Small molecule compounds targeting the inhibition of prolyl hydroxylase activity

Abstract

The application discloses small-molecule compounds for targeted inhibition of proline hydroxylase activity, including Aristolone, Isosakuranin, Butein and Brevilin A. The small-molecule compounds screened by the application can be combined with the active site of PHD, and molecular biology experiments prove that the small-molecule compounds can inhibit the enzyme activity of PHD, activate the HIF-1 signal pathway, and cause the expression of a target gene Glut-1 to increase. The small-molecule compounds lay a foundation for future research and development of related drugs by inhibiting the enzyme activity of PHD.

Description

Technical Field

[0001] This invention belongs to the field of targeted protein inhibitors, specifically relating to a group of small molecule compounds that target and inhibit proline hydroxylase and their applications. Background Technology

[0002] Hypoxia-inducible factor (HIF) is a key transcription factor for cellular adaptation to hypoxic environments. It is stably expressed in tissue anemia and inflammatory diseases, playing a crucial role in alleviating these conditions. HIF consists of the HIF-α and HIF-β subunits. The HIF-α subunit is oxygen-sensitive, and its expression within the cell is controlled by oxygen concentration; while the HIF-β subunit is a structural subunit. The stability of HIF is primarily regulated by the post-translational prolyl hydroxylation of the α subunit, a reaction catalyzed by proline hydroxylase domain (PHD).

[0003] PHDs are a class of oxygen-dependent, 2-oxogluatarate (2-OG) and Fe... 2+ There are four catalytically active dioxygenase subtypes: PHD1, PHD2, PHD3, and PHD4. These four subtypes share high sequence homology in their C-terminal catalytic domains, but their distribution and function within cells and tissues differ. PHD1 is widely expressed in the nucleus, PHD2 is mainly expressed in the cytoplasm, while PHD3 is expressed in both the nucleus and cytoplasm. PHD4 has been less studied. In terms of tissue distribution, PHD-1 is mainly found in the testes, but also in the brain, kidneys, heart, and liver; PHD-2 is found in most tissues; and PHD-3 is mainly found in the heart. PHD2 is the most important proline hydroxylase subtype in the hypoxia signaling pathway. PHD has two domains: a variable N-terminal domain and a conserved C-terminal catalytic domain. The core of the PHD2 C-terminal catalytic domain consists of eight β-chains from a double-stranded β-helix (DSBH) fold motif, with three α-helices and other β-chains and loops stacked along the nucleus. The DSBH core fold motif is a key structural feature of 2-OG-dependent dioxygenases, typically composed of major and minor β-sheets, with an open terminal containing Fe. 2+ And 2-OG binding sites (active sites). The N- or / and C-termini of the DSBH core, plus other β-chains, α-helices, or loops, play a role in stabilizing DSBH folds and recognizing and binding substrates. The Fe required for catalysis... 2+Typically, the three highly conserved residues of the conserved HXD / EXH motif are chelated. In the four-dimensional structure, the crystal structure of the PHD2 C-terminal catalytic domain is a homotrimer, with the C-terminal α-helix of each monomer having intermolecular contact with the residues surrounding the active site of the adjacent monomer.

[0004] Under hypoxic conditions or in the presence of PHD inhibitors, PHD hydroxylation activity decreases, hindering HIF-1α degradation and leading to stable expression and accumulation of HIF-1α. This, in turn, activates downstream target genes, improving anemia, local tissue ischemia, and tissue damage-related diseases. Therefore, the development of PHD inhibitors has become one of the important strategies for treating related diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a group of small molecule compounds that target and inhibit proline hydroxylase and their applications.

[0006] Small molecule compounds that target and inhibit proline hydroxylase activity, characterized in that they include Aristolone, Isosakuranin, Butein, and Brevilin A.

[0007] The application of the small molecule compound in the preparation of inhibitors targeting proline hydroxylase.

[0008] A drug comprising the small molecule compound.

[0009] A drug precursor comprising the small molecule compound.

[0010] Preferably, it also includes one or more pharmaceutically acceptable excipients or carriers.

[0011] Preferably, the excipients or carriers include diluents, excipients, fillers, binders, wetting agents, absorption promoters, surfactants, adsorbent carriers, or lubricants.

[0012] Preferably, the drug or drug precursor can be formulated into dosage forms such as tablets, capsules, granules, powders, dispersible tablets, oral liquids, pills, or injections.

[0013] The beneficial effects of this invention: The small molecule compounds Aristolone, Isosakuranin, Butein, and Brevilin A screened in this invention can bind to the active site of PHD. Molecular biology experiments have confirmed that they can inhibit the enzymatic activity of PHD, activate the HIF-1 signaling pathway, and lead to increased expression of its target gene Glut-1. Their inhibition of PHD enzyme activity lays the foundation for future drug development. Attached Figure Description

[0014] Figure 1The change in PHD2 enzyme activity under different dosage treatment conditions is represented by the amount of change.

[0015] Figure 2 The expression level of Glut-1 mRNA under different treatment doses was determined. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0017] Example 1: Computer Simulation Screening

[0018] 1. Based on preliminary research, a computer-based virtual screening was conducted targeting the active sites of human PHD2 proteins. Data was collected from the RCSBPDB database (…). http: / / www.rcsb.org / Download the crystal structure of the PHD2 protein. Use the Protein Preparation Wizard module to perform peptide chain analysis, structure optimization, and energy optimization.

[0019] 2. The 2D structures of the BCL-116K compound library are processed through hydrogen bond adjustment, structure optimization, and energy optimization to output 3D structures for virtual screening.

[0020] 3. Virtual Screening: Import the compounds. First, use the High Throughput Screening (HTVS) mode in the Glide module to screen the small molecule compounds in BCL-116K. Select the top 30% of the small molecule compounds by score and use the Standard (SP) mode for the second round of screening. Then, select the top 30% by score and use the High Precision (XP) mode for the third round of screening. Output the screening results.

[0021] Five small molecule compounds were screened, including Aristolone, Isosakuranin, Butein, and Brevilin A (Table 1).

[0022] Table 1

[0023]

[0024] Aristolone's structural formula:

[0025] The structural formula of Isosakuranin:

[0026] Butein's structural formula:

[0027] The structural formula of Brevilin A:

[0028] Example 2. Detection of PHD2 enzyme activity

[0029] 1. Reagent Preparation: Aristolone (catalog number: HY-N1464A), Isosakuranin (catalog number: HY-4296), Butein (catalog number: HY-16558), and Brevilin A (catalog number: HY-N2959) were all purchased from MedChemExpress (MCE). All compounds were dissolved in DMSO solution to prepare a 50 mM preservation solution. The PHD2 activity kit (catalog number: MBS7607905) was purchased from Mybiosource.

[0030] 2. Cell treatment: HepG2 cells in logarithmic growth phase were trypsinized, then counted and seeded in 6-well plates at a density of 2 × 10⁵ cells / well. One day later, cells were treated with drugs at doses of 5, 10, and 20 μM; control cells were treated with an equal volume of the small molecule compound solvent DMSO. Drug treatment lasted for 12 hours.

[0031] 3. Preparation of cell lysis buffer: Aspirate the supernatant and add pre-chilled PBS once. Then, add 0.5 ml of RIPA lysis buffer (NP-40 lysis buffer) and gently scrape away adherent cells with a cell scraper. Transfer the suspension to centrifuge tubes and lyse on ice for 30 minutes, intermittently shaking the tubes to ensure complete protein lysis. Determine protein concentration using the BCA method and homogenize the protein content of each group.

[0032] 4. Prepare fresh biotin-labeled antibody working solution and HRP-streptavidin working solution. Set up standard and test sample wells in a pre-coated 96-well plate, add biotin-labeled antibody working solution to each well, and incubate at 37°C for 90 min. Then add HRP-labeled streptavidin working solution and react at 37°C for 30 min. Next, add TMB substrate and react for 10-20 minutes (the reaction time can be shortened or extended according to the actual color change, but should not exceed 30 minutes). Finally, add the reaction stop solution and immediately measure the absorbance at 450 nm using a microplate reader and calculate the enzyme activity value.

[0033] Example 3. Detection of HIF-target gene Glut-1 in HepG2 cells

[0034] 1. Cell treatment: HepG2 cells in logarithmic growth phase were trypsinized, and then the cells were counted at a concentration of 2 × 10⁻⁶. 5Cells were seeded at a density of cells / well in 6-well plates. One day later, cells were treated with drugs at doses of 5 μM, 10 μM, and 20 μM. Control cells were treated with an equal volume of the small molecule compound solvent DMSO for 12 hours.

[0035] 2. Total RNA was collected from cells using the Trizol method and quantified using an ultra-micro UV-Vis spectrophotometer. 1 μg of total RNA was used for cDNA synthesis using the HiScript RT SuperMix for qPCR reverse transcription kit. Real-time quantitative PCR was then performed on a C1000 Touch Thermal Cycler PCR instrument using the ChamQ™ SYBR qPCR Master Mix reaction system. The forward primer for Glut-1 was 5'-gtggccttctttgaagtggg-3', and the reverse primer was 5'-aagacgtagggaccacacag-3'. The forward primer for the internal control β-Actin was 5'-catccgcaaagacctgtacg-3', and the reverse primer was 5'-cctgcttgctgatccacatc-3'. The PCR amplification program was: 95℃ for 30 s, 95℃ for 5 s, and 60℃ for 30 s, for a total of 40 cycles. Each experiment was independently repeated three times. Using β-Actin as an internal reference gene, 2 -ΔΔCt The relative expression level of Glut-1 in each treatment group was calculated using the method.

[0036] The above experiments confirm that ( Figure 1 and Figure 2 Four small molecule compounds—Aristolone, Isosakuranin, Butein, and Brevilin A—can bind to the active site of PHD, and subsequent molecular biology experiments have confirmed that they can inhibit PHD enzymatic activity, activate the HIF-1 signaling pathway, and lead to increased expression of its target gene Glut-1. Their inhibition of PHD enzyme activity lays the foundation for future drug development.

[0037] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of Aristolone in the preparation of inhibitors targeting proline hydroxylase, characterized in that, The structural formula of Aristolone is: .

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