Use of leukadherin-1 in the preparation of a medicament for preventing and / or treating sars-cov-2 infection

Abstract

The application discloses application of Leukadherin-1 in preparation of a medicine for preventing and / or treating SARS-CoV-2 infection. The application uses a luciferase pseudovirus encoding SARS-CoV-2 spike protein for evaluation, and it is found for the first time that Leukadherin-1 can effectively inhibit infection of SARS-CoV-2 pseudovirus particles (SARS2pp), and exhibits strong antiviral activity.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and pharmaceuticals, specifically relating to the application of Leukadherin-1 (Lek-1) as a COVID-19 inhibitor in the treatment of severe acute respiratory syndrome, and particularly to the application of Leukadherin-1 in the preparation of drugs for the prevention and treatment of SARS-CoV-2 mediated diseases. Background Technology

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogen that caused the 2019 coronavirus disease (COVID-19) pandemic and is still evolving globally. SARS-CoV-2 is the third known highly pathogenic coronavirus in humans, the first two being Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), which broke out in 2002-2003, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which has been breaking out since 2012. COVID-19 is a respiratory illness caused by a virus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is an RNA virus that enters the human body through droplets, primarily causing infection through the respiratory tract. The virus has a spike protein that binds to the ACE2 receptor on human cells, allowing the virus to enter the cell and replicate. After infection, people will experience symptoms such as fever, cough, fatigue, and difficulty breathing. Severe cases may lead to pneumonia, acute respiratory distress syndrome (ARDS), and organ failure.

[0003] Currently, treatments for COVID-19 are still under active research and development, mainly including antiviral drugs, immunomodulatory drugs, and other therapeutic agents. Remdesivir is a broad-spectrum antiviral drug initially used to treat Ebola virus infection. It has been granted emergency use authorization in some countries and has shown some efficacy in clinical trials, particularly in severe and critically ill patients. Favipiravir is another antiviral drug initially used to treat influenza. It has been approved in some countries and is undergoing clinical trials, with preliminary results showing some clinical efficacy. Glucocorticoid anti-inflammatory drugs such as dexamethasone have been shown to reduce mortality in severely ill patients, but are not suitable for mild and moderate cases. Interleukin-6 (IL-6) receptor antagonists such as tocilizumab are used to treat severe cytokine release syndrome (CRS). Regarding plasma therapy, there are options involving the extraction of antibodies from the plasma of recovered patients for the treatment of severely ill patients. Some antibody drugs, such as bamlanivimab and casilimbab / imdevimab, have received emergency use authorization for the treatment of mild to moderate cases. It should be noted that the efficacy and safety of these drugs are still under further investigation and evaluation. Furthermore, the development and distribution of vaccines are also crucial means of controlling the COVID-19 pandemic. However, due to the increasing variability and potential drug resistance of SARS-CoV-2, the search for safe and effective antiviral drugs has become particularly urgent.

[0004] Leukadherin-1 is a small molecule compound with a molecular weight of 421.49, possessing a unique chemical structure and biological activity. Leukadherin-1 regulates leukocyte adhesion and migration by binding to adhesion protein-1 (LFA-1) on the surface of leukocytes. LFA-1 is an integrin expressed on the surface of leukocytes, involved in the regulation of inflammatory responses and the activation of immune cells. LFA-1 mediates leukocyte adhesion and migration by binding to intercellular adhesion molecule (ICAM). During inflammation, leukocytes bind to ICAM on the surface of cells in damaged tissues or sites of inflammation via LFA-1, thereby triggering an inflammatory response. Leukadherin-1 contains a specific ring structure that interacts with the binding site of LFA-1. This interaction can alter the binding ability of LFA-1 to ICAM, thereby regulating leukocyte adhesion and migration. Research results show that Leukadherin-1 can inhibit leukocyte adhesion and infiltration, reducing inflammatory responses and tissue damage. Leukadherin-1 can also inhibit the activation of immune cells and reduce the release of inflammatory factors. Studies have found that Leukadherin-1 can inhibit the activation of the NF-κB signaling pathway and reduce the production of inflammatory factors. NF-κB is a key transcription factor involved in inflammatory responses and the activation of immune cells. By inhibiting NF-κB activation, Leukadherin-1 can alleviate inflammatory responses and tissue damage. Leukadherin-1 shows potential in the treatment of inflammatory diseases. Inflammatory diseases are a class of chronic diseases caused by inflammatory responses, such as rheumatoid arthritis and inflammatory bowel disease. Research results show that Leukadherin-1 can reduce inflammatory responses and tissue damage, and improve disease symptoms. In animal models, Leukadherin-1 can reduce intestinal inflammation and infiltration, and decrease the production of inflammatory factors. In addition, Leukadherin-1 has also been studied for the prevention of transplant rejection. During organ transplantation, the patient's immune system may attack the transplanted organ, leading to rejection. Leukadherin-1 can prevent organ rejection and improve the survival rate of transplanted organs by regulating the activation and adhesion of immune cells. Research results indicate that Leukadherin-1 has the potential to treat inflammatory diseases and may become a novel therapeutic agent. However, Leukadherin-1 is still in the research and development stage and has not yet been widely used in clinical practice. Summary of the Invention

[0005] The purpose of this invention is to provide new pharmaceutical uses for Leukadherin-1 (Lek-1).

[0006] The novel pharmaceutical use of Leukadherin-1 (Lek-1) provided by this invention is its application in the preparation of the following products:

[0007] 1) Drugs for treating COVID-19 infection;

[0008] 2) COVID-19 inhibitors;

[0009] 3) Drugs for the prevention and / or treatment of SARS-CoV-2 infection;

[0010] 4) Medications for the prevention and / or treatment of SARS-CoV-2-mediated diseases;

[0011] In 4) above, the SARS-CoV-2-mediated diseases include respiratory diseases caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), more specifically COVID-19.

[0012] In the application described, the concentration of Leukadherin-1 (Lek-1) is greater than 8 μM, preferably greater than 10 μM, and more preferably 10 μM-40 μM.

[0013] Leukadherin-1, CAS No.: 344897-95-6, molecular formula: C 22 H 15 NO4S2, English name: Leukadherin-1, has the following chemical structural formula:

[0014]

[0015] This invention uses fluorescein pseudovirus encoding the SARS-CoV-2 spike protein for evaluation and is the first to discover that Leukadherin-1 can effectively inhibit the infection of SARS-CoV-2 pseudovirus particles (SARS2pp), demonstrating strong antiviral activity. Attached Figure Description

[0016] Figure 1This indicates that Leukadherin-1 inhibits SARS-CoV-2 virus infection. (A) Cell counting kit 8 (CCK-8) was used to evaluate the viability of HEK-293T-ACE2 cells treated with different doses of Leukadherin-1 for 24 hours. (BC) HEK-293T-ACE2 cells were treated with Leukadherin-1 2.5 hours before infection with SARS-CoV-2 pseudovirus particles (SARS2pp). The medium was then changed, and cells were infected with pseudovirus particles with an MOI of 0.3 for 24 hours. Cell viability was detected by CCK-8 assay (A), and luciferase activity (B) and immunofluorescence intensity (C) were detected by immunofluorescence assay. Scale bar: 100 μm.

[0017] Data are presented as mean ± SEM of biological replicates (CCK-8: n=3; immunofluorescence: n=6). ***p<0.001 and ns: not significant (one-way ANOVA and Dunnett's post-hoc test). Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0020] Materials and methods

[0021] Experimental Design

[0022] This study primarily evaluated the effects of Leukadherin-1 (Lek-1) on SARS-CoV-2 infection. The efficacy of Leukadherin-1 against SARS-CoV-2 infection was assessed using immunofluorescence assay.

[0023] Pseudoviruses, cells, and reagents

[0024] The SARS-CoV-2 XBB.1.16 was provided by Professor Wang Xuejun of the AMMS Bioinformatics Center, with Li Tian assisting in the related experiments.

[0025] HEK-293T-ACE2 cell line was maintained in a humidified 5% CO2 incubator at 37°C in Dulbecco modified Eagle medium (DMEM; Gibco, 04242) supplemented with 10% fetal bovine serum (FBS; Gibco, FND500) and 1% penicillin-streptomycin (Macgene).

[0026] Leukadherin-1 (MedChemExpress, HY-15701) was dissolved in dimethyl sulfoxide (DMSO; Innochem, D3855) at a concentration of 2 mM and stored at -80°C until use. Before use, it was diluted with DMEM to different concentrations for CCK-8 cytotoxicity assays; it was also diluted with opti-MEM (Gibco, 31985-070) to different concentrations for antiviral drug intervention assays; Steady-Lumi... TM The activity of firefly luciferase reporter gene assay kit (Beyotime, RG058M) was detected.

[0027] Statistical analysis

[0028] For comparisons of multiple groups, one-way ANOVA and Dunnett's post-hoc test were used. Data are presented as mean ± SEM.

[0029] Example 1: Leukadherin-1 Cytotoxicity Study

[0030] Cell culture and toxicity testing

[0031] HEK-293T-ACE2 cells were used at a rate of 3 × 10⁻⁶ 5 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated overnight at 37°C. Cells were then treated with different concentrations of Leukadherin-1 (0-80 μM) at 37°C for 24 hours. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Bimake, C6005M) according to the product instructions. In short, 70 μL of DMEM-diluted CCK-8 (10%) reagent was added to each well of the cell culture plate and incubated with the cells at 37°C for 2 hours, followed by measurement of optical density (OD) at 450 nm.

[0032] To monitor the effect of different concentrations of Leukadherin-1 on cell viability, we evaluated the effect of different concentrations of Leukadherin-1 on cell viability. Cell viability assays showed that Leukadherin-1 did not exhibit any cytotoxicity in HEK-293T-ACE2 cells at doses below 40 μM, and the 50% concentration (CC50) of Leukadherin-1 was 58.96 μM (HEK-293T-ACE2 cells). Figure 1 (A)

[0033] Example 2: Leukadherin-1 inhibits SARS-CoV-2 virus infection.

[0034] Pseudoviral infection and drug intervention

[0035] In the pseudovirus particle infection experiment, HEK293 / hACE cells were infected at a rate of 3 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated overnight at 37°C. Cells were then infected with pseudovirus particles at an MOI of 0.3 for 24 hours. Steady-Lumi was used. TM II. Luciferase activity was detected using a luciferase assay kit (Beyotime). For experiments involving invasion inhibitors and treatment time, HEK-293T-ACE2 cells were treated with Leukadherin-1 2.5 hours before infection with SARS-CoV-2 pseudovirus particles (SARS2pp).

[0036] To identify the inhibitory effect of Leukadherin-1 on SARS-CoV-2 infection, we infected HEK-293T-ACE2 cells with SARS2pp and measured the effect. We found that at concentrations of 4 μM, 6 μM, 8 μM, and 10 μM, Leukadherin-1 inhibited SARS2pp infection by approximately 47%, 81%, 92%, and 99%, respectively. Figure 1 (B, C)

[0037] The results of pseudovirus infectivity assays showed that Leukadherin-1 significantly inhibited SARS-pp infection at safe concentrations, with a median effective concentration (EC50) of 4.15 μM, indicating that Leukadherin-1 may be an effective inhibitor of SARS-pp infection. Figure 1 (Middle BC). The calculated selectivity index of Leukadherin-1 was 14, indicating a wide range of working concentrations available (generally, compounds with a selectivity index greater than 1 are considered promising because they exhibit low cytotoxicity while effectively inhibiting the target activity).

[0038] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of Leukadherin-1 in the preparation of a medicament for preventing and / or treating SARS-CoV-2 infection.

2. Use according to claim 1, characterized in that, In the use, the concentration of Leukadherin-1 is greater than 8 μM.

3. Use according to claim 2, characterized in that, In the use, the concentration of Leukadherin-1 is 10 μM-40 μM.

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