KR20120002119A - Inhibitor for phd2 - Google Patents

Abstract

PURPOSE: A PHD2(HIF-1alpha specific proline hydroxylase domain 2) inhibitor peptide is provided to suppress hydration of HIF-1alpha and to prevent or treat ischemic diseases. CONSTITUTION: A peptide with PHD2-specific suppression contains 556th to 575th amino acids of human HIF-1alpha protein. The 564th amino acid has a proline in which 4th carbon is substituted with hetero atom of S, O, N, or P. A proline variant is thiazolidine carboxylic acid, dehydroproline, methanoproline, or oxazolidine carboxylic acid. The peptide has an amino acid sequence of sequence number 2(DLDLEALAXYIPADDDFQLR). A composition for preventing or treating ischemic diseases contains the peptide as an active ingredient.

Description

PHD2 inhibitors {Inhibitor for PHD2}

The present invention relates to inhibitor peptides comprising amino acid analogs that inhibit the hydroxylation of HIF-1α by PHD2.

HIF-1α (hypoxia inducible factor-1 alpha) is an important regulator of the regulation of hypoxia by regulating the expression of genes involved in energy metabolism, vascular control, neovascularization and apoptosis. It is a protein that plays a role. HIF-1α, which is degraded by proteosomes in normal oxygen conditions, stabilizes and accumulates in hypoxic states. The regulatory mechanism by proteosomes is ubiquitin- after HIF-1α is hydroxylated by PHD2. It is degraded by binding to pVHL (von Hippel-Lindau tumour suppressor) protein, a substrate recognition protein of ubiquitin-ligase complex. Therefore, inhibition of HIF-1α-induced PHD2-induced hydroxylation inhibits HIF-1α degradation in normal oxygen environment, thereby facilitating cell cycle progression, angiogenesis, or cell survival. And ischemic conditions such as vascular insufficiency (US 6,787,326 B1). On the other hand, promoting the activity of PHD2 can inhibit the growth of cancer tissues by inhibiting angiogenesis in hypoxic state, which is very useful for cancer treatment research (Amato G. et al., Nature Reviews 2: 1-9, 2003).

The hydroxylation reaction of HIF-1α is catalyzed by prolyl hydroxylase domain proteins (PHDs) such as PHD1, PHD2, PHD3. In particular, PHD2 is known to be an important enzyme for controlling the decomposition process of HIF-1α based on the detection of oxygen. It is an enzyme that hydroxylates the proline group located at 402 or 564th amino acid sequence. The PHD2 enzyme reacts in the presence of oxygen, iron, and cofactor 2-oxoglutarate, which is used to prevent fast deactivation of the enzyme itself by oxidation. (ascorbate) is required (Ivan M. et al., PNAS 99 (21): 13459-13464, 2002). Through this hydroxylation reaction, HIF-1α-mediated hypoxia-induced protein transcription and HIF-1α formation of mechanisms that regulate the hydrolysis (proteolytic destruction) is involved in various pathological mechanisms are involved.

In order to analyze the effects of HHD-1α-induced PHD2 on physiological phenomena associated with HIF-1α and to inhibit the activity of PHD2, the development of a related disease therapeutic agent that is directly regulated by HIF-1α activity is required. Inhibitors are required. However, inhibitors of PHD2 known to date are mostly low molecular weight substances that bind PHD2 while competing with 2-oxoglutarate. Inhibitors targeting these 2-oxoglutarate binding sites selectively inhibit PHD2 as well as other inhibitors of HIF (factor inhibiting HIF), another HIF activity regulator that uses 2-oxoglutarate as a cofactor. In terms of disadvantages. One solution to this problem is to use inhibitors that target HIF-1α binding sites in the active pocket of PHD2. However, there are no reports of inhibitors targeting HIF-1α binding sites in PHD2 activity pockets, as well as at the small organic molecule level.

Accordingly, the present inventors have completed the present invention by developing an inhibitor peptide that targets the binding site of HIF-1α in PHD2.

One embodiment of the present invention provides an inhibitor peptide that inhibits the hydroxylation reaction of HIF-1α by HIF-1α specific proryl hydroxylase 2 (PHD2).

Another example provides a method for preparing the inhibitor peptide.

Another example provides a composition for the prevention or treatment of ischemic diseases comprising the inhibitor peptide as an active ingredient.

The inhibitor peptide developed in the present invention was synthesized by substituting the proline group, which is the 564th amino acid in which hydroxylation occurs, as an analog of the HIF-1α fragment peptide consisting of the 556th to 575th amino acid sequences of the HIF-1α protein. The substituted amino acid has a carbon ring structure similar to that of proline, but the C-4 position of the proline is modified to bind to PHD2, but the reaction is not progressed. The inhibitor peptide thus synthesized can be used to activate HIF-1α by specifically inhibiting only the HIF-1α hydroxylation reaction by PHD2 without affecting FIH, another HIF hydroxylase, and thus specific inhibition on PHD2. Can be usefully used to determine how HIF-1α activated by HF-1 affects physiological phenomena and diseases associated with HIF protein.

Hereinafter, the present invention will be described in detail.

One example of the present invention is an inhibitor peptide that inhibits the hydroxylation reaction of HIF-1α by HIF-1α specific proryl hydroxylase-2 (PHD2). To provide.

The inhibitor peptide is a peptide derived from HIF-1α, and is selected from the group consisting of peptides that inhibit PHD2 by inserting another functional group into the proline ring, thereby facilitating the binding reaction with PHD2 but not allowing the hydroxylation reaction to proceed.

The inhibitor peptide is a HIF-1α fragment peptide consisting of the 556th to 575th amino acid sequence of the HIF-1α protein. The proline group, which is the 564th amino acid in which hydroxylation occurs, may be substituted with another amino acid. The HIF-1α protein may be a mammalian, such as human, HIF-1α protein (AAF20139, 561th amino acid methionine may be substituted with alanine for oxidation).

The 556 th to 575 th amino acid sequence of the HIF-1α protein may be the sequence of SEQ ID NO: 1 (556-DLDLEALA P YIPADDDFQLR-575, the underlined amino acid indicates the proline 564 th position).

The amino acid substituting the proline group has a carbon ring structure similar to that of proline, but the C-4 position of the proline is modified to bind to PHD2, but it is designed to exhibit a mechanism of inhibiting PHD2 by not reacting. And specifically inhibits only the hydroxylation reaction of HIF-1α by PHD2 without affecting other HIF hydroxylases (eg, factor inhibiting HIF). The more specific principle of the inhibitory effect of PHD2 by the modification of the proline is that when the peptide binds to PHD2, the hydroxyl reaction proceeds to position 4 carbon while maintaining the ring conformation of the 564th proline as C4-endo. By designing all types of proline variants with chemical modifications to prevent them from being replaced, the modified proline is replaced at position 564. Such proline analogue is that the carbon at position 4 is substituted with a heteroatom selected from the group consisting of S, O, N, P, etc., or the carbon at position 4 forms a double bond with the carbon at position 3, or the position 4 And carbon in position 3 may form a bicyclic form by forming a ring with an alkyl group of C1-C3. For example, the proline variant may be thiazolidine carboxylic acid (eg, L-thiazolidine-4-carboxylic acid (Thz)), dehydroproline (eg, 3,4-dehydro-L-proline ( DhP)), metanoproline (methanoproline; for example 3,4-trans-L-methanoproline (BcP)), oxazolidine carboxylic acid; for example, L-oxazolidine-4-carboxylic acid (Oxa) It may be selected from the group consisting of.

Thus, the inhibitor peptide can be represented by the following SEQ ID NO: 2

(SEQ ID NO: 2)

DLDLEALA X YIPADDDFQLR

In the above sequence, X is a proline analog designed to maintain C4-endo but not to undergo a hydroxylation reaction at position 4 carbon, and the carbon at position 4 of proline is selected from the group consisting of S, O, N, and P. Substituted with a hetero atom, carbon at position 4 forms a double bond with carbon at position 3, or carbon at position 4 and carbon at position 3 forms a ring with an ethyl group of C1-C3 to form a bicyclic ), Such as thiazolidine carboxylic acid, dehydroproline, metanoproline, or oxazolidine carboxylic acid.

In one embodiment of the invention, the inhibitor peptide consists of the 556th to 575th amino acid sequence of the HIF-1α protein, the 564th amino acid is substituted with Thz (L-thiazolidine-4-carboxylic acid) (DLDLEALA-Thz-YIPADDDFQLR, SEQ ID NO: 3), substituted with DhP (3,4-dehydro-L-proline) (DLDLEALA-DhP-YIPADDDFQLR, SEQ ID NO: 4), BcP (3,4-trans- L-methanoproline) (DLDLEALA-BcP-YIPADDDFQLR, SEQ ID NO: 5), Oxa (L-oxazolidine-4-carboxylic acid) substituted (DLDLEALA-Oxa-YIPADDDFQLR, SEQ ID NO: 6) and the like.

In one embodiment, the peptide designed as described above can be synthesized by using a peptide synthesis method on a general solid support using Fmoc protected amino acid. The synthesized Thz peptide (SEQ ID NO: 3) and the DhP peptide (SEQ ID NO: 4) were confirmed using a MALDI-TOF mass spectrometer.

In order to measure the degree of inhibition in vitro, the PHD2 reaction was performed with inhibitor peptides, and then MALDI-TOF MS method was used for reaction analysis. In the MALDI-TOF MS analysis, when the PHD2 reaction was sent without the inhibitor peptide (A of FIG. 2), a hydroxylated substrate peptide was produced in which the molecular weight of 16 dalton was increased compared to the biotin-attached substrate peptide (SEQ ID NO: 1). When the PHD2 reaction with Thz and DhP peptides was hardly produced, it was confirmed that the inhibitory peptides effectively inhibited the PHD2 reaction due to the fact that little hydroxylated substrate peptides were generated (Figs. 2B and C).

To determine the degree of quantitative inhibition of this inhibitor peptide, IC ₅₀ values were obtained by increasing the concentration of Thz and DhP peptides and measuring the inhibition of PHD2 reaction. For this, a method using a previously reported fluorescence polarization was used (H. Cho, H. Park, and E. Yang, Biochem . Biophy , Res . Comm . 337: 275-280. 2005). When the fluorinated substrate peptide (SEQ ID NO: 1) is hydroxyproline 564 hydroxylated by PHD2 to hydroxyproline 564, the substrate peptide and the VBC protein bind to each other to increase the fluorescence polarization compared to the non-hydroxylated substrate peptide. The extent of PHD2 response was measured. As a result, the IC ₅₀ values for the PHD2 response of the Thz and DhP peptides were determined to be 58.6 and 11.7 μM, respectively (FIG. 3).

In order to confirm the selectivity of PHD2 inhibitory effect by Thz and DhP peptides, inhibition of FIH, another HIF-1α hydroxylase, was confirmed by using inhibitor peptides of constant concentration (100 μM). The degree of inhibition of the FIH reaction was also analyzed using fluorescence polarization. In this case, the fluorinated FIH substrate peptide (SEQ ID NO: 5) showed increased fluorescence polarization by binding to the p300 protein, but when bound to the p300 protein. As a result, the decrease in fluorescence polarization was used in the analysis as the amount of hydroxylation progressed, in contrast to the case of PHD2. As a result, when the PHD2 reaction did not increase the fluorescence polarization in the presence of inhibitory peptides, both Thz and DhP peptides showed the effect of inhibiting the PHD2 response (Fig. 4, A).

On the other hand, in the case of the FIH reaction, the reaction in the presence of inhibitor peptides did not show a difference compared to the case in which the fluorescence polarization was not (in the presence of DMSO), indicating that the Thz and DhP peptides did not inhibit the FIH reaction. (Figure 4, B). This is in contrast to the fact that baicalein, one of the known PHD2 inhibitors, inhibits not only PHD2 but also FIH. Baicalein when seen that known to exhibit an inhibitory effect, by the 2-oxoglutarate in the PHD2 and the FIH active pocket prevents the iron 2 in the enzyme combined with the ion (Fe ^{2 +),} inhibitor peptides of the present invention and 2-oxoglutarate Irrespective of the inhibitory effect by the interaction between PHD2 and HIF-1α.

In order to prove that the inhibitor peptides of the present invention inhibit PHD2 irrespective of the 2-oxoglutarate binding site, the degree of inhibition of PHD2 by DhP peptide was measured by competition in varying concentrations of 2-oxoglutarate. Baicalein has shown that the inhibition of PHD2 was alleviated with increasing concentrations of 2-oxoglutarate in the same kind of competitive experiments in the literature (Cho et al. Mol. Pharm. 2008, 74, 70-81). On the other hand, DhP peptide constantly inhibited PHD2 regardless of the concentration of 2-oxoglutarate (FIG. 5). Therefore, it can be seen that the inhibitor peptides of the present invention, Thz and DhP peptides exhibit an inhibitory effect by interfering with interaction between PHD2 and HIF-1α regardless of 2-oxoglutarate.

In order to confirm the PHD2 inhibitory effect of the inhibitor peptide of the present invention in cells, the amount of HIF-1α expression in HeLa cells grown in the medium treated with the inhibitor peptide was analyzed and compared with the normal cells without the inhibitor. As a result, an increase in the amount of HIF-1α expression was confirmed, which effectively inhibited the PHD2-induced hydroxylation in the cell, thereby preventing HIF-1α destruction in the proteasome by ubiquitination that proceeds after the hydroxylation. It can be seen that the act was the result. Therefore, it can be seen that the inhibitor peptides (Thz and DhP peptides) of the present invention effectively inhibit PHD2 not only in vitro but also in cells.

As described above, since the inhibitor peptide of the present invention has excellent inhibitory effect of PHD2, it inhibits the hydroxylation reaction of HIF-1α by PHD2, thereby inhibiting HIF-1α degradation in a normal oxygen environment, thereby promoting the progress of the cell cycle or vascular Since the formation of the hyperlipidemia or cell viability is enhanced, it has a prophylactic and / or therapeutic effect of ischemic disease and a neuroprotective effect.

Accordingly, another aspect of the present invention provides a composition for preventing and / or treating and / or neuroprotective ischemic diseases containing the inhibitor peptide as an active ingredient. The composition is effective for ischemic diseases, and the ischemic disease includes ischemic brain (vascular) disease, ischemic heart disease, and the like, for example, cerebral ischemic attack, stroke, anemia, brain failure, vascular insufficiency, coronary insufficiency, etc. It may have a prophylactic and / or therapeutic effect on one or more diseases selected from the group consisting of.

The content of the inhibitor peptide as an active ingredient in the composition may be appropriately adjusted according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, and the like, for example, 0.0001 to 99.9% by weight, preferably 0.001 to 50, based on the total weight of the composition. The weight% is preferably, but is not limited thereto.

The composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, and oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like according to conventional methods It may be used in the form of a dosage form, an external preparation, a suppository, or a sterile injectable solution.

When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one or more excipients and / or lubricants. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.

The preferred dosage of the composition depends on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. For a more preferable effect, the dosage of the composition of the present invention may be about 0.1 to 100 mg / kg per day based on the active ingredient, for example, 1 to 10 mg / kg per day, but is not limited thereto. It doesn't happen. Administration may be administered once a day or may be divided several times. The compositions of the present invention can be administered by various routes to an animal, preferably a mammal, including a human. All modes of administration can be expected, for example, can be administered orally, intravenously, intramuscularly, subcutaneously, etc., preferably, intravenously. The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt of the active ingredient, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

As described above, the inhibitor peptide according to the present invention inhibits the HIF-1α hydroxylation by PHD2, so that the decomposition mechanism of HIF-1α protein is not inhibited in the normal oxygen state, thereby preventing coronary artery failure and brain failure. And it can be useful not only for the prevention or treatment of ischemic diseases such as vascular insufficiency, but also for studying the intracellular physiological phenomena that may be associated with the activity of HIF-1α.

1 is a diagram showing the amino acid sequence of inhibitor peptides and the structure of amino acids substituted for HIF-1α proline 564 (Pro564),
2 is a result of analyzing the inhibition of PHD2 by inhibitor peptides using MALDI-TOF MS,
FIG. 3 shows the results of analyzing IC50 values of inhibitor peptides by PHD2 inhibition by fluorescence polarization.
FIG. 4 shows the results of analyzing PHD2 inhibition selectivity of peptides by assay using fluorescence polarization by measuring A) PHD2 and B) FIH responses by inhibitor peptides, respectively (Bc is baicalein).
FIG. 5 shows that the inhibitor peptide DhP peptide uses the fluorescence polarization of the competition reaction by changing the concentration of 2-oxoglutarate to show that the inhibitory effect does not affect the portion of 2-oxoglutarate binding in the active pocket of PHD2. Analysis results,
Figure 6 is the result of analysis by measuring the amount of HIF-1α remaining undegraded by Western blot the degree of inhibition of the inhibitor peptide Thz peptide PHD2 in HeLa cells. Thz: Thz peptide treated with HeLa cells by electroporation using 400V or 800V electricity. DFO: Treated HeLa cells with 10 μM deferoxamine farm. Control: HeLa cells that did not process anything.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

< Example  1> Inhibitor Peptide  Produce

To prepare inhibitor peptides that inhibit the hydroxylation of HIF-1α by PHD2, a method using a solid support phase was performed. Link amide MBHA resin (Novabiochem, Switzerland) was used as a solid support phase. Peptide synthesis used a commonly used Fmoc coupling protocol.

Specifically, the solid phase reaction was carried out using 25 mM resin and amino group protected amino group with Fmoc, the resin was de-protected with 20% piperidine for 20 minutes and then 1 mM dimethylformamide. Washed five times with (N, N'-dimethylforamide, DMF). Fmoc-protected amino acid or biotin and 3 equivalents of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBop), 3 equivalents of Nhydroxylbenzotriazole (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: H2O = 95: 2.5: 2.5)] to remove the peptide from the solid support, and then the supernatant was transferred to another container. The precipitate was added by adding 5 times diethyl ether. 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.

Reversed Phase HPLC Conditions

Buffer A: 0.1% TFA in H2O,

Buffer B: 0.1% TFA in acetonitrile

The first 10 minutes use a gradient that increases the percentage of B solution by 3% per minute for 100% A and then 10-40 hours.

Flow rate: 1 mL / min.

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 shown in Table 1 below, and the modified proline amanoic acid structure is shown in FIG. 1 (FIG. 1).

order Substrate peptide Biotin-DLDLEALA-P-YIPADDDFQLR-NH ₂ (C-terminal) (Biotin-SEQ ID NO: 1) Inhibitor peptides DLDLEALA-Thz-YIPADDDFQLR-NH ₂ (SEQ ID NO: 3, Thz Peptide) Inhibitor peptides DLDLEALA-DhP-YIPADDDFQLR-NH ₂ (SEQ ID NO: 4, DhP Peptide)

* Thz = L-thiazolidine-4-carboxylic acid)

* DhP = 3,4-dehydro-L-proline

< Example  2> PHD2  Protein manufacturing

In order to prepare a PHD2 protein, in this example, the amino acid sequence of the 184th to 426th amino acid sequence of the human PHD2 gene (GeneBank accession number: Q9GZT9) was cloned into pET32a (GE Healthcare Life Sciences, USA) to express the pET32a-PHD2 expression vector. After preparation, the strains were transformed into BL21 (DE3) (Novagen) strain. BamH I and Sal I (New England Biolabs) were used as restriction enzymes. The transformed cells were inoculated in LB medium (Sigma-Aldrich) containing 50 μg / ml of ampicillin, respectively, and then cultured at 37 ° C. until the absorbance was 0.6 at 600 nm.

After incubation, 0.25 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce protein expression and incubated at 18 ° C. for 15 hours. After removing the supernatant by centrifugation, 30 mL of washing buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, 10 mM imidazole) was added and suspended. 40 μL of 100 mM PMSF (phenylmethylsulfonyl fluoride), 200 μL of 100 mg / μL lysozyme, 30 μL of 2-mercaptoethanol, and 2.5 mL of 60% glycerol were added to the cell solution, followed by sonication at 4 ° C.

Triton X-100 was added to the cell extracts, mixed well, and then placed on ice for 10 minutes, followed by centrifugation at 13,000 rpm for 30 minutes. The separated supernatant was mixed with Ni-NTA resin (Amersham Biosciences), stirred at 4 ° C. for 2 hours, and 20 mL of washing buffer was added to the reaction mixture, followed by centrifugation at 1,000 rpm for 2 minutes. 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) to prepare 10 mL of washing buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, 10). filtered off with mM imidazole to remove non-adsorbed material. A buffer solution containing 50 mM NaH ₂ PO ₄ , 500 mM NaCl, and 300 mM imidazole was flowed to the column to obtain PHD2, which was eluted to the bottom of the column, and glycerol was added to -70 in a form containing 20% glycerol. Stored at ° C. At this time, the obtained protein was checked for purity using SDS-PAGE, and quantified by Bradford protein analysis (Bio-Rad). The protein quantified as above was 2.6 mg / mL.

< Example  3> In in vitro in PHD2  Inhibitor Peptide HIF -1α hydroxylation inhibition effect analysis

The hydroxylation reaction by PHD2 of the substrate peptide consisting of the 556th to 575th amino acid sequence of the biotinylated HIF-1α consisting of SEQ ID NO: 1 of Table 1 was performed by NETN buffer (20 mM Tris pH8.0, 100 mM NaCl). , 1 mM EDTA, 0.5% Nonidet P40, 1 mM PMSF, Sigma-Aldrich) and the reaction was performed with or without adding an inhibitor peptide consisting of SEQ ID NO: 3 or SEQ ID NO: 4.

200 μM ascorbic acid, 20 μM 2-oxoglutarate (2-OG) and 0.3 mg / ml PHD2 in NETN buffer (20 mM Tris pH8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P40, 1 mM PMSF) (From amino acid 184 to amino acid 426), 10 μM substrate peptide of PHD2 (biotin-SEQ ID NO: 1) and 100 μM inhibitor peptide (Thz: SEQ ID NO: 3 or DhP: SEQ ID NO: 4) were reacted at room temperature for 2 hours. I was. After desalting using ZipTip C18 (Millipore), the reaction results were analyzed using MALDI-TOF MS (Voyager, Applied Biosystem, USA) and the results are shown in FIG. 2.

As can be seen in Figure 2, when the inhibitor peptide was not added (A: 2 vol% DMSO instead of the inhibitor peptide in Figure 2), the mass of the molecule to which the substrate peptide is hydroxylated was detected, the inhibitor peptide is added In the reaction, the molecular weight of the hydroxylated substrate peptide was not detected (see B and C in Fig. 2).

To obtain the IC ₅₀ value for the PHD2 enzyme reaction of Thz and DhP, the same conditions as in the mass spectrometry were used, but the substrate peptide (FITC-AHA-DLDLEALA-P-YIPADDDFQLR-NH2 (C-terminal) (AHA) was used. = 6-aminohexanoic acid, FITC = fluorescein isothiocyanate), FITC-AHA-SEQ ID NO 1) using 1 μM and varying the concentration of inhibitor peptide (Thz (unit: μM): 1, 6.2, 31, 62, 124, 310, 620, 992; DhP (unit: μM): 0.1, 0.5, 1, 5, 10, 30, 50, 100) at room temperature for 30 minutes. After standard to stop the reaction and heated at 95 ℃ 5 bungan fluorescence polarization method shown in the literature by the IC ₅₀ (Cho et al. Biochem Biophys Res Commun. 2005, 337, 275-80) The value was determined and the results are shown in FIG. 3. As confirmed in FIG. 3, IC ₅₀ values of the inhibitor peptides Thz and DhP of the present invention were determined to be 11.7 μM and 58.6 μM, respectively (FIG. 3).

< Example  4> PHD2  Inhibitor Peptide  Inhibition Specificity Assay

To determine the specificity of inhibition of Thz and DhP, the PHD2 inhibitor peptides according to the present invention, whether the PHD2 inhibitor peptides Thz and DhP inhibit the activity of FIH (factor inhibiting HIF, NP_060372), another hydroxylase of HIF-1α. Experiments were performed to confirm.

To compare with FIH, inhibition of PHD2 was compared with baicalein (Bc, Sigma-Aldrich), a known PHD2 inhibitor. For PHD2 inhibition experiments, each inhibitor (Thz, DhP or Bc) was used at a concentration of 100 μM and performed using a fluorescence polarization assay under the conditions described in Example 3. Inhibition experiments on FIH were performed in FIH reaction buffer (20 mM Tris, pH 7.5, 5 mM KCl, 1.5 mM MgCl ₂ , 400 μM ascorbic acid, 100 μM 2-OG) in FIH (0.1 mg / ml, FEBS Letters 581 ( 2007) see 1542-1548), FIH substrate peptide (FITC-AHA-SMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN-NH2 (AHA = 6-aminohexanoic acid, FITC = fluorescein isothiocyanate), FITC-AHA- (SEQ ID NO: 7); 1 μM), and inhibitors Thz, DhP, or Bc: 100 μM) was mixed and reacted at room temperature for 30 minutes.

The reaction was stopped by heating at 95 ° C. for 5 minutes, followed by mixing 250 ml of EBC buffer buffer (50 mM Tris pH 8.0, 120 mM NaCl, 0.5% Nonidet P40) with 0.4 mg / ml of p300 protein. Fluorescence polarization was measured using a method known from (Cho et al. FEBS Letters, 2007, 581, 1542-1548).

The obtained result is shown in FIG. As shown in FIG. 4, baicalein inhibited both PHD2 and FIH, but Thz and DhP, which are the PHD2 inhibitor peptides of the present invention, inhibited only PHD2 but did not inhibit FIH.

< Example  5> PHD2  Inhibitor Peptide On inhibition  About 2- OG Impact analysis

To determine whether 2-oxoglutarate (2-OG) affects the inhibitory activity of PHD2 of the inhibitor peptide of the present invention, first, ascorbic acid (200 μM) and substrate peptide (1 μM, FITC-bound SEQ ID NO: 1), PHD2 (0.3 mg / ml, AJ310543), DhP (100 μM, DhP peptide (SEQ ID NO: 4)) and mix with 2-OG by concentration (0.02, 0.5, 1, 2 μM) and react for 20 minutes at room temperature I was. Thereafter, the reaction was stopped by boiling at 95 ° C. for 5 minutes, and 250 ml of EBC buffer and 1 mg / ml of VBC protein were added thereto, and fluorescence polarization was measured as in <Example 3>. As a result, the DhP peptide was shown to inhibit PHD2 regardless of the 2-OG concentration (see FIG. 5).

< Example  6> within the cell PHD2  Inhibitor Peptide HIF -1α hydroxylation inhibition effect analysis

In order to determine the intracellular efficacy of the PHD2 inhibitor peptide of the present invention, the same electroporation method as reported in Ahn et al. Bioorg. Med. Chem. Lett., 2009, 19, 4403-4405 was used. The inhibitor peptide was transferred into HeLa cells, and the cells were incubated for 16 hours in a CO ₂ cell incubator at 37 ° C. Thereafter, the cells were disrupted and precipitated by centrifugation, and the supernatant was developed by SDS-PAGE to analyze the amount of HIF-1α protein in the cells through Western Blot (see FIG. 6).

Detailed experimental method is as follows.

After incubating the HeLa cells (American Type Culture Collection) for one day in a 5% CO ₂ incubator maintained at 37 ° C, if more than 90% of the plate area is occupied, the medium is removed, washed with PBS buffer, and then Trypsin / EDTA. 1 ml of each solution (Sigma-Aldrich) was added to separate cells from the plate. The isolated cells were collected in a test tube through a 1200 rpm centrifuge. The collected cells were washed once more with PBS, and then the cells were released in a buffer containing Thz peptide (SEQ ID NO: 3, 1 mM) and buffered with 10% DMSO (dimethyl sulfoxide) (Resuspension buffer (R buffer), Invitrogen After releasing the remaining two cells, electroporation was performed under conditions of 400V, 35ms, 10pulse or 800V, 35ms, 10pulse. After electroporation, the cells were incubated in DMEM buffer (Dulbecco's modified eagle medium, Sigma-Aldrich) in a 5% CO ₂ incubator maintained at 37 ° C for 16 hours.

For comparison, cells were cultured under the same conditions in DMEM buffer containing 100 μM deferoxamine (DFO) and DMSO. After incubation, cells were pulverized in RIPA buffer (Sigma-Aldrich) to obtain only the supernatant via 13000 rpm centrifuge. The obtained supernatant was added to SDS-gel development buffer and heated at 95 ° C. for 5 minutes, followed by 12% SDS-PAGE. After the SDS-PAGE, the protein was transferred to the PDVF membrane (Millipore) (100 V, one hour), and the membrane was blocked in 3% skimmed milk TBST buffer (Biosesang) (at room temperature, one hour). After immersing the membrane in 3% skimmed milk TBST (1: 2000 (w / w), BD Biosciences) containing HIF-1α antibody at 4 ° C. for one day, the membrane was washed three times with TBST. In a 3% skimmed milk TBST (1: 5000 (w / w), Ab Cam) containing an anti-mouse IgG antibody, reacted at room temperature for 2 hours, washed three times with TBST, and added chemiluminescence HRP substrate (Immobilon Western Chemiluminescent HRP substrate, Millipore). After the reaction, the membrane was exposed to X-ray film for 15 seconds.

The results thus obtained are shown in FIG. 6. As can be seen in FIG. 6, since the HIF-1a protein is oxidized by PHD2 and destroyed by ubiquitination in the normal oxygen state, the expression level is low as shown in the control band (5 vol% DMSO instead of peptide). On the other hand, when the well-known PHD2 inhibitor deferoxamine (DFO band) to the cell treatment PHD2 is inhibited and as a result it can be seen that the HIF-1a protein remains unbroken. When treated with Thz, the peptide inhibitor of the present invention, it was confirmed that HIF-1a could be stabilized by PHD2 inhibition similarly to DFO (400V or 800V Thz band-electroporation using 400V and 800V conditions). To treat cells with Thz inhibitor peptides).

<110> Korea Institute of Science and Technology <120> Inhibitor for PHD2 <130> DPP20102572KR <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment from 556th to 575th positions of HIF1-alpha          protein <400> 1 Asp Leu Asp Leu Glu Ala Leu Ala Pro Tyr Ile Pro Ala Asp Asp Asp   1 5 10 15 Phe Gln Leu Arg              20 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> PD223 inhibitor peptide <220> <221> MOD_RES <222> (9) <223> X represents a modified proline wherein 4-C is substituted by S,          O, N, or P; H at 4-C is removed, generating double bond between          4-C and neighboring C; or 3-C and 4-C form a ring with C1-C3          alkyl, generating bicyclic form <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> 20 <212> PRT <213> Artificial Sequence <220> <223> PHD2 inhibitor peptide <220> <221> MOD_RES <222> (9) X is Thz (L-thiazolidine-4-carboxylic acid) <400> 3 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> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> PHD2 inhibitor peptide <220> <221> MOD_RES <222> (9) <223> X is DhP (3,4-dehydro-L-proline) <400> 4 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> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> PD223 inhibitor peptide <220> <221> MOD_RES <222> (9) X is BcP (3,4-trans-L-methanoproline) <400> 5 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> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> PD223 inhibitor peptide <220> <221> MOD_RES <222> (9) <223> X is Oxa (L-oxazolidine-4-carboxylic acid) <400> 6 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> 7 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> substrate for FIH <400> 7 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 Gln Val Asn          35 40

Claims (7)

Consisting of the 556th to 575th amino acid sequence of human HIF-1α protein,
The 564th amino acid is substituted with a heteroatom selected from the group consisting of S, O, N, and P at position 4 of proline, carbon at position 4 forms a double bond with carbon at position 3, or Carbon in position and carbon in position 3 are proline modifications forming a bicyclic form by forming a ring with an alkyl group of C1-C3,
Proryl hydroxylase-2 (PHD2) specific inhibitory activity,
Peptides.
The peptide of claim 1, wherein the proline variant is thiazolidine carboxylic acid, dehydroproline, metanoproline, or oxazolidine carboxylic acid.
The peptide of claim 1 consisting of the amino acid sequence of SEQ ID NO:
<SEQ ID NO 2>
DLDLEALAXYIPADDDFQLR
In the sequence, X in position 9 is substituted with a heteroatom selected from the group consisting of S, O, N, and P in the position 4 carbon of proline, or the position 4 carbon double bonds with the position 3 carbon Or, the peptide of position 4 and carbon of position 3 is a proline variant to form a bicyclic (bicyclic) form a ring with an alkyl group of C1-C3.
The peptide of claim 3, wherein X is thiazolidine carboxylic acid, dehydroproline, metanoproline, or oxazolidine carboxylic acid.
The method of claim 3, wherein SEQ ID NO: 3 (DLDLEALA-Thz-YIPADDDFQLR, Thz = L-thiazolidine-4-carboxylic acid), SEQ ID NO: 4 (DLDLEALA-DhP-YIPADDDFQLR, DhP = 3,4-dehydro-L-proline) , Amino acid sequence of SEQ ID NO: 5 (DLDLEALA-BcP-YIPADDDFQLR, BcP = 3,4-trans-L-methanoproline), or SEQ ID NO: 6 (DLDLEALA-Oxa-YIPADDDFQLR, Oxa = L-oxazolidine-4-carboxylic acid) Consisting of peptides.
A composition for the prevention or treatment of ischemic diseases containing the peptide of any one of claims 1 to 5 as an active ingredient.
The composition of claim 6, wherein the ischemic disease is cerebral ischemic attack, stroke, anemia, coronary insufficiency, brain insufficiency, or vascular insufficiency.

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