CN117442616A - Application of Leukadherin-1 in the preparation of drugs for preventing and/or treating SARS-CoV-2 infection - Google Patents

Abstract

The invention discloses an application of Leukadherein-1 in preparing a medicine for preventing and/or treating SARS-CoV-2 infection. The invention uses the fluorescein pseudovirus encoding SARS-CoV-2 spike protein for evaluation, and discovers that Leukadherein-1 can effectively inhibit SARS-CoV-2 pseudovirus particle (SARS 2 pp) infection for the first time, and shows strong antiviral activity.

Description

Application of Leukadherein-1 in preparation of medicine for preventing and/or treating SARS-CoV-2 infection

Technical Field

The invention belongs to the field of biomedicine and medicines, and particularly relates to application of Leukadherein-1 (Lek-1) as a novel coronavirus inhibitor in treating severe acute respiratory syndrome diseases, in particular to application of Leukadherein-1 in preparing medicines for preventing and treating SARS-CoV-2 mediated diseases.

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic of coronavirus disease (COVID-19) in 2019, and is currently evolving continuously worldwide. SARS-CoV-2 is the third highly pathogenic coronavirus known to humans, the first two being severe acute respiratory syndrome coronavirus (SARS-CoV) that has exploded in 2002 to 2003 and middle east respiratory syndrome coronavirus (MERS-CoV) that has exploded since 2012, respectively. Covd-19 is a respiratory disease caused by a virus known as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). SARS-CoV-2 is an RNA virus that is transmitted into the human body by droplets, causing infection primarily through the respiratory tract. The virus is structured with spike proteins (spike proteins) that bind to ACE2 receptors on human cells, allowing the virus to enter the cell and replicate. After infection, the human body can have symptoms such as fever, cough, hypodynamia, dyspnea and the like, and severe cases can lead to pneumonia, acute Respiratory Distress Syndrome (ARDS) and organ failure.

Currently, therapeutic drugs against covd-19 are still under active research and development, mainly including antiviral drugs, immunomodulatory drugs, and other therapeutic drugs. Remdesivir (Remdesivir) is a broad-spectrum antiviral drug that was originally used to treat Ebola virus infection. Emergency use authorities have been obtained in some countries and have shown a certain efficacy in clinical trials, especially in critically ill and critically ill patients. Favipiravir is another antiviral drug that was originally used to treat influenza. Approval has been obtained in some countries and clinical trials are underway, with preliminary results showing a certain clinical outcome. Glucocorticoids anti-inflammatory drugs such as Dexamethasone (Dexamethasone) have been shown to have mortality reducing effects in critically ill patients, but are not suitable for mild and moderate cases. Interleukin-6 (IL-6) receptor antagonist drugs such as tolizumab (Tocilizumab) are used to treat severe Cytokine Release Syndrome (CRS). Regarding the plasma treatment, there is a scheme for treating severe patients by extracting antibodies from the plasma of a convalescent person. Some antibody drugs such as balanoximab (Bamlanivimab) and cassiabab Li Mbu (Casirivimab/Imdevimab) have been given emergency authorization for use for the treatment of mild to moderate cases. It is noted that the efficacy and safety of these drugs is still under further investigation and evaluation. In addition, vaccine development and popularization are also one of the important means for controlling the epidemic situation of the COVID-19. However, due to the variability of SARS-CoV-2 and the potential increase in drug resistance, the search for safe and effective antiviral drugs has become particularly urgent.

Leukadherein-1 is a small molecular compound with a molecular weight of 421.49 and has unique chemical structure and biological activity. Leukadherein-1 regulates leukocyte adhesion and migration by binding to leukocyte surface adhesion protein-1 (LFA-1). LFA-1 is an integrin expressed on the surface of leukocytes and is involved in the regulation of inflammatory responses and in the activation of immune cells. LFA-1 mediates leukocyte adhesion and migration through binding to intercellular adhesion molecules (ICAM). During inflammation, leukocytes can bind to ICAM on the cell surface of damaged tissue or inflammation sites via LFA-1, thereby triggering an inflammatory response. Leukadherein-1 contains a specific cyclic structure in its structure that can interact with the binding site of LFA-1. This interaction can alter LFA-1 binding capacity to ICAM, thereby modulating leukocyte adhesion and migration. The research result shows that Leukadherein-1 can inhibit the adhesion and infiltration of leucocytes and reduce inflammatory reaction and tissue injury. Leukadherein-1 also inhibits immune cell activation and reduces inflammatory factor release. The research shows that Leukadherein-1 can inhibit the activation of NF- κB signal path and reduce the production of inflammatory factors. NF- κB is a critical transcription factor involved in inflammatory reactions and immune cell activation. Leukadherein-1 can reduce inflammatory response and tissue damage by inhibiting NF- κB activation. Leukadherein-1 has shown potential in the treatment of inflammatory diseases. Inflammatory diseases are a type of chronic diseases caused by inflammatory reactions, such as rheumatoid arthritis, inflammatory bowel disease, etc. The results of the study showed that Leukadherein-1 can reduce inflammatory response and tissue damage and ameliorate disease symptoms. In animal models, leukadherein-1 can reduce intestinal inflammation and infiltration, and reduce inflammatory factor production. In addition, leukadherein-1 was also studied for preventing rejection of transplanted organs. During organ transplantation, the patient's immune system may attack the transplanted organ, resulting in rejection reactions. Leukadherein-1 can prevent organ rejection reaction and improve survival rate of transplanted organs by regulating activation and adhesion of immune cells. Research results show that Leukadherein-1 has potential capability of treating inflammatory diseases and can become a novel therapeutic drug. However, leukadherein-1 is still in the research and development stage and has not yet been widely used clinically.

Disclosure of Invention

The invention aims to provide a novel medical application of Leukadherein-1 (Lek-1).

The novel medical application of the Leukadherein-1 (Lek-1) provided by the invention is the application of the Leukadherein-1 (Lek-1) in the preparation of the following products:

1) A drug against a new coronavirus infection;

2) A novel coronavirus inhibitor;

3) A medicament for preventing and/or treating SARS-CoV-2 infection;

4) A medicament for preventing and/or treating SARS-CoV-2 mediated disease;

the SARS-CoV-2 mediated disease of 4) above comprises a respiratory disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), more specifically, COVID-19.

In such applications, the concentration of Leukadherein-1 (Lek-1) is greater than 8. Mu.M, preferably greater than 10. Mu.M, more preferably from 10. Mu.M to 40. Mu.M.

Leukadherein-1, CAS number: 344897-95-6 with molecular formula of C ₂₂ H ₁₅ NO ₄ S ₂ English name: leukadherein-1 has the chemical structural formula shown below:

the invention uses the fluorescein pseudovirus encoding SARS-CoV-2 spike protein for evaluation, and discovers that Leukadherein-1 can effectively inhibit SARS-CoV-2 pseudovirus particle (SARS 2 pp) infection for the first time, and shows strong antiviral activity.

Drawings

FIG. 1 shows that Leukadherein-1 inhibits infection with SARS-CoV-2 virus. (A) Cell counting kit 8 (CCK-8) was used to assess viability of HEK-293T-ACE2 cells treated with varying doses of Leukadherein-1 for 24 hours. (B-C) HEK-293T-ACE2 cells were subjected to Leukadherein-1 treatment 2.5 hours prior to infection with SARS-CoV-2 pseudovirion (SARS 2 pp). Then, the liquid is changed, cells are infected by pseudovirus particles with MOI of 0.3, the cell viability (A) is detected by a CCK-8 method for 24 hours, the luciferase activity (B) and the immunofluorescence intensity (C) are detected by an immunofluorescence method, and the ratio scale is: 100 microns.

Data are expressed as mean ± SEM of biological replicates (CCK-8:n =3; immunofluorescence: n=6). * P <0.001 and n.s.: not significant (one-way analysis of variance with Dunnett's post hoc test).

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Materials and methods

Design of experiment

The present study mainly evaluates the effect of Leukadherein-1 (Lek-1) on the infection of new coronaviruses. The effect of Leukadherein-1 on SARS-CoV-2 virus infection was evaluated by immunofluorescence.

Pseudoviruses, cells and agents

SARS-CoV-2 XBB.1.16 is provided by the teaching of AMMS bioinformatics center Wang Xuejun, li Tian to assist in the relevant experiments.

HEK-293T-ACE2 cell line supplemented with 10% fetal bovine serum (FBS; gibco, FND 500)) And 1% of a dual antibiotic (penicillin-streptomycin; macgene) Dulbecco modified Eagle medium (DMEM; gibco, 04242) in 37 ℃ wet 5% CO ₂ Culture was maintained in an incubator.

Leukadherein-1 (MedChemExpress, HY-15701) was dissolved in dimethyl sulfoxide (DMSO; innochem, D3855) at a concentration of 2mM and stored at-80℃until use, diluted with DMEM to different concentrations for use in CCK-8 cytotoxicity assay; when in use, the composition is diluted into different concentrations by opti-MEM (Gibco, 31985-070) for antiviral drug intervention experiments; using Steady-Lumi ^TM The firefly luciferase reporter assay kit (Beyotime, RG 058M) was used for activity detection.

Statistical analysis

For comparison of the groups, one-way anova with Dunnett's post hoc test (GraphPad Prism, usa) was used. Data are expressed as mean ± SEM.

EXAMPLE 1 Leukadherein-1 cytotoxicity Studies

Cell culture and toxicity detection

HEK-293T-ACE2 cells at 3X 10 ⁵ The individual cells/ml were seeded at a density of 100 μl per well in 96-well plates and incubated at 37 ℃ overnight. The cells were then treated with varying concentrations of Leukadherein-1 (0-80. Mu.M) at 37℃and incubated for 24 hours. Cell viability was measured by cell counting kit-8 (CCK-8; bimake, C6005M) and was performed according to the product instructions. Briefly, CCK-8 (10%) reagent diluted with DMEM was added to the cell culture plate at a volume of 70. Mu.L per well and incubated with the cells in an incubator at 37℃for 2 hours, and then the optical density (OD value) was measured at a wavelength of 450 nm.

To monitor the effect of varying concentrations of Leukadherein-1 on cell viability, we assessed the effect of varying concentrations of Leukadherein-1 on cell viability, and cell viability assays showed that Leukadherein-1 did not exhibit any cytotoxicity at doses below 40. Mu.M in HEK-293T-ACE2 cells, and that the 50% cell concentration (CC 50) of Leukadherein-1 was 58.96. Mu.M (HEK-293T-ACE 2 cells) (A in FIG. A).

Example 2 Leukadherein-1 inhibits infection with SARS-CoV-2 Virus

Pseudovirus infection and pharmaceutical intervention

HEK293/hACE cells were used in 3X 10 in pseudovirion infection experiments ⁵ The density of individual cells per milliliter was 100 microliters per well, seeded in 96-well plates, incubated at 37 ℃ overnight. Cells were then infected with pseudovirions with an MOI of 0.3 for 24 hours. Using Steady-Lumi ^TM II luciferase assay kit (Beyotime) detects luciferase activity. For the invasion inhibitor and treatment time experiments, HEK-293T-ACE2 cells were subjected to Leukadherein-1 treatment 2.5 hours prior to SARS-CoV-2 pseudovirion (SARS 2 pp) infection.

To identify the infection inhibitory effect of Leukadherein-1 on SARS-CoV-2, we used the assay of SARS2 pp-infected HEK-293T-ACE2 cells, and found that compound Leukadherein-1 was able to achieve a SARS2 pp-infection inhibition of about 47%, 81%, 92%, 99% respectively (B, C in FIG. 1) at concentrations of 4. Mu.M, 6. Mu.M, 8. Mu.M, 10. Mu.M.

The results of the pseudovirus infectivity assay showed that Leukadherein-1 significantly inhibited SARS2pp infection at a safe concentration with an effector concentration (EC 50) of 4.15. Mu.M, indicating that Leukadherein-1 may be an effective SARS2pp blocking infection inhibitor (B-C in FIG. 1). The calculated selectivity index for Leukadherein-1 is 14, indicating a wide range of working concentrations available for selection (generally, a selectivity index greater than 1 is considered a potential compound because of their low cytotoxicity to cells while effectively inhibiting the activity of interest).

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims (8)

1. Use of leukadherin-1 for the preparation of a medicament against a new coronavirus infection.
2. Use of leukadherin-1 in the preparation of a novel coronavirus inhibitor.
3. Use of leukadherin-1 for the preparation of a medicament for the prevention and/or treatment of SARS-CoV-2 infection.
4. Use of leukadherin-1 for the preparation of a medicament for the prevention and/or treatment of SARS-CoV-2 mediated diseases.
5. 5. The use according to claim 4, wherein the SARS-CoV-2 mediated disease comprises a respiratory disease caused by severe acute respiratory syndrome coronavirus type 2.
6. 6. The use according to claim 4, wherein the SARS-CoV-2 mediated disease is COVID-19.
7. 7. The use according to any one of claims 1 to 6, wherein the concentration of Leukadherin-1 in the use is greater than 8 μm.
8. 8. The use according to any one of claims 1 to 6, wherein the concentration of Leukadherin-1 in the use is 10 μm to 40 μm.