Application of compound as PCSK9 inhibitor

Abstract

The invention provides an application of a compound as a PCSK9 inhibitor, relates to the technical field of biological medicines, and comprises an application of a compound with a structure shown in a formula I as the PCSK9 inhibitor. The compound with the structure shown in the formula I can be combined with an active pocket of PCSK9, and can be combined with 66th-site histidine and 145th-site glutamic acid of the PCSK9 to form the PCSK9 inhibitor. The histidine at the 295th site forms a conservative hydrogen bond, so that the combination of PCSK9 and LDLR is reduced, the LDLR protein expression is remarkably enhanced, and the level of LDL-C cholesterol in blood can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of a compound as a PCSK9 inhibitor. Background Art

[0002] In traditional lipid-lowering treatment programs, statins are the main drugs used for lipid-lowering treatment. This type of drug can reduce the risk of cardiovascular events by lowering LDL-C. However, under the current traditional lipid-lowering drugs, especially statins, patients still have substandard blood lipids. Summary of the invention

[0003] The purpose of the present invention is to provide a compound for use as a PCSK9 inhibitor to improve the above problems. In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0004] The present application provides a compound having a structure of Formula I for use as a PCSK9 inhibitor;

[0005]

[0006] The present invention also provides the use of the compound having the structure of formula I for preparing a drug for inhibiting the binding of PCSK9 to low-density lipoprotein receptor-related protein.

[0007] The low-density lipoprotein receptor-related protein is a low-density lipoprotein receptor-related protein of hepatocytes.

[0008] The present invention also provides the use of the compound having the structure of formula I for preparing a drug for treating or alleviating a disease by inhibiting the activity of PCSK9.

[0009] The disease is hyperlipidemia.

[0010] The disease is hypercholesterolemia.

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

[0012] The present invention uses a compound having a structure of Formula I to bind to the active pocket of PCSK9, and form conservative hydrogen bonds with histidine at position 66, glutamic acid at position 145, and histidine at position 295 of PCSK9, thereby reducing the binding between PCSK9 and LDLR, thereby significantly enhancing LDLR protein expression, thereby being able to reduce the level of LDL-C cholesterol in the blood.

[0013] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or be understood by implementing the embodiments of the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the written description, claims, and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 It is a schematic diagram of the binding of the molecule of the compound having the structure of Formula I in the present invention to the active pocket of the PCSK9 molecule.

[0016] Figure 2 This is a diagram showing the SDS-PAGE electrophoresis results of a Western blot experiment of the EPG2 cell protein standard at different dosage concentrations in Example 3 of the present invention.

[0017] Figure 3 This is a bar graph of LDLR content at different dosage concentrations in a Western blot experiment of the EPG2 cell protein standard in Example 3 of the present invention.

[0018] Figure 4 This is a diagram showing the SDS-PAGE electrophoresis results of a protein blotting experiment of a normal group and a drug-treated group in Example 3 of the present invention.

[0019] Figure 5 It is a bar graph of LDLR content in the normal group and the drug-treated group in Example 3 of the present invention in a Western blot experiment.

[0020] Markings in the figure: 1-compound molecule having the structure of formula I, 2-active pocket of PCSK9 molecule. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] It should be noted that similar numbers and letters represent similar items in the following figures, so once an item is defined in one figure, it does not need to be further defined and explained in the subsequent figures. At the same time, in the description of the present invention, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance. "LDLR" in the text means low-density lipoprotein receptor-related protein. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0023] The present application provides a compound having a structure of Formula I for use as a PCSK9 inhibitor;

[0024]

[0025] The present invention also provides the use of the compound having the structure of formula I for preparing a drug for inhibiting the binding of PCSK9 to low-density lipoprotein receptor-related protein.

[0026] The low-density lipoprotein receptor-related protein is a low-density lipoprotein receptor-related protein of hepatocytes.

[0027] The present invention also provides the use of the compound having the structure of formula I for preparing a drug for treating or alleviating a disease by inhibiting the activity of PCSK9.

[0028] The disease is hyperlipidemia.

[0029] The disease is hypercholesterolemia.

[0030] Example 1

[0031] Study on the molecular docking of the compound molecule with the structure of formula I and PCSK9

[0032] The compound molecule with the structure of Formula I was obtained using scifinder, and the compound molecule with the structure of Formula I was docked with the PCSK9 molecule using the Discovery studio software and the CDOKER module. The results are as follows Figure 1 As shown, from Figure 1 It can be seen that the compound molecule 1 with the structure of Formula I can enter the active pocket 2 of the PCSK9 molecule and form conservative hydrogen bonds with the 66th histidine, 145th glutamic acid, and 295th histidine of the active pocket 2 of the PCSK9 molecule. This verifies the feasibility of docking the compound molecule with the structure of Formula I with the PCSK9 molecule, and provides a theoretical basis for the mechanism of action of the compound molecule with the structure of Formula I and PCSK9.

[0033] When the drug of the present invention is used, the compound having the structure of formula I is used as the only active ingredient, or the compound having the structure of formula I is used as one of the active ingredients, and can be mixed with one or more pharmaceutically acceptable carriers to form a pharmaceutical dosage form for different administration routes. The compound having the structure of formula I can be obtained through commercial routes. The composition of the compound and the carrier can preferably be a capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

[0034] Example 2

[0035] Study the effect of compounds with formula I on the binding inhibition rate of PCSK9-LDLR

[0036] Preparation of stock solutions for experimental and control groups

[0037] Experimental group: The lyophilized powder of the compound having the structure of Formula I was dissolved by dimethyl sulfoxide and prepared into a 20 mM mother solution of the compound having the structure of Formula I for storage.

[0038] Control group: Pep2-8 lyophilized powder was dissolved in double deionized water to prepare 2 mM Pep2-8 stock solution.

[0039] The experimental steps of this embodiment are as follows:

[0040] Prepare a reaction buffer containing 80 ng / mL recombinant His-tagged PCSK9;

[0041] Using the reaction buffer, continuously diluting a 20 mM stock solution of the compound having the structure of Formula I to a final concentration and mixing well to obtain a diluted solution of the compound having the structure of Formula I;

[0042] The reaction buffer is used to continuously dilute the 2 mM Pep2-8 stock solution to the final concentration and mix well to obtain a diluted Pep2-8 solution;

[0043] The diluted solution of the compound having the structure of Formula I and the diluted Pep2-8 solution were pre-incubated at room temperature for 1 hour to obtain an experimental group reaction mixture and a control group reaction mixture, respectively;

[0044] 100 μL of the experimental group reaction mixture and 100 μL of the control group reaction mixture were added to the well plate coated with the EGF (AB) peptide of LDLR;

[0045] The absorbance of the test wells of the well plate was measured at dual wavelengths of 450 / 540 nm using an EnVision multimode plate reader (EnSpire, PerkinElmer);

[0046] The percentage inhibition of the experimental group reaction mixture was calculated from the standard His-tagged PCSK9 dose-absorption curve after correction for the vehicle control IC 50 Value, IC 50 The value is 3.25 μM, indicating that the compound with the structure of Formula I has a good inhibitory effect on the binding of PCSK9-LDLR.

[0047] Example 3

[0048] The study showed that the compound with the structure of formula I has a promoting effect on the expression of LDLR

[0049] Preparation of protein standards

[0050] Preparation of EPG2 cell protein standard: HEPG2 cells were cultured to a density of 80%, and then divided into three groups after changing to serum-free DMEM medium. The drug concentrations (the drug concentrations here and below refer to the concentrations of PCSK9 inhibitors with the compound having the structure of Formula I) were 0, 10 μM, and 20 μM, respectively. After 12 hours of cell culture, protein extract RIPA was added to collect the total protein to obtain HEPG2 cell protein standard.

[0051] Preparation of animal liver cell protein standards: Collect liver cells from dosed mice (the dosed mice referred to here and below refer to mice dosed with a PCSK9 inhibitor having a compound of Formula I, and the dose concentration is 100 mg / kg mouse body weight) and normal mice, freeze them in liquid nitrogen and then grind them, collect proteins, and obtain liver cell protein standards from dosed mice and normal mice, respectively.

[0052] The HEPG2 cell protein standard with the above-mentioned drug concentrations of 0, 10 μM, and 20 μM was subjected to Western blotting experiments;

[0053] Similarly, Western blotting experiments were performed on the liver cell protein standards of the above-mentioned drug-treated mice and the liver cell protein standards of normal mice.

[0054] The Western blot experiment process is as follows:

[0055] Protein standard treatment: dilute 5mg / mL BSA to 0.5mg / mL BSA with PBS. Add PBS to a 96-well plate at 20μl, 18μl, 16μl, 14μl, 12μl, 8μl, 4μl, and 0μl. Then add protein standards to PBS at 0μl, 2μl, 4μl, 6μl, 8μl, 12μl, 16μl, and 20μl, so that the concentrations of protein standards are 0mg / mL, 0.05mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, and 0.5mg / mL, respectively. Set up 2 replicate wells for each concentration.

[0056] Sample processing: dilute the sample 10 times, add 18μl PBS to each 96-well plate, then take 2μl of the sample and add it to PBS, set up 2 replicate wells for each concentration; finally, add the BCA working solution prepared with BCA reagent A and reagent B in a volume ratio of 50:1 to the well plate, add 200μl BCA working solution to each well, put it in a 37℃ incubator and incubate it for 30 minutes, use an enzyme reader to measure the absorbance at a wavelength of 562nm, draw a protein standard curve, and calculate the protein content of the sample based on the absorbance.

[0057] The experimental operation steps of this embodiment are as follows:

[0058] (1) Leak test: Clean the quartz glass plate (1.0 mm), edge strips, sample comb, SDS-PAGE gel caster and other accessories, place them flat on the table, and wipe them with anhydrous ethanol; align the bottom ends of the long and short quartz glass plates, place them in the gel caster in sequence, align the bottom ends again, gently clamp the gel caster, fix it with a plastic clamp, add double distilled water between the glass plates until the short glass plate is flush, and let it stand for 2-3 minutes;

[0059] (2) Preparation of separation gel: Pour off the double distilled water and gently absorb the water along the edge with filter paper / paper; prepare 10% separation gel, add each component in turn, mix well, add to 2 / 3 along the glass plate, add double distilled water to seal, let stand at room temperature for about 30 minutes to solidify, and pour off the double distilled water;

[0060] (3) Preparation of concentrated gel: Prepare 5% separation gel, add each component in sequence, mix well, fill the glass plate, gently insert a comb, fill the edge of the comb with the remaining concentrated gel, and let it stand at room temperature for about 20 minutes to solidify;

[0061] (4) Prepare samples: Add 5× SDS-PAGE Loading Buffer to the sample at a ratio of 4:1 protein: Loading Buffer, mix well, boil the sample in a metal bath at 100°C for 10 min, and immediately cool it in ice water. Divide each concentration sample into several tubes, use a portion of the sample for the experiment, and store the rest in a -20°C refrigerator;

[0062] (5) Electrophoresis: Gently pull out the comb vertically upward, take out the glass plate from the gel casting machine, wash the glass plate with tap water, install the glass plate in the electrophoresis card holder, put the electrophoresis card holder into the electrophoresis tank, add electrophoresis buffer along the middle of the glass plate, and cover the bottom of the glass plate with the outside, load 20 μg of total protein, 3 μl of marker, and fill both sides of the sample well with Loading Buffer; connect the power supply and start electrophoresis, 60V for the concentrated gel, about 30 min, 120V for the separation gel, about 60 min;

[0063] (6) Transfer: Cut the NC membrane, cut off the front and back marks on the upper right corner of the membrane, cut filter paper of the same size, prepare a tray, add pre-cooled transfer buffer, and soak the transfer clip, sponge, filter paper, and NC membrane; take out the glass plate, cut the gel in the area where the target protein is located according to the protein marker, place the gel on the filter paper, place the membrane on the gel, then put the filter paper, close the transfer clip, put it in the transfer tank, and add all the transfer buffer; put the transfer tank in ice, connect the power supply to start transfer, and the transfer conditions are 200mA, 2.5h;

[0064] (7) Blocking: Take out the NC membrane, put it into 5% skimmed milk powder, shake it slowly on a shaker, and block it at room temperature for 1 h;

[0065] (8) Antibody incubation: Place the NC membrane in TBST, separate the membrane target protein from the internal reference, dilute the LDLR antibody and β-actin antibody, and incubate at 4°C overnight; wash the NC membrane three times in TBST, each time for 10 min; dilute the horseradish peroxidase-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled goat anti-mouse IgG secondary antibodies at 1:5000 and incubate at room temperature for 1 h;

[0066] (9) Development: Wash the NC membrane three times in TBST, each time for 10 min; mix luminescent solution A and B in a ratio of 1:1, add an appropriate amount onto the NC membrane, and use a chemiluminescent gel imaging system for protein development.

[0067] The experimental results of this embodiment are as follows Figure 2-5 shown.

[0068] like Figure 2-3The results of the western blot experiment of the EPG2 cell protein standard showed that the color of the protein band at a drug concentration of 10 μM was significantly darker than that of the protein band at a drug concentration of 0, and the color of the protein band at a drug concentration of 20 μM was significantly darker than that of the protein band at a drug concentration of 10 μM. This shows that the higher the drug concentration, the darker the LDLR protein band. Therefore, it can be known that the LDLR content in HEPG2 cells increases significantly with the increase of the drug concentration, which shows that the compound with the structure of Formula I has a promoting effect on the expression of LDLR in EPG2 cells.

[0069] like Figure 3-4 The results of western blotting experiments on the liver cell protein standards of the dosed mice and the liver cell protein standards of the normal mice showed that the color of the LDLR protein band in the western blotting experiment of the liver cell protein standards of the dosed mice was significantly darker than that of the LDLR protein band in the western blotting experiment of the liver cell protein standards of the normal mice, indicating that the compound with the structure of Formula I has a promoting effect on the expression of LDLR in the liver cells of mice.

[0070] In summary, the compound having the structure of Formula I has a promoting effect on the expression of LDLR.

[0071] Example 4

[0072] LDL enzymatic assay

[0073] Sample preparation: Prepare naturally coagulated serum from treated mice and naturally coagulated serum from normal mice.

[0074] Required equipment: ELISA reader, biochemical analyzer, visible light spectrophotometer. The best working wavelength is 550-555nm. If this wavelength is not available, 570, 530, and 490nm are recommended. The optical diameter of the cuvette is 1cm.

[0075] Standard dilution: 5mM LDL standard was diluted with anhydrous ethanol in the following ratios: 2500, 1250, 625, 312.5, 156, 78, and 36 μmol / L.

[0076] The steps for LDL separation are as follows:

[0077] Take 50 μL of naturally coagulated serum from the treated mice and 50 μL of naturally coagulated serum from the normal mice;

[0078] Add an equal volume of 50 μL of LDL capture agent and vortex to mix.

[0079] Incubate at room temperature for 10 minutes. Let sediment for 10-30 minutes at room temperature or 4°C. Centrifuge at 2000g for 20 minutes. Centrifuge at 4-22°C. The sediment contains LDL. Carefully remove the supernatant, centrifuge again at 2000g for 10 minutes, and discard the supernatant.

[0080] Add 100 μL of PBS or saline (twice the volume of the initial plasma sample) and resuspend the precipitate by vortexing.

[0081] LDL determination steps:

[0082] Prepare the assay working solution: Mix 4 ml of reagent R1 with 1 ml of reagent R2 in a 4:1 ratio and use immediately.

[0083] Take 190 μl of the assay working solution and add it to the microplate wells.

[0084] Add 10 μL of the supernatant prepared in step 1.6. The supernatant contains LDL. If there is flocculent after standing, centrifuge again. The amount of standard sample added to the standard sample tube is also 10 μL. The total reaction volume is 200 μL.

[0085] 37℃ for 20min (15~30min), or 25℃ for 30min, but the sensitivity is slightly lower. After the reaction is balanced, the color is stable within 60min.

[0086] The above LDL enzymatic assay experimental steps were repeated three times, firstly adjusting the blank tube to zero with distilled water, and then measuring the LDL OD550 value of each tube as shown in Table 1.

[0087] Table 1

[0088]

[0089] It can be seen from the LDL enzymatic assay experimental results in Table 1 that the LDL content in the serum of normal mice is significantly higher than that in the serum of the dosed mice. This indicates that under the action of the compound having the structure of Formula I as PCSK9, the binding of PCSK9 to LDLR is inhibited in the dosed mice, which promotes the binding of LDL to LDLR (i.e., LDL receptor).

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0091] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of a compound having a structure of formula I as a PCSK9 inhibitor; 2. Use of the compound having the structure of formula I as claimed in claim 1 for preparing a drug for inhibiting the binding of PCSK9 to low-density lipoprotein receptor-related protein.

3. The use according to claim 2, characterized in that: The low-density lipoprotein receptor-related protein is a low-density lipoprotein receptor-related protein of hepatocytes.

4. Use of the compound having the structure of formula I as claimed in claim 1 for preparing a drug for treating or alleviating a disease by inhibiting the activity of PCSK9.

5. The use according to claim 4, characterized in that: The disease is hyperlipidemia.

6. The use according to claim 4, characterized in that: The disease is hypercholesterolemia.

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