Use of navitoclax (ABT263) in the preparation of broad-spectrum antiviral inhibitors

Abstract

The application relates to the field of biological medicine, and particularly relates to application of Navitoclax (ABT263) in preparation of a broad-spectrum antiviral inhibitor. The application of the compound Navitoclax (ABT263), an analogue or a salt thereof shown in formula (I) in preparation of a broad-spectrum antiviral inhibitor. It is found in the application that the compound has broad-spectrum antiviral activity, including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, the compound can act on the virus entry stage, and the virus is resisted outside the target cell. In addition, the compound has a high selection index (CC50 / IC50) for different viruses, and has considerable safety. The application provides a broad-spectrum antiviral small-molecule compound, which has the advantages of simple preparation method, low cost, easy transportation and storage, high safety and good activity, and has a broad prospect in the fields of biological medicine and response to new emerging infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of Navitoclax (ABT263) in the preparation of a broad-spectrum antiviral inhibitor. Background Technology

[0002] Similar to SARS-CoV, SARS-CoV-2 belongs to the beta-coronavirus family and exhibits high transmissibility and pathogenicity in humans. The continuous emergence of numerous SARS-CoV-2 variants, accompanied by significant immune escape phenomena, particularly the Omicron mutant strain which has evolved into several sublineages and caused numerous reinfections in recovered COVID-19 patients and breakthrough infections from vaccines, poses a serious challenge to existing vaccines and antiviral drugs in clinical practice. Therefore, there is an urgent need to develop broad-spectrum antiviral drugs targeting current SARS-CoV-2 and its variants, as well as other coronaviruses.

[0003] Small molecule antiviral drugs have unique advantages such as low preparation cost, good oral bioavailability, mature production process, and good in vivo safety. Therefore, the development of new small molecule broad-spectrum antiviral drugs has obvious advantages in responding to the occurrence of future emerging infectious diseases. Summary of the Invention

[0004] The purpose of this invention is to provide an application of Navitoclax (ABT263) in the preparation of broad-spectrum antiviral inhibitors.

[0005] To achieve the above objectives, the following technical solution is adopted:

[0006] The application of Navitoclax (ABT263) in the preparation of broad-spectrum antiviral inhibitors, such as the application of compounds of formula (I), Navitoclax, analogues or salts thereof in the preparation of broad-spectrum antiviral inhibitors.

[0007]

[0008] Preferably, the compound Navitoclax (ABT263) as shown in formula (I) is used in the preparation of broad-spectrum antiviral inhibitors.

[0009] The viruses mentioned include type I enveloped viruses, type II enveloped viruses, and type III enveloped viruses. These include, but are not limited to, coronaviruses and influenza viruses.

[0010] A broad-spectrum antiviral inhibitor, the formulation comprising the compound Navitoclax (ABT263) as shown in formula (I), an analogue or a salt thereof.

[0011] The formulation uses the compound Navitoclax (ABT263) of formula (I), its analogues or salts thereof as the active pharmaceutical ingredient, and prepares an inhibitor with a pharmaceutically acceptable adjuvant; wherein the active ingredient accounts for 0.01-99% of the mass of the formulation.

[0012] The dosage form of the preparation is injection, capsule, tablet, oral liquid, granule, ointment, aerosol or suppository.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. The compounds provided by this invention are small molecule compounds that can effectively inhibit broad-spectrum viruses, and have unique advantages in dealing with emerging and re-emerging infectious diseases caused by enveloped viruses;

[0015] 2. The method for preparing compounds provided by this invention has a short preparation cycle, low cost, and the resulting compounds have stable properties. Attached Figure Description

[0016] Figure 1 This describes the effect of the compound in Example 2 of this invention on the formation of hexahelices in the HR1 and HR2 regions of coronaviruses. A. Native-PAGE detection; B. Band grayscale scanning and statistical analysis;

[0017] Figure 2 The compound in Example 2 of this invention exhibits anti-SARS-CoV-2 pseudovirus infection activity;

[0018] Figure 3 This demonstrates the activity of the compound in Example 2 of this invention against SARS-CoV-2 omeprazole and its major circulating subvariant pseudovirus infection;

[0019] Figure 4 The results are the Time of addition experiment (A), cell safety (B), and drug selection index (SI) statistics (C) of the compound in Example 3 of this invention;

[0020] Figure 5 The inhibitory activity of the compound in Example 4 of this invention on the infection activity of SARS-CoV(A) and MERS-CoV(B) pseudoviruses on Caco-2 cells was detected by Luciferase assay and drug selection index (SI) statistics (C).

[0021] Figure 6 The compound in Example 4 of this invention inhibits live Omeprone virus BA.2. Immunofluorescence was used to detect the viral N protein (A), and RT-PCR was used to detect the N gene (B).

[0022] Figure 7The results of the compound in Example 5 of this invention inhibiting H5N1 pseudovirus infection (A), cell safety (B), and drug selection index (SI) statistics (C) are shown. Detailed Implementation

[0023] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] Unless otherwise defined, all terms used in this invention (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms, such as those defined in common dictionaries, shall be interpreted as having the meaning consistent with their meaning in the context of the relevant art and shall not be interpreted in an idealized or overly formal sense, unless expressly so defined in this invention.

[0025] This invention provides the application of Navitoclax (ABT263) in the preparation of broad-spectrum antiviral inhibitors, including but not limited to SARS-CoV-2, SARS-CoV, and MERS-CoV. This compound acts on the viral entry phase, resisting the virus outside target cells. Furthermore, this compound exhibits a high selectivity index (CC50 / IC50) against various viruses and demonstrates considerable safety. This invention provides a broad-spectrum antiviral small molecule compound with advantages such as simple preparation method, low cost, easy transportation and storage, high safety, and good activity, showing broad prospects in the biomedical field and in responding to emerging and re-emerging infectious diseases.

[0026] The viruses used in the following examples are all known and can be purchased from relevant departments and prepared with reference to relevant literature.

[0027] Example 1: Structural Characterization of Compounds

[0028] Typically, this embodiment includes a compound called Navitoclax (ABT263) with the structure shown in the following formula; and the compound was purchased from TagerMol China.

[0029]

[0030] Example 2: Native-PAGE detection of the effect of compounds on hexahelical formation in the HR1 and HR2 regions of coronaviruses

[0031] Compounds diluted with DMSO to final concentrations of 50, 100, and 150 μM were incubated with an equal volume of SARS-CoV-2 HR1P (300 μM) at 37 °C for 1 hour. Then, SARS-CoV-2 HR2P (50 μM) was added and incubated at 37 °C for 0.5 hours to obtain a mixture. HR1P, HR2P, and the mixture were spotted onto an 18% Tris-glycine gel. After electrophoresis for 3 hours, the gel was stained with Coomassie Brilliant Blue. Figure 1 ).

[0032] This embodiment is provided by Figure 1 It is evident that the compound can compete with SARS-CoV-2HR2P to influence the formation of the hexahelix.

[0033] Example 3: Inhibitory effect of the compound on SARS-CoV-2 pseudovirus

[0034] In this embodiment, the backbone plasmid (pNL4-3.Luc.RE-) was provided by the US NIH AIDS Reagents and References Project, catalog number 3418. The pcDNA3.1-SARS-CoV-2-S plasmid was constructed and preserved in our laboratory (Emerg MicrobesInfect.2023,12:2178241).

[0035] Plasmids containing SARS-CoV-2 and its mutant S protein (pcDNA3.1-SARS-CoV-2-S-D614G / BA.1 / BA.3 / BA.2.12.1 / BA.2.75 / BA.5.2 / BA4.6 / BF.7 / XBB / XBB.1.5 / CH.1.1, all constructed in our laboratory JMed Virol.2023,95:e28641) and backbone plasmid (pNL4-3.Luc.RE-) were co-transfected into 293T cells. After 48 hours, the cell supernatant was collected, centrifuged at 3000 rpm for 10 min, aliquoted and frozen at -80°C, and the pseudovirus titer was measured on Caco-2 target cells.

[0036] To determine the antiviral activity of the compound, it was dissolved in DMSO and serially diluted in 96-well plates with serum-free medium. An equal volume of SARS-CoV-2 (D614G) pseudovirus was then added, and the mixture was incubated at 37°C for 45 minutes. Finally, 1 x 10⁻⁶ ppm of the solution was added to the solution. 4 In Caco-2 cells at / wells, the experiment included a peptide drug control (EK1), a cell control (cells only, without virus or drug), and a virus control (without drug). After incubation at 37°C for 48 hours, the inhibitory activity of the compounds was detected by a Luciferase assay. Figure 2Using the same method described above, the inhibitory activity of the compounds against different mutant pseudoviruses of SARS-CoV-2 was detected, such as... Figure 2 and Figure 3 As shown, the compounds exhibit varying degrees of inhibitory effects on pseudovirus infection by SARS-CoV-2 mutants.

[0037] Caco-2 cells were seeded in 96-well plates (1 × 10⁶ cells per well). 4 Cells were cultured (at 12 hours prior to infection). The compound was added at 0.5 hours before infection, 0 hours (with pseudovirus added simultaneously), and 0.5 hours, 1 hour, 2 hours, 4 hours, and 8 hours after infection, with a final compound concentration of 18 μM. After 12 hours of pseudovirus infection, the culture medium was replaced with fresh medium and cultured for another 48 hours. As previously described, luminescence detection was then performed using a luciferase detection system (Promega, Madison, WI, USA). Figure 4 As shown in Figure A, the compound's effect in inhibiting pseudovirus entry gradually decreased over time, indicating that the compound may act on the entry phase of the virus.

[0038] The above-mentioned series of infectious compounds were serially diluted in 96-well plates with serum-free medium and added to a solution containing 1x10⁻⁶ compounds. 4 In Caco-2 cells at / well, a cell-free and drug-free group (DMEM group) and a drug-free group were simultaneously set up as controls. After incubation at 37°C for 48 hours, the safety of the compound on different cell types was tested according to the CCK-8 kit instructions. Figure 4 As shown in B and C, the compounds only exhibit some cytotoxicity under high concentration conditions, but still have a relatively safe selectivity index (SI = CC50 / IC50).

[0039] Example 4: Inhibitory effect of compounds against SARS-CoV and MERS-CoV pseudoviruses

[0040] In this embodiment, the pcDNA3.1-SARS-CoV-S plasmid and pcDNA3.1-MERS-S plasmid were constructed and preserved in our laboratory (construction process can be found in Cell Res. 2020, 30:343-355). The backbone plasmid is the same as in Example 3. The packaging method for SARS-CoV and MERS-CoV pseudoviruses is the same as in Example 3.

[0041] 293T cells were co-transfected with pcDNA3.1-SARS-CoV-S plasmid or pcDNA3.1-MERS-S plasmid and pNL4-3.Luc.RE- plasmid. After 48 hours, the cell supernatant was collected, centrifuged at 3000 rpm for 10 min, and aliquoted and stored at -80°C. The titers of SARS-CoV and MERS-CoV pseudoviruses were measured on Caco-2 cells. Using a method similar to that in Example 3, the inhibitory effects of the compounds on SARS-CoV and MERS-CoV pseudoviruses were detected on Caco-2 cells. Figure 5 ).

[0042] Depend on Figure 5 It is evident that the compound exhibits varying degrees of inhibitory effects against SARS-CoV and MERS-CoV pseudovirus infection, and possesses a relatively safe selectivity index.

[0043] Example 5: Inhibitory effect of compounds against live SARS-CoV-2 virus

[0044] Caco-2 cells were seeded into 96-well plates. Twenty-four hours later, 0.01 μL of multiplicity of infection (MOI) OmicronBA.2 live virus was incubated with serially diluted Navitoclax at 37°C for 30 minutes, and the mixture was transferred to Caco-2 cells. Forty-eight hours later, the collected Caco-2 cells were fixed with 4% paraformaldehyde and immersed in 0.2% Triton X-100. Non-specific binding sites were blocked with 2% skim milk for 2 hours, followed by incubation at 37°C for 2 hours with rabbit anti-SARS-CoV-2N protein antibody. Subsequently, the cells were incubated with polyclonal sheep anti-rabbit IgG antibody (FITC-labeled) at 37°C for 1 hour and counterstained with DAPI staining solution. Fluorescence images were acquired under a fluorescence microscope (Thermo Fisher Scientific). Figure 6 A).

[0045] The supernatant was collected from the SARS-CoV-2 live virus inhibition assay described above for the quantification of SARS-CoV-2 copies. Figure 6 B). After extracting RNA from the viral supernatant, qPCR experiments were performed using a One-Step PrimeScrip RT-PCR kit (Takara, Tokyo, Japan) to detect the SARS-CoV-2N gene copy number. The primers and probes used to detect SARS-CoV-2N gene mRNA are as follows: Forward primer:

[0046] 5'-GGGGAACTTCTCCTGCTAGAAT-3'; Reverse primer:

[0047] 5'-CAGACATTTTGCTCTCAAGCTG-3'; and probe: 5'-FAM-TTGCTGCTGCTTGACAGATTTAMRA-3'.

[0048] Depend on Figure 6 It is evident that the compound can effectively inhibit the infection of live Omeprazole virus BA.2.

[0049] Example 6: Inhibitory effect of compound on influenza pseudovirus H5N1

[0050] The plasmids used to package the H5N1 pseudovirus were: the H5N1 HA plasmid pVKD-HA, the H5N1 NA plasmid pVKD-NA, and the HIV backbone plasmid pNL4-3.Luc.R containing the luciferase reporter gene. - E - The HA and NA genes encoded by the pVKD-HA and pVKD-NA plasmids were obtained from influenza virus A / Shanghai / 4664T / 2013 (H5N1) via RT-PCR (both plasmids were available from the New York Blood Center or could be purchased directly). The plasmids containing the HA gene and the plasmid containing the NA gene were then compared with pNL4-3.Luc.R. - E - The three plasmids were co-transfected into 293T cells. After 48 hours, the cell supernatant was collected, centrifuged at 3000 rpm for 10 min, and the resulting supernatant was aliquoted and frozen at -80℃. The titer of H5N1 pseudovirus was then measured on MDCK cells.

[0051] Using a method similar to that in Example 3, the inhibitory effect of the compound on H5N1 pseudovirus was detected in MDCK cells. Figure 7 A) MDCK cells, purchased from the Cell Bank of the Chinese Academy of Sciences.

[0052] The compound Navitoclax (ABT263) was serially diluted in a 96-well plate with serum-free medium and added to a solution containing 1 x 10⁻⁶ mg / L. 4 In MDCK cells at / well, a cell-free and drug-free group (i.e., DMEM group) and a drug-free group were set up as controls. After incubation at 37°C for 48 hours, the cytotoxicity of the compounds to different cell types was detected according to the CCK-8 kit instructions. Figure 7 B).

[0053] Depend on Figure 7 It is evident that the compound exhibits certain inhibitory activity against the influenza pseudovirus H5N1 and has a relatively safe selectivity coefficient.

[0054] In summary, the compound Navitoclax (ABT263) of this invention can serve as a broad-spectrum antiviral agent, capable of broadly inhibiting enveloped viruses, including SARS-CoV-2, SARS-CoV, and MERS-CoV. The antiviral spectrum of the compound should include, but is not limited to, the enveloped viruses listed above. This type of compound exhibits high activity and a high selectivity index (CC50 / IC50), and is expected to effectively combat infections caused by emerging and re-emerging viral infectious diseases or cross-infection by multiple viruses.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a compound of formula (I) Navitoclax (ABT263) or a salt thereof in the preparation of an antiviral inhibitor, characterized in that: The virus is SARS-CoV-2 live virus Omicron BA.2 or influenza pseudovirus H5N1; The compound Navitoclax (ABT263) is shown as formula (I), Formula (I).

2. The use according to claim 1, characterized in that: The compound Navitoclax (ABT263) shown as formula (I) or a salt thereof is used as a pharmaceutical active ingredient to prepare a preparation with a pharmaceutically acceptable adjuvant; wherein the active ingredient accounts for 0.01-99% of the mass of the preparation.

3. The use according to claim 2, characterized in that: The dosage form of the preparation is injection, capsule, tablet, oral liquid, granule, ointment, aerosol or suppository.

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