High-throughput screening method of CCL5 / CCR5 biological axis targeting antagonist

Abstract

The invention relates to a high-throughput screening method of a CCL5 / CCR5 biological axis targeting antagonist, which is used for detecting the signal change of the interaction of a chemotactic factor and a receptor thereof through a microwell plate before and after the action of the antagonist so as to realize the screening of the CCL5 / CCR5 biological axis targeting antagonist. According to the high-throughput screening model of the targeted antagonist based on the biological axis of the microporous pore plate, analysis of combination of various cell surface receptors and corresponding ligands and high-throughput screening of corresponding antagonists are realized. The vitepofen obtained through test screening is a targeted antagonist of a CCL5 / CCR5 biological axis, a molecular probe and a disease treatment drug related to the target.

Description

Technical Field

[0001] The present invention relates to a technology in the field of antagonist screening, specifically a high-throughput screening method for CCL5 / CCR5 biological axis targeting antagonists. Background Art

[0002] The CCL5 / CCR5 biological axis is a therapeutic target for many diseases, including allergic diseases, inflammation, immune diseases, malignant tumors, and COVID-19. It is closely related to a variety of malignant tumors, such as triple-negative breast cancer. Antagonists targeting this biological axis are considered to be potential therapeutic molecules for these malignant diseases. However, at present, there are still no reports of specific antagonists targeting this biological axis. Summary of the invention

[0003] In view of the deficiency of the prior art that only CCR5 single target targeted antagonists are used, the present invention proposes a high-throughput screening method for CCL5 / CCR5 biological axis targeted antagonists. By constructing a high-throughput screening model for biological axis targeted antagonists based on microporous plates, the analysis of the binding of various cell surface receptors to corresponding ligands and the high-throughput screening of their corresponding antagonists are achieved.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention relates to a high-throughput screening method for a CCL5 / CCR5 biological axis targeting antagonist, which realizes the screening of antagonists targeting the CCL5 / CCR5 biological axis by detecting the signal changes of the interaction between a chemokine and its receptor before and after the action of the antagonist through a microplate.

[0006] The antagonists include: small molecule compounds, polypeptides and / or antibodies.

[0007] The antagonist effect refers to: sequentially adding the antagonist candidate to be screened and the His-CCL5 purified protein to human embryonic kidney cells (HEK293T-CCR5) overexpressing human CCR5 that are solidified and sealed in a microplate.

[0008] The signal of the interaction between the chemokine and its receptor refers to: amplifying the signal of the binding of CCL5 and CCR5 by adding HRP (horseradish peroxidase) or fluorescence-coupled anti-His antibody to the microplate, and further generating a color product by adding HRP substrate ABTS or directly detecting the fluorescent signal.

[0009] The direct detection of the signal refers to: calculating the inhibition efficiency of the antagonist candidate in the experimental group on the binding of the chemokine to its receptor based on the absorbance value at 405nm or the fluorescent signal of the fluorescent antibody To achieve the identification of its targeted antagonist, the experimental group contains antagonist candidates, the negative control group contains a DMSO solution with the same concentration, and the blank control group contains a PBS solution with an equal volume to his-CCL5. Technical Effects

[0010] The present invention uses microplates to analyze the binding of cell surface receptors to their corresponding ligands and high-throughput screening of their corresponding targeted antagonists, thereby realizing high-throughput screening of antagonist libraries such as small molecule compounds, peptides or antibodies, and screening various types of antagonists targeting the CCL5 / CCR5 biological axis. In the present invention, 7555 compounds from multiple compound libraries have been screened to screen targeted antagonists of the CCL5 / CCR5 biological axis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a flow chart of the present invention;

[0012] Figure 2 It is the principle diagram of the present invention;

[0013] Figure 3 The results of the screening of 7555 compounds by high-throughput screening to screen the CCL5 / CCR5 biological axis targeted antagonists are shown in the figure;

[0014] Figure 4-Figure 7 It is a schematic diagram of the effect of the embodiment. DETAILED DESCRIPTION

[0015] like Figure 1 As shown, the present invention relates to a method for screening a targeted antagonist of the CCL5 / CCR5 biological axis, comprising the following steps:

[0016] Step 1) Human embryonic kidney cells (HEK293T-CCR5) overexpressing human CCR5 were seeded into a 96-well plate at a density of 10,000 cells per well and cultured at 37° C. overnight;

[0017] Step 2) Take out the cells, remove the culture medium, and wash once with 50 μL PBS solution. Add 50 μL 4% paraformaldehyde (Servicebio, G1101-500ML) to each well and fix at room temperature in the dark for 20 minutes;

[0018] Step 3) remove the paraformaldehyde in the wells and add 50 μL PBS solution to each well for washing once;

[0019] Step 4) Add PBS solution containing 5% BSA to each well, adjust the shaker to the lowest speed, shake horizontally, and block at room temperature for 1 hour;

[0020] Step 5) Remove the blocking solution and add 50 μL PBS solution to each well for washing once;

[0021] Step 6) After adding PBS solution and candidate antagonists to be screened into the wells, place them on a shaker and shake horizontally, and incubate at room temperature for 1-3 hours. The wells with antagonists added are experimental wells, 0.5% DMSO is a negative control well, and 100 μM 2,7-dibromo-4-hydroxymercury fluorescent red disodium salt (mercury red) (Sigma-Aldrich, M7011) is a positive control well;

[0022] The candidate antagonists, such as 7555 compounds to be screened, include: 1,840 compounds in FDA library I (screening concentration is 20 μM), 1,700 compounds in FDA library II (screening concentration is 100 μM), 480 compounds in European FDA library (screening concentration is 100 μM), 879 compounds in NCI mechanism library (screening concentration is 10 μM), 1,856 compounds in NCI library (screening concentration is 100 μM) and 800 compounds in natural product library (screening concentration is 100 μM).

[0023] Step 7) Add His-CCL5 purified protein at a final concentration of 30 μg / mL directly to each well, place on a shaker and shake horizontally, and incubate at room temperature for 1-3 hours. The wells with an equal amount of PBS solution are blank control wells;

[0024] Step 8) Pour out the solution in the wells and wash with PBST solution containing 1 / 2000 Tween-20 for 3 times, 10 min each time;

[0025] Step 9) HRP-conjugated anti-His primary antibody (Proteintech, HRP-66005) was diluted with PBS solution at a ratio of 1:5000, 50 μL was added to each well, and incubated overnight at 4°C on a shaker;

[0026] Step 10) Wash the 96-well plate three times with PBST solution containing 1 / 2000 Tween-20, each time for 10 min;

[0027] Step 11) Add 100 μL ABTS solution (ThermoFisher Scientific, 002024) to each well, place on a shaker, and incubate at room temperature in the dark for 30 min;

[0028] Step 12) Place the plate in a full-wavelength microplate reader and read the absorbance at 405 nm.

[0029] Step 13) Figure 3As shown, the interaction between CCL5 and its receptor CCR5 was detected in the microwells (using HRP-coupled anti-His antibody as a signal detection unit), and the screening of its targeting antagonists was performed respectively.

[0030] like Figure 4 and Figure 5 As shown, after screening 7555 compounds from different compound libraries and comparing them with the existing CCR5 antagonist maraviroc, verteporfin was obtained to be a highly effective and specific antagonist targeting the CCL5 / CCR5 biological axis. Figure 3 The antagonist verteporfin targeting the CCL5 / CCR5 biological axis obtained by the screening process has an inhibitory efficiency IC of CCL5 / CCR5 biological axis. 50 The intensity is at least 200 times higher than that of maraviroc. Compared with the known CCR5 antagonist maraviroc, it can more significantly interrupt the green fluorescence of CCL5 binding to CCR5 as determined by fluorescence confocal microscopy.

[0031] like Figure 6 As shown, by the present invention Figure 3 The IC values ​​of verteporfin, a highly effective small molecule antagonist targeting the CCL5 / CCR5 biological axis, were detected by the screening process to detect the inhibitory efficiency of CCL5 and other four cytokine ligands that can bind to CCR5 on CCR5. 50 The results showed that the verteporfin obtained by the method of the present invention can effectively and specifically interrupt the CCL5 / CCR5 biological axis.

[0032] like Figure 7 As shown, by the present invention Figure 3 The IC values ​​of verteporfin, a highly effective small molecule antagonist targeting the CCL5 / CCR5 biological axis, were detected by the screening process to detect the inhibitory efficiency of the binding of CCL5 to CCR5 and other three receptors. 50 The results showed that the verteporfin obtained by the method of the present invention can effectively and specifically interrupt the CCL5 / CCR5 biological axis.

[0033] In summary, the present invention realizes the detection of ligand-receptor binding signals and high-throughput screening of corresponding antagonists by adding cells that overexpress receptors, ligand units, signal amplification units, and optical signal detection units. The screened verteporfin is a highly efficient, specific, and efficient inhibitor targeting the CCL5 / CCR5 biological axis.

[0034] The above-mentioned specific implementation can be partially adjusted in different ways by those skilled in the art without departing from the principle and purpose of the present invention. The protection scope of the present invention shall be based on the claims and shall not be limited by the above-mentioned specific implementation. Each implementation scheme within its scope shall be subject to the constraints of the present invention.

Claims

1. A high-throughput screening method for CCL5 / CCR5 biological axis targeting antagonists, characterized in that: The signal changes of the interaction between chemokines and their receptors before and after the action of antagonists are detected by microplate, so as to screen antagonists targeting the CCL5 / CCR5 biological axis. The antagonists include: small molecule compounds, polypeptides and / or antibodies; The antagonist effect refers to: sequentially adding the antagonist candidate to be screened and the His-CCL5 purified protein to human embryonic kidney cells (HEK293T-CCR5) overexpressing human CCR5 that are solidified and sealed in a microplate.

2. The high-throughput screening method for CCL5 / CCR5 biological axis targeting antagonists according to claim 1, characterized in that: The signal of the interaction between the chemokine and its receptor refers to: amplifying the signal of the binding of CCL5 and CCR5 by adding HRP or fluorescence-coupled anti-His antibody to the microplate, and further generating a color product by adding HRP substrate ABTS or directly detecting the fluorescent signal.

3. The high-throughput screening method for CCL5 / CCR5 biological axis targeting antagonists according to claim 2, characterized in that: The direct detection of the signal refers to: calculating the inhibition efficiency of the antagonist candidate in the experimental group on the binding of the chemokine to its receptor based on the absorbance value at 405nm or the fluorescent signal of the fluorescent antibody To achieve the identification of its targeted antagonist, the experimental group contains antagonist candidates, the negative control group contains a DMSO solution with the same concentration, and the blank control group contains a PBS solution with an equal volume to his-CCL5.

4. The high-throughput screening method for CCL5 / CCR5 biological axis targeting antagonists according to claim 1, characterized in that: Antagonist candidates of small molecule compounds of the experimental cases of the present invention can be detected but are not limited to, including: 1,840 compounds in FDA library I, 1,700 compounds in FDA library II, 480 compounds in European FDA library, 879 compounds in NCI mechanism library, 1,856 compounds in NCI library and 800 compounds in natural product library.

5. The high-throughput screening method for CCL5 / CCR5 biological axis targeting antagonists according to any one of claims 1 to 4, characterized in that: include: Step 1) Human embryonic kidney cells (HEK293T-CCR5) overexpressing human CCR5 in good growth state were seeded into a 96-well plate at a density of 10,000 cells per well and cultured at 37° C. overnight; Step 2) The cells were removed, the culture medium was removed, and 50 μL of PBS solution was added to wash once, and 50 μL of 4% paraformaldehyde was added to each well, and fixed at room temperature in the dark for 20 minutes; Step 3) remove the paraformaldehyde in the wells and add 50 μL PBS solution to each well for washing once; Step 4) Add PBS solution containing 5% BSA to each well, adjust the shaker to the lowest speed, shake horizontally, and block at room temperature for 1 hour; Step 5) Remove the blocking solution and add 50 μL PBS solution to each well for washing once; Step 6) After adding PBS solution and candidate antagonists to be screened into the wells, place them on a shaker for horizontal shaking and incubate at room temperature for 1-3 hours; the wells with antagonists added are experimental wells, 0.5% DMSO is a negative control well, and 100 μM 2,7-dibromo-4-hydroxymercury fluorescent red disodium salt is a positive control well; Step 7) directly add His-CCL5 purified protein at a final concentration of 30 μg / mL to each well, place on a shaker for horizontal shaking, and incubate at room temperature for 1-3 hours; the wells added with an equal amount of PBS solution are blank control wells; Step 8) Pour out the solution in the wells and wash with PBST solution containing 1 / 2000 Tween-20 for 3 times, 10 min each time; Step 9) Dilute the HRP-conjugated anti-His primary antibody with PBS solution at a ratio of 1:5000, add 50 μL to each well, and incubate overnight at 4°C on a shaker; Step 10) Wash the 96-well plate three times with PBST solution containing 1 / 2000 Tween-20, each time for 10 min; Step 11) Add 100 μL ABTS solution to each well, place on a shaker, and incubate at room temperature in the dark for 30 min; Step 12) Place the plate in a full-wavelength microplate reader and read the absorbance at 405 nm; Step 13) using HRP-coupled anti-His antibody as a signal detection unit, the interaction between CCL5 and its receptor CCR5 is detected in the microwells, and its targeted antagonist is screened.

6. An application of verteporfin targeting the CCL5 / CCR5 biological axis, characterized in that: It is used as a molecular probe targeting the CCL5 / CCR5 biological axis or a small molecule compound for the physiological and medical mechanism of action and disease treatment of the CCL5 / CCR5 axis.

Images