Inhibitors of ace2 expression and their use in ace2-mediated diseases

Abstract

This invention provides ACE2 expression inhibitors and their application in ACE2-mediated diseases. It uses human RXRα as a target protein for virtual drug screening and employs computer molecular docking technology to screen compounds with high affinity for the human RXRα receptor. Combined with cell experiments, it screens compounds that inhibit ACE2. The ACE2 expression inhibitors are at least one of ten drugs selected from ursolic acid, phlorizin, resveratrol, triamcinolone, budesonide, fluticasone propionate, vitamin D2, vitamin D3, calcitriol, and alfacalcidol. The ACE2 inhibitors screened by this invention can serve as targeted drugs for ACE2-mediated diseases, providing more drug options for the prevention or treatment of these diseases.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, specifically to ACE2 expression inhibitors and their application in ACE2-mediated diseases. Background Technology

[0002] ACE2 (angiotensin-converting enzyme 2) is a key receptor for the novel coronavirus (also known as "SARS-CoV-2 virus" or "COVID-19") to infect the host. SARS-CoV-2 infection and entry into the host depend on the binding of its spike S protein to the ACE2 receptor. The ACE2 receptor is widely expressed in organs such as the respiratory system, digestive tract, liver, kidneys, heart, and brain. The expression of the ACE2 receptor in patients is positively correlated with susceptibility, severity, and mortality from SARS-CoV-2. To date, several vaccines and monoclonal antibodies have been developed to neutralize the spike S protein. However, newly emerging mutant strains often mutate in the spike S protein, making them unrecognizable by vaccines and monoclonal antibodies. Therefore, strategies to prevent or block viral entry by downregulating ACE2 in host cells are more advantageous than strategies targeting only the viral spike S protein. As a key receptor for SARS-CoV-2 virus infection of host cells, the regulation of ACE2 expression levels is of great significance for the prevention and treatment of COVID-19 (the disease caused by the SARS-CoV-2 virus is named COVID-19).

[0003] The farnesoid X receptor (FXR) is an important member of the nuclear receptor family, and its functions involve multiple aspects such as bile acid metabolism, lipid and carbohydrate metabolism, and inflammation regulation. Previous studies (non-patent literature 1) have shown that drugs containing "FXR inhibitors" can shut down the ACE2 receptor, effectively blocking viral entry into cells and potentially preventing SARS-CoV-2 infection.

[0004] The retinoic X receptor (RXR) is another important member of the nuclear receptor family, playing a crucial role in multiple physiological processes, including cell growth and differentiation, metabolic regulation, morphogenesis, and embryonic development. As a ligand-regulated transcription factor, RXR can regulate signaling pathways related to metabolism and the cell cycle at the transcriptional level. RXR exists in three isoforms: α, β, and γ. The FXR / RXR heterodimer is a complex formed by the FXRα and RXRα nuclear receptors. The FXR / RXR heterodimer can bind to FXR response elements (FXREs) on target gene promoters, regulating the transcription of numerous downstream genes. However, given the numerous factors influencing the transcriptional metabolism of intracellular substances and the highly complex regulation of FXR, it remains unclear whether there is a necessary link between RXR and the inhibition of ACE2 transcriptional expression, as well as the prevention and / or treatment of SARS-CoV-2 infection.

[0005] Furthermore, the available ACE2 expression inhibitors are very limited.

[0006] Ursolic acid, also known as ursolic acid, is a pentacyclic triterpenoid compound widely found in various plants. It possesses diverse biological activities, including anti-inflammatory, antioxidant, antibacterial, and antitumor effects. The association between ursolic acid and inhibition of ACE2 transcriptional expression, as well as its role in the prevention and / or treatment of SARS-CoV-2 infection, has not yet been reported.

[0007] Phloridzin, the glucoside form of phlorizin, belongs to the dihydrochalcone class of substances and is widely found in nature. Phloridzin possesses various biological activities, such as antioxidant, anti-inflammatory, and anti-diabetic effects. The association between phloridzin and inhibition of ACE2 transcriptional expression, as well as its role in the prevention and / or treatment of SARS-CoV-2 infection, has not yet been reported.

[0008] Polydatin, also known as resveratrol glycoside, is extracted from the dried rhizome and root of Polygonum cuspidatum. Polydatin possesses various medicinal effects, including dispelling wind and dampness, dispersing blood stasis, relieving pain, and relieving cough and phlegm. The association between polydatin and inhibition of ACE2 transcriptional expression, as well as its role in the prevention and / or treatment of SARS-CoV-2 infection, has not yet been reported.

[0009] Triamcinolone, budesonide, and fluticasone propionate are all corticosteroids with significant anti-inflammatory, anti-allergic, and immunosuppressive effects. They effectively alleviate symptoms of various inflammatory diseases by inhibiting the activity of inflammatory cells and the production of inflammatory mediators. The association between triamcinolone, budesonide, and fluticasone propionate and the inhibition of ACE2 transcriptional expression, as well as the prevention and / or treatment of SARS-CoV-2 infection, has not yet been reported.

[0010] Vitamin D2, vitamin D3, calcitriol, and alfacalcidol are all fat-soluble open-ring sterols with broad biological activities in the body and can be used to treat osteoporosis, rickets, etc. The association between vitamin D2, vitamin D3, calcitriol, and alfacalcidol and the inhibition of ACE2 transcriptional expression, as well as the prevention and / or treatment of SARS-CoV-2 infection, has not yet been reported.

[0011] Non-patent literature

[0012] Non-patent literature 1: Nature, 2023, Vol. 615, No. 7950, pp. 134-142;

[0013] Non-patent literature 2: Journal of Ginseng Research, 2019, Vol. 43, No. 3, pp. 442-451;

[0014] Non-patent literature 3: Molecular Nutrition & Food Research, 2020, Vol. 6, No. 12, e2000034;

[0015] Non-patent literature 4: BMC Complementary and Alternative Medicine, 2016, Vol. 16, 514;

[0016] Non-patent literature 5: Biology of Reproduction, 2006, Vol. 74, No. 1, pp. 23-28;

[0017] Non-patent literature 6: Scientific Report, 2017, Vol. 12, No. 7, 40593. Summary of the Invention

[0018] One objective of this invention is to provide a novel ACE2 (angiotensin-converting enzyme 2) expression inhibitor.

[0019] ACE2 expression inhibitors are drugs obtained by screening using a rapid screening method for ACE2 expression inhibitors.

[0020] The rapid screening method for ACE2 expression inhibitors includes the following steps:

[0021] Selecting a ligand database: Selecting structural analogues of compounds that have a regulatory effect on RXR (retinoic acid X receptor) or RXR dimers to form a ligand database;

[0022] Preliminary screening: Obtain the three-dimensional structure of the human RXRα receptor (human retinoic acid X receptor α) protein, and use computer molecular docking technology to simulate docking of the ligands in the ligand database of step (1) with the three-dimensional structure of the human RXRα receptor protein. From the ligand database, ligands with docking binding free energy of less than -5 kcal / mol with the three-dimensional structure of the human RXRα receptor protein are initially screened as candidate drugs.

[0023] Secondary screening: Using HepG2 cells as recipient cells, the candidate drugs obtained in step (2) are co-cultured with the recipient cells. After co-culture, the cells are collected and the mRNA expression level of ACE2 in the cells is detected. Drugs that inhibit the transcriptional expression of ACE2 are screened from the candidate drugs and used as ACE2 expression inhibitors.

[0024] The ACE2 expression inhibitors obtained through the screening in step (3) above are at least one of the following 10 drugs: ursolic acid, phlorizin, polygalactoside, triamcinolone, budesonide, fluticasone propionate, vitamin D2, vitamin D3, calcitriol, and alfacalcidol.

[0025] The inventors are the first to use computer molecular docking technology to screen ACE2 inhibitors. Furthermore, they were the first to attempt to use human RXRα as a target protein for virtual drug screening and, through computer molecular docking, screen compounds with high affinity for the human RXRα receptor. Combined with cell experiments, they screened 10 drugs that inhibit ACE2: ursolic acid, phlorizin, resveratrol, triamcinolone, budesonide, fluticasone propionate, vitamin D2, vitamin D3, calcitriol, and alfacalcidol. No prior art reports have been found regarding the correlation between these 10 drugs and the inhibition of ACE2 transcriptional expression. In other words, the inventors have developed a novel inhibitor capable of inhibiting ACE2 transcriptional expression, achieving unexpected technical effects. Moreover, some of these 10 drugs are structural analogs of RXR agonists, and some are structural analogs of RXR regulators (whether their effect on RXR inhibition or agonism is unclear), but all have shown significant inhibitory effects on ACE2 receptor transcriptional expression. The ACE2 inhibitors screened by this invention can be used as targeted drugs for ACE2-mediated diseases, providing more drug options for the prevention or treatment of ACE2-mediated diseases.

[0026] Furthermore, the structure in step (1) is similar to a small molecule compound with a molecular weight of less than 1000 Da, either natural or synthetic.

[0027] Furthermore, the computer molecular docking software used in the computer molecular docking technology described in step (2) is AutoDock Vina.

[0028] Furthermore, the docking centers selected in the computer molecular docking technology described in step (2) are: center_x = 49.192, center_y = 63.991, center_z = -7.522, the docking box sizes are: size_x = 19.5, size_y = 19.5, size_z = 19.5, and the number of docking operations is 15.

[0029] Furthermore, in step (3) of the rapid screening method for expression inhibitors, recipient cells that are not co-cultured with candidate drugs are used as the negative control group for cell experiments, and the screening condition is "the significance of the mRNA expression level of ACE2 in cells compared with the negative control group of cell experiments is <0.05 (n=3)" to screen for drugs that have a significant inhibitory effect on the transcriptional expression of ACE2.

[0030] Furthermore, the ACE2 expression inhibitor obtained after the second screening in step (3) was also verified in mouse experiments. In the mouse experiments, mice that were not given the drug were used as the negative control group, and the verification condition was "the significance of the mRNA expression level of ACE2 in mouse tissue cells after drug administration compared with the negative control group of the mouse experiment was <0.05 (n=3)".

[0031] The second objective of this invention is the application of the ACE2 expression inhibitor described in the first objective of this invention in the prevention and / or treatment of ACE2-mediated diseases.

[0032] Furthermore, the disease mediated by ACE2 is pneumonia (COVID-19) caused by SARS-CoV-2 infection.

[0033] Meanwhile, the ACE2 expression inhibitors can be used to prepare targeted drugs for the prevention and / or treatment of ACE2-mediated diseases.

[0034] Furthermore, the targeted drug is any one of the ACE2 expression inhibitor, a pharmaceutically acceptable salt of the ACE2 expression inhibitor, or a pharmaceutically acceptable mixture containing the ACE2 expression inhibitor. When the targeted drug is a pharmaceutically acceptable mixture containing the ACE2 expression inhibitor, the targeted drug may further comprise any one or more of the following: a pharmaceutically acceptable dressing, a carrier, an active factor that enhances human immunity, or other drugs against SARS-CoV-2.

[0035] Furthermore, the dosage form of the targeted drug is any one of tablets, capsules, nasal sprays, pills, powders, or granules. Attached Figure Description

[0036] Figure 1 These are the test results of cell experiment re-screening of each drug in the ACE2 expression inhibitors of this invention;

[0037] Figure 2 These are the test results of mouse experiments (mixed administration of mouse feed) verifying the various drugs in the ACE2 expression inhibitors of this invention;

[0038] Figure 3 These are the test results of mouse experiments (intranasal administration to mice) verifying the various drugs in the ACE2 expression inhibitors of this invention. Detailed Implementation

[0039] The following detailed description, in conjunction with the accompanying drawings, illustrates the specific implementation of the ACE2 expression inhibitor of the present invention and its application in ACE2-mediated diseases:

[0040] The ACE2 expression inhibitor of the present invention is a drug obtained by screening using a rapid screening method for ACE2 expression inhibitors;

[0041] The rapid screening method for ACE2 expression inhibitors includes the following steps:

[0042] Ligand Database Selection: A ligand database was formed by selecting structural analogs of existing compounds with RXR-regulating effects. Specifically, the structural data of the ligands were obtained from the PubChem database. The selection principle was to choose structural analogs (natural or synthetic small molecule compounds with a molecular weight less than 1000 Da) of compounds that have been reported to regulate RXR or RXR dimers. Ligands included: ① Pentacyclic triterpenoids: Since Saba et al. found that ginsenoside Rg3 (a pentacyclic triterpenoid saponin) can exert anti-inflammatory effects by stimulating the heterodimers of RXRα and PPARγ (Non-Patent Literature 2), indicating that pentacyclic triterpenoids may have a good regulatory effect on RXRα, this invention selected a series of other structurally similar pentacyclic triterpenoids as ligands for molecular docking, such as oleanolic acid, ursolic acid, and corosolic acid. ② Flavonoids and polyphenols: Studies by Little R et al. have shown that quercetin, a representative flavonoid compound, can exert lipid regulation by acting on the heterodimers of RXRα and PPARα (Non-Patent Literature 3). Studies by Wang Q et al. have shown that salvianolic acid (a polyphenol compound) can improve lipid metabolism in mice with ischemic heart disease by acting on RXRα (Non-Patent Literature 4). The above studies show that flavonoids and polyphenols may have a good regulatory effect on RXRα. Therefore, this invention selected a series of other structurally similar flavonoids and polyphenols as ligands for molecular docking, such as silymarin, gallic acid esters, and chlorogenic acid. ③ Adrenal glucocorticoids: Studies by Hewitt et al. have shown that a certain dose of dexamethasone can significantly increase the expression of RXRα and PPARγ in the placenta and fetus of pregnant rats (Non-Patent Literature 5), indicating that glucocorticoid compounds have a good regulatory effect on RXRα. Therefore, this invention selected a series of other structurally similar glucocorticoid compounds as ligands for molecular docking, such as cortisone, hydrocortisone, and budesonide. ④ Vitamin D: Studies by Sylvia et al. have shown that 1,25-dihydroxyvitamin D3 can exert a variety of physiological functions by regulating the heterodimers of RXRα and VDR (Non-Patent Literature 6), indicating that vitamin D substances may have a good regulatory effect on RXRα. In summary, this invention selected natural or synthetic compounds, including pentacyclic triterpenoids, flavonoid polyphenols, glucocorticoids, and vitamin D derivatives, to form a ligand database for molecular docking. These compounds are structurally similar to previously reported compounds that regulate RXRα or RXRα dimers. Since previously reported compounds regulate RXRα, the ligand library molecules selected in this invention may also regulate RXRα. The aim is to virtually screen compounds with high RXRα molecular docking activity to investigate the effect of structural analogs of RXRα regulators on ACE2 expression.

[0043] Preliminary screening: The three-dimensional structure of the human RXRα receptor protein was obtained. Using computer molecular docking technology, ligands from the ligand database in step (1) were simulated to dock with the three-dimensional structure of the human RXRα receptor protein. Compounds with a docking binding free energy lower than -5 kcal / mol to the human RXRα receptor were preliminarily screened from the ligand database as candidate drugs. That is, the present invention uses human RXRα as the target protein for virtual drug screening. Specifically, the data of the three-dimensional structure of the human RXRα receptor protein in the present invention comes from the RCSB PDB database, and its PDB ID is: 3A9E. The software used for the computer molecular docking technology of the present invention is AutoDock Vina, and its graphical operation tools and result analysis software AutoDock Tools 1.5.7 are used in combination. Before docking, the target receptor protein and small molecule chemical ligands are pretreated, including protein hydrogenation, charge calculation, and atom type addition. The selected docking centers for the computer-aided molecular docking technique were: center_x = 49.192, center_y = 63.991, center_z = -7.522; the docking box sizes were: size_x = 19.5, size_y = 19.5, size_z = 19.5; and the number of docking operations was 15. Based on this, virtual drug screening was performed, and the results were scored and ranked. Finally, the top 10 compounds in each category were selected from the ligand database, as shown in Table 1. These initially screened compounds (i.e., candidate drugs) with different structural types were used for further screening in subsequent cell experiments.

[0044] Table 1

[0045]

[0046] (3) Secondary screening: Human HepG2 cells (HepG2 cells are a commonly used cell line in experiments, originally derived from liver cancer tissue of a 15-year-old liver cancer patient, which can secrete various plasma proteins and is mainly used for experimental research on liver cancer) were used as recipient cells. Different candidate drugs were co-cultured with the recipient cells. After co-culture, the cells were collected, and the mRNA expression level of ACE2 in the cells was detected. Drugs that inhibit the transcriptional expression of ACE2 were screened from the candidate drugs and used as ACE2 expression inhibitors. Specifically, human HepG2 cell line was used as recipient cells and cultured in DMEM cell culture medium. 1 mL of HepG2 cell solution with a concentration of 1×105 cells / mL was inoculated into each well of a 6-well cell culture plate and cultured at 37°C and 5% CO2 for 24 h. The test drug concentration was the highest concentration that did not produce cytotoxicity (a preliminary experiment was conducted using 96-well plates to determine the cytotoxic concentration for each test drug). Serum-free medium was used, and after changing the medium, 1 mL of drug solution was added to each well, with three replicates for each drug solution. Incubation continued for 24 h. Cells were then digested with trypsin and collected. Total RNA was extracted from the cells, and the obtained total RNA was subjected to reverse transcription. The relative transcription level of ACE2 in each group of cells was determined using real-time quantitative PCR (qPCR). The primer sequences for ACE2 and the internal control GAPDH are shown in Table 2 below.

[0047] Table 2

[0048]

[0049] Results processing was performed using the ΔΔCT method:

[0050] A = CT(target gene, sample to be tested) - CT(internal standard gene, sample to be tested)

[0051] B = CT(target gene, control sample) - CT(internal standard gene, control sample)

[0052] K=AB

[0053] Expression multiple = 2 - K

[0054] The results of this experiment are as follows: Figure 1 As shown, Figure 1 In the text, "*" and "**" indicate that the relative levels of ACE2 mRNA compared to the control group were P<0.05 and P<0.01, respectively; n=3. Furthermore, from... Figure 2The results show that among the initially screened ligand compounds, only 10 compounds—ursolic acid, phlorizin, resveratrol, triamcinolone, budesonide, fluticasone propionate, vitamin D2, vitamin D3, calcitriol, and alfacalcidol—significantly reduced ACE2 levels. Combined with the results of the molecular docking virtual screening, there was no linear relationship between the strength of the compound's RXRα docking activity and its inhibitory effect on ACE2 expression. These results, through further screening at the cellular level, identified a group of compounds that can inhibit ACE2 transcriptional expression via the RXR pathway.

[0055] Furthermore, the inventors also conducted mouse experiments to verify the ACE2 expression inhibitor obtained after the secondary screening in step (3). The mouse experiments included a mouse diet-mixed administration experiment and a mouse intranasal administration experiment.

[0056] The mouse feed-mixed administration experiment specifically involved using ICR clean-grade male mice as experimental subjects. The administration method was feed mixing, with the animals taking the medication naturally. The effect of the test substances on the expression levels of ACE2 in the major organs of mice was investigated. Ursolic acid, phlorizin, and polygalactosin were mixed into the feed powder at a mass ratio of 1%, processed into shape, and dried. The dosages of other compounds (triamcinolone, budesonide, fluticasone propionate, vitamin D2, vitamin D3, calcitriol, and alfacalcidol) were 50 times the recommended human dose, mixed into the feed powder, processed into shape, and dried. Five mice were used in each group, with free access to food and water, and the experiment lasted for 7 days. After the experiment, the animals were anesthetized and euthanized, and the lung and small intestine tissues were collected and stored at -80°C for subsequent experiments. The stored tissues were ground in liquid nitrogen, and total RNA was extracted. The obtained total RNA was then subjected to reverse transcription. The transcriptional level of ACE2 was measured using real-time quantitative PCR (qPCR). This study investigated the effects of oral administration of each test drug on the transcriptional expression levels of ACE2 in various tissues and organs. The results are attached. Figure 2 As shown. Figure 2 In the figure, “*” and “**” indicate that the relative levels of ACE2 mRNA in mouse lung tissue compared with the control group are P<0.05 and P<0.01, respectively; n=3. Figure 2 In the table, "#" and "##" represent the relative levels of ACE2 mRNA in mouse small intestinal tissue compared to the control group (P<0.05, P<0.01, n=3). The results indicate that, except for triamcinolone, budesonide, and fluticasone propionate, the other tested drugs (ursolic acid, phlorizin, resveratrol, vitamin D2, vitamin D3, calcitriol, and alfacalcidol) significantly reduced the relative mRNA levels of ACE2 in lung and small intestinal tissues when administered orally.

[0057] The mouse intranasal administration experiment was conducted as follows: ICR clean-grade male mice were used as experimental subjects, and the administration method was intranasal instillation. The test drug was prepared with physiological saline to a concentration of 1 mg / mL and sterilized through a 0.22 μm filter membrane. Mice were lightly anesthetized with isoflurane, and 20 μL of the drug solution was carefully instilled slowly into both nostrils using a pipette. The drug was administered once daily, with free access to food and water, for 7 days. After the experiment, the animals were euthanized under anesthesia, dissected, and nasal turbinates and lung tissues were collected and stored at -80°C for subsequent experiments. The stored tissues were ground in liquid nitrogen, and total RNA was extracted. The total RNA was then subjected to reverse transcription. ACE2 transcription levels were measured using real-time quantitative PCR (qPCR). The effect of oral administration of the test drugs on ACE2 transcriptional expression levels in various tissues and organs was investigated. The results of this experiment are attached. Figure 3 As shown. Figure 3 In the text, “*” and “**” indicate that the relative levels of ACE2 mRNA in mouse nasal turbinate tissue were significantly different from those in the control group (P<0.05, P<0.01); n=3. Figure 3 In the table, "#" and "##" represent the relative levels of ACE2 mRNA in mouse lung tissue compared to the control group (P<0.05, P<0.01, n=3). The results indicate that the test drug, administered via nasal drops, significantly reduced the relative mRNA levels of ACE2 in respiratory tissues such as the nasal turbinates and lungs.

[0058] The conclusion drawn from the experimental results of the above embodiments of the present invention is that the group of compounds screened by the present invention—ursolic acid, phlorizin, polygalactoside, triamcinolone acetonide, budesonide, fluticasone propionate, vitamin D2, vitamin D3, calcitriol, and alfacalcidol—can all significantly downregulate the expression level of ACE2 through the RXR pathway, and have good clinical application prospects. Targeted drugs for the prevention and / or treatment of ACE2-mediated diseases can be prepared using the ACE2 expression inhibitors described above.

[0059] The applications of the compounds screened in this invention include, but are not limited to, pneumonia caused by SARS-CoV-2 infection (COVID-19).

[0060] Furthermore, the targeted drug may be any one of the ACE2 expression inhibitor, a pharmaceutically acceptable salt of the ACE2 expression inhibitor, or a pharmaceutically acceptable mixture containing the ACE2 expression inhibitor. The compounds screened in this invention may contain acidic groups, which can form pharmaceutically acceptable salts with suitable bases. Representative examples of pharmaceutically acceptable salts include, but are not limited to: salts formed with inorganic ions, such as sodium, potassium, calcium, aluminum, ammonium, and zinc salts; and salts formed with organic bases, such as methylamine, ethylamine, triethylamine, and meglumine.

[0061] When the targeted drug is a pharmaceutically acceptable mixture containing the ACE2 expression inhibitor, the targeted drug may further include any one or more of the following: a pharmaceutically acceptable dressing, a carrier, an active factor that enhances human immunity, or other drugs against the novel coronavirus SARS-CoV-2.

[0062] Furthermore, the dosage form of the targeted drug is any one of tablets, capsules, nasal sprays, pills, powders, or granules.

[0063] For those skilled in the art, without departing from the concept of this invention, several simple deductions or substitutions can be made, and all such deductions or substitutions should be considered to fall within the scope of protection of this invention.

Claims

1. An angiotensin-converting enzyme 2 expression inhibitor, which is a drug obtained by screening using a rapid screening method for angiotensin-converting enzyme 2 expression inhibitors; characterized in that, The rapid screening method for angiotensin-converting enzyme 2 expression inhibitors includes the following steps: Selecting a ligand database: Selecting structural analogues of compounds that have a regulatory effect on retinoic acid X receptor or retinoic acid X receptor dimer to form a ligand database; Preliminary screening: Obtain the three-dimensional structure of the human retinoic acid X receptor α protein and use computer molecular docking technology to simulate docking of the ligands in the ligand database of step (1) with the three-dimensional structure of the human retinoic acid X receptor α protein. From the ligand database, ligands with docking binding free energy lower than -5 kcal / mol with the three-dimensional structure of the human retinoic acid X receptor α protein are initially screened as candidate drugs. Secondary screening: HepG2 cells were used as recipient cells. The candidate drugs obtained in step (2) were co-cultured with the recipient cells. After co-culture, the cells were collected and the mRNA expression level of angiotensin-converting enzyme 2 was detected. Drugs that inhibit the transcriptional expression of angiotensin-converting enzyme 2 were screened from the candidate drugs and used as the expression inhibitors of angiotensin-converting enzyme 2. The angiotensin-converting enzyme 2 expression inhibitors obtained through the screening in step (3) above are at least one of the following 10 drugs: ursolic acid, phlorizin, polygalactoside, triamcinolone, budesonide, fluticasone propionate, vitamin D2, vitamin D3, calcitriol, and alfacalcidol.

2. The angiotensin-converting enzyme 2 expression inhibitor according to claim 1, characterized in that: The structure in step (1) of the rapid screening method for expression inhibitors is similar to a small molecule compound with a molecular weight of less than 1000 Da, either natural or synthetic.

3. The angiotensin-converting enzyme 2 expression inhibitor according to claim 1, characterized in that: The computer molecular docking software used in step (2) of the rapid screening method for expression inhibitors is AutoDock Vina; the docking centers are: center_x = 49.192, center_y = 63.991, center_z = -7.522, the docking box sizes are: size_x = 19.5, size_y = 19.5, size_z = 19.5, and the number of docking operations is 15.

4. The angiotensin-converting enzyme 2 expression inhibitor according to claim 1, characterized in that: In step (3) of the rapid screening method for expression inhibitors, recipient cells that are not co-cultured with candidate drugs are used as the negative control group for cell experiments, and drugs that significantly inhibit the transcriptional expression of angiotensin-converting enzyme 2 are screened with the screening condition that "the mRNA expression level of angiotensin-converting enzyme 2 in cells is significantly different from that in the negative control group of cell experiments (P value < 0.05 (n=3)".

5. The angiotensin-converting enzyme 2 expression inhibitor according to claim 1, characterized in that: The angiotensin-converting enzyme 2 expression inhibitor obtained after the second screening in step (3) of the rapid screening method for the expression inhibitor was further verified in mouse experiments. In the mouse experiments, mice that were not given the drug were used as the negative control group of the mouse experiments, and the verification condition was "the significance of the mRNA expression level of angiotensin-converting enzyme 2 in mouse tissue cells after drug administration compared with the negative control group of the mouse experiments was <0.05 (n=3)".

6. The use of any one of the angiotensin-converting enzyme 2 expression inhibitors according to claims 1 to 5 in the preparation of targeted drugs for the prevention and / or treatment of angiotensin-converting enzyme 2-mediated diseases.

7. The application according to claim 6, characterized in that: The angiotensin-converting enzyme 2-mediated disease is pneumonia caused by COVID-19 infection.

8. The application according to claim 7, characterized in that: The targeted drug is any one of the angiotensin-converting enzyme 2 expression inhibitor, a pharmaceutically acceptable salt of the angiotensin-converting enzyme 2 expression inhibitor, or a pharmaceutically acceptable mixture containing the angiotensin-converting enzyme 2 expression inhibitor.

9. The application according to claim 7, characterized in that: The dosage form of the targeted drug is any one of tablets, capsules, nasal sprays, pills, powders, or granules.

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