Novel prolyl hydroxylase 2 inhibitor

Abstract

The invention relates to a new function of an aromatic organic acid compound, and belongs to the technical field of biology. By utilizing a prolyl hydroxylase 2 small molecule compound inhibitor screening system, the aromatic organic acid compound is found to be capable of effectively inhibiting the activity of prolyl hydroxylase 2. The aromatic organic acid compound improves the protein level of an intracellular hypoxia-inducible factor HIF-1alpha by inhibiting the activity of prolyl hydroxylase 2.

Description

Technical Field

[0001] The present invention relates to an aromatic organic acid compound as a novel inhibitor of prolyl hydroxylase 2. Background Art

[0002] Hypoxia Induced Factor (HIF) is an important control factor in the hypoxia signaling pathway. The Hypoxia Induced Factor family (HIFs) includes three subtypes: HIF-1, HIF-2, and HIF-3. Among them, HIF-1 is the most characteristic subtype in the HIFs family that responds to the hypoxic environment. It consists of a 120 kDa regulatory HIF-1α subunit and a 91 - 94 kDa constitutive HIF-1β subunit, forming a heterodimer to form the transcription factor HIF-1, which belongs to the members of the helix-loop-helix / Per-Arnt-Sim transcription factor family. Under hypoxic conditions, the stabilization of the HIF-1α protein level triggers a series of hypoxia adaptation responses in the body, mainly focusing on the cascade upregulation of downstream gene transcription. This adaptation is very important for protecting body tissues.

[0003] Prolyl hydroxylase 2 (PHD2) is encoded by the EGLN1 gene, contains 426 amino acids, and has a molecular weight of approximately 46 kDa. Human PHD2 protein can be detected in organs such as the heart, liver, kidney, lung, brain, and skeletal muscle of adults and fetuses, and is highly expressed in the brain, heart, and skeletal muscle. HIF-1α is a specific substrate of PHD2. Under normal oxygen levels, PHD2 recognizes and hydroxylates the proline residues Pro402 and Pro564 on HIF-1α, and then promotes the degradation of the HIF-1α protein through the ubiquitination degradation pathway. Under hypoxic conditions, the hydroxylation activity of PHD2 is inhibited, resulting in the blockage of the HIF-1α protein degradation process and promoting the accumulation of the HIF-1α protein, thereby improving the related physiological responses induced by hypoxia. Therefore, prolyl hydroxylase 2 inhibitors have the effect of stabilizing the HIF-1α protein and promoting hypoxic adaptation. Summary of the Invention

[0004] The present invention utilizes the mechanism that PHD2 specifically recognizes and hydroxylates the proline residues Pro402 and Pro564 on HIF-1α. Through a fluorescence polarization technology platform, small molecule inhibitors that specifically inhibit the hydroxylation activity of PHD2 are screened, and the ability of the screened small molecules to regulate the HIF-1α protein level by inhibiting the hydroxylation activity of PHD2 is verified at the cellular level. The results show that the obtained small molecules can specifically inhibit the activity of PHD2 and upregulate the HIF-1α protein level in cells.

[0005] To solve the above technical problems, the technical solution of the present invention is:

[0006] An aromatic organic acid compound, 3-(3,4-dihydroxyphenyl)-2-acrylic acid, has the following structural formula:

[0007]

[0008] The above aromatic organic acid compound is used as an inhibitor of prolyl hydroxylase 2.

[0009] Preferably, the aromatic organic acid compound can effectively inhibit the activity of prolyl hydroxylase 2.

[0010] The above aromatic organic acid compound targets and inhibits the activity of prolyl hydroxylase 2, thereby stabilizing the protein level of HIF-1α in cells.

[0011] The beneficial effects of the present invention are as follows:

[0012] By screening a small molecule compound library using a prolyl hydroxylase 2 small molecule inhibitor screening system, a small molecule compound was discovered. The small molecule compound is an aromatic organic acid compound and can effectively inhibit the activity of prolyl hydroxylase 2. Immunoblotting experiments showed that this compound can effectively stabilize the protein level of HIF-1α in cells. The above results indicate that this compound can stabilize the protein level of HIF-1α in cells by inhibiting the activity of prolyl hydroxylase 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the result of screening inhibitors using the PHD2 small molecule inhibitor screening system of the present invention. The results show that the screened small molecule compound can effectively inhibit the activity of PHD2.

[0014] Figure 2 This is the experimental result of the half inhibitory concentration of the small molecule compound of the present invention in vitro experiments. IC 50 is 0.16 μM.

[0015] Figure 3 This is to detect the effect of the small molecule compound on the protein level of HIF-1α in cells using immunoblotting. The results show that the small molecule compound can significantly increase the protein level of HIF-1α in cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The present invention will be described below with reference to specific embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention and do not limit the protection scope of the present invention in any way. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used are all commercially available products unless otherwise specified.

[0017] Example 1

[0018] Screening for small molecule compounds that inhibit PHD2 activity

[0019] ① According to the PHD2 small molecule inhibitor screening system, add each component: Tris-HCl 20 mM (pH 8.0); PHD2 protein 0.5 μM; Vc 2 mM; α-KAD 20 μM, and the total volume of the system is 5 μl.

[0020] ② Add the small molecules in the small molecule compound library to the reaction system respectively, mix well, and let stand at room temperature for 30 min.

[0021] ③ Add the substrate of PHD2 (FITC-DLDLEALAPYIPADDDFQLR) to the reaction system to 100 nM, mix well in the dark, and let stand at room temperature for 60 min.

[0022] ④ Add the VBC protein complex to the reaction system to 700 nM, and at the same time make up the final system to 50 μl with 20 mM Tris-HCl (pH 8.0) buffer solution.

[0023] ⑤ Use a fluorescence polarization spectrophotometer, set the excitation light wavelength to 530 nm; the emission light wavelength to 460 nm. Detect the fluorescence polarization value of the screening system.

[0024] As Figure 1 The results showed that 3-(3,4-dihydroxyphenyl)-2-acrylic acid could effectively inhibit the hydroxylase activity of PHD2.

[0025] Example 2

[0026] Determination of the half inhibitory concentration of 3-(3,4-dihydroxyphenyl)-2-acrylic acid on PHD2 hydroxylase activity.

[0027] According to the in vitro detection system of PHD2 small molecule inhibitors, add each component (20 mM Tris-HCl (pH 8.0); PHD2 protein 0.5 μM; Vc 2 mM; α-KAD 20 μM, total volume 5 μl). Set concentration gradients for 3-(3,4-dihydroxyphenyl)-2-acrylic acid: (0.3125 μM, 0.15625 μM, 0.125 μM, 0.09375 μM, 0.0625 μM, 0.046875 μM, 0.03125 μM, 0.015625 μM, 0.007813 μM). Use a fluorescence polarization spectrophotometer to detect the polarization value. Statistically analyze the data with Graph pad.

[0028] As shown in Result 2, 3-(3,4-dihydroxyphenyl)-2-acrylic acid inhibited the hydroxylase activity of PHD2 in vitro, and its half inhibitory concentration (IC 50 ) was 0.16 μM.

[0029] Example 3

[0030] Effect of 3-(3,4-dihydroxyphenyl)-2-acrylic acid on the intracellular HIF-1α level in human neuroblastoma cell line SH-SY5Y

[0031] ① Culturing SH-SY5Y cells in 6-cm culture dishes: Collect SH-SY5Y cells in the logarithmic growth phase, inoculate the cells at 7.5×10 5 cells / dish, culture overnight at 37°C and 5% CO2 until the cells grow to 80%.

[0032] ② Treating SH-SY5Y cells with 3-(3,4-dihydroxyphenyl)-2-acrylic acid: Add 3-(3,4-dihydroxyphenyl)-2-acrylic acid to the culture medium to a concentration of 250 nM and 500 nM respectively, and culture at 37°C and 5% CO2 for 24 h.

[0033] ③ Extracting the nuclear protein of SH-SY5Y cells: Collect SH-SY5Y cells and extract the nuclear protein of SH-SY5Y cells.

[0034] ④ Detecting by Western blotting: Using Histon as an internal reference, detect the content of HIF-1α protein in the nucleus of SH-SY5Y cells.

[0035] As Figure 3 shown in the results, compound I can stabilize and increase the level of HIF-1α protein in the nucleus of SH-SY5Y cells.

Claims

1. This patent relates to an aromatic organic acid compound as an inhibitor of prolyl hydroxylase 2, and the compound is 3-(3,4-dihydroxyphenyl)-2-acrylic acid.

2. The compound according to claim 1, wherein Specifically inhibit the activity of prolyl hydroxylase 2.

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