阅读说明：本技术 一种小分子抑制剂在制备靶向p2x7受体中的用途 (Application of small molecule inhibitor in preparation of targeting P2X7 receptor ) 是由 陶金辉 李晓玲 于 2021-09-26 设计创作，主要内容包括：本发明属于P2X7R拮抗剂筛选技术领域,具体涉及一种小分子抑制剂在制备靶向P2X7受体中的用途,通过预测P2X7R蛋白结构建模及变构位点确定P2X7R蛋白变构抑制剂的候选位点,然后筛选小分子变构抑制剂,通过对化合物库中小分子进行预处理和构象生成,分析结合模式获得小分子抑制剂。本发明筛选获得的靶向P2X7受体的小分子抑制剂特异性高,安全性好,没有任何毒性,副作用低,能够应用于痛风关节炎的防治。(The invention belongs to the technical field of P2X7R antagonist screening, and particularly relates to application of a small molecule inhibitor in preparation of a targeted P2X7 receptor, wherein candidate sites of a P2X7R protein allosteric inhibitor are determined by predicting P2X7R protein structure modeling and allosteric sites, then the small molecule allosteric inhibitor is screened, and the small molecule inhibitor is obtained by performing pretreatment and conformation generation on small molecules in a compound library and analyzing a binding mode. The small molecule inhibitor targeting the P2X7 receptor obtained by screening has high specificity, good safety, no toxicity and low side effect, and can be applied to the prevention and treatment of gouty arthritis.)

1. The use of a small molecule inhibitor for preparing a targeting P2X7 receptor, wherein the molecular structural formula of the small molecule inhibitor is as follows:

2. the use of the small molecule inhibitor according to claim 1 for preparing a targeting P2X7 receptor, wherein the screening process of the small molecule inhibitor comprises the following steps:

the first step is as follows: prediction of P2X7R protein structural modeling and allosteric site: firstly, modeling the protein structure of P2X7R, then predicting an allosteric site, and finally scoring the allosteric site to determine a candidate site of the P2X7R protein allosteric inhibitor;

the second step is that: screening of small molecule allosteric inhibitors: firstly, a P2X7R protein crystal compound 5U1X is downloaded, the protein is processed by adopting hydrogenation, dehydration and protein structure optimization in a Glide protein pretreatment process, a small molecule library is screened, molecules containing a PAINS structure are removed, pretreatment and conformation generation are carried out on the small molecules, Schordinger software is used for docking to obtain a docking conformation, the small molecules are graded and screened, the small molecules graded and ranked at the top 2000 are selected, the small molecules ranked at the top 30 are screened from the small molecules, the binding mode of the small molecules is analyzed, the small molecules are screened by highest precision docking and the binding mode of the small molecules is analyzed, and finally the small molecule inhibitor for docking is obtained.

3. Use of a small molecule inhibitor according to claim 2 for the preparation of a targeting P2X7 receptor, characterized in that in a first step the protein structure of P2X7R is modeled using the protein modeling program I-tessar.

4. Use of a small molecule inhibitor according to claim 3 for the preparation of a targeting P2X7 receptor, wherein in the first step the allosteric site of the protein is predicted using the protein allosteric site prediction program allositeproro software.

5. Use of a small molecule inhibitor according to claim 4 for the preparation of a targeting P2X7 receptor, characterized in that in the first step the three allosteric sites of the P2X7R protein are scored using the AlloSite program.

6. Use of a small molecule inhibitor according to claim 5 for the preparation of a targeting P2X7 receptor, wherein in the first step the three allosteric sites of the P2X7R protein are scored by the AlloSite program on the basis of Pocket Volume, AlloSite Score and pertubation Score;

wherein, when the AlloSite program shows that the Pocket Volume is greater than or equal to 659.833, the AlloSite Score is greater than or equal to 0.630 and the pertubation Score is greater than or equal to 0.110, the site can be used as a candidate site of the P2X7R protein allosteric inhibitor.

7. The use of a small molecule inhibitor according to claim 6 for the preparation of a targeting P2X7 receptor, wherein in the second step the screening of the library of small molecules is performed using a virtual screen provided by the ceramic chemistry.

8. Use of a small molecule inhibitor according to claim 7 for the preparation of a targeted P2X7 receptor, characterized in that in the second step the small molecule is pre-treated and conformationally generated using the ligaprep module in schrodinger.

9. The use of a small molecule inhibitor according to claim 1 for the preparation of a medicament targeting the P2X7 receptor, wherein the small molecule inhibitor is used for the preparation of a medicament for the treatment of gouty arthritis.

Technical Field

The invention belongs to the technical field of P2X7R antagonist screening, and particularly relates to an application of a small molecule inhibitor in preparation of a targeted P2X7 receptor.

Background

The purinoceptors P2X7(P2X7R) are ATP-gated ion channels that play an important role in innate immunity in humans, and are ubiquitously expressed in immune cells of almost all tissues and organs, such as dendritic cells, T lymphocytes, B lymphocytes, and neutrophils, and are highly expressed on monocytes or macrophages. P2X7R can be activated by ATP to form nonselective cation channel, causing Na + and Ca2+ influx and K + efflux to change cell ion homeostasis, and has the ability to form multiple nonselective membrane pores under stimulation of high concentration of ATP, sufficient to allow substances with molecular weight of 900kDa to pass through macrophage membrane, resulting in cell death. In addition, P2X7R is the most inflammatory response-related receptor in the P2X family, and is thought to be involved in the development of various diseases such as pain, neurodegeneration and inflammatory diseases, including crohn's disease, gout, rheumatoid arthritis and osteoarthritis, by activating NLRP3 inflammasome activation and promoting IL-1 β release. Therefore, antagonism of P2X7R can effectively prevent and treat occurrence and development of various diseases, and has important clinical application value;

the P2X7 receptor can be blocked by specific biological agents, including small molecule compounds, monoclonal antibodies, nanobodies, and the like. Traditional P2X7R specific antagonists, such as Brilliant Blue G, stimulate some intracellular signaling enzymes, and have lethal toxicity which is not suitable for human body research; the small molecule compound A438079 can affect the pannexin-1 channel and inhibit the output of intracellular ATP; in addition, many inhibitors affect other P2 receptors besides inhibiting P2X7R, and have poor specificity, so that the screening of small-molecule inhibitors with high specificity and low side effects is significant.

Disclosure of Invention

The invention provides application of a small molecule inhibitor in preparation of a targeted P2X7 receptor, and particularly relates to a small molecule inhibitor which is high in specificity, good in safety, free of any toxicity and low in side effect by predicting P2X7R protein structure modeling and allosteric sites and screening the small molecule allosteric inhibitor, and is a high-efficiency allosteric inhibitor aiming at an ATP receptor P2X7R, and an ATP-purine receptor P2X ligand-gated ion channel 7(P2X7R) signal channel is blocked by an allosteric effect, so that activation of NLRP3 inflammasome is inhibited.

The first purpose of the invention is to provide the use of a small molecule inhibitor in the preparation of a targeting P2X7 receptor, wherein the molecular structural formula of the small molecule inhibitor is as follows (as shown in figure 8):

further, the screening process of the small molecule inhibitor comprises the following steps:

the first step is as follows: prediction of P2X7R protein structural modeling and allosteric site: firstly, modeling the protein structure of P2X7R, then predicting an allosteric site, and finally scoring the allosteric site to determine a candidate site of the P2X7R protein allosteric inhibitor;

the second step is that: screening of small molecule allosteric inhibitors: firstly, downloading a crystal compound 5U1X of P2X7R protein, and adopting hydrogenation, dehydration and protein structure optimization in the Glide protein pretreatment process; screening a small molecule library, removing molecules containing a PAINS structure, preprocessing and conformationally generating the small molecules, docking by using Schordinger software to obtain docking conformations, scoring and screening the small molecules, selecting the small molecules which are scored at the top 2000, screening the small molecules ranked at the top 30, analyzing the binding mode of the small molecules, screening the small molecules by means of highest-precision docking and analyzing the binding mode of the small molecules, and finally obtaining the small molecule inhibitor for docking.

Further, in the first step, the protein structure of P2X7R was modeled using the protein modeling program I-tessar.

Further, in the first step, the allosteric site of the protein was predicted using the protein allosteric site prediction program allositeproro software.

Further, in the first step, the protein allosteric site prediction algorithm is a prediction program based on feature extraction and machine learning algorithms of all known allosteric sites proved by crystal experiments.

Further, in the first step, the three allosteric sites of P2X7R protein were scored using the AlloSite program.

Further, in the first step, the three allosteric sites of P2X7R protein were scored by the AlloSite program based on Pocket Volume, AlloSite Score and pertubation Score;

wherein, when the AlloSite program shows that the Pocket Volume is greater than or equal to 659.833, the AlloSite Score is greater than or equal to 0.630 and the pertubation Score is greater than or equal to 0.110, the site can be used as a candidate site of the P2X7R protein allosteric inhibitor.

Further, in the second step, the screening of small molecule libraries is performed using virtual screening provided by the ceramic chemistry.

Further, in the second step, the small molecule was pre-treated and conformationally generated using the ligaprep module (Epik mode) in schrodinger.

Furthermore, the small molecule inhibitor is used for preparing a medicine for treating gouty arthritis.

Compared with the prior art, the invention has the following beneficial effects:

1. the P2X7 receptor small-molecule inhibitor obtained by screening has high specificity, good safety, no toxicity and low side effect, is a high-efficiency ATP receptor P2X 7R-directed inhibitor, blocks an ATP-purine receptor P2X ligand-gated ion channel 7(P2X7R) signal channel, and further inhibits NLRP3 inflammatory corpuscle activation.

2. The small molecule inhibitor of the P2X7 receptor obtained by screening can be applied to the prevention and treatment of gouty arthritis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a three-dimensional modeling structural diagram of the P2X7R protein in the invention;

FIG. 2 is a diagram of the predicted allosteric site map of the P2X7R protein of the invention;

FIG. 3 is a conformational diagram of a small molecule inhibitor of the present invention and P2X7R protein;

FIG. 4 is a diagram of the binding pattern of the small molecule inhibitor and the docking structure of P2X7R protein in the present invention;

FIG. 5 is a graph showing the effect of small molecule inhibitors on the channel function of HEK-293T cells transfected with wild-type human P2X7R by ATP induction;

FIG. 6 is a graph showing the effect of different doses of small molecule inhibitors on BzATP-induced inflammation of THP-1-derived macrophages;

FIG. 7 is a graph showing the effect of the small molecule inhibitor of the present invention on ATP-induced inflammatory response of mouse bone marrow macrophages.

FIG. 8 is a structural formula of the small molecule inhibitor of the present invention.

FIG. 9 shows the effect of small molecule inhibitors of the invention on the inflammatory response of PBMC-derived macrophages in patients with gout.

FIG. 10 is an apparent representation of the effect of different treatment groups on the right ankle and the right dorsum of the foot of a rat in the invention;

wherein, the figure A is the appearance of the effect of injecting PBS on the right ankle joint and the right dorsum of the foot of the rat;

FIG. B is an apparent representation of the effect of MSU injection on the right ankle and the right dorsum of the foot in rats;

FIG. C is an appearance of the effect of MSU + ATP injection on the right ankle and the right dorsum of the foot of a rat;

panel D is an appearance of the effect of Z1456467176+ MSU injection on the right ankle and right instep of rats.

FIG. 11 is a graph of the effect of small molecule inhibitors of the invention on anti-inflammatory effects in a gout rat model;

wherein Panel A shows the ankle circumference of a rat;

panel B shows the ankle swelling index in rats.

FIG. 12 is a graph showing the effect of different treatment groups on IL-1. beta. in rat serum in the present invention.

FIG. 13 is an optical microscope image of articular tissue sections under different treatment groups;

wherein, panel a is an optical microscope image of a joint tissue section injected with PBS;

figure B is an optical microscope image of a MSU injected articular tissue section;

panel C is an optical microscope image of an ATP + MSU injected joint tissue section;

panel D is an optical microscopy image of a joint tissue section injected with Z1456467176+ MSU.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.

Example 1

A method for screening a small molecule inhibitor targeting a P2X7 receptor, comprising the following steps:

step one, P2X7R protein structure modeling and allosteric site prediction

The P2X7R protein has 595 amino acids in sequence and no known crystal structure. The inventors first modeled the protein structure of P2X7R using the protein modeling program I-tessar (shown in figure 1);

then, using a protein allosteric site prediction program AlloSitePro software to predict the allosteric site of the protein; the protein allosteric site prediction algorithm is a prediction program based on feature extraction and machine learning algorithms of all known allosteric sites proved by crystal experiments at present, and can dynamically process a protein structure and identify the most possible allosteric sites (as shown in fig. 2). The three allosteric sites predicted using this program were designated I, II, and III, respectively.

Finally, the three allosteric sites of the P2X7R protein were scored using the AlloSite program. The AlloSite program evaluates allosteric sites primarily by their Pocket Volume, AlloSite Score, and Perturbation Score. When the AlloSite program shows that the Pocket Volume is greater than or equal to 659.833, the AlloSite Score is greater than or equal to 0.630 and the Perturbation Score is greater than or equal to 0.110, the site can be used as a candidate site of the P2X7R protein allosteric inhibitor.

The results show that the predicted allosteric sites I and II have enough size, higher allosteric probability and disturbance intensity (as shown in Table 1), so that the two sites can be used as candidate sites of the P2X7R protein allosteric inhibitor.

TABLE 1 predicted allosteric sites and their attributes

Allosteric site	Pocke Volume	AlloSite Score	Perturbation Score
				I	2735.119	0.802	1.000
II	1978.654	0.830	0.887
				III	659.833	0.630	0.110

And step one, the action target is found for high-flux screening of the small-molecule allosteric inhibitor in the next step.

Step two, primarily screening the targeted P2X7R small molecule allosteric inhibitor

Virtual screening was performed using the Glide module of the commercial software Schordinger software. Firstly, we downloaded P2X7R crystal 5U1X from PBD database, and protein treatment was optimized by hydrogenation- > dehydration- > protein structure in Glide protein pretreatment procedure. The virtual screening of small molecules adopts a virtual screening small molecule library (the number is 200 ten thousand +) provided by ceramic chemistry, then the library is handed over to Shanghai Dai Biotechnology Limited to operate, molecules containing PAINS structures are removed, and finally the number of molecules subjected to butt joint calculation is 180 ten thousand +. We then used the ligaprep module (Epik mode) in schrodinger for small molecule pretreatment and conformation generation. The lattice file was selected centered at F108, and the 18 angstrom residues around it were selected as docking pockets. Docking with Glide to obtain a docked conformation, which is: 9.50271 in its docked conformation (FIG. 3), which was analyzed for the binding pattern of small molecule inhibitors to P2X7R protein (FIG. 4), suggesting that hydrogen bonding between small molecule inhibitors and VAL, ASP, LYS was formed.

The screening was continued according to the above method, and the first 2000 small molecules were screened. Then, the 2000 small molecules are screened out by XP precision (highest precision docking) to obtain the first 30 small molecules, the binding mode of the small molecules is analyzed, and finally, a docking-enabled small molecule file is obtained, wherein the number of the small molecule inhibitor is Z1456467176, and the structural formula of the small molecule inhibitor is shown in FIG. 8.

The molecular composition of the small molecule inhibitor is as follows:

Cl.CC(CC＝1C＝CC＝C(C1)C(F)(F)F)C(＝O)NC＝2C＝CC＝C(C2)S(＝O)(＝O)NCCN。

the specific results of the analysis in conjunction with fig. 4 are recorded in tables 2-5.

TABLE 2 hydrophobic interactions

Index	Residue	AA	Distance	Ligand Atom	Protein Atom
						1	88A	PHE	3.35	4788	798
2	95A	PHE	3.85	4799	895
						3	103A	PHE	3.69	4798	1019
4	108A	PHE	3.98	4789	1103
						5	295A	TYR	3.93	4795	3832
6	310A	ILE	3.82	4790	4090

TABLE 3 Hydrogen bonding interactions

TABLE 4 Pi-Stacking

TABLE 5 π -C actions interactions

Example 2

First, the effect of small molecule inhibitors targeting P2X7 receptors on channel function of HEK-293T cells transfected with wild-type P2X7R induced by ATP was investigated.

The method specifically comprises the following steps:

(1) construction of a lentiviral vector overexpressing human P27 XR:

both h-P2RX7 (wild type) and empty viral-type overexpressing lentiviral vectors were synthesized directly by Henan biosciences, Inc., Shanghai, Japan. The main process is as follows: selecting a lentiviral vector, and designing a target fragment PCR primer, wherein the target fragment PCR primer comprises an upstream primer LV-h-P2RX7-E/B-F, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer LV-h-P2RX7-E/B-R is shown as SEQ ID NO. 2; selecting restriction enzymes EcoRI and BamHI to carry out enzyme digestion on the vector, and recycling agarose gel to obtain a purified linearized vector; carrying out PCR of a target fragment according to the designed primer, and recovering agarose gel to obtain the target fragment with the correct size; connecting the linearized vector and the target fragment according to a homologous recombination or T4 connection method; transforming competence DH5a or stbl3, plating with bacterial liquid, and culturing for 12-16 h; selecting a monoclonal for proceeding colony verification; selecting positive clones with correct colony verification for sequencing; carrying out plasmid extraction on a clone sample with correct sequencing;

LV-h-P2RX7-E/B-F(SEQ ID NO.1)：

TAGAGGATCTATTTCCGGTGAATTCGCCACCATGCCGGCCTGCTGCAGC

LV-h-P2RX7-E/B-R(SEQ ID NO.2)

TCACTTAAGCTTGGTACCGAGGATCCGTAAGGACTCTTGAAGCCACTGT

(2) establishing HEK-293T cells expressing wild-type P2X 7R:

culturing HEK-293T cells with good growth in a carbon dioxide incubator at 37 deg.C for 24 hr (2 x 10)⁵Cells/well).

The respective transfected wild-type P2X7R lentiviruses with MOI values of 10 were cultured in a carbon dioxide incubator at 37 ℃ for 24 hours. HEK-293T cells were harvested 24 hours after transfection by adding small molecule inhibitors (10, 50. mu.M) for 30min, control wells were incubated with an equal volume of DMSO for 30 min.

The cells from each well were aspirated, washed once, and resuspended in 0.5ml of HEPES buffered medium containing KCl at 37 ℃ and collected with a flow tube;

(3) ethidium bromide was added, and HEK-293T was tested for ethidium bromide uptake before and after the stimulant:

ethidium bromide (25. mu.M) was added to each tube, and cells were collected using a CytoFlex flow cytometer at 1000 cells per second, once every 5 seconds of sample, for 40 seconds, after which ATP (1.0mM) was added. 1000 cells per second were collected, once every sample for 5 seconds for a total of 5 minutes (300 seconds);

(4) inhibition of ATP-activated channel function by small molecule inhibitors:

the sample data was analyzed using CytExpert software, and the mean fluorescence intensity value for each sample was read and plotted versus time. HEK-293T was compared to changes in ethidium bromide uptake after ATP addition.

As shown in FIG. 5, the uptake of ethidium bromide by cells was used as a method for detecting the strength of the opening function of P2X7R pore channel induced by ATP. After addition of a small molecule inhibitor (Z1456467176), 293T cells transfected with hP2X7R showed dose-dependent effects with reduced uptake of ethidium bromide induced by ATP.

In fig. 5, Control: uptake of ethidium bromide induced by ATP by 293T cells transfected with hP2X 7R;

z1456467176 (10. mu.M) 293T cells transfected with hP2X7R were incubated with inhibitor (10. mu.M) and ATP-induced uptake of ethidium bromide

Z1456467176 (50. mu.M) 293T cells transfected with hP2X7R were incubated with inhibitors (50. mu.M) and ATP-induced uptake of ethidium bromide.

The experimental method comprises the following steps: flow cytometry

The abscissa: time of ingestion

Ordinate: mean fluorescence intensity of ethidium bromide.

Secondly, the influence of the small molecule inhibitor on the generation of inflammatory reaction of THP-1 derived macrophage induced by BzATP is discussed.

The method comprises the following specific steps:

(1) establishing a human macrophage model with a THP-1 source:

THP-1 cells were plated at 1X 10⁵Density per well was seeded in 24-well plates. Stimulating THP-1 cells with 100ng/mL propylene glycol methyl ether acetate (phorbol-12-myrisitate-13-acetate, PMA) overnight, withdrawing the solution after THP-1 is attached to differentiate into macrophage, washing with PBS once, stimulating with 50ng/mL Lipopolysaccharide (LPS) for 3 hours, withdrawing the solution, washing with PBS once, and carrying out subsequent experiments;

(2) after addition of small molecule inhibitor and BzATP (100. mu.M), expression of IL-1. beta. in THP-1 cell culture supernatant was detected by ELISA:

small molecule inhibitors (1, 10, 50, 100. mu.M) were added for 30min of incubation, control wells were added with an equal volume of DMSO for 30min of incubation, and 100. mu.M BzATP was added for 30min of incubation. Culture supernatants were collected, centrifuged at 400g for 5 minutes, reagents required for ELISA were equilibrated at room temperature, diluted proportionally, and 100. mu.L of diluted cell supernatants were added to each well of the sample. An IL-1. beta. standard curve was prepared. The IL-1 beta standard solution is diluted for 6 times in sequence according to the proportion of 1:1, and the IL-1 beta standard solution needs to be fully mixed each time. The concentrations were 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91pg/mL in this order, and the dilutions were used as the base points of the calibration curve. Plates were sealed with a sealing plate membrane and incubated at room temperature for 2 hours. The plate was washed 4 times with 200. mu.L of 1 Xwash solution, left for 4 minutes each time, and then patted dry with absorbent paper. 200 μ L of horseradish peroxidase-labeled streptavidin was added to each well, membrane-sealed with a fresh plate, membrane-sealed with a plate seal, and incubated at room temperature for 2 hours. And repeating the plate washing steps. To each well was added 200. mu.L of chromogenic substrate TMB and incubated for 30min at room temperature in the dark. The reaction was stopped by adding 50. mu.L of stop solution to each well. The absorbance values at 450nm and 570nm (reference wavelength) were measured with a multifunctional microplate reader, respectively. Each group of OD values is obtained by subtracting the absorbance value of 570nm from the absorbance value of 450nm and then subtracting the OD value in the blank control group, and the numerical value of each group is calculated through a standard curve;

results figure 6 shows that small molecule inhibitors (Z1456467176) can dose-dependently inhibit BzATP-induced macrophage IL-1 β secretion in THP-1 cells.

Secondly, the influence of the small molecule inhibitor on ATP-induced mouse bone marrow macrophage inflammatory response is discussed.

The method comprises the following specific steps:

establishing a mouse bone marrow macrophage model:

② C57BL/6 mouse cervical dislocation and disinfection by soaking in 75% alcohol for 10 min.

And thirdly, separating the thighbone and the shinbone on the two sides of the mouse under the aseptic condition, removing the attached tissues such as the muscle and the like, and soaking the tissues in 75 percent of alcohol.

Cutting off two ends of femur and tibia, washing marrow cavity with sterile PBS, washing marrow cells to 15ml centrifugal tube, centrifuging for 5 min at 400g, discarding supernatant, adding 1ml erythrocyte lysate, re-suspending, standing for 5 min, adding PBS, stopping centrifugation at 400g for 5 min, and discarding supernatant.

Adding DMEM cell culture medium containing 20% L929 supernatant to resuspend cells according to 1x 10⁶Cells/well plated at 1: 100 add double antibody. Changing the liquid after 2-3 days.

Sixthly, on the 7 th day, washing with PBS once, stimulating with Lipopolysaccharide (LPS) of 50ng/mL for 3 hours, removing the solution, washing with PBS once, and carrying out subsequent experiments;

seventhly, adding Z1456467176(1, 10, 50, 100 mu M) to incubate for 30 minutes, adding an equal volume of DMSO to incubate for 30 minutes in a control well, and adding 1mM ATP to incubate for 30 minutes.

Eighthly, collecting culture solution supernatant, centrifuging for 5 minutes at 400g, balancing reagents required by ELISA at room temperature, diluting the reagents in proportion, and adding 100 mu L of diluted cell supernatant of 3 to each sample hole. An IL-1. beta. standard curve was prepared. The IL-1 beta standard solution is diluted for 6 times in sequence according to the proportion of 1:1, and the IL-1 beta standard solution needs to be fully mixed each time. The concentrations were 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91pg/mL in this order, and the dilutions were used as the base points of the calibration curve. Plates were sealed with a sealing plate membrane and incubated at room temperature for 2 hours. The plate was washed 4 times with 200. mu.L of 1 Xwash solution, left for 4 minutes each time, and then patted dry with absorbent paper. 200 μ L of horseradish peroxidase-labeled streptavidin was added to each well, membrane-sealed with a fresh plate, membrane-sealed with a plate seal, and incubated at room temperature for 2 hours. And repeating the plate washing steps. To each well was added 200. mu.L of chromogenic substrate TMB and incubated for 30min at room temperature in the dark. The reaction was stopped by adding 50. mu.L of stop solution to each well. The absorbance values at 450nm and 570nm (reference wavelength) were measured with a multifunctional microplate reader, respectively. Each group of OD values is obtained by subtracting the absorbance value of 570nm from the absorbance value of 450nm and then subtracting the OD value in the blank control group, and the numerical value of each group is calculated through a standard curve;

the results are shown in FIG. 7, and the small molecule inhibitor (Z1456467176) can inhibit ATP-induced macrophage IL-1 β secretion in BMDM cells in a dose-dependent manner.

Thirdly, the influence of the small molecule inhibitor on the inflammatory reaction of PBMC derived macrophages of gout patients is discussed

(1) Establishing a macrophage model derived from PBMC (peripheral blood mononuclear cell) of a gout patient: a. extracting peripheral anticoagulated PBMC (2 ml) of gout patient with 1 × 10 cells⁶Density of/well was inoculated in 12-well plates overnight, withdrawal of fluid, PBS washing once, stimulation with 50ng/mL LPS for 3 hours, withdrawal of fluid, PBS washing once, for subsequent experiments;

(2) expression of IL-1. beta. in PBMC cell culture supernatants was detected by ELISA after addition of small molecule inhibitor allosteric inhibitor and BzATP (100. mu.M): small molecule inhibitors (1, 10, 50, 100. mu.M) were added for 30min of incubation, control wells were added with an equal volume of DMSO for 30min of incubation, and 100. mu.M BzATP was added for 30min of incubation. The subsequent ELISA procedure was as above;

the results are shown in fig. 9, and the small molecule inhibitor (Z1456467176) inhibits BzATP-induced secretion of IL1 β from peripheral blood PBMC culture supernatant of gout patients.

(3) Establishing a rat gout model: a. after the rats were labeled and weighed, they were divided into 4 control groups, MSU (urate), ATP + MSU (adenosine triphosphate + urate), inhibitor + MSU (small molecule inhibitor + urate), and control groups, each group containing 5-7 rats, according to a random principle. ATP + MSU group 500. mu.L of normal saline containing ATP (10mM) is intraperitoneally injected, inhibitor + MSU group (Z1456467176+ MSU) 500. mu.L of normal saline containing small molecule inhibitor (50mg/kg) is intraperitoneally injected, and MSU group and control group are intraperitoneally injected with normal saline 500. mu.L respectively. After half an hour, chloral hydrate (0.3mL/100g) is injected into the abdominal cavity, the state of the rat is observed after 10 minutes, and the initial circumferences of the right ankle joint and the right instep of the rat are measured after the muscle strength of the rat is reduced. By reference to the classic Coderre modeling method: approximately 4mg of urate (MSU) crystals were dissolved in 100 μ L sterile PBS and injected into the right ankle cavity of animals (MSU group, ATP + MSU group, inhibitor + MSU group) via subcutaneous route, and the control group was injected with an equal volume of PBS into the right ankle cavity. After 12 hours, the circumferences of the right ankle joint and the right instep are measured by using a tape measure, and the clinical performance of the right ankle joint of the model mouse is evaluated by using an ankle joint swelling index. Ankle swelling index ═ circumference of ankle after treatment-initial circumference)/initial circumference. b. Blood samples of rats are obtained by a method of taking eyeballs and blood, 1ml of blood of the eyeballs of the rats is taken and kept stand for 30 minutes at room temperature, 500g of blood is centrifuged for 5 minutes, and supernatant is kept for ELISA detection of serum IL-1 beta. c. The right ankle joint of the rat is left and fixed in 10% formalin for 1 day, and then placed in decalcification solution, and after 1-2 weeks of decalcification, the right ankle joint is cut along the sagittal plane of the joint, embedded in paraffin, and rat ankle joint tissue sections are prepared, stained with H & E, and images are collected by an optical microscope (Olympus, Tokyo, Japan).

The results are as follows:

as can be seen in fig. 10, intraperitoneal ATP injection aggravated MSU-induced local joint swelling, and small molecule inhibitors alleviated ATP-induced joint symptoms.

As can be seen from fig. 11, after ATP + MSU injection, the ankle circumference and ankle swelling index were more increased than those of the MSU group, and after small molecule inhibitor + MSU injection, the ankle circumference and ankle swelling index were decreased than those of the MSU group.

As can be seen in FIG. 12, after ATP + MSU injection, IL-1. beta. levels in rat serum were elevated compared to the MSU group, and after small molecule inhibitor + MSU injection, IL-1. beta. levels were reduced.

As can be seen from FIG. 13, the degree of inflammatory cell infiltration of rat ankle tissue sections was increased after ATP + MSU injection compared with that of the MSU group, and decreased after small molecule inhibitor + MSU injection.

It should be noted that when referring to numerical ranges in the present invention, it should be understood that both endpoints of each numerical range and any number between the endpoints can be selected, and in order to prevent redundant description, the preferred embodiments of the present invention have been described, but once the basic inventive concept is known, other changes and modifications can be made to the embodiments by those skilled in the art. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<110> pottery Jinhui Xiaolingling

Application of small molecule inhibitor in preparation of targeting P2X7 receptor

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