GLYR1 target specific small molecule compound and application thereof

Abstract

The invention relates to a specific small molecule compound of a GLYR1 target spot and application thereof, and belongs to the field of biological medicine. The invention provides a small molecule compound C20H32O5, which is specifically combined with 27185-285amino acid motifs of glyoxylate reductase homologue-1 (GLYR1) protein, so that the combining capacity of the GLYR1 and chromosome DNA in a cell nucleus is reduced, and gene transcription is inhibited; meanwhile, the retention of GLYR1 in cytoplasm is prolonged, and the phosphorylation levels of ERK and MAPK are reduced, so that the transcription up-regulation of proinflammatory cytokines in macrophages is inhibited. The compound rapidly inhibits inflammatory storm caused by coronavirus infection, and significantly reduces severe illness and death risks; experiments prove that the survival rate in an SARS-CoV-2 infected mouse model is increased to 100% from 63.5%, the survival rate in an MHV-3 infected model is remarkably increased, and tissue pathological injuries of lungs and spleens are relieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to specific small molecule compounds targeting GLYR1 and their applications. Background Technology

[0002] A cytokine storm (CS) is a systemic inflammatory response syndrome (SIRS) triggered by pathogenic microbial infection, mechanical injury, cancer, autoimmune diseases, etc. When a virus invades the body, the immune system is activated, producing an immune response that releases a series of cytokines. These cytokines then act on the site of infection and the systemic circulatory system to clear the virus and maintain homeostasis. However, excessive activation of the inflammatory response during this process can lead to a cytokine storm, causing inflammatory diseases and, in severe cases, shock and multiple organ dysfunction syndrome (MODS). Therefore, early clinical interventions targeting cytokine storms are crucial for timely treatment and improved prognosis in patients with viral infections. Increasing evidence suggests that a severe cytokine storm is a significant contributing factor to the accelerated progression of disease after viral infection, particularly evident in severe cases of COVID-19.

[0003] COVID-19 is an infectious disease caused by the SARS-CoV-2 virus, affecting the respiratory tract and multiple organs throughout the body, and has infected more than 200 million people worldwide. Most COVID-19 patients experience mild symptoms, but approximately 20% of cases progress to severe illness, including acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), and a cytokine storm. SARS-CoV-2 is a single-stranded positive-sense RNA virus that activates the host's RIG-I-like receptor to produce interferon and inflammatory factors. Clinical studies have shown that the levels of cytokines such as IL-2 and TNF-α in the plasma of severely ill COVID-19 patients are significantly upregulated, indicating the presence of a cytokine storm during the course of the disease. Currently reported cytokines involved in the COVID-19 cytokine storm include: IL-1, IL-6, IL-8, IL-10, IL-18, etc.; the interferon family IFN-γ / -λ; TNF-α; and monocyte chemoattractant protein-1 (MCP-1). These factors can activate downstream kinases such as JAK-STATs and NF-κB pathways, leading to severe illness.

[0004] Besides SARS-CoV-2, the strain-dependent fulminant hepatitis model established by inbred mice infected with Murine hepatitis virus strain-3 (MHV-3) has also become an important animal model for studying the mechanism of "cytokine storm." MHV-3 is a hepatotropic single-stranded RNA virus and also belongs to the coronavirus family. Susceptible mouse strains such as Balb / c, DBA / 1, and C57 show severe lesions throughout the liver 48 hours after viral infection, manifested as thrombus formation in the hepatic sinusoids, capillaries, and venules, with platelets and fibrin as the main components. Furthermore, these infected mice also show a large accumulation of cytokines in their plasma, such as IL-1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1. Therefore, infection of mice with coronavirus MHV-3 can also lead to cytokine storm and cause severe illness or death.

[0005] The glyoxylate reductase-1 homolog gene (GLYR1), also known as cytokine-like nuclear factor N-PAC (NPAC) or nuclear protein NP60 (NP60), is also called 3-hydroxyisobutyrate dehydrogenase-like protein, nucleosome-destabilizing factor (NDF), and putative oxidoreductase GLYR1. GLYR1 proteins are primarily located in the cell nucleus. The protein encoded by GLYR1 contains the PWWP functional motif, which has a conserved aromatic cage for histone methyl lysine recognition and synergistic binding to histones and DNA, contributing to their nucleosome binding ability and chromatin localization. Studies have confirmed that the GLYR1 protein binds to histone H3K36me3, thereby catalyzing the demethylation of histone H3K4 and affecting gene transcription, indicating that GLYR1 plays a crucial role in epigenetic regulation. Research shows that excessive and aberrant expression of the GLYR1 gene can promote tumor growth and spread. Furthermore, GLYR1 can dephosphorylate JAK2 and STAT3, inhibiting the proliferation of hepatocellular carcinoma cells and contributing to cell apoptosis; GLYR1 may also promote the chemosensitivity of colorectal cancer by blocking p38 / PI3K / AKT signaling. With further research into GLYR1, it is hoped that it can be used as a target for intervention and that therapeutic drugs can be developed, opening new avenues for the prevention and treatment of related diseases.

[0006] In recent years, computer-aided drug design has been widely applied to molecular design research targeting specific molecules. This not only significantly shortens the drug development cycle but also offers the advantages of low cost and high efficiency. Computer-aided drug design is divided into two categories: novel drug design and virtual screening. Further, based on different principles, it can be divided into novel drug design / virtual screening based on receptors or ligands. Receptor-based methods mainly utilize the 3D structural information of target protein molecules for novel molecular design or screening, including methods such as molecular docking and molecular dynamics simulations. Molecular docking primarily predicts the structural conformation of a compound on the target and the affinity of the compound based on the ligand-receptor interaction energy. Molecular dynamics simulations study intermolecular interaction modes, interaction energies, and other physicochemical properties through molecular dynamics behavior in solvated systems. Since the GLYR1 protein has multiple functions, this provides a foundation for computer-aided drug design targeting GLYR1. Summary of the Invention

[0007] In view of this, one objective of the present invention is to provide a small molecule compound specifically targeting GLYR1, and another objective is to provide an application of the small molecule compound specifically targeting GLYR1.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a specific small molecule compound targeting GLYR1, wherein the small molecule compound is C 20 H 32 O5;

[0010] Preferably, the structural formula of the small molecule compound is shown below:

[0011] Furthermore, the application of the GLYR1-target-specific small molecule compounds in the preparation of antiviral drugs;

[0012] Preferably, the virus is the novel coronavirus SARS-CoV-2 or the mouse coronavirus strain MHV-3.

[0013] Preferably, the in vitro concentration of the small molecule compound is 5 μM;

[0014] Preferably, the in vivo concentration of the small molecule compound is 10 mg / Kg;

[0015] Preferably, the specific small molecule compound is used in the preparation of a treatment for "cytokine storm" caused by coronavirus infection.

[0016] The beneficial effects of this invention are as follows:

[0017] Using GLYR1 protein as a target, we screened for small molecule compounds C that specifically bind to it. 20 H 32 O5, the aforementioned small molecule compounds specifically and tightly bind to amino acids 271-285 of the GLYR1 protein in macrophages, thereby inhibiting gene transcription and reducing the binding ability of GLYR1 to chromosomal DNA in the cell nucleus; simultaneously, it can downregulate ERK and MAPK phosphorylation. It inhibits the transcriptional upregulation of pro-inflammatory cytokines, C 20 H 32 O5 effectively inhibits coronaviruses (including SARS-CoV-2 and...)

[0018] The transcription of various pro-inflammatory cytokines (such as IL-1β, IL-6, TNF-α, and MCP-1) in macrophages is upregulated in response to MHV-3 infection. These small molecule compounds can rapidly inhibit macrophage-derived inflammation induced by coronavirus infection, thereby reducing severe illness and / or death.

[0019] Confirmed through animal models (SARS-CoV-2 and MHV-3 infected mice), C 20 H 32 O5 treatment significantly improved survival rates (e.g., the survival rate of SARS-CoV-2 infected mice increased from 63.5% to 100%, and the survival rate of MHV-3 infected mice was significantly improved) and reduced histopathological damage (e.g., reduced inflammatory cell infiltration and fibrin deposition in the lungs and spleen).

[0020] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0022] Figure 1 This is a protein structure model of GLYR1 constructed using alphaFold2 software in Example 1;

[0023] Figure 2 This is a list of potential small molecule compounds that may bind to GLYR1 in Example 1;

[0024] Figure 3 Candidate small molecule compound C in Example 220 H 32 Structural diagram of O5;

[0025] Figure 4 Example 2C 20 H 32 Affinity assessment and binding kinetic analysis of O5 with human GLYR1 protein showed that it exhibited a very high binding affinity.

[0026] Figure 5 C in Example 3 20 H 32 O5 significantly improved the survival rate of mice infected with SARS-CoV-2 (M17 strain); A: Histopathological changes in spleen and lungs observed by H&E; B: Statistical analysis of survival rate;

[0027] Figure 6 C in Example 4 20 H 32 O5 significantly improved the survival of MHV-3 infected mice; A: weight change; B: statistical analysis of survival rate;

[0028] Figure 7 C in Example 5 20 H 32 O5 can significantly downregulate the phosphorylation levels of MAPK and ERK induced by LPS stimulation in macrophages. Detailed Implementation

[0029] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0030] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0031] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0032] Example 1: Small molecule lead compounds that can bind to GLYR1 protein were screened using molecular simulation techniques.

[0033] Since there are currently no reports on the structure of human and mouse GLYR1 proteins, this embodiment uses alphaFold 2 to predict the structure of human interfibroblast GLYR1 protein; further, a protein structure model of the GLYR1 catalytic site is constructed.

[0034] The application website for Alphafold2 is: (AlphaFold Protein Structure Database, https: / / alphafold.ebi.ac.uk / entry). See attached file. Figure 1 The characteristics of the human GLYR1 protein structure predicted using Alphafold2 software.

[0035] Based on the catalytic subunit structure of the obtained GLYR1 protein, virtual screening was performed using molecular docking technology with Schrodinger Glide software. Protein-small molecule binding affinity was evaluated across approximately 3 million small molecules from compound libraries such as Specs and ChemDiv, and drug-like structures were screened based on the five rules of drug development and the PAINS rule. Analysis of structural diversity and binding modes was conducted. The inventors constructed methods based on surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), enzyme activity experiments, and crystallography to clarify the binding activity of the virtually screened small molecules with GLYR1. Based on the small molecule binding affinity and structure-activity relationship data indicated by molecular docking, medicinal chemistry methods were used to modify the small molecules, discovering novel, highly selective, and specific GLYR1 lead compounds, providing new chemical entities for subsequent functional studies and drug development of GLYR1.

[0036] Thirty-seven candidate molecules were identified through screening from a small molecule compound library (see appendix). Figure 2 Appendix Figure 2 The candidate molecules obtained may not bind to GLYR1 in real-world scenarios, or even if they do bind, their interference effect may be weak or nonexistent. Therefore, it is necessary to further concentrate the number of candidate small molecule compounds and verify their interference effects.

[0037] Example 2 Small molecule lead compound C 20 H 32 Affinity assay of O5 binding to GLYR1 protein

[0038] To screen for small molecule compounds with strong affinity for recombinant GLYR1 protein, we used biointerference technology (BLI) to evaluate the affinity and perform binding kinetic analysis on the screened candidate small molecules. The specific steps are as follows:

[0039] (1) Use PBST buffer to prepare a 500uM stock solution of small molecule compounds screened by flow cytometry, ensuring that the DMSO concentration is 0.5%, and then use PBST + 0.5% DMSO buffer to dilute the samples.

[0040] (2) The concentrations of small molecule compounds were set to 500, 250, 125, 62.5, 31.25, and 15.625 μM, and placed on ice for later use;

[0041] (3) Prepare biotinylated GLYR1 protein sample: Take 100 μL of mouse-derived recombinant GLYR1 protein solution into a centrifuge tube, add 0.3 μL of biotinylation reagent, mix well and react at room temperature for 30-60 min;

[0042] (4) Pretreatment of the desalting column: fix the gravity column vertically, remove the protective cap, equilibrate the desalting column with at least 10 ml of PBS, and discard the liquid flow-through.

[0043] (5) Add 100 μL of the biotinylated target solution to the desalting column. After the sample has completely entered the desalting column, add 0.4 mL of PBS for flow through without collecting. Add 0.4 mL of PBS and collect the eluent. This eluent is the solution of biotinylated recombinant GLYR1 protein.

[0044] (6) Using the Sartorius Octet RED96e molecular interaction analysis system, select the SSA sensor and pre-wet in PBS for 10 min.

[0045] (7) The diluted small molecule compound and the biotinylated GLYR1 protein were injected into a 96-well flat-bottom black plate, with 200 μL in each well;

[0046] (8) Set up two sets of SSA sensors. The first set is a protein solidification detection sensor, and the second set is a blank control sensor. Both sets of SSA sensors use the same batch, and the running steps and running time of each step are the same.

[0047] (9) Monitor the binding process of GLYR1 protein to small molecule compounds in real time and record the binding curves;

[0048] (10) Based on the binding and dissociation curve data, use the corresponding software to analyze the binding rate constant (Ka), dissociation rate constant (Kd), affinity constant (KD), and steady-state fitting curve shape, and screen out candidate small molecule compounds with good performance;

[0049] (11) Small molecule compound C 20 H 32 The concentrations of O5 were set to 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μM, and placed on ice for later use.

[0050] (12) Repeat the experiment to ensure the reliability and reproducibility of the data, and record all experimental conditions and results.

[0051] Experimental results show that, after screening, candidate small molecule compound C 20 H 32 O5 has received significant attention, and its chemical structure is as follows: Figure 3As shown. Analysis using the Sartorius Octet RED96e molecular interaction analysis system revealed C... 20 H 32 O5 exhibited a very high binding affinity to human recombinant GLYR1 protein. Figure 4 Specifically, the compound exhibited a low dissociation constant (KD) in binding experiments with recombinant human GLYR1 protein, indicating its strong binding ability; furthermore, statistical analysis of the binding experiment results showed that the candidate small molecule compound C... 20 H 32 The binding capacity of O5 at different concentrations showed a good linear relationship, indicating its potential for application in organisms.

[0052] Example 3 Small molecule lead compound C 20 H 32 Inhibitory effect of O5 on death caused by novel coronavirus infection

[0053] To study C 20 H 32 Can O5 influence downstream cytokine storms and thus improve disease progression through GLYR1 activity? We used the recent COVID-19 outbreak as a case study, analyzing C in vivo... 20 H 32 The regulatory role of O5 in severe illness or death caused by novel coronavirus infection. The specific implementation steps in this case are as follows:

[0054] (1) The novel coronavirus mouse strain MA17 was obtained from the P3 laboratory of Guangzhou Medical University; C57BL / 6 mice were bred in this laboratory.

[0055] (2) Animal grouping and infection model preparation: To simulate the severe phenotype caused by novel coronavirus infection in the elderly, we cultured C57BL / 6 mice in IVC for 8 months to establish an aged mouse model. The mice were divided into 3 groups: Group 1, uninfected; Group 2: 500 PFU MA17 infection + 1% DMSO; Group 3: 500 PFU MA17 infection + C 20 H 32 O5 (10 mg / kg). Ten mice were used in each group, and the experiment was repeated three times. The infection experiment was conducted in the P3 laboratory of Guangzhou Medical University.

[0056] (3) Record the body weight and survival status of mice from D0 infection for 15 consecutive days.

[0057] (4) Fixation, gradient dehydration, and preparation of paraffin sections of mouse lung, spleen, and kidney tissues: Lung, spleen, and kidney tissues were collected from infected mice on day 4 and fixed in 4% paraformaldehyde for 24 hours. Gradual alcohol dehydration was then performed: 50% alcohol, 20 min; 75% alcohol, 20 min; 80% alcohol, 20 min; 95% alcohol, 20 min; 100% anhydrous ethanol (No. 1), 20 min; 100% anhydrous ethanol (No. 2), 20 min. Xylene clearing: Xylene (No. 1), 5 min; Xylene (No. 2), 5 min. The tissues were then completely immersed in 60℃ liquid paraffin (No. 1) for 2 hours; subsequently, the tissues were placed in fresh liquid paraffin (No. 2) and left overnight. Embedding was performed at room temperature; sections were prepared using a microtome to a thickness of approximately 3–5 μm.

[0058] (5) Mouse lungs, kidneys, and spleen were H&E stained paraffin sections and baked overnight in a 60℃ oven. Dewaxing was performed with xylene: xylene 1, 10 min; xylene 2, 10 min; xylene 3, 10 min. Gradient alcohol rehydration was performed: 100% anhydrous ethanol 1, 5 min; 100% anhydrous ethanol 2, 5 min; 95% ethanol, 5 min; 75% ethanol, 5 min; 50% ethanol, 5 min; ddH2O, 5 min. Hematoxylin staining solution was added to the slide for 1 min, and the staining was stopped by rinsing with running water. 0.4% ammonia solution was used to reverse blue for a few seconds until the cell nuclei turned blue, and the staining was stopped by tap water. Eosin staining solution was added to the slide to stain the cytoplasm for 1 min, and the staining was stopped by rinsing with running water. Gradient dehydration was performed, and the slides were mounted with neutral resin for observation.

[0059] (6) Staining of mouse lung, kidney, and spleen fibrosis paraffin sections: Following the routine steps in 1.4, dewax the sections to ddH2O and stain using the Solarbio modified Masson trichrome staining kit according to the kit instructions. A brief description is as follows: Azurite blue and hematoxylin were used to lightly stain cell nuclei; aniline blue was used to stain collagen fibers to make them blue; and ponceau red was used to stain muscle fibers, cytoplasm, cellulose, keratin, and erythrocytes to make them red. The fibrosis status in mouse tissues was determined by observing the blue collagen fibers.

[0060] (7) Immunohistochemistry, H&E staining, and Masson staining results were analyzed using an M8 scanner. Representative images of the average level were selected for presentation. Prism software was used to statistically analyze the survival rate of infected mice and plot the body weight change curve.

[0061] Aged mice were infected with SARS-CoV-2 M17 (500 PFU). Results were as follows: Figure 5 As shown in Figure A, on day 5 of infection, we collected lungs and spleens from infected mice. H&E staining revealed severe lung and spleen damage and inflammatory cell infiltration in mice treated with 1% DMSO, along with significant fibrin (blue) deposition. Meanwhile, C... 20 H32 The O5 (10 mg / Kg) treatment group showed a significant reduction ( Figure 5 A). For example Figure 5 As shown in B, statistical analysis indicates that during the 17-day continuous observation period, the mortality rate of mice treated with 1% DMSO was 63.5%, while in C... 20 H 32 Mice treated with O5 had 100% survival (no deaths), and statistical analysis showed that C 20 H 32 O5 significantly improved the survival rate of mice compared to 1% DMSO treatment. This indicates that C... 20 H 32 O5 can counteract death caused by novel coronavirus infection.

[0062] Example 4 Small molecule lead compound C 20 H 32 The effect of O5 on the improvement of pneumonia caused by MHV-3 virus infection

[0063] Influenza virus infection can also trigger a "cytokine storm," leading to severe illness or death in patients. For research on C... 20 H 32 Can O5 influence the cytokine storm induced by influenza virus and thus improve disease progression through GLYR1 activity? We investigated this using mouse coronavirus MHV-3 strain infection as the research subject, and analyzed C in vivo. 20 H 32 The regulatory role of O5 in severe illness or death caused by viral infection. The specific implementation steps in this case are as follows:

[0064] (1) Sixty male SPF-grade C57BL / 6 mice, >8 weeks old, were used as experimental animals. During the experiment, the mice were fed complete pelleted food, had free access to water, and were exposed to light and darkness for 12 hours each, with good ventilation. The animal experiments followed the 3R principle.

[0065] (2) Grouping and Modeling Experiment: The experiment was divided into 3 groups: 1: normal control group; 2: MHV-3 (100 PFU) infection + 1% DMSO; 3: MHV-3 (100 PFU) infection + C 20 H 32 O5 (10 mg / kg) treatment group. Observed continuously for 15 days.

[0066] (3) Observation indicators: (a) Clinical observation: Observe the activity status, back fur, respiration, spontaneous activity, etc. of animals in each group. (b) Viral load: Crystal violet staining method. (c) Histopathological examination: On day 7, the left lung tissue and liver of animals in each close contact transmission group were collected, fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes and scored.

[0067] (4) Statistical analysis The experimental data were analyzed using SPSS 22.0 statistical software. The mean ± standard deviation (x ± s) was used. The t-test was used to compare data between two groups. The homogeneity of variance and normality tests were performed to compare data among multiple groups. p < 0.05 was used to indicate that there was a significant difference.

[0068] During the experiment, mice in the normal control group exhibited good activity levels, glossy fur on their backs, and stable respiratory rates without significant abnormalities. In the virus-infected group, piloerection, rapid breathing, and reduced spontaneous activity were observed; as the modeling period lengthened, some animals became lethargic. Figure 6 As shown in A, viral infection + C 20 H 32 The weight loss in mice in the O5 (10 mg / kg) group was not significant compared to that in mice treated with 1% DMSO. Furthermore, viral infection +C 20 H 32 The survival rate of mice in the O5 group was significantly higher than that of mice treated with 1% DMSO. Figure 6 B) indicates that C 20 H 32 O5 can suppress severe illness or death caused by MHV-3 virus.

[0069] Example 5 Compound C 20 H 32 Inhibitory effect and mechanism of O5 on LPS-induced pro-inflammatory cytokines in macrophages

[0070] The implementation steps of this embodiment are as follows:

[0071] (1) Cell culture: THP-1 cells were cultured in RPMI 1640 medium containing 10% FBS at 37°C and 5% CO2 saturated humidity for 2-3 days. The medium was changed and passaged at a ratio of 1:2. The third generation cells were used for subsequent experiments.

[0072] (2) LPS stimulation was used to obtain logarithmic growth phase THP-1 cells and seeded into 6-well plates at 5×10⁶ cells / wells. 5 Cells were incubated at 37°C for 24 h with 0 and 0.2 μg / mL LPS respectively. The mRNA transcription levels of TNF-α, IL-1β, and IL-6 genes in cells were detected by qRT-PCR. Total RNA was extracted from cells using Trizo reagent, and cDNA was synthesized by reverse transcription using a reverse transcription kit. cDNA was then used as a template for PCR amplification. PCR amplification conditions were: 95°C for 10 s; 95°C for 5 s, 64°C for 10 s, 72°C for 10 s, for a total of 40 cycles; 72°C for 5 min. Each PCR reaction was performed in triplicate, with GAPDH as an internal control. -ΔΔCt The transcriptional level of each gene's mRNA was calculated using this method.

[0073] (3) Whole RNA sequencing: Using transcriptome sequencing to analyze LPS+C 20 H 32 Bulk RNA sequencing was performed on THP-1 cells treated with O5 (10 μM) or LPS + 0.1% DMSO. Whole-transcriptional library construction, subsequent experiments, and preliminary single-cell sequencing upstream analysis were all performed by Bio-EasyCare Pharmaceutical Technology Co., Ltd. Differentially expressed genes were extracted, volcano plots were generated using R studio, and preliminary GO and KEGG analyses were conducted.

[0074] (4) Western blot analysis of protein phosphorylation level: THP-1 cells were stimulated with LPS and C20H32O5 (10 μM) or 0.1% DMSO were added. After 10 or 30 min, the cell supernatant was discarded, and the cells were rinsed twice with 1 ml / well PBS. The cells were then treated with cell lysis buffer and lysed on a shaker at 4°C for 15 min. The cells were then centrifuged at 12000 rpm at 4°C for 15 min and the supernatant was collected. First, protein concentration was determined using the Bradford method, followed by SDS-PAGE electrophoresis and wet transfer onto a 0.25 μm PVDF membrane. The PVDF membrane was then blocked in 5% skim milk powder for 1 h at room temperature, followed by TBST washing for 3 min / time, and then washed 3 times. Next, the membrane was incubated overnight at 4 °C in GAPDH (1:10000), p65, ERK, p38MAPK, JNK, and various phosphorylated antibodies (1:2000) dilution buffer, followed by TBST washing for 3 times, 5 min / time. Horseradish peroxidase (HRP)-conjugated secondary antibody was then incubated at room temperature for 1 h, followed by TBST washing for 3 times, 5 min / time. Finally, the membrane was imaged using Thermo chemiluminescence reagent in a Bio-rad chemiluminescence detector.

[0075] like Figure 7 As shown in Figure A, stimulation of THP-1 cells with 0.2 μg / mL LPS significantly increased the mRNA transcription levels of various pro-inflammatory cytokine genes. However, the cells showed improved mRNA transcription levels after the addition of C... 20 H 32 Cells were cultured at O5 (5 μM) for an extended period. Bulk-RNA sequencing confirmed that, regardless of the polarization conditions in M1 and M2, this treatment significantly downregulated the mRNA transcription levels of multiple pro-inflammatory cytokine genes. Western blot analysis showed that cells stimulated with 0.2 μg / mL LPS for 5 min were significantly reduced by the addition of C... 20 H 32 O5 can significantly inhibit the phosphorylation levels of p-ERK, p-p38, p-JAK, and p-p65. Figure 7 B). These results indicate that C 20 H 32O5 can downregulate the production of macrophage inflammatory factors by inhibiting the phosphorylation level of kinases.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A small molecule compound specifically targeting GLYR1, characterized by: The small molecule compound is C. 20 H 32 O5.

2. The GLYR1-target-specific small molecule compound according to claim 1, characterized in that: The structural formula of the small molecule compound is shown below:

3. The application of the GLYR1-target-specific small molecule compound according to claim 2 in the preparation of antiviral drugs.

4. The application according to claim 3, characterized in that, The virus in question is either the novel coronavirus SARS-CoV-2 or the mouse coronavirus strain MHV-3.

5. The application according to claim 3 or 4, characterized in that: The in vitro concentration of the small molecule compound is 5 μM.

6. The application according to claim 3 or 4, characterized in that: The in vivo concentration of the small molecule compound is 10 mg / kg.

7. The use of the specific small molecule compound of claim 1 or 2 in the preparation of a treatment for "cytokine storm" caused by coronavirus infection.

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