KR101856032B1 - Composition for inhibiting prolyl hydroxylase including a coordination complex of zinc and using thereof - Google Patents

Abstract

A composition for inhibiting prolyl hydroxylase, comprising a zinc complex salt thereof, a pharmaceutically acceptable salt thereof, or a derivative thereof, and uses thereof. According to this, zinc coordination complexes, pharmaceutically acceptable salts thereof, or derivatives thereof selectively inhibit a prolyl hydroxylase such as prolyl hydroxylase 3 and inhibit ischemic diseases, cancer, degenerative brain Diseases, inflammation, or a combination of these can be effectively prevented or treated.

Description

[0001] The present invention relates to a composition for inhibiting prolyl hydroxylase, which comprises a coordination complex of zinc, and a use thereof.

Zinc, and a use thereof. The present invention also relates to a composition for inhibiting pro-hydroxylase activity, and a use thereof.

Changes in oxygen partial pressure are associated with various diseases such as myocardial infarction, stroke, and cancer. When hypoxic conditions are induced in the body, changes such as red blood cell formation and neovascularization are induced. This process is mediated by the hypoxia inducible factor 1-α (HIF1-α), a transcription factor, and HIF1 lt; / RTI > of the amino acid sequence of the < RTI ID = 0.0 > Hydroxylation of the proline residue of HIF1-a is mediated by prolyl hydroxylase (PHD), and HIF1-alpha, in which the proline residue is hydroxylated, binds to the von Hippel-Lindau tumor suppressor (pVHL). As a result, HIF1-a is degraded through proteasome-related degradation and the function of the transcription factor is inhibited. HIF1-α is useful in the study of ischemia by regulating the expression of various genes in hypoxic conditions and promoting the expression of VEGF (Vascular Endothelial Growth Factor). HIF-1? Is useful for the treatment of ischemic diseases such as coronary artery dysfunction, brain injury and angiopathy by accelerating cell cycle progression, hypertrophication of hypertrophy, or cell survival function in a hypoxic state, Inhibits the growth of cancer tissues through inhibition of the formation of cancer cells, and thus is useful for prevention or treatment of cancer, or prevention or treatment of inflammation.

Three types of prolyl hydroxylase (PHD), mammalian PHD1 to PHD3, are known. PHD2 plays a crucial role in maintaining the unstable state of HIF1-α continuously under normal oxygen conditions, and PHD3 is required for nerve cell apoptosis induced by nerve growth factor loss. Recently, studies have been conducted to find out the target proteins of PHD other than HIF1-alpha and to elucidate their functions. The activity of PHD can be confirmed by the interaction of hydroxylated HIF1-a with the VHL protein. Inhibition of PHD activity not only regulates HIF1-a function by inducing a hypoxia-mimic state but also regulates the function of target protein of newly reported PHD.

Therefore, there is a need to develop a composition for inhibiting PHD that inhibits the activity of PHD in order to prevent or treat diseases such as ischemic diseases, cancer, and inflammation by controlling the function of the target protein of PHD.

A composition for inhibiting prolyl hydroxylase is provided.

Ischemic diseases, cancer, degenerative brain diseases, inflammation, or a combination thereof.

Ischemic diseases, cancer, degenerative brain diseases, inflammation, or a combination thereof.

One aspect provides a composition for inhibiting prolyl hydroxylase comprising a coordination complex of zinc, a pharmaceutically acceptable salt thereof, or a derivative thereof.

The coordination complex refers to a complex formed by coordination bonds. Conjugate complexes are compounds consisting mainly of central atoms, which are metal atoms, or binding molecules or ions, also known as ions and ligands. A variety of metals, especially compounds containing transition metals, may be coordination complexes.

The coordination complex salt of zinc may be a complex in which zinc is a central atom and bonds to surrounding molecules or ions by coordination bonds. The coordination complex salt of zinc may be bis (2-pyridylthio) zinc, 1,1'-dioxide, pyrithione-zinc or zinc pyrithione. Pyrithione-zinc is a compound in which zinc is the central atom and the oxygen atom and sulfur atom of pyrithione are coordinated to zinc.

The term "pharmaceutically acceptable salts" refers to the inorganic and organic acid addition salts of the compounds. Acid addition salts can be obtained from inorganic acids or non-toxic organic acids. Inorganic acids are, for example, hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid and phosphorous acid. Non-toxic organic acids include, for example, oxalic, maleic, fumaric, malic, tartaric, citric, benzoic, aliphatic mono- and dicarboxylates, phenyl- substituted alkanoates, , Aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include, for example, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates The present invention relates to a process for the preparation of a compound of formula (I) wherein R is selected from the group consisting of chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, But are not limited to, but are not limited to, nitrate, nitrate, succinate, sorateate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, , Hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, claw Phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-2-sulfonate, 1-sulfonate, naphthalene-2-sulfonate or mandelate.

The term " derivative "refers to a compound obtained by substituting a part of the structure of the above compound with another atom or an atomic group.

The prolyl hydroxylase (PHD) may also be referred to as procollagen-proline dioxygenase. Prolyl hydroxyl sila agent is 2-oxoglutarate (oxoglutarate) - dependent dioxygenase as one of the 2-oxoglutarate, Fe ^{+ 2,} and the organic substrate through a mechanism that requires ascorbate (ascorbate) To form (2S, 4R) -4-hydroxyproline. The prolyl hydroxylase may be PHD1, PHD2, or PHD3. PHD2 may also be referred to as a hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2) or a prolyl hydroxylase domain-containing protein 2 (PHD2) . PHD2 can be encoded by the EGLN1 gene. PHD2 is an enzyme consisting of an N-terminal domain that is homologous to the MYND zinc finger domain and a C-terminal domain that is homologous to 2-oxoglutarate dioxygenase. In the normal oxygen state, PHD2 or PHD3 hydroxylates proline, the 564th amino acid residue and the 402th amino acid residue in the amino acid sequence of HIF1-α, to convert hypoxia-inducible factor 1-alpha (HIF1- - can be degraded through proteasome related degradation pathways.

The term " inhibiting composition "refers to a substance which is interchangeable with the term" inhibitor "and which reduces the activity of the enzyme. The activity of the enzyme can be reduced by blocking binding of the enzyme to the substrate or target substance by binding to the enzyme for inhibition composition. The binding of the inhibitory composition to the enzyme may be reversible or irreversible.

Another aspect provides a pharmaceutical composition for the prevention or treatment of diseases of ischemic diseases, cancer, degenerative brain diseases, inflammation, or a combination thereof, comprising a zinc complex salt, a pharmaceutically acceptable salt thereof, or a derivative thereof .

The coordination complexes, pharmaceutically acceptable salts and derivatives of zinc are as described above.

The zinc coordination complex, its pharmaceutically acceptable salt, or derivative thereof may inhibit the prolyl hydroxylase to control the function of the substrate protein or target protein of the prolyl hydroxylase. The substrate protein or target protein of the prolyl hydroxylase may be, but is not limited to, for example, HIF1-a.

The ischemia refers to a state in which blood supply to the tissue or organ is short or absolute relative to the oxygen demand of the organ. Ischemic diseases refer to diseases caused by ischemia, and ischemic diseases may be, for example, coronary artery dysfunction, brain dysfunction, vascular insufficiency, or a combination thereof. Coronary artery insufficiency can be, for example, myocardial infarction or angina. Brain failure can be, for example, an ischemic stroke. Vascular insufficiency can be, for example, arterial insufficiency or venous insufficiency.

The cancer may be, for example, lung cancer, stomach cancer, colon cancer, liver cancer, breast cancer, head and neck cancer, esophageal cancer, biliary cancer, pancreatic cancer, gynecological cancer, urological cancer, blood cancer, brain tumor, osteosarcoma, or skin cancer.

The degenerative brain disease may be, for example, Alzheimer's disease, Parkinson's disease, or a combination thereof.

The term " prevention "refers to any action that inhibits disease or delays onset by administration of the composition. The term " treatment "refers to any action that improves or alleviates a symptom of a disease by administration of the composition.

The pharmaceutical composition comprises from about 0.0001% to about 10%, or from about 0.001% to about 1%, by weight of a zinc complex salt, or a pharmaceutically acceptable salt thereof, or derivative thereof, based on the total weight of the total composition .

Each of the pharmaceutical compositions may be formulated into preparations for oral administration or parenteral administration according to conventional methods.

In the above pharmaceutical composition, the solid preparation for oral administration may be, for example, a tablet, a pill, a light / soft capsule, a liquid, a suspension, an emulsifier, a syrup, a granule or an elixir. The solid preparation may further comprise an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid preparation may further comprise a lubricant such as magnesium stearate or talc. In the above pharmaceutical composition, the liquid preparation for oral administration may be a suspension, a solution, an emulsion, or a syrup. The liquid formulation may comprise water, or liquid paraffin. The liquid preparations may contain excipients such as wetting agents, sweetening agents, perfumes, or preservatives.

In the above pharmaceutical composition, the preparation for parenteral administration may be, for example, a sterilized aqueous solution, a non-aqueous solvent, a suspension, an oil, a lyophilization, a suppository or an inhaler. Non-aqueous solvents or suspensions may include vegetable oils or esters. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The suppository base may be witepsol, macrogol, tween 61, cacao paper, laurin, or glycerogelatin. Inhalants may be prepared by bringing the active compound into a powder or liquid form, blending it into an inhalable propellant and / or carrier, and charging the mixture into a suitable vaporizer. When the active compound is a powder, a conventional mechanical powder vaporizer may be used. When the compound is a liquid, a vaporizer such as a nebulizer may be used. In addition, the inhalant may optionally contain a conventionally used surfactant, oil, flavoring, cyclodextrin or a derivative thereof, if necessary.

The pharmaceutical composition may further comprise a carrier, an excipient or a diluent. Carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Vaginal cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The dosage of the pharmaceutical composition varies depending on the condition and the weight of the patient, the severity of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. For example, zinc coordination complexes, pharmaceutically acceptable salts, or derivatives thereof may be administered at a dose of about 0.0001 mg / kg body weight to about 100 mg / kg body weight, or about 0.001 mg / kg body weight to about 10 mg / kg body weight The dose can be administered once a day, several times a day, or once a day. The pharmaceutical composition may be administered to the mammal, including mice, mice, dogs, cows, horses, and humans, in a variety of routes. The method of administration may be, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injection.

The pharmaceutical composition may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A solubilizing agent such as a flavoring agent can be used. In addition to the active ingredient, the pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier and may be preferably formulated into the pharmaceutical composition.

Acceptable pharmaceutical carriers for compositions that are formulated in a liquid solution include sterile water and sterile water suitable for the body such as sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical preparation forms of the pharmaceutical composition may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, .

Another aspect provides a method of preventing or treating a disease of an ischemic disease, cancer, degenerative brain disease, inflammation, or a combination thereof, comprising administering to the subject a zinc complex salt, a pharmaceutically acceptable salt thereof, or a derivative thereof. ≪ / RTI >

The coordination complexes, pharmaceutically acceptable salts, derivatives, ischemic diseases, cancers, degenerative brain diseases, prevention and treatment of zinc are as described above.

The term "individual" includes mammals including rats, mice, dogs, cows, horses, and humans.

Zinc coordination complexes, pharmaceutically acceptable salts thereof, or derivatives thereof may be administered to a subject either orally or parenterally once a day, several times a day, or once in several days.

According to one aspect of the present invention, there is provided a composition for inhibiting proliferation of zinc containing a complex salt of zinc, and a composition for inhibiting proliferation of the prophylactic hydratase, which comprises selectively inhibiting prolyl hydroxylase such as prolyl hydroxylase 3, Can be used to prevent or treat diseases of ischemic diseases, cancer, degenerative brain diseases, inflammation, or a combination thereof by controlling the function of a target protein of a hydroxylase.

1 is a graph showing inhibition rate (%) of PHD3 by a compound library.
Figs. 2A to 2C are graphs showing the fluorescence polarization degree according to the concentration (nM or mu M) of PHD2F, PHD2CT, and PHD3, respectively).
3 is a graph showing the fluorescence polarization degree according to the concentration (2M) of 2-oxoglutarate (2OG) in the presence of PHD2F, PHD2CT, or PHD3.
4A to 4C are graphs showing fluorescence polarization degrees by PHD2F, PHD2CT, and PHD3, respectively, according to the concentration (μM) of plied on-zinc.
5A to 5C are graphs showing fluorescence polarization degrees by PHD2F, PHD2CT, and PHD3, respectively, according to the concentration (μM) of the pity on.

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

Example 1. Screening of a prolyl hydroxylase inhibitor

1.1 VBC  Preparation of protein

Prolyl hydroxylase (PHD) 3 is an enzyme that hydrolyzes the proline residue, the 564th amino acid of the amino acid sequence of hypoxia inducible factor 1-alpha (HIF1-alpha), and PHD 3 hydrolyzes the proline residue of HIF1-?, The hydroxylated HIF1-? Binds to the von hippel-Lindau (VHL) protein, after which the HIF1-a protein hydrolyzes through the ubiquitin system . Therefore, in order to screen the inhibitors of PHD 3, a complex of VHL protein, Elongin B protein, and Elogin C protein (VBC protein) was prepared.

Specifically, a polynucleotide fragment corresponding to the 54th to 213st amino acid sequence of the VHL protein in a polynucleotide (GenBank Accession No. NM_000551.3) encoding human VHL protein (GenBank Accession No: NP_000542.1) A polynucleotide fragment corresponding to the first to 118th amino acid sequence of Elongin B protein in a polynucleotide (GenBank Accession No. NM_007108.3) encoding B protein (GenBank Accession No: NP_009039.1) was designated pGEX-4T-1 GE Healthcare Life Sciences) was prepared as the plasmid pGEX4T-VHL-EB. In addition, a polynucleotide fragment corresponding to the 17th to 112th amino acid sequence of Elogin C protein in a polynucleotide (GenBank Accession No. NM005648.3) encoding human Elogin C protein (GenBank Accession No: NP_005639.1) was designated as pET29b Novagen) to prepare the plasmid pDEV-EC. The prepared pGEX4T-VHL-EB and pET29b were transformed with the transforming Escherichia coli BL21 (DE3) (Enzynomic), which was made ready to accept DNA. At this time, heat shock was applied to the mixture of plasmid and BL21 (DE3) cells to transform.

The transformed cells were inoculated into LB medium (Sigma-Aldrich) containing 100 μg / ml of ampicillin (Sigma-Aldrich) and 100 μg / ml of kanamycin (Sigma-Aldrich) lt; RTI ID = 0.0 > 37 C. < / RTI > 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich) was added to the culture, followed by incubation at 18 ° C. for 15 hours to induce the expression of the transformed proteins. The cultured cells were obtained by centrifugation. To the obtained pellet were added 110 mM NaCl (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 0.2 mM PMSF (phenylmethylsulfonyl fluoride) (Phosphate-based saline buffer; PBS) containing 10 μg / ml lysozyme (Sigma-Aldrich) and sonicated at 4 ° C. to dissolve E. coli. After adding 1% (v / v) Triton X-100 (Sigma-Aldrich) to the lysate and mixing well, the reaction solution was incubated at 4 ° C for 10 minutes and 10,000 0.0 > rpm < / RTI > for 30 minutes.

The separated supernatant was mixed with glutathione-sepharose 4B (GE Healthcare), stirred at 4 ° C for 2 hours, and then centrifuged at 4 ° C at a speed of 2,000 rpm for 5 minutes. After removing the supernatant, the phosphate buffer solution (137 mM NaCl, 2.7 mM KC, 10 mM Na ₂ HPO ₄ , 2 mM KHPO ₄ ), phosphate buffer containing 1 M NaCl, and 50 mM Tris The proteins not bound to glutathione-sepharose 4B resin were removed using a -PH8.0 solution. The reaction was added to Bio-Spin Disposable Chromatography Columns (Bio-Rad) and washed with 10 ml of 50 mM Tris-pH 8.0 solution. The GST-VBC protein was then eluted with a 50 mM Tris-pH 8.0 solution containing 10 mM Glutathione (GE healthcare).

The obtained GST-VBC protein was confirmed by SDS-PAGE and quantified using Bradford protein analysis (Bio-Rad).

1.2 Ploll Of the hydroxylase (PHD)  Ready

In order to screen inhibitors that selectively inhibit PHD3, PHD3 proteins were prepared. The PHD2 full-length protein as a comparative group and the PHD2 protein having only the C-terminal domain, which is a catalytic domain, were removed from the N-terminal domain of PHD2 in a structure similar to that of PHD3.

Specifically, the coding for the human egl-9 family hypoxia-inducible factor 1 protein (PHD2 protein, GenBank Accession No: CAC42509.1) was carried out in the polynucleotide (GenBank Accession No: AJ310543.1) A polynucleotide fragment corresponding to the sequence (PHD2 full-length protein; PHD2F) or a polynucleotide fragment corresponding to the 184th to 426th amino acid sequence (PHD2 protein having only the C-terminal domain; PHD2CT) of the PHD2 protein was prepared. The prepared polynucleotide fragment was inserted into pET21b (Novagen) to prepare plasmids pET21b-PHD2F and pET21b-PHD2CT. In addition, the amino acid sequence corresponding to the first to the 225th amino acid sequence of a polynucleotide (GenBank Accession No: AJ310545.1) encoding the human egl-9 family hypoxia-inducible factor 3 protein (PHD3 protein, GenBank Accession No: CAC42511.1) The polynucleotide fragment was inserted into pET22b (Novagen) to prepare plasmid pET22b-PHD3. The prepared plasmids pET21b-PHD2F, pET21b-PHD2CT, and pET22b-PHD3 were transformed with Escherichia coli BL21 (DE3) (Enzynomic), each of which was made ready to accept DNA. At this time, heat shock was applied to the mixture of plasmid and BL21 (DE3) cells to transform.

The transformed cells were inoculated into LB medium (Sigma-Aldrich) containing 100 / / ml of ampicillin (Sigma-Aldrich) and cultured at 37 째 C until the absorbance was 0.6 at a wavelength of 600 nm. 1 mM IPTG (Sigma-Aldrich) was added to the culture, followed by incubation at 18 ° C for 15 hours to induce the expression of the transformed proteins. The cultured cells were obtained by centrifugation. To the obtained pellet, 0.014 mM 2-mercaptoethanol (Sigma-Aldrich), 0.2 mM PMSF (phenylmethylsulfonyl fluoride) (Sigma-Aldrich), and 1 mg / 10 ml lysozyme (50 mM Na ₂ PO ₄ , 500 mM NaCl, and 10 mM imidazole (Sigma-Aldrich), and sonicated at 4 ° C to dissolve 1% (v / v) Triton X-100 (Sigma-Aldrich) was added and mixed well. The reaction mixture was incubated at 4 ° C for 10 minutes, centrifuged at 4 ° C at 10,000 rpm for 30 minutes Respectively.

The separated supernatant was mixed with Ni-NTA agarose (Quiagen), stirred at 4 ° C for 1 hour, and then centrifuged at 4 ° C for 5 minutes at a rate of 2,000 rpm. After removal of the supernatant, a washing solution (50 mM Na ₂ PO ₄ , 500 mM NaCl, 20 mM imidazole) 10 times the volume of the supernatant was added and centrifuged at 200 rpm for 5 minutes to remove the supernatant. . The reaction was added to Bio-Spin Disposable Chromatography Columns (Bio-Rad) and washed with 10 ml of wash solution. Then, elution solutions (50 mM Na ₂ PO ₄ , 500 mM NaCl, 300 mM imidazole) were used to obtain PHD2F, PHD2CT, and PHD3 proteins, respectively. The obtained protein was identified by SDS-PAGE and quantified using Bradford protein analysis (Bio-Rad).

1.3 Fluorescence Labeled HIF1 Preparation of -α polypeptide

In order to screen for a compound that inhibits the activity of PHD3 using a fluorescence polarization-based assay, the 556th through 575th amino acid sequences of HIF1-a which contains proline, which is the 564th amino acid residue hydroxylated by PHD, Was prepared and a polypeptide obtained by binding a fluorescent probe FITC (fluorescein isothiocyanate) (Invitrogen) to the N-terminal of the prepared polypeptide using an aminohexanoic acid linker (Sigma-Aldrich) (F-p564). In addition, a peptide (F-Hyp564) in which the 564th proline-hydroxylated peptide of the F-P564 peptide was synthesized for use as a positive control.

1.4 Screening of inhibitors

Inhibitors that inhibit the activity of PHD3 were screened using 1,040 biologically active compounds (NINDS Custom Collection II, Microsource Discovery Systems).

To the compound at a final concentration of 20 [mu] M, 0.2 to 600 nM of PHD3 prepared in Example 1.2 was added and mixed well. Thereafter, a solution of 2 mM ascorbic acid (Sigma-Aldrich), 1 mM 2-oxoglutarate (Sigma-Aldrich), and buffer solution (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.5% (v / v) NP-40 (Sigma-Aldrich)). Hydroxylation was induced by adding 1 μM of the final concentration of the F-P564 peptide prepared in Example 1.3, followed by incubation at room temperature for 1 hour. To the reaction was added a binding solution (50 mM Tris, 120 mM NaCl, 0.5% (v / v) NP-40 (Sigma-Aldrich)) containing 250 nM of the VBC protein prepared in Example 1.1 and mixed at room temperature And then transferred to a 96-well plate. Fluorescence polarization degree was measured using VICTOR (Perkin-Elmer).

The inhibition rate (%) of PHD3 calculated by measuring the fluorescence polarization degree by each compound is shown in Fig. The percent inhibition of PHD3 is defined as the percent activity of PHD3 reduced by the compound based on the fluorescence polarization value measured upon binding of 100 nM F-HyP564 polypeptide already hydrolyzed with proline to 250 nM VBC protein Respectively. Twelve compounds inhibiting PHD3 activity by about 60% or more by the treated compounds were identified, and one of them was pyrithione-Zn.

Example  2. Pirichion - by zinc PHD  Identification of active inhibition

2.1 PHD Determination of the concentration of

To determine the concentration of PHD to be used to analyze the degree of inhibition of PHD activity by pyridinium-zinc, the concentration of PHD required to 90% hydroxylate F-P564 protein at a final concentration of 1 [mu] M was determined.

For PHD2CT and PHD2F, 0 μM to 4.5 μM PHD was added to 20 μM 2-oxoglutarate and mixed well. The F-p564 polypeptide prepared in Example 1.3 was dissolved in a buffer solution (20 mM Tris-HCl, pH 7.5, 5 mM KCl, 1.5 mM MgCl ₂ ) to prepare 1 μM of F-p564 polypeptide. To the reaction was added 200 μM ascorbic acid (Sigma-Aldrich) and 1 μM F-p564 polypeptide, followed by incubation at 30 ° C. for 2 hours to induce the hydroxylation reaction of the F-p564 polypeptide. For PHD3, 0 μM to 4.5 μM PHD was added to 1 mM 2-oxoglutarate and mixed well. The F-p564 polypeptide prepared in Example 1.3 was dissolved in a buffer solution (20 mM Tris-HCl, pH 7.5, 5 mM KCl, 1.5 mM MgCl ₂ ) to prepare 1 μM of F-p564 polypeptide. 2 mM ascorbic acid (Sigma-Aldrich) and 1 μM F-p564 polypeptide were added to the reaction, followed by incubation at 30 ° C for 2 hours to induce hydroxylation of F-p564 polypeptide. 200 nM of the binding solution containing the VBC protein prepared in Example 1.1 was added to each reaction and then transferred to a 96-well plate. Fluorescence polarization was measured using Appliskan (Thermo Scientific), and PHD The fluorescence polarization degree by PHD2F, PHD2CT, and PHD3 according to the concentration (0 to 30 [mu] M) of fluorescence intensity is shown in Figs. 2A to 2C, respectively.

As shown in FIGS. 2A-2C, it was confirmed that about 400 nM of PHD2F, about 50 nM of PHD2CT, and about 0.83 μM of PHD3 were required to hydrolyze about 90% of the final concentration of 1 μM of F-P564 polypeptide.

2.2 PHD  2- Oxoglutarate  Determination of concentration

The hydroxylation of the proline residue of HIF1-a by PHD requires a co-factor 2-oxoglutarate (2OG). In order to compare the activity inhibition levels of the various types of PHD prepared in Example 1.2, the pyridione-zinc screened in Example 1-4 under the same conditions, the PHD2F, PHD2CT, and PHD3 proteins required 2 -Oxo glutarate was determined.

400 nM of PHD2F protein (Sigma-Aldrich), a final concentration of 0 μM to 1000 μM of 2-oxoglutarate (Sigma-Aldrich) and a species identified to hydrolyze about 1 μM of substrate peptide (F-P564) , 50 nM of PHD2CT protein, or 0.83 [mu] M of PHD3 were prepared and PHD prepared in each concentration of 2-oxoglutarate was added and mixed well.

The F-p564 polypeptide prepared in Example 1.3 was dissolved in a buffer solution (20 mM Tris-HCl, pH 7.5, 5 mM KCl, 1.5 mM MgCl ₂ ) to prepare 1 μM of F-p564 polypeptide. To the reaction was added 200 μM ascorbic acid (Sigma-Aldrich) and 1 μM F-p564 polypeptide, followed by incubation at 30 ° C. for 2 hours to induce the hydroxylation reaction of the F-p564 polypeptide. 200 nM of the binding solution containing the VBC protein prepared in Example 1.1 was added to the reaction and then transferred to a 96-well plate and the fluorescence polarization was measured using Appliskan (Thermo Scientific), and 2-oxo The fluorescence polarization degree by PHD2F, PHD2CT and PHD3 according to the concentration of glutarate is shown in Fig.

As shown in Fig. 3, it was confirmed that both PHD2F, PHD2CT and PHD3 proteins had activity in the presence of 300 μM 2-oxoglutarate.

2.3 Pirichion - by zinc PHD2F , PHD2CT , or PHD3  Quantitative analysis of active inhibition

To quantitatively analyze the degree of inhibition of PHD activity by pyridinium-zinc, 300 μM of 2-oxoglutarate, the concentration determined in Example 2.2, was added to the amount of PHD determined in Example 2.1 and mixed well. After the hydroxylation reaction of the F-p564 polypeptide was induced in the same manner as in Example 2.1 in the presence of pyridium-zinc at a final concentration of 0 mM to 8 mM, the fluorescence polarization degree was measured, and the concentration of the pyridium- The fluorescence polarization degree by PHD2F, PHD2CT, and PHD3 according to the present invention is shown in Figs. 4A to 4C.

The concentration of pyridon-zinc, which inhibits the activity of PHD by 50% from the fluorescence polarization degree by PHD2F, PHD2CT, and PHD3 depending on the concentration of pyridinium-zinc, was calculated using SigmaPlot (TM) program (Systat Software Inc.) . As a result, the 50% inhibitory concentration (IC ₅₀ ) of pityrich-zinc for PHD2F, PHD2CT, and PHD3 was about 8300.77 μM, about 1024.22 μM, and about 2.47 μM, respectively. PHD2F and PHD2CT blood is rich on-but hardly inhibited by zinc, IC ₅₀ value of the PHD3 was low about 3360 times higher than the IC ₅₀ value of PHD2F. Therefore, it was confirmed that pyridinium-zinc selectively inhibited PHD3.

2.4 Pirichiona  by PHD3  Quantitative analysis of active inhibition

In order to confirm whether pliotic on-zinc selectively inhibits PHD3, inhibition of the activity of PHD3 by zinc free pyridinone was confirmed.

After the hydroxylation reaction of the F-p564 polypeptide was induced in the same manner as in Example 2.1 in the presence of a final concentration of 0 mM to 1 mM of picilion (Sigma-Aldrich), the fluorescence polarization degree was measured, Fluorescence polarization degree by PHD2F, PHD2CT, and PHD3 depending on the concentration is shown in Figs. 5A to 5C.

The concentration of pyridon which inhibits the activity of PHD by 50% from the fluorescence polarization degree by PHD2F, PHD2CT, and PHD3 depending on the concentration of pyridium was calculated using SigmaPlot (TM) program (Systat Software Inc.). As a result, the 50% inhibitory concentration (IC ₅₀ ) of picrichone against PHD2F, PHD2CT, and PHD3 was about 590.99 μM, about 318.98 μM, and about 20.36 μM, respectively. IC ₅₀ values of the blood rich-on to PHD3 was low about 30 times the IC ₅₀ value of PHD2F. In addition, IC ₅₀ values of the blood-rich whole blood for PHD3 is rich on for PHD3 - was about 8 times the IC ₅₀ value of zinc. Therefore, it was confirmed that pyrichion had a lower selectivity for inhibiting PHD3 activity than pyridinium-zinc.

Claims (8)

A composition for inhibiting prolyl hydroxylase (PHD) 3 in vitro comprising pyrithione-zinc or a pharmaceutically acceptable salt thereof. delete delete A pharmaceutical composition for preventing or treating degenerative brain diseases comprising pyruvate-zinc or a pharmaceutically acceptable salt thereof. delete delete The pharmaceutical composition according to claim 4, wherein the degenerative brain disease is Alzheimer's disease, Parkinson's disease, or a combination thereof. A method of preventing or treating degenerative brain disease comprising administering to a mammal other than a human a pityrich-zinc or a pharmaceutically acceptable salt thereof.

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