Application of compound in preparation of medicine for treating related diseases and / or symptoms caused by new coronavirus infection

Abstract

The invention relates to application of a compound in preparation of a medicine for treating related diseases and / or symptoms caused by new coronavirus infection. The compound has efficient activity of inhibiting new coronavirus infection in vivo and in vitro, and is low in cytotoxicity. Meanwhile, as a host AXL receptor inhibitor, the SARS-CoV-2 inhibitor blocks the binding site of the SARS-CoV-2 and the receptor AXL of the SARS-CoV-2, and as an interferon inducer, the SARS-CoV-2 inhibitor can strongly up-regulate the expression of I-type interferon, and double-targeting host receptor and interferon pathway can exert strong anti-neocoronavirus activity. Therefore, the compound disclosed by the invention is based on a novel anti-new coronavirus double-targeting strategy of blocking AXL receptor effect and up-regulating interferon response, is relatively safe to host cells, and can be used for preventing and treating new coronavirus infection.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the use of a compound in the preparation of a medicament for treating diseases and / or symptoms caused by COVID-19 infection. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) causes COVID-19. In the past three years, the development of specific antiviral drugs targeting RNA-dependent RNA polymerase (RdRp) and main protease (Mpro, also known as 3C-like protease, 3CLpro) inhibitors of SARS-CoV-2 has been relatively successful. Six antiviral drugs have been launched in China, including azvudine tablets, monoravir capsules, deuterated remdesivir hydrobromide tablets, nelmatvir / ritonavir combination packs, cenostatin / ritonavir tablets, and leretvir tablets. However, each has its advantages and disadvantages, including high prices, the need for early-stage treatment, and unclear adverse reactions such as abnormal kidney and liver function. Most patients infected with the novel coronavirus still experience prolonged symptoms such as fatigue, shortness of breath, cough, and decreased sense of smell or taste during the recovery period. Some may still experience discomfort such as muscle aches, sore throat, palpitations, and hoarseness. Therefore, the global demand for safe and effective antiviral drugs against COVID-19 is extremely urgent and cannot be delayed. More antiviral drugs are urgently needed for clinical trials. The focus of new drug development for COVID-19 lies in the design and discovery of drugs targeting key factors in the virus's replication cycle, including the interaction between the virus and the host. Viral adsorption to host receptors is the first step in viral invasion, making these receptors highly effective antiviral targets. With the discovery and confirmation of the receptors or co-receptors of COVID-19 (including ACE2, TMPRSS2, and AXL), drug development targeting these receptors has become a hot area in antiviral drug development. ACE2 is not only a necessary receptor for COVID-19 infection, but its physiological function also involves targeting Ang II, demonstrating protective effects on the lungs and cardiovascular system. Studies have reported that decreased ACE2 levels after COVID-19 infection are a key factor contributing to lung damage and failure. Therefore, drug development strategies that reduce or inhibit ACE2 carry a high risk and may worsen the condition.

[0003] Activation of the host's innate immune system (especially the interferon pathway) is the first line of defense against viral invasion, and innate immune agonists are a hot area for the development of low-toxicity antiviral drugs. Stimulating interferon gene protein (STING) is an important positive regulator of interferon production. Current research has found that the novel coronavirus can delay or antagonize the interferon production pathway by blocking STING signal transduction, leading to innate immune escape and ultimately resulting in long-term high viral load in the host. Therefore, STING pathway agonists could be highly promising candidate drugs for the treatment of COVID-19. However, more complexly, recent studies have found that elevated interferon levels are associated with the worsening of COVID-19 infection. This may be because interferon can induce the expression of ACE2 or ACE2 cutoff, and the novel coronavirus can utilize an interferon-induced transmembrane protein, IFITM, as its own penetration factor to enhance infection; therefore, interferon is a double-edged sword for COVID-19 infection.

[0004] Ascomlactone A is a novel nine-membered ring lactone derivative compound derived from a marine mangrove endophytic fungus isolated by the inventors' collaborative team. Ascomycota sp .SK2YWS-L Ascomlactone A has a novel structure and has been granted a national invention patent (patent number: ZL201710253375.2). This invention discloses a nine-membered ring lactone derivative derived from marine fungi, its preparation method, and its applications. It exhibits significant α-glucosidase inhibitory activity and has a promising market prospect in the preparation of hypoglycemic drugs, especially for the treatment of type II diabetes. However, there are currently no publicly reported reports on the antiviral activity of Ascomlactone A. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of a compound in the preparation of a medicament for treating diseases and / or symptoms caused by COVID-19 infection.

[0006] The first objective of this invention is to provide the use of compounds with structures as shown in formula (I) in the preparation of medicaments for treating COVID-19.

[0007] A second objective of this invention is to provide the use of compounds with structures as shown in formula (I) in the preparation of medicaments for treating diseases and / or symptoms caused by the novel coronavirus.

[0008] A third objective of this invention is to provide the use of pharmaceutically acceptable salts of compounds with structures as shown in formula (I) in the preparation of medicaments for treating COVID-19.

[0009] A fourth object of the present invention is to provide the use of pharmaceutically acceptable salts of compounds with the structure shown in formula (I) in the preparation of medicaments for treating diseases and / or symptoms caused by the novel coronavirus.

[0010] A fifth object of the present invention is to provide the use of compounds with structures as shown in formula (I) in the preparation of type I interferon inducers.

[0011] A sixth object of the present invention is to provide the use of compounds with structures as shown in formula (I) in the preparation of type I interferon agonists.

[0012] This invention focuses on the key receptor AXL, which is widely expressed in the respiratory system. AXL can mediate viral invasion and infection by binding to the N-terminal NTD region of the SARS-CoV-2 spike protein, independent of ACE2. Based on two levels of targets—the crystal structure target protein of the AXL receptor and key factors in the interferon pathway—high-throughput, high-content screening was conducted using computer-aided virtual drug screening technology, resulting in a novel marine-derived target compound, Ascomlactone A (ALA). Further experiments demonstrated that Ascomlactone A exerts strong anti-SARS-CoV-2 activity by blocking the binding site of SARS-CoV-2 to its receptor AXL and simultaneously acting as an interferon inducer to strongly upregulate the expression of type I interferon, thus targeting both the host receptor and the interferon pathway. This indicates that Ascomlactone A is a novel multi-targeted anti-SARS-CoV-2 drug.

[0013] To achieve the above objectives, the present invention is implemented through the following technical solution: The application of compounds with structures shown in formula (I) in the preparation of drugs against the novel coronavirus. .

[0014] The use of compounds with structures as shown in formula (I) in the preparation of drugs for treating diseases and / or symptoms caused by the novel coronavirus. .

[0015] The application of pharmaceutically acceptable salts of compounds with structures shown in formula (I) in the preparation of drugs against COVID-19. .

[0016] The use of pharmaceutically acceptable salts of compounds with the structure shown in formula (I) in the preparation of drugs for treating diseases and / or symptoms caused by the novel coronavirus. .

[0017] Preferably, the compound with the structure shown in formula (I) acts as a host AXL receptor inhibitor, blocking the binding site of SARS-CoV-2 to its receptor AXL.

[0018] Preferably, the compound with the structure shown in formula (I) inhibits the SARS-CoV-2 infection activity.

[0019] Preferably, the compound with the structure shown in formula (I) promotes the production of type I interferon in the host cell.

[0020] Preferably, the dosage form of the drug is tablets, oral liquids, oral pills, oral granules, oral powders, injections, eye drops, nasal drops, aerosols, or inhalers.

[0021] The application of compounds with structures shown in formula (I) in the preparation of type I interferon inducers.

[0022] Formula (I).

[0023] The application of compounds with structures shown in formula (I) in the preparation of type I interferon agonists.

[0024] Formula (I).

[0025] Compared with the prior art, the present invention has the following beneficial effects: The Ascomlactone A described in this invention exhibits highly efficient in vivo and in vitro inhibitory activity against SARS-CoV-2 infection with low cytotoxicity. Furthermore, this invention discloses that Ascomlactone A, as a host AXL receptor inhibitor, blocks the binding site of SARS-CoV-2 to its receptor AXL, and simultaneously acts as an interferon inducer, strongly upregulating the expression of type I interferon, thus exerting strong anti-SARS-CoV-2 activity by targeting both the host receptor and the interferon pathway. Therefore, this invention proposes a novel anti-SARS-CoV-2 strategy based on blocking the AXL receptor effect and upregulating the interferon response using Ascomlactone A, which is relatively safe for host cells and can be used for the prevention and treatment of SARS-CoV-2 infection. Attached Figure Description

[0026] Figure 1 To investigate the cytotoxic effects of Ascomlactone A on SARS-CoV-2-susceptible cells 293T-hAXL and Vero E6.

[0027] Figure 2 Ascomlactone A was used to inhibit the production of viral particles of SARS-CoV-2 (including pseudoviruses of different variants of SARS-CoV-2 Delta, BA.1, or D614G, or live SARS-CoV-2 BA.1 virus) in SARS-CoV-2 susceptible cells 293T-hAXL and to measure its half-maximal inhibitory effect (EC50) in SARS-CoV-2 susceptible cells 293T-hAXL. 50 .

[0028] Figure 3To investigate the protective effect of Ascomlactone A on the survival of RD cells during SARS-CoV-2 infection.

[0029] Figure 4 To detect the direct interaction between Ascomlactone A and human AXL recombinant protein (rhAXL) using surface plasmon resonance assays (Biacore molecular interaction assay system).

[0030] Figure 5 This study aims to detect the in vivo anti-SARS-CoV-2 activity of Ascomlactone A.

[0031] Figure 6 Ascomlactone A can promote the production of type I interferon in 293T cells and influence the expression level of its downstream factor, interferon-stimulated genes.

[0032] Figure 7 Treatment with Ascomlactone A resulted in a dose-dependent increase in phosphorylation levels of TBK1 and IRF3 in 293T-hAXL cells. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0034] ALA compound is Ascomlactone A, a nine-membered ring lactone compound with the structure shown in formula (I). , The preparation method is described in "CN107056761B A nine-membered ring lactone derivative derived from marine fungi and its preparation method and application".

[0035] Example 1: The cytotoxic effect of Ascomlactone A I. Experimental Methods Selected SARS-CoV-2 susceptible cells 293T-hAXL (1×10⁻⁶) 4 / hole) and Vero E6 (1×10 4Cells were seeded in 96-well plates and treated with different concentrations of ALA compound (final concentrations: 0, 10, 20, 40, 80, 160 μM) for 48 h. Cell viability was measured using the MTS colorimetric assay to detect the effect of ALA on the growth of 293T-hAXL and Vero E6 cells.

[0036] II. Experimental Results See results Figure 1 The effect of ALA on the growth of 293T-hAXL and Vero E6 cells was detected using the MTS colorimetric assay. The results showed that up to the highest dose of 160 μM, ALA did not have a significant growth inhibitory effect on either 293T-hAXL or Vero E6 cells. These results indicate that ALA has low cytotoxicity.

[0037] Example 2: Effects of Ascomlactone A on SARS-CoV-2 replication I. Experimental Methods (1) The effect of compound ALA in inhibiting the entry of SARS-CoV-2 into cells was evaluated using a pseudovirus infection system for SARS-CoV-2. The specific method was as follows: different doses of ALA (final concentrations: 0.1, 0.5, 1, 5, 10, 20 μM) were mixed with pseudoviruses of different variants of SARS-CoV-2 Delta, BA.1, or D614G (all with a titer of 10 μM). 3 TCID 50 Premixed with 293T-hAXL cells (2 × 10⁶ / ml) in 24-well culture plates, then mixed with the premixed ... 4 Cells were co-incubated in wells ( / well) for 48 hours. Cell lysates were then collected to measure luciferase activity in order to evaluate the adsorption and penetration efficiency of SARS-CoV-2 pseudoviruses.

[0038] (2) 293T-hAXL cells (2×10 4 Cells were seeded in 24-well plates and treated with different concentrations of ALA compounds (final concentrations: 0.1, 0.5, 1, 5, 10, 20 μM) for 2 h after 24 h. Then, the cells were infected with SARS-CoV-2 BA.1b virus solution (MOI=0.5) for 24 h (the corresponding concentration of ALA was added during the infection process, and the concentration remained unchanged during the preservation treatment). The cell culture supernatant was collected, and the effect of different concentrations of ALA on the amount of live virus in the cell supernatant was determined by plaque assay.

[0039] II. Experimental Results See results Figure 2Different doses of ALA were premixed with pseudoviruses of different SARS-CoV-2 Delta, BA.1, or D614G variants, and then co-incubated with susceptible 293T-hAXL cells. Cell lysates were collected and luciferase activity was measured to evaluate the adsorption and penetration efficiency of SARS-CoV-2 pseudoviruses. The results showed that ALA strongly inhibited the adsorption and penetration of pseudoviruses of the Delta, BA.1, and D614G variants in a dose-dependent manner. The IC50 values ​​for each group were [not specified in the original text]. 50 The values ​​were 2.61±0.23 μM, 3.81±0.55 μM, and 3.13±0.43 μM, respectively. This indicates that ALA can inhibit the invasion of the novel coronavirus into host cells by blocking the receptor AXL.

[0040] Different doses of ALA were premixed with live SARS-CoV-2 BA.1 virus and then co-incubated with 293T-hAXL. Quantification was performed using a plaque formation assay based on Vero E6 cells. The results showed that ALA had strong anti-SARS-CoV-2 activity, with a half-maximal effective concentration (EC50) of [missing value]. 50 The value was 1.66 ± 0.30 μM.

[0041] Example 3: Protective effect of Ascomlactone A on host cell survival during the process I. Experimental Method 293T-hAXL cells (1×10) 4 Cells were seeded in 96-well plates and treated with different concentrations of ALA compound (final concentrations: 0, 0.1, 0.5, 1, 5, 10, 20 μM) for 2 hours after 24 hours. Cells were then infected with SARS-CoV-2 solution for 24 hours (with corresponding concentrations of ALA added during the infection process). Cell viability was measured using the MTS assay to detect the protective effect of ALA on host cell survival during SARS-CoV-2 infection.

[0042] II. Experimental Results See results Figure 3 The effect of ALA on the cytopathic effect (CPE) of 293T-hAXL cells infected with SARS-CoV-2 BA.1 was detected by MTS colorimetric assay. The results showed that ALA significantly reduced CPE induced by SARS-CoV-2 BA.1 infection in 293T-hAXL cells in a dose-dependent manner.

[0043] Example 4: Ascomlactone A is an AXL receptor inhibitor I. Experimental Methods Biacore biomolecular interaction analysis (BIA) is a biosensing and analysis technique based on the physical optical phenomenon of surface plasmon resonance (SPR) to track the interactions between biomolecules in real time.

[0044] The target protein AXL recombinant protein was immobilized on the surface of a sensor chip, and the target compound ALA, which interacts with it, was dissolved in a solution and flowed through the surface of the NTA photosensitive chip. The detector can track and detect the changes in the binding and dissociation of molecules in the solution with molecules on the chip surface throughout the entire process. This was used to identify the affinity of candidate compounds ALA for AXL. Monorapvir was used as a negative pair.

[0045] The simplified experimental procedure is as follows: Using BIA technology, when the isoelectric point (PI) of the sample is unknown, sodium acetate solutions with pH values ​​of 5.5, 5.0, 4.5, and 4.0 were used for testing in sequence. The concentration of human AXL recombinant protein (rhAXL) was selected from 10 to 50 μg / ml, the injection time was selected as 180 seconds, and 50 mM NaOH was used as the washing solution. Based on the results, an appropriate pH concentration was selected as the coupling condition. ALA samples were dissolved in buffer solutions of different pH values ​​to give them different amounts of charge, and the samples were flowed through the chip surface for preliminary experiments. The binding curve between the samples and the chip was observed to determine the conditions that are conducive to the adsorption of protein molecules onto the chip surface. Based on the preliminary experimental results, human AXL recombinant protein (rhAXL) was immobilized on the Biacore CM5 chip using the amino coupling method. The corresponding AXL recombinant protein was diluted with a prepared buffer solution, centrifuged, and then loaded onto the chip. The sample buffer solution was kept as consistent as possible with the system carrier buffer solution. The changes in the binding and dissociation process of molecules in the detection solution with molecules on the chip surface were observed. Based on the obtained affinity experimental data between small molecules and target proteins, the direct interaction between ALA and AXL receptor proteins was identified.

[0046] II. Experimental Results The direct interaction between compound ALA and recombinant human AXL protein (rhAXL) was detected using surface plasmon resonance assays (Biacore molecular interaction assay system). The results showed that ( Figure 4 Its equilibrium dissociation constant K D It is 1.13 × 10 -6This result indicates that ALA can directly bind to the rhAXL protein, while the negative control, Molnupiravir, has no binding effect on rhAXL. Furthermore, in the constructed 293T cells (293T-hAXL) that stably and highly express the hAXL receptor, the intracellular AXL expression level did not change significantly under different concentrations of ALA treatment.

[0047] Example 5: Ascomlactone A in vivo against SARS-CoV-2 infection I. Experimental Methods Using an ACE2 humanized mouse model (hACE2-All CDS-B6J, product number: C001191, available from Cyagen Biosciences Co., Ltd.) as the infection subject, a solvent control group and groups with different concentrations of ALA compound (0, 5, 10 mg / kg) were set up. After anesthesia, SARS-CoV-2 virus (1×10⁻⁶ mg / kg) was injected intranasally. 4 Mice were weighed on days 0, 1, 2, 3, 4, and 5 using PFU (Proteinized ALA). ACE2 humanized mice were sacrificed on day 5, and lung or bronchial tissue was collected and preserved for quantitative viral detection and analysis. The effect of different concentrations of ALA on the viral nucleic acid content in mouse lung or bronchial tissue was determined using absolute real-time quantitative RT-PCR (primers are shown in Table 1).

[0048] Table 1 Primers and probes for SARS-CoV-2 virus N gene nucleic acid detection

[0049] II. Experimental Results The in vivo anti-SARS-CoV-2 activity of ALA was evaluated using an hACE2 transgenic mouse model. Results showed ( Figure 5 On day 5, the average body weight of mice treated with 5 mg / kg and 10 mg / kg ALA was significantly higher than that of the solvent control group, indicating that ALA can slow down weight loss caused by SARS-CoV-2 infection. Absolute real-time quantitative RT-PCR was used to quantify the viral genome copy number, and the results showed that the average viral load in the lung tissue of the solvent control mice was 7.04 × 10⁻⁶. 6 In mice treated with ALA at a dose of 5 mg / kg, viral load in lung tissue decreased to 1.47 × 10 copies / g. 4 In mice treated with ALA at a dose of 10 mg / kg, the viral load in the lung tissue decreased to 6.43 × 10 copies / g. 2 The results indicate that ALA possesses strong in vivo anti-SARS-CoV-2 activity.

[0050] Example 6: Ascomlactone A, as an interferon inducer, promoted the production of type I interferon in host cells. I. Experimental Methods The human embryonic kidney cell line 293T (which is commonly used in the promoter-luciferase dual reporter gene system experiment, so this invention will also conduct this experiment in 293T cells; the 293T cell line is preserved by the inventor's research group) was used as the infection target.

[0051] While infecting cells with live SARS-CoV-2 BA.1 virus, 293T cells were treated with different concentrations (0, 0.5, 1, 2 μM) of ALA compound for 24 h. Cell lysates were collected, and the IFN-β-Luc dual reporter gene system was used to detect whether ALA promoted IFN-β promoter transfection in cells. Cell culture supernatant was collected, and ELISA was used to detect the effect of ALA treatment on the expression level of intracellular IFN-β (Proteintech, catalog number: KE00195). Real-time quantitative RT-PCR (primers shown in Table 2) was used to detect the expression level of host cells. IFNB and interferon-stimulated genes ISGs (including OAS1 , MX1 and Tetherin The expression of TBK1 and IRF3 was increased; analysis by immunoblotting showed that the phosphorylation levels of TBK1 and IRF3 in 293T-hAXL cells treated with different concentrations of ALA increased in a dose-dependent manner.

[0052] Table 2 Primers related to gene detection

[0053] II. Experimental Results Detection using the interferon gene promoter-luciferase dual reporter system revealed that ALA promotes transcriptional activation of the IFN-β reporter gene. ELISA analysis showed that ALA promotes increased IFN-β secretion in cells. Real-time quantitative RT-PCR analysis revealed... Figure 6 ALA can induce increased expression of IFN-β and ISGs (OAS1, MX1, and Tetherin). These results indicate that ALA can promote the production of type I interferon.

[0054] Analysis using immunoblotting revealed ( Figure 7Treatment with different concentrations of ALA resulted in a dose-dependent increase in the phosphorylation levels of TBK1 and IRF3 in 293T-hAXL cells. Nuclear-cytoplasmic separation combined with Western blotting analysis revealed that ALA promotes the translocation of IRF3 from the cytoplasm to the nucleus, thereby activating the transcription of type I interferon. These results preliminarily indicate that ALA enhances the production of type I interferon by activating the TBK1-IRF3 signaling pathway.

Claims

1. Use of a compound having a structure as shown in formula (I) in the manufacture of a medicament for the treatment of a novel coronavirus, 。 2. Use of a compound having a structure as shown in formula (I) in the manufacture of a medicament for the treatment of a disease and / or a symptom caused by a novel coronavirus, 。 3. Use of a pharmaceutically acceptable salt of a compound having a structure as shown in formula (I) in the manufacture of a medicament for the treatment of a novel coronavirus, 。 4. Use of a pharmaceutically acceptable salt of a compound having a structure as shown in formula (I) in the manufacture of a medicament for the treatment of a disease and / or a symptom caused by a novel coronavirus, 。 5. Use according to any one of claims 1 to 4, characterized in that, The compound having a structure as shown in formula (I) is a host AXL receptor inhibitor, which blocks the binding site of SARS-CoV-2 to its receptor AXL.

6. Use according to any one of claims 1 to 4, characterized in that, The compound having a structure as shown in formula (I) inhibits the infectivity of a novel coronavirus.

7. Use according to any one of claims 1 to 4, characterized in that, The compound having a structure as shown in formula (I) promotes the production of type I interferon in host cells.

8. Use according to any one of claims 1 to 4, characterized in that, The dosage form of the medicament is a tablet, an oral liquid, an oral pill, an oral granule, an oral powder, an injection, an eye drop, a nose drop, an aerosol or an inhalant.

9. Use of a compound having a structure as shown in formula (I) in the manufacture of a type I interferon inducer, 。 10. Use of a compound having a structure as shown in formula (I) in the manufacture of a type I interferon agonist, 。

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