie, hydroxylation of HIF-1a peptide. The amino acid proline may be a synthetic peptide substituted with another amino acid.

The substitutable amino acid may be a natural amino acid or an artificially synthesized amino acid, and preferably has a 556 th to 575 th amino acid sequence of HIF-1a (Genebank Accession No. u22431), and a proline, 564 th amino acid, may be used. It may be an amino acid selected from the group consisting of an amino acid having a cyclic hydrophobic functional group, an amino acid having a hydrogen bonding functional group, and an amino acid made by inserting a functional group other than a hydroxyl group in the proline ring.

More specifically, the amino acid having an acyclic hydrophobic functional group may be selected from the group consisting of valine, Val, leucine, Leu, and isoleucine, lle,

The amino acid having a functional group capable of hydrogen bonding may be selected from the group consisting of homoserine (homoserine, Hse), threonine (threonine, Thr), and asparagine (Asn),

The amino acid formed by inserting a functional group other than a hydroxyl group into the proline ring is composed of 4-fluoroproline (4-fluoroproline), 4-aminoproline (4-aminoproline), and 4-thioproline (4-thioproline). It may be selected from the group.

More preferably, the peptide in which the proline is substituted with another amino acid may have one amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 10, in particular, SEQ ID NO: 3 or SEQ ID NO: More preferably, it has an amino acid sequence of 8 (see Table 1).

In addition, in the embodiment of the present invention, in order to determine whether the peptides thus prepared can inhibit the formation of the complex between the proline group-hydroxylated HIF-1a peptide and the VBC protein, quantitative analysis through changes in the previously reported fluorescence polarization values (Korean Patent Application No. 2005-0066881; Domestic Patent Application No. 2007-0007036; H, Cho, et al., Biochem. Biophy. Res. Comm., 337, 275-280, 2005) It was.

Specifically, the present invention

1) Fluorescence polarization value was measured after reacting a mixture of a fluorescent label (F-HyP564, SEQ ID NO: 2) and a VBC protein containing an already hydroxylated proline residue located at at least 564 of the amino acid sequence of HIF-1a Making;

2) measuring the fluorescent polarization value after adding at least one synthetic peptide selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 10 to the reaction solution before measuring the fluorescent polarization value; And

3) When the value of step 2) is reduced based on the value of step 1) by comparing the fluorescence polarization values measured in steps 1) and 2), the peptide may be a hydroxylated HIF-1a peptide (FF) for VBC protein. Determining an inhibitor peptide that competitively interacts with -HyP564, SEQ ID NO: 2) to inhibit complex formation between the F-HyP564 and the VBC protein.

It provides a method for measuring the inhibition of the inhibitor peptide on the binding of the proline group hydroxylated HIF-1a and VBC protein.

In an embodiment of the present invention described below, the degree of inhibition of the peptides to the binding of the F-HyP564 peptide and the VBC protein using the measurement method was measured by the fluorescent polarization value, concentration-dependently fluorescence by the peptide It was confirmed that the polarization value was reduced (see FIGS. 3A and 3B).

Thus, in another aspect, the peptide of the present invention is a cardiac disease, including heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction and angina, and ischemic cardiovascular disease, including stroke and peripheral vascular disease, more preferably. May be used as an active ingredient of a composition for the prevention or treatment of one or more ischemic diseases selected from the group consisting of coronary artery failure, brain failure, and vascular failure.

Accordingly, the present invention provides a composition for preventing or treating at least one ischemic disease selected from the group consisting of coronary failure, brain failure, and vascular failure, comprising the peptide as an active ingredient. The dosage of the composition may generally be 0.01 to 100 mg / kg, more preferably 0.5 to 10 mg / kg, based on the active ingredient content.

The composition may further include one or more additives selected from the group consisting of pharmaceutically acceptable excipients, disintegrants, binders and lubricants in addition to the active ingredient, wherein the type of the additives is not particularly limited. In addition, the composition may be used orally, for example, it may be prepared as a warning agent, granules, powders, tablets, capsules and the like. The dosage of the composition is appropriately adjusted according to the use, the purpose of use, the condition of the patient, age, sex, weight, prescription drug and disease, and preferably once to several times a day, more preferably 3 It can be administered in divided doses.

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

< Example  1> Inhibitor Peptide  Produce

In order to prepare an inhibitor peptide that specifically inhibits the binding of proline group-hydroxylated HIF-1a to VBC protein, a method using a solid support phase was performed. As a solid support phase, Link amide MBHA resin (Novabiochem, Switzerland) was used. Used. Peptide synthesis used a commonly used Fmoc coupling protocol.

Specifically, the solid phase reaction was carried out using 25 mM resin and amino group with amino group protected by Fmoc, and the resin was de-protected for 20 minutes with 20% of piperidine, followed by 1 mM dimethylformamide (N , N'-dimethylforamide, DMF) was washed five times. Fmoc-protected amino acid and 3 equivalents of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBop), 3 equivalents of N-hydroxylbenzotriazole (HOBT), and 6 equivalents of diisopropylethylamine ( Coupling reaction was carried out for 3 hours after the addition of DIPEA). Completion of the reaction was confirmed through a Kaiser test. The resin was reacted at room temperature for 2 hours in 1 mL of mixed solution [TFA (Trifluoroacetic acid): thioanisole: H ₂ O = 95: 2.5: 2.5)] to remove the peptide from the solid support phase, and then the supernatant was It was transferred to a vessel and precipitated by the addition of 5 times the volume of diethylether. After removing the supernatant, the precipitate was washed once with 5 times the volume of diethyl ether, and the precipitate obtained was dried in vacuo, dissolved in water and purified using reversed phase HPLC. Each purified peptide was identified by amino acid sequence using a MALDI-TOF (Voyager, Applied Biosystem, USA) mass spectrometer. The sequence of each peptide thus synthesized is described in SEQ ID NOs: 3 to 10 as shown in Table 1 below (Table 1), and the structure of the peptide having such substituted amano acid is illustrated in FIG. 1 (FIG. 1).

SEQ ID NO: synthesis Peptide One FITC-AHA-DLDLEALA-P-YIPADDDFQLR-NH ₂ (F-P564) 2 FITC-AHA-DLDLEALA-Hyp-YIPADDDFQLR-NH ₂ (F-HyP564) 3 DLDLEALA-4FP-YIPADDDFQLR-NH2 (4FP = 4-trans-fluoro-L-proline) 4 DLDLEALA-Leu-YIPADDDFQLR-NH ₂ 5 DLDLEALA-Ile-YIPADDDFQLR-NH ₂ 6 DLDLEALA-Asn-YIPADDDFQLR-NH ₂ 7 DLDLEALA-Asp-YIPADDDFQLR-NH ₂ 8 DLDLEALA-Hse-YIPADDDFQLR-NH2 (Hse = L-homoserine) 9 DLDLEALA-Val-YIPADDDFQLR-NH ₂ 10 DLDLEALA-Thr-YIPADDDFQLR-NH ₂

< Example  2> Fluorescently labeled HIF -1a Peptide  And VBC  Protein manufacturing

<2-1> Fluorescently labeled HIF -1a Peptide  Produce

To attach fluorescent material to the HIF-1a peptide, hydroxylation in the amino acid sequence of a site known to bind VBC protein in human HIF-1a protein (Min, JH, et al., Science, 296, 1886-1889, 2002) The 556-575th amino acid site including the proline group that induces the reaction was selected, and the N-terminus of the HIF-1a peptide was attached with an aminocaproic acid linker, followed by FITC (fluorescein isothiocyanate). A peptide consisting of three parts labeled was synthesized by Angen (Korea). The synthesized peptide was named F-P564 (FITC-ACA-DLDLEALAPYIPADDDFQLR, SEQ ID NO: 1), and the 564th proline group (9th amino acid of the amino acid sequence of SEQ ID NO: 1) of the F-P564 peptide was a hydroxylated peptide. F-HyP564 (FITC-ACA-DLDLEALAHyPYIPADDDFQLR, SEQ ID NO: 2) was synthesized respectively (Table 1).

<2-2> VBC  Protein manufacturing

In order to prepare VBC (von Hippel-Lindau-Elongin B-Elongin C) protein, in this embodiment, the 54-213 amino acid sequence of the Human von Hippel-Lindau gene (GeneBank accession number: NM000551) and the Human Elongin B gene (GeneBank) The 1-118th amino acid sequence of registration number: NM007108) was cloned into pGEX-4T-1 (GE Healthcare Life Sciences, USA) to prepare a pGEX4T-VHL-EB expression vector, and the Human Elongin C gene (GeneBank accession number: NM005648 Cloned into the pET29b (Novagen) to prepare pDEV-EC expression vectors, respectively, and transformed simultaneously into BL21 (DE3) (Novagen) strain.

The transformed cells were inoculated in LB medium containing 50 μg / ml of ampicillin, respectively, and cultured at 37 ° C. until the absorbance became 0.8-0.9. After incubation, 0.5 mM of IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce protein expression and incubated at 18 ° C. for 15 hours. 10 mM phosphate buffer solution (PBS, pH 7.4) in which PMSF (phenylmethylsulfonyl fluoride, Sigma) and lysozyme (Sigma) were added to the cells obtained by centrifugation so that their final concentrations were 0.2 mM and 1 mg / ml, respectively; Suspended in 110 mM NaCl, 1 mM DTT) and triturated by ultrasound at 4 ° C.

2% Triton X-100 was added to the obtained cell extracts, mixed well, and then placed on ice for 10 minutes, followed by centrifugation at 13,000 rpm for 30 minutes. 1 mM of DTT was added to the separated supernatant, mixed with glutathione-sepharose 4B resin (Bio-Rad), and then stirred at 4 ° C. for 2 hours. A volume of phosphate buffer solution was added, centrifuged at 2,100 rpm for 5 minutes, and then the supernatant was removed. The procedure was repeated three times in succession to remove the supernatant, and then the reaction resin mixture was placed in Bio-Spin ^® Disposable Chromatography Columns (Bio-Rad), filtered with 5 ml of PBS, and filtered with 2 ml of 1 M NaCl solution. Unnecessary reactants were removed. The column was eluted with 10 mM glutathione (GSH) to yield GST-VBC protein. At this time, the obtained protein was confirmed purity by SDS-PAGE, and quantified by Bradford protein analysis (Bio-Rad).

< Example  3> HIF -1a and VBC  Inhibitors of protein binding Peptide  Inhibitory efficacy analysis

The above-mentioned fluorescence polarization measurement method was used to measure the inhibitory effect of the inhibitor peptide synthesized in Example 1 on the binding of the proline group-hydroxylated F-HyP564 peptide to the VBC protein. To this end, first, by reacting the VBC protein prepared in Example 2-2 and the F-HyP564 (SEQ ID NO: 2) peptide synthesized in Example 2-1 to measure the change in fluorescence polarization value according to the complex formation of the protein-ligand It was. The results are shown in FIG.

As shown in FIG. 2, as a result of increasing the concentration of VBC to F-Hyp564 at a constant concentration of 100 nM, the formation of the complex of the fluorescently labeled F-Hyp564 peptide and the VBC protein was increased to increase the fluorescence polarization value. From this graph, it was found that the dissociation constant (Ki) value between the VBC protein and the HIF-1a peptide was 50 nM (FIG. 2).

Based on the above results, in order to investigate the degree of inhibition of the inhibitor peptide on the formation of HIF-1a and VBC protein complex, NP-40 (nonidet P-40) was added so that the final concentration was 0.5% without adding the inhibitor peptide. ) Was added to the buffer solution (50 mM Tris, 120 mM NaCl, pH 8.0) to form a complex by adding 100 nM of F-Hyp564 peptide and 154 nM of VBC protein, and then measured the value of fluorescence polarization. Used as a value. In addition, the inhibitor peptide synthesized in the present invention was added to the complex solution before fluorescence polarization measurement at 25 ° C. while increasing the concentration of 0-500 mM, respectively, and then the fluorescence polarization value according to the inhibitor peptide concentration was measured. In this case, the fluorescence polarization value was used with a fluorometer (Perkin-Elmer), the size of the slit was set to 5 nm, the integration time was set to 5 seconds. The results are shown in Figures 3a and 3b.

As shown in Figures 3a and 3b, the addition of the inhibitor peptide decreased the concentration of fluorescence polarization in a concentration-dependent manner compared to the case without addition (Figs. 3a and 3b), and the dissociation constant (Ki) between these inhibitor peptides and the VBC protein ) Values were lowest in Hse (Ki = 3.4 uM) and 4FP (Ki = 5.8 uM) as shown in Table 2 (Table 2).

Inhibitor peptides 4FP Hse Leu lle Val Asn Asp Thr Ki (uM) 5.8 3.4 58 67 16 13 15 18

From these results, it can be seen that the inhibitor peptide inhibits the complex formation of the F-HyP564 peptide and the VBC protein by interacting with the F-HyP564 peptide with respect to the VBC protein. In other words, the inhibitor peptides act as inhibitors that inhibit the interaction between HIF-1a and the VBC protein.

As described above, the inhibitor peptide according to the present invention specifically inhibits the binding of the proline group-hydroxylated HIF-1a and VBC protein, so that the HIF-1a protein hydroxylated in the normal oxygen state is not decomposed, coronary artery failure, In addition to being useful for the prevention or treatment of ischemic diseases such as brain and vascular insufficiency, it may be useful for studying intracellular physiological phenomena that may be associated with hydroxylated HIF-1a.

Attach sequence list electronic file  

Claims (8)

Has the amino acid sequence 556 to 575 of HIF-1a (Genebank Accession No. u22431), Proline, the 564th amino acid, Amino acids having acyclic hydrophobic functional groups selected from the group consisting of valine, Val, leucine, Leu, and isoleucine, lle; Amino acids having a functional group capable of hydrogen bonding selected from the group consisting of homoserine (Hse), threonine (Th), and asparagine (Asn); And Made by inserting a functional group other than a hydroxyl group into a proline ring selected from the group consisting of 4-fluoroproline, 4-aminoproline, and 4-thioproline Substituted with an amino acid selected from the group consisting of amino acids, Inhibitor peptides that inhibit the binding of HIF-1a and VBC proteins. delete According to claim 1, wherein the peptide is a peptide having an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 3 to SEQ ID NO: 10. 1) has the 556th to 575th amino acid sequence of HIF-1a (GeneBank Accession No. u22431), Amino acid having an acyclic hydrophobic functional group selected from the group consisting of valine (Val), leucine (Leu), and isoleucine (lle); Amino acids having a functional group capable of hydrogen bonding selected from the group consisting of homoserine (Hse), threonine (Th), and asparagine (Asn); And Made by inserting a functional group other than a hydroxyl group into a proline ring selected from the group consisting of 4-fluoroproline, 4-aminoproline, and 4-thioproline Synthesizing by substituting any one amino acid selected from the group consisting of amino acids; And 2) separating and purifying the synthesized peptide, A method for preparing an inhibitor peptide that inhibits binding of HIF-1α and VBC protein. delete Claim 1 or claim 3 comprising the peptide of the active ingredient, the composition for the prevention or treatment of ischemic diseases. The composition for preventing or treating ischemic disease of claim 6, wherein the ischemic disease is selected from the group consisting of coronary artery failure, brain failure, and vascular failure. The composition for preventing or treating ischemic disease of claim 6, wherein the composition further comprises one or more additives selected from the group consisting of pharmaceutically acceptable excipients, disintegrants, binders and glidants.

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