Application of 2'-Cl-2'-dAMP, a chlorinated nucleoside monophosphate molecule, in the preparation of anti-coronavirus drugs

Abstract

The present invention provides an application of the chlorinated nucleoside monophosphate molecule 2'-Cl-2'-dAMP in the preparation of anti-coronavirus drugs. It has obvious inhibitory effects on the wild strain of the novel coronavirus, the mutant strains of the novel coronavirus (Beta strain, Delta strain, Omicron strain), and other coronaviruses such as human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, and murine coronavirus MHV, laying a foundation for the clinical treatment of coronaviruses and having great clinical significance and promotion value.

Description

Technical Field

[0001] This invention patent relates to the field of biomedical or biopharmaceutical technology, and particularly to the application of the catalytic product of halogenase AdeV, the chlorinated nucleoside monophosphate molecule 2'-Cl-2'-dAMP, in the preparation of anti-coronavirus drugs. Background Art

[0002] The novel coronavirus (SARS-CoV-2) belongs to the genus β coronavirus. The main transmission routes are through respiratory droplets and close contact. In relatively enclosed environments, it can be transmitted through aerosols, and infection may also occur after contact with virus-contaminated items. The general population is susceptible.

[0003] The novel coronavirus is a positive-sense single-stranded RNA virus. Its genome consists of at least 10 open reading frames and some regulatory genes, encoding structural proteins (nucleocapsid protein N, transmembrane protein M, envelope protein E, spike protein S) and non-structural proteins (protease 3CLpro, protease PLpro, helicase, RNA-dependent RNA polymerase RdRp) respectively. These non-structural proteins are key enzymes in the virus life cycle. Nucleoside drugs are an important class of drugs used clinically to treat viral infectious diseases, and nearly 50% of the currently used antiviral drugs are nucleoside drugs. Currently, some research work has screened some nucleoside antiviral drugs effective against the novel coronavirus through the strategy of "repurposing old drugs", such as Sofosbuvir, Galidesvir, Favipiravir, Ribavirin, Azudine, etc. However, the inhibitory activity of these drugs against the novel coronavirus is generally weak or has obvious cytotoxicity, and the treatment effect is limited.

[0004] Therefore, it is necessary to develop an innovative and effective anti-coronavirus drug. Summary of the Invention

[0005] In medicinal chemistry, the modification of biomolecules with halogens (Cl, Br, I, and F) is a research hotspot, and approximately 50% of the best-selling drugs contain covalently linked halogen atoms. Halogens have the characteristics of increasing molecular lipophilicity, improving the permeability of lipid membranes, and the electronegativity of halogens can increase the biological activity of the central molecule. For example, the introduction of strong electron-withdrawing groups such as F can enhance binding, metabolic stability, changes in physical properties, and selective activity. In addition, there are also literature reports that through the modification of pyrimidine rings, purine rings, and riboses with halogens, nucleic acid derivatives exhibit stronger antiviral, antimetabolic, antibacterial, and other properties. For example, 8-haloadenine nucleoside compounds have been used in the development of drugs for the treatment of viral infections such as HIV and HBV.

[0006] Most of the halogenated nucleoside molecules currently in use are synthesized chemically, and only a few naturally occurring halogenated nucleoside molecules are isolated from microorganisms. Recently, a halogenase AdeV from actinomycetes was found to be involved in the biosynthesis of adechlorin. AdeV can catalyze the chlorination of 2'-dAMP to generate 2'-Cl-2'-dAMP, which is a key intermediate in the biosynthetic pathway of adechlorin. AdeV is the first halogenase that can catalyze the selective halogenation of nucleotides and exhibits limited substrate specificity for several 2'-dAMP analogs. We resolved the complex crystal structure of the AdeV complex and elucidated the molecular mechanism of the interaction between AdeV and 2'-dAMP and its analogs through molecular docking, mutagenesis, and biochemical analysis, providing data support for a better understanding of the catalytic mechanism of AdeV. At the same time, we also found that the catalytic product 2'-Cl-2'-dAMP of halogenase AdeV has the ability to significantly inhibit virus replication activity and can be used as a potential nucleoside antiviral drug candidate.

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. Thus, in the first aspect of the present invention, the present invention provides the use of 2'-Cl-2'-dAMP or its pharmaceutically acceptable salt in the preparation of an anti-coronavirus drug.

[0008] The structure of 2'-Cl-2'-dAMP is shown as follows:

[0009]

[0010] In one or more embodiments of the present invention, the coronavirus includes one or more of human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, murine coronavirus MHV, and novel coronavirus.

[0011] In one or more embodiments of the present invention, the coronavirus is the novel coronavirus.

[0012] In one or more embodiments of the present invention, the novel coronavirus is selected from one or more of the novel coronavirus wild strain, novel coronavirus variant Beta strain, novel coronavirus variant Delta strain, and novel coronavirus variant Omicron strain.

[0013] In the second aspect of the present invention, the present invention provides the use of a pharmaceutical preparation in the preparation of an anti-coronavirus drug. The active ingredient of the pharmaceutical preparation includes 2’-Cl-2’-dAMP or its pharmaceutically acceptable salt, and the concentration of the 2’-Cl-2’-dAMP or its pharmaceutically acceptable salt is 2 to 10 μM, preferably 10 μM. When the final concentration of 2’-Cl-2’-dAMP or its pharmaceutically acceptable salt reaches 10 μM, the inhibition rate of virus replication can reach more than 90%.

[0014] The present invention proposes that 2’-Cl-2’-dAMP has low cytotoxicity and is basically non-toxic to cells at a final concentration below 10 μM. The cells tested include African green monkey kidney cells VERO E6, human lung fibroblasts MRC-5, human colorectal epithelial cells HCT-8, and mouse liver cells NCTC clone 1469.

[0015] Specifically, 2’-Cl-2’-dAMP is obtained by catalyzing 2’-dAMP with the actinomycete-derived halogenase AdeV.

[0016] The beneficial effects of the present invention are as follows:

[0017] The present invention provides a nucleoside analogue 2’-Cl-2’-dAMP with a broad-spectrum inhibitory effect on coronaviruses, which is a natural catalytic product of the actinomycete-derived halogenase AdeV. The 2’-Cl-2’-dAMP provided by the present invention has an obvious inhibitory effect on the replication activities of the wild type of the novel coronavirus, the variants of the novel coronavirus (Beta strain, Delta strain, Omicron strain), and other coronaviruses (human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, murine coronavirus MHV), laying a foundation for the clinical treatment of novel coronavirus infection and having great clinical significance and promotion value. Description of the Drawings

[0018] Figure 1 It is a graph showing the results of cytotoxicity determination of 2’-Cl-2’-dAMP in Example 3 of the present invention;

[0019] Figure 2 It is a graph showing the inhibitory effect of 2’-Cl-2’-dAMP on the wild type of SARS-CoV-2 in Example 4 of the present invention;

[0020] Figure 3 It is a graph showing the inhibitory effect of 2’-Cl-2’-dAMP on the replication ability of different variants of SARS-CoV-2 in Example 5 of the present invention;

[0021] Figure 4This is the inhibitory effect diagram of 2’-Cl-2’-dAMP on the replication ability of different variants of SARS-CoV-2 in Example 5 of the present invention;

[0022] Figure 5 This is the inhibitory effect diagram of 2’-Cl-2’-dAMP on the replication ability of other coronaviruses in Example 6 of the present invention. Detailed implementation manners

[0023] The present invention will be further described below in conjunction with specific embodiments and drawings. However, the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those conditions not specified in the following embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The methods used, unless otherwise specified, are all conventional methods well-known in the art. The consumables and reagents used, unless otherwise specified, are all commercially available. Unless otherwise stated, the professional and scientific terms used herein have the same meaning as those familiar to skilled personnel in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0024] Example 1

[0025] Design and synthesis of primers and probes involved in the embodiments of the present invention:

[0026] The primer and probe sequences for detecting the SARS-CoV-2 N gene are from the sequences recommended by the Chinese Center for Disease Control and Prevention. The upstream primer (SEQ ID NO.1) is 5’-ggggaacttctcctgctagaat-3’, the downstream primer (SEQ ID NO.2) is 5’-cagacattttgctctcaagctg-3’, and the probe (SEQ ID NO.3) is 5’-FAM-ttgctgctgcttgacagatt-BHQ1-3’. All primers and probes were synthesized by a conventional biological synthesis company.

[0027] Preparation of 2’-Cl-2’-dAMP

[0028] According to the genomic sequence information of actinomycetes (KP025768.1), the orf9 gene sequence was synthesized in a conventional gene synthesis service company (GenScript), and its protein product is AdeV halogenase. The orf9 gene sequence is shown as SEQ ID NO.4.

[0029] The synthesized orf9 gene sequence was inserted into the prokaryotic expression vector pET28-a and transferred into BL21(DE3) bacteria for cultivation. When the OD value of the bacterial solution reached 0.6, IPTG with a final concentration of 0.1 mM was added for induction at a temperature of 18 °C for 20 hours. After the induction, the bacterial cells were collected by centrifugation, and the cell pellet was resuspended in a lysis buffer (20 mM Tris-HCl, 50 mM NaCl, 1 mM TECP) and sonicated at 4 °C. The sonicated bacterial lysate was centrifuged to obtain a precipitate, and the supernatant was passed through a Ni-NTA column. The column was rinsed with 50 mM imidazole, and finally, the target protein, namely AdeV halide, was eluted with 200 mM imidazole. The eluate was desalted using an HP-10 column and finally stored at -80 °C.

[0030] The purified AdeV halide was used for the synthesis reaction of 2'-Cl-2'-dAMP. The reaction buffer contained 30 μM AdeV halide, 0.1 mM Fe 2+ 、1 mM Cl - 、0.5 mM α-KG, and 0.15 mM 2'-dAMP. After the reaction, an equal volume of absolute ethanol was added to terminate the reaction, and the target product was purified by HPLC to obtain 2'-Cl-2'-dAMP.

[0031] Example 2

[0032] The cell culture methods involved in the examples of the present invention are as follows:

[0033] VERO E6 cells (African green monkey kidney cells, ATCC CRL-1586) were cultured using MEM medium (product of Gibco), and fetal bovine serum (product of Gibco) with a final concentration of 10% was added to the medium to provide the necessary nutrients for cell growth. These cells were used for SARS-CoV-2 virus and NL63 virus-related infection experiments.

[0034] MRC-5 cells (human lung fibroblasts, ATCC CCL-171) were cultured using MEM medium (product of Gibco), and fetal bovine serum (product of Gibco) with a final concentration of 10% was added to the medium to provide the necessary nutrients for cell growth. These cells were used for 229E virus-related infection experiments.

[0035] HCT-8 cells (human colorectal epithelial cells, ATCC CCL-244) were cultured using RPMI-1640 medium (product of Gibco), and fetal bovine serum (product of Gibco) with a final concentration of 10% was added to the medium to provide the necessary nutrients for cell growth. These cells were used for OC43 virus-related infection experiments.

[0036] NCTC clone 1469 cells (mouse liver cells, ATCC CCL-9.1) were cultured using DMEM medium (product of Gibco), and fetal bovine serum (product of Gibco) with a final concentration of 10% was added to the medium to provide the necessary nutrients for cell growth. These cells were used for MHV virus-related infection experiments.

[0037] All cells were cultured in an incubator environment with 5% CO 2 at 37°C.

[0038] Example 3: Cytotoxicity assay of 2’-Cl-2’-dAMP

[0039] VERO E6 cells, MRC-5 cells, HCT-8 cells, and NCTC clone 1469 cells were seeded into 96-well plates, with one plate for each type of cell. The seeding density was 3000 cells per well, and the medium volume was 100 μL. 2’-Cl-2’-dAMP was added to the wells to make the final concentrations 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM respectively. There were 8 replicates for each concentration, and control wells (added with the same volume of sterile water) were set up. After incubation for 48 hours, 10 μL of CCK-8 reagent was added to each well. After incubation for 1 hour, the absorbance at 450 nm was measured. When the final concentrations of 2’-Cl-2’-dAMP were 0 μM, 2 μM, 5 μM, and 10 μM respectively, the activities of different cells were measured (detected using CCK8 reagent), and there were 3 replicate samples for the activity detection at each concentration. The results are as Figure 1 shown. It can be seen from the experimental results that when the concentration of 2’-Cl-2’-dAMP was controlled within 10 μM, there was no obvious cytotoxicity to VERO E6 cells, MRC-5 cells, HCT-8 cells, and NCTC clone 1469 cells.

[0040] Example 4: Inhibitory effect of 2’-Cl-2’-dAMP on wild-type SARS-CoV-2

[0041] In this example, all infection experiments related to live SARS-CoV-2 virus were carried out in a biosafety level 3 (BSL-3) facility authorized by the National Health Commission of the People's Republic of China. All infected samples were inactivated using the protocol recommended in the "Prevention and Control Plan for Coronavirus Disease 2019 (Eighth Edition)" and were tested after being taken out of the BSL-3 facility.

[0042] VERO E6 cells were seeded in 12-well plates. When the cell density reached 50 - 60%, the cells were infected with wild-type SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1. After 2 hours of infection, the cell supernatant was discarded, and fresh DMEM medium was replaced. 2’-Cl-2’-dAMP was added to make its final concentrations 2 μM, 5 μM, and 10 μM respectively. Three replicate wells were set for each concentration, and control wells (added with the same volume of sterile water) were also set up. After 60 hours of infection, the cell supernatant was discarded, and cell RNA was collected using TRIzol (a product of Thermo Fisher). Total RNA from each well was extracted and reverse transcribed into cDNA using MMLV reverse transcriptase (a product of Promega) with random primers. Finally, the ratio of the expression levels of the SARS-CoV-2 N gene and the internal reference gene (GAPDH) in the cDNA was detected by fluorescence quantitative PCR. This ratio was the inhibition rate of 2’-Cl-2’-dAMP against wild-type SARS-CoV-2, and the inhibitory effects of 2’-Cl-2’-dAMP at different concentrations were calculated. The results are shown in Table 1, Figure 2 as follows. When the final concentrations of 2’-Cl-2’-dAMP were 0 μM, 2 μM, 5 μM, and 10 μM respectively, the ratio of the intracellular viral RNA to the expression level of the internal reference gene GAPDH was measured, and the inhibition rate at different concentrations was calculated compared with the control group.

[0043] Table 1 Inhibitory effect of 2’-Cl-2’-dAMP on wild-type SARS-CoV-2

[0044] Final concentration of compound (μM) Inhibition rate 0 0％ 2 44.3％ 5 63.7％ 10 76.3％

[0045] From the above experimental results, it can be seen that 2’-Cl-2’-dAMP has an inhibitory effect on the replication of wild-type SARS-CoV-2 at different concentrations (0 μM, 2 μM, 5 μM, 10 μM). When the final concentration reaches 10 μM, the inhibitory activity is 76.3%, showing a relatively obvious inhibitory effect.

[0046] Example 5: Inhibitory effect of 2’-Cl-2’-dAMP on the replication ability of different variants of SARS-CoV-2

[0047] VERO E6 cells were seeded in 12-well plates. When the cell density reached 50 - 60%, the cells were infected with different variants of SARS-CoV-2 (novel coronavirus variants (Beta strain, Delta strain, Omicron strain)) at a multiplicity of infection (MOI) of 0.1. After 2 hours of infection, the cell supernatant was discarded, and fresh DMEM medium was replaced. 2’-Cl-2’-dAMP was added to make its final concentrations 2 μM, 5 μM, and 10 μM respectively. Three replicate wells were set for each concentration, and control wells (added with the same volume of sterile water) were set up simultaneously. After 60 hours of infection, the cell supernatant and cells in each well were collected respectively. RNA in the supernatant was extracted using TRIZOL LS (product of Thermo Fisher), and cell RNA was collected using TRIZOL (product of Thermo Fisher). For the detection of the virus content in the supernatant, TaqMan Fast Virus 1-Step Master Mix kit (product of Thermo Fisher) was used for TaqMan RT-PCR one-step detection, with the target being the SARS-CoV-2 N gene. For the detection of the virus content in cells, cDNA was reverse transcribed by using MMLV reverse transcriptase plus random primers. Finally, the ratio of the expression levels of the SARS-CoV-2 N gene and the internal reference gene (GAPDH) in the cDNA was detected by fluorescence quantitative PCR, and this ratio was the inhibition rate of 2’-Cl-2’-dAMP on different variants of SARS-CoV-2 (novel coronavirus variants (Beta strain, Delta strain, Omicron strain)). When the final concentrations of 2’-Cl-2’-dAMP were 0 μM, 2 μM, 5 μM, and 10 μM respectively, the copy number of the virus in the supernatant was measured. The results are as Figure 3 shown. When the final concentrations of 2’-Cl-2’-dAMP were 0 μM, 2 μM, 5 μM, and 10 μM respectively, the ratio of the intracellular virus RNA to the expression level of the internal reference gene GAPDH was measured. The results are as Figure 4 shown. The inhibitory effect of 2’-Cl-2’-dAMP on the replication ability of different variants of SARS-CoV-2 is shown in Table 2.

[0048] Table 2 Inhibitory effect of 2’-Cl-2’-dAMP on the replication ability of different variants of SARS-CoV-2

[0049]

[0050] Conclusion: 2’-Cl-2’-dAMP has an inhibitory effect on the replication of multiple variants of SARS-CoV-2 at different concentrations. When the final concentration reaches 10 μM, the inhibitory activity exceeds 75%, showing a relatively obvious inhibitory effect.

[0051] Example 6: Inhibitory effect of 2’-Cl-2’-dAMP on the replication ability of other coronaviruses (229E, OC43, NL63, MHV)

[0052] For the human coronavirus 229E-related infection experiment, MRC-5 cells were used; for the human coronavirus OC43-related infection experiment, HCT-8 cells were used; for the human coronavirus NL63-related infection experiment, VERO E6 cells were used; and for the murine coronavirus MHV-related infection experiment, NCTC clone 1469 cells were used.

[0053] In a 12-well plate, susceptible host cells of various coronaviruses were inoculated. When the cell density reached 50-60%, they were infected with the corresponding coronaviruses at an MOI of 0.1. After 2 hours of infection, the cell supernatant was discarded, and fresh DMEM medium was replaced. 2’-Cl-2’-dAMP was added to a final concentration of 10 μM, and a control well (added with the same volume of sterile water) was set up. After 72 hours of infection, the cell status was observed under an optical microscope. After 2 hours of infection of different coronaviruses (human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, murine coronavirus MHV) with their corresponding susceptible host cells, the cells were incubated with 2’-Cl-2’-dAMP at final concentrations of 0 μM and 10 μM. After 72 hours of infection, the cell status was observed under an optical microscope. The results are as Figure 5 shown.

[0054] Conclusion: Treating cells with 2’-Cl-2’-dAMP at a concentration of 10 μM can effectively inhibit the replication ability of the corresponding coronavirus in the cells and reduce the cytopathic effect caused by the virus on the cells.

[0055] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, and all should be included within the protection scope of the present invention.

Claims

1. Use of 2'-Cl-2'-dAMP or its pharmaceutically acceptable salt in the preparation of an anti-coronavirus drug, characterized in that, the coronavirus includes one or more of human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, murine coronavirus MHV, SARS-Cov-2, and the structure of 2'-Cl-2'-dAMP is shown as follows:

2. The use according to claim 1, characterized in that, the SARS-Cov-2 is selected from one or more of the novel coronavirus wild strain, novel coronavirus variant Beta strain, novel coronavirus variant Delta strain, and novel coronavirus variant Omicron strain.

3. Use of a pharmaceutical preparation in the preparation of an anti-coronavirus drug, characterized in that, the active ingredient of the pharmaceutical preparation includes 2'-Cl-2'-dAMP or its pharmaceutically acceptable salt, the concentration of the 2'-Cl-2'-dAMP or its pharmaceutically acceptable salt is 2-10 μM, the coronavirus includes one or more of human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, murine coronavirus MHV, SARS-Cov-2, and the structure of 2'-Cl-2'-dAMP is shown as follows:

4. The use according to claim 3, characterized in that, the concentration of the 2'-Cl-2'-dAMP or its pharmaceutically acceptable salt is 10 μM.

5. The use according to any one of claims 1-4, characterized in that, 2'-Cl-2'-dAMP is obtained by catalyzing 2'-dAMP with the actinomycete-derived halogenase AdeV.

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