CN113855694A - Application of adenosine kinase inhibitor in preparing anti-coronavirus preparation - Google Patents

Abstract

The invention discloses application of an adenosine kinase inhibitor in preparation of an anti-coronavirus preparation, wherein the adenosine kinase inhibitor is 5-iodotubercidin. The invention discovers for the first time that the 5-iodotubercidin can obviously inhibit the activity of RNA polymerase dependent on coronavirus and shows good resistance to SARS-CoV-2 exonuclease, and further provides the application of the 5-iodotubercidin in preparing the coronavirus-dependent RNA polymerase antagonist and the coronavirus resistant medicament. The invention provides a small molecular compound which can effectively resist coronavirus, thereby further enriching the treatment means of coronavirus.

Description

Application of adenosine kinase inhibitor in preparing anti-coronavirus preparation

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of an adenosine kinase inhibitor in preparation of an anti-coronavirus preparation.

Background

The new type of coronavirus pneumonia (cova virus disease 2019, covi-19) seriously threatens human health and is caused by the new type of coronavirus (SARS-CoV-2). The discovery of new drugs for resisting the novel coronavirus diseases is an important component of prevention and control measures, and the discovery of the new drugs provides an important treatment scheme for clinically preventing and treating the novel coronavirus diseases.

The gene sequencing result shows that SARS-CoV-2 belongs to the same coronavirus beta genus as SARS-CoV and MERS-CoV, and belongs to the same S genus as SARS-CoVThe gene sequence homology of ARS-CoV is 75% -80%, and the homology with bat SARS-like coronavirus (bat-sl-coVZC45) is over 85%. Compared with SARS-CoV virus, SARS-CoV-2 virus has stronger infectivity and infection coefficient (R)₀) Can reach 5.7(SARS-CoV is about 2.0), and its latent period is 7-14 days, and is greatly greater than SARS-CoV (2-7 days), and has lots of asymptomatic infectors with infection capacity, and the virus control is faced with great challenge.

Vaccines and antiviral drugs are the most effective means for the control of new coronaviruses. The new use of old medicine is an important idea for searching anti-SARS-CoV-2 virus medicine, and the WHO provides 4 most promising new coronary pneumonia treatment schemes: ritexivir, chloroquine and hydroxychloroquine, lopinavir/ritonavir and lopinavir/ritonavir/arbidol. Most nucleoside analogs, when incorporated into viral RNA, are cleaved by the non-structural protein nsp14 exonuclease expressed by coronaviruses (nsp 14-Exon), but Reidesvir is resistant to nsp14-Exon, which makes it superior to other nucleoside analogs. However, the subsequent clinical trial research shows that the medicines have great side effect and insignificant curative effect and are not suitable for the wide anti-SARS-CoV-2 treatment. Therefore, there is still a need to develop new effective therapeutic drugs against neocoronary infections.

The SARS-CoV-2 virus is a positive strand single-stranded RNA virus, and the SARS-CoV-2 genome length is 29.8 kb-29.9 kb. The life cycle is currently thought to involve viral entry by adsorption, decortication, transcription and replication of the genome, synthesis of viral proteins, assembly and release. The virus encodes 16 non-structural proteins (nsp 1-nsp 16). Some of these 16 nsps are enzymes essential for SARS-CoV-2 replication. Including papain-like protease (nsp3), chymotrypsin-like protease (3CL protease, nsp5), primer-enzyme complex (nsp7-nsp8), RNA-dependent RNA polymerase RdRp (nsp12), helicase (nsp13), and exonuclease (nsp14), which are potential targets for anti-SARS-CoV-2 drug development.

The RNA dependent RNA polymerase (RdRp) of the new coronavirus is mainly responsible for the replication of the viral RNA and is a target point for researching broad-spectrum antiviral drugs. In one aspect, the RNA polymerase of the novel coronaviruses is a RNA-dependent RNA polymerase encoded by the virus itself, which is completely different from DNA-dependent RNA polymerase in mammalian cells; in addition, the gene sequences of each subunit of the new coronavirus coding polymerase are highly conserved among different new coronavirus strains, so that the new coronavirus RNA polymerase is an anti-new coronavirus drug target with great potentiality. Studies have shown that the formation of RdRp requires the involvement of the viral accessory factors nsp7 and nsp 8. Nucleoside analogs act as broad-spectrum inhibitors of RdRp, and act as antiviral agents by blocking RNA synthesis by occupying the nucleoside binding space through viral RdRp binding. A novel nucleoside analog prodrug, Reidesivir (GS-5734), developed by Gilidard (Gilead) Inc. in the United states, is metabolized in vivo to GS-441542 upon intravenous administration, and GS-441542 is further triphosphoric to a pharmacologically active Nucleoside Triphosphate (NTP). Upon entry of the virus into the cell, RdRp and the cell compete for binding of the nucleoside. Since the RNA polymerase of the cells cannot recognize nucleoside analogs such as Reidsivir, it is not affected. Similarly, other nucleoside analogs can only be metabolized to triphosphorylation in vivo to have antiviral effects. The complex action mechanism seriously restricts the development of an enzyme activity analysis method and directly influences the screening and research and development of the medicine taking the new crown RdRp as a target spot. Although active RdRp can be isolated from purified virions in basic studies, this costly and low-yield assay is difficult to apply to large-scale drug screening and drug efficacy evaluation.

5-Iodotubercidin (ITU) is a broad class of kinase inhibitors, including Cdc 2-like kinase (CLKs), dual-specific tyrosine (Y) phosphorylation-regulated kinases (DYRKs), and the like, and particularly inhibitors of Adenosine kinase (ADK), which can have an effect on the proliferation and survival of cells. 5-iodotubercidin is widely used as an inhibitor of adenosine kinase. Intracellular adenylate kinase is a key enzyme for adenosine reuptake and metabolism, regulates the intracellular adenosine level by converting intracellular adenosine into AMP, and can effectively improve the adenosine level in the microenvironment of cells by inhibiting the activity of adenosine kinase. Intervention targeted at adenosine kinase can result in an imbalance in adenosine homeostasis, which plays an important role in epilepsy, degenerative neurological diseases, and psychiatric disorders. Zhang Xiaomin et al (Zhang Xiaomin. 5-iodotubercidin induces neural tube malformation and mechanism research [ D ] Shanxi university of medicine) found that 5-iodotubercidin can cause DNA damage of colon cancer cell HCT-116, activate AMP-p53 pathway, cause up-regulation of p53 gene expression, and promote apoptosis of colon cancer cell; meanwhile, the application of 5-iodotubercidin can also enable the colon cancer tumor of a nude mouse to be rapidly reduced, while the control group still can grow normally; therefore, the 5-iodotubercidin is supposed to be a new gene poison medicine and has the potential of chemotherapy effect. Studies on the demethylation of the DLC-1 gene of HT-29 cells by 5-iodotubercidin (Zhang sensitively, Wang Asia Xue, Xiekai, et al. [ J ]. third military medical science report, 2015,37(002):106-110) found that 5-iodotubercidin can inhibit the proliferation of colon cancer cells HT-29, induce the apoptosis of colon cancer cells, and predict that 5-iodotubercidin may be used as a new chemotherapeutic drug. In addition, the research on the effect of the Monauspicious (the research on the effect of Iodotubarcidin loaded by the CD44 targeted nanoliposome on breast cancer tumor stem cells [ D ]. southern KAI university) finds that the 5-Iodotubercidin can reduce the proportion of side group cells in breast cancer cells MDA-MB-231 and lung cancer cells H460, and possibly plays a role in inhibiting breast cancer stem cells. This suggests that 5-iodotubercidin has different biological activities and that compounds effective against coronaviruses are still under development.

Disclosure of Invention

The invention aims to provide application of an adenosine kinase inhibitor in preparing an anti-coronavirus preparation.

In order to achieve the object of the present invention, in a first aspect, the present invention provides the use of an adenosine kinase inhibitor which is 5-iodotubercidin, having the structure shown in formula I:

such coronaviruses include SARS-CoV-2, such as HCoV-OC43 and HCoV-NL63, and the like.

The invention discovers a compound capable of effectively inhibiting SARS-CoV-2RdRp, successfully establishes a cell level CoV-RdRp-Gluc reporting system by utilizing Gaussia luciferase (Gluc) reporting gene, and carries out quantitative analysis and verification on the activity of the new coronavirus RdRp by detecting fluorescence intensity through the established cell level SARS-CoV-2RdRp high-flux reporting system. Changes of the levels of Gluc positive strand RNA and Gluc negative strand RNA in the CoV-RdRp-Gluc system are detected through qRT-PCR experiments, and 5-iodotubercidin is found to be capable of remarkably reducing the levels of the Gluc positive strand RNA and the Gluc negative strand RNA. Then, the invention further verifies by utilizing a SARS-CoV-2RdRp exonuclease model, and finds that the compound still can show good resistance to SARS-CoV-2 exonuclease nsp 14. Thus, the effectiveness of the compounds of formula I for coronavirus inhibition is ultimately demonstrated.

In a second aspect, the invention provides an anti-coronavirus medicament, the active ingredient of which is 5-iodotubercidin.

Further, the medicament also comprises pharmaceutically acceptable auxiliary agents.

The auxiliary agent comprises one or more of carrier, excipient or diluent.

Carrier materials include, but are not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), sparingly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, and carboxymethylcellulose, etc.). Water-soluble carrier materials are preferred. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. For example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents, such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil, etc.; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. For example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, talc, and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. Such as polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like. In order to prepare the unit dosage form into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, sweeteners, or other materials may be added to the pharmaceutical preparation according to actual needs. The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; for luminal administration, such as rectally and vaginally; administration to the respiratory tract, e.g., nasally; administration to the mucosa. The above administration route is preferably oral administration or injection (intravenous injection).

The invention discovers for the first time that the 5-iodotubercidin can inhibit the activity of RNA polymerase dependent on SARS-CoV-2RNA of the new coronavirus so as to inhibit the replication of the new coronavirus, and can effectively inhibit the replication of the coronavirus HCoV-OC43 and HCoV-NL63, thereby providing key data for developing novel anti-new coronavirus lead compounds and providing theoretical basis for developing effective treatment medicaments of COVID-19. Can also be used for preparing medicines for resisting various types of coronavirus.

In a third aspect, the invention provides the use of an adenosine kinase inhibitor against coronavirus.

The adenosine kinase inhibitor is 5-iodotubercidin,

such coronaviruses include SARS-CoV-2, such as HCoV-OC43 and HCoV-NL63, and the like.

Compared with the prior art, the invention has at least the following advantages:

the 5-iodotubercidin can show good activity of inhibiting the new coronavirus SARS-CoV-2RdRp under the condition of low cytotoxicity, and the inhibition effect is verified on the level of CoV-RdRp-Gluc RNA. Therefore, the compound is expected to be developed into a small molecule inhibitor of SARS-CoV-2RdRp clinically. In addition, the compound is found to be capable of effectively inhibiting the replication of HCoV-OC43 and HCoV-NL63 viruses.

Drawings

Fig. 1 is a schematic diagram of a report system construction principle and a verification result diagram according to embodiment 1 of the present invention; wherein A is a principle schematic diagram constructed by using a CoV-RdRp-Gluc report system; the left graph in B is the expression result of nsp12, nsp7 and nsp8 proteins in a CoV-RdRp-Gluc report system detected by Western Blot; the right panel in B is a microplate reader Centro XS³LB 960 test CoV-RdRp-Gluc reportExpression results of each group of glucs in the system; c is a Z' factor detection result of a CoV-RdRp-Gluc reporting system for high-throughput detection.

FIG. 2 is a graph showing the inhibitory effect of SARS-CoV-2 RNA-dependent RNA polymerase in example 2 of the present invention; wherein A is a result graph of the inhibition rate of 5-iodotubercidin and Reidesvir to SARS-CoV-2 RNA-dependent RNA polymerase; b is the EC of compound 5-iodotubercidin on SARS-CoV-2RNA dependent RNA polymerase₅₀A result graph; EC of SARS-CoV-2 RNA-dependent RNA polymerase with Ryscerivir C as positive compound₅₀And (5) a result chart.

FIG. 3 is a graph showing the effect of the transcriptional activity of SARS-CoV-2 RNA-dependent RNA polymerase in example 3 of the present invention; wherein A is a result chart of the influence of compound 5-iodotubercidin on the transcription activity of SARS-CoV-2 RNA-dependent RNA polymerase at the mRNA level; b is a graph of the effect of the positive compound, Reidcisvir, on the transcription activity of SARS-CoV-2 RNA-dependent RNA polymerase at the mRNA level.

FIG. 4 is a graph showing the results of an experiment in example 4 of the present invention; wherein, A is the expression result of nsp12, nsp7, nsp8, nsp14 and nsp10 proteins in a report system for detecting SARS-CoV-2 exonuclease nsp14 by using Western Blot; b is Centro XS using a microplate reader³LB 960 detects the expression result of Gluc in a SARS-CoV-2 exonuclease nsp14 report system; c is a resistance result graph of the compound 5-iodotubercidin against SARS-CoV-2 exonuclease nsp 14; d is a result chart of the resistance of positive compound Ridexilvir to SARS-CoV-2 exonuclease nsp14, and E is a result chart of the resistance of negative compound ribavirin to SARS-CoV-2 exonuclease nsp 14.

FIG. 5 is a graph showing the inhibitory effect of 5-iodotubercidin and Reddesivir (Remdesivir) as compounds in example 5 of the present invention against coronavirus HCoV-OC43(A) and HCoV-NL63(B) strains.

Detailed Description

The present invention provides a compound which is effective against coronavirus.

The invention adopts the following technical scheme: the invention provides application of an adenosine kinase inhibitor in preparation of a coronavirus dependent RNA polymerase antagonist, wherein the adenosine kinase inhibitor is 5-iodotubercidin, namely a compound shown in a formula I.

The compound shown in the formula I can inhibit the activity of RNA polymerase dependent on new coronavirus SARS-CoV-2RNA so as to inhibit the replication of the new coronavirus, can effectively inhibit the replication of coronavirus HCoV-OC43 and HCoV-NL63, provides data support for the development of a novel anti-new coronavirus lead compound, provides a theoretical basis for the development of an effective treatment medicament of COVID-19, and can also be used for preparing medicaments for resisting various types of coronaviruses.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The eukaryotic codon-optimized plasmid pCOVID19-nsp7, eukaryotic codon-optimized plasmid pCOVID19-nsp8, eukaryotic codon-optimized plasmid pCOVID19-nsp10, eukaryotic codon-optimized plasmid pCOVID19-nsp12, eukaryotic codon-optimized plasmid pCOVID19-nsp14 referred to in the following examples are plasmids obtained by seamless cloning of nsp7, nsp8, nsp10, nsp12, nsp14 genes into pCMV6-entry vector (purchased from ORIGEN) by XhoI, respectively.

nsp7, nsp8, nsp10, nsp12, nsp14 genes are found in Ren LL, Wang YM, Wu ZQ, Xiang ZC, Guo L, Xu T, Jiang YZ, Xiong Y, Li YJ, Li XW, Li H, Fan GH, Gu XY, Xiao Y, Gao H, Xu JY, Yang F, Wang XM, WuC, Chen L, Liu YW, Liu B, Yang J, Wang XR, Dong J, Li L, Huang CL, Zhao JP, Hu Y, CheZS, Liu LL, an Qin ZH, Qin C, Jin Q, Cao B, Waiveg JW.entification of a nova ronavirus cage culture photovoltaic cell in a. 00: 00-00. doi:10.1097/CM 9.0000000000000722.

Example 1 establishment of cellular level CoV-RdRp-Gluc reporter System

In this example, a luciferase reporter system specifically initiated by SARS-CoV-2RdRp, CoV-RdRp-Gluc for short, was established. The pCoV-Gluc plasmid was constructed by inserting Gaussia luciferase (Gluc) coding sequence (as shown in SEQ ID NO: 1) between the 5 'UTR and 3' UTR of the novel coronavirus, then inserting it between pRetrox-light-Pur (Clontech) vectors BamHI-HF (# R3136L, NEB) and Notl (NotI-HF, NEB) sites, using forward primer (5'-GGCGGATCCATTAAAGGTTTATAC-3') and reverse primer (5'-TTAGCGGCCGCGTCATTCTCCTAAGAA-3'). Under the action of the CMV promoter, Gluc (mRNA) of the positive strand is transcribed and translated to produce Gluc protein. When a novel coronavirus RNA-dependent RNA polymerase (nsp12) is simultaneously expressed in the system, RdRp first synthesizes negative-strand vRNA using positive-strand Gluc as a template, and then the vRNA is transcribed into positive-strand Gluc (mrna) and finally translated into Gluc protein. Thus, the increased Gluc after addition of RdRp reflects the activity of SARS-CoV-2 RdRp. The schematic construction is shown in A in FIG. 1.

In addition, the involvement of nsp7 and nsp8 is required for nsp12 to function. Thus, the present example was provided with three sets of tests for comparison: CoV-Gluc (pCoV-Gluc plasmid constructed above) (10ng) was expressed alone, CoV-Gluc (10ng) and eukaryotic codon-optimized plasmid pCOVID19-nsp12(nsp12) (200ng) were co-expressed, and CoV-Gluc (10ng), nsp12(200ng), eukaryotic codon-optimized plasmid pCOVID19-nsp7(nsp7) (600ng) and eukaryotic codon-optimized plasmid pCOVID19-nsp8(nsp8) (600ng) were co-expressed. In order to ensure that the total transfection amount of the plasmids is the same in the experimental process, the empty vector plasmid pCMV6-entry is added to each group for supplement. See B in fig. 1 for specific results. The left graph in B is the expression result of nsp12, nsp7 and nsp8 proteins in a CoV-RdRp-Gluc report system detected by Western Blot in example 1 of the invention; the right panel in B shows the microplate reader Centro XS used in example 1 of the present invention³LB 960 examined the expression results of each group of glucs in the CoV-RdRp-Gluc reporter system.

Western Blot、Centro XS³The specific detection method of LB 960 is as follows:

(1) western Blot experiment

Mixing 2.5X 10⁵HEK 293T cell suspension per mL was seeded at 2mL per well in 6-well plates. The culture media used by the HEK 293T cells are DMEM culture media containing 10% Fetal Bovine Serum (FBS); the cells were cultured in a constant temperature incubator containing 5% carbon dioxide at 37 ℃. When the cells grow to 80%, plasmid transformation is carried out on each hole of the HEK 293T cell group according to the design of the experimental groupAnd (6) dyeing. The medium was changed to DMEM medium containing 10% Fetal Bovine Serum (FBS) 4 hours after transfection; the culture was continued for 24 hours. The medium was discarded, 80. mu.L of RIPA lysate was added to each well, the lysate was transferred to 1.5ml EP tubes for 20 minutes on ice, 20. mu.L of 5 Xprotein loading buffer (loading buffer) was added to each tube, and the tubes were metal-bathed at 100 ℃ for 30 minutes. SDS-PAGE gel electrophoresis separation, and Western Blot for detecting the expression levels of nsp12, nsp7 and nsp 8. The detection result is shown in the left panel B in FIG. 1.

(2) Gaussia luciferase Activity assay

Dissolving 250 μ g of substrate Coelenterazine-h lyophilized powder in 600 μ L of anhydrous ethanol to obtain substrate mother liquor with concentration of 1.022mM, and storing at-20 deg.C; before measurement, the stock solution was diluted in PBS at a ratio (volume ratio) of 1:60 to prepare a substrate working solution. Standing at room temperature for 30min to stabilize the working solution, and performing light-shielding treatment in the whole process due to unstable substrate light; mu.L of cell culture supernatant (cell supernatant after 24 hours of culture after transfection in the Western Blot experiment described above) was transferred to a white opaque 96-well plate and applied to a microplate reader Centro XS³LB 960 Autosampler the photophobically incubated substrate working solution was added well by well at a rate of 60. mu.L per well, and the signal was collected for 0.5 sec and measured in Relative Light Units (RLU). Three parallel sets were set up and statistical analyses were performed, where P < 0.01 and P < 0.001 was referenced to the individual CoV-Gluc expression set. Experimental data on

Expressed and plotted using GraphPad Prism5.0 and statistically analyzed. The detection result is shown in the right diagram B in FIG. 1.

The experimental results showed that the groups co-expressing CoV-Gluc and nsp12 and the groups CoV-Gluc, nsp12, nsp7 and nsp8 were 2-fold and 38-fold, respectively, higher than the group expressing CoV-Gluc alone. The above results show that the expression of Gluc protein in the CoV-RdRp-Gluc system constructed by the present invention is specifically dependent on the novel coronavirus RdRp.

In this embodiment, the CoV-RdRp-Gluc report system is further subjected to Z' factor detection, and the detection method is as follows:

mixing 2.5X 10⁵A suspension of HEK 293T cells (2 mL/mL) was plated in 6-well plates and cultured for 24h before transfection. The method is carried out in two groups: cells expressing plasmid CoV-Gluc (10ng) were used as a negative control (supplemented with 1.4. mu.g of plasmid pCMV6-entry, purchased from ORIGEN), and cells co-expressing plasmids CoV-Gluc (10ng), nsp12(200ng), nsp7(600ng) and nsp8(600ng) were used as a positive control. After 12h of transfection, two groups of cells were digested with pancreatin, added with DMEM medium to prepare cell suspension, and inoculated on a 96-well plate (1X 10)⁵one/mL), 100. mu.l per well. Luciferase activity was measured after 24h incubation. The calculation formula is as follows: the Z' factor is 1- (3 × positive control relative fluorescence value SD +3 × negative control relative fluorescence value SD)/(positive control relative fluorescence value average-negative control relative fluorescence value average).

The detection result is shown as C in figure 1, which is the detection result of the Z 'factor of the CoV-RdRp-Gluc reporting system used for high-throughput screening in the embodiment 1 of the invention, and the Z' factor is 0.73 according to the result, which meets the requirement of high-throughput screening.

Example 2 adenosine kinase inhibitor (5-iodotubercidin) was tested using the CoV-RdRp-Gluc reporter System

Mixing 2.5X 10⁵HEK 293T cell suspension per mL was seeded at 2mL per well in 6-well plates. When the cells were 80% long, the HEK 293T cell group was co-transfected with 10ng of pCoV-Gluc constructed in example 1, 200ng of eukaryotic codon-optimized plasmid pCOVID19-nsp12, 600ng of eukaryotic codon-optimized plasmid pCOVID19-nsp7 and 600ng of eukaryotic codon-optimized plasmid pCOVID19-nsp8 per well. The medium was changed to DMEM medium containing 10% Fetal Bovine Serum (FBS) 4 hours after transfection, and the culture was continued for 12 hours. Digesting the cells in a six-well plate to prepare a cell suspension, and preparing the cell suspension according to the proportion of 1.0 multiplied by 10⁴HEK 293T cells were seeded at 100. mu.L/well in 96-well plates per ml. mu.L of compound 5-iodotubercidin (compound of formula I) and Reidesciclovir, at a concentration of 10. mu.M, was added to each well, respectively, followed by culturing the cells for 24 hours. Wherein the negative control group was added with 1. mu.L DMSO (dimethyl sulfoxide) per well. Finally, fluorescence was measured according to the method of example 1. Three groups of parallels are set up in the experiment and statistical analysis is carried out, and the data are compared with the value of DMSO groupThe percent treatment is carried out. Experimental data on

Expressed and plotted using GraphPad Prism5.0 and statistically analyzed. The test results showed that compound 5-iodotubercidin exhibited more than 85% activity against SARS-CoV-2RdRp compared to the negative control group (DMSO group) (see a in fig. 2, wherein P < 0.001 is with DMSO group as reference). The inhibition rate is (negative control group-sample group)/(negative control group-positive control group) × 100%.

This example continues the validation of the SARS-CoV-2RdRp activity assay for compound 5-iodotubercidin to detect EC inhibition₅₀. Experimental procedures As described above, the final concentrations of compound 5-iodotubercidin and Reidesciclovir were 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 μ M, respectively. Experimental groups were run in triplicate, each with reference to the DMSO group of each group. Experimental data on

Expressed and plotted using GraphPad prism5.0 and statistically analyzed. The results are shown in FIG. 2, where B is the EC of the compound 5-iodotubercidin against SARS-CoV-Gluc RdRp₅₀Results are shown in the figure, C is the EC of positive compound Reidesvir against SARS-CoV-Gluc RdRp₅₀And (5) a result chart. The experimental result shows that the compound 5-iodotubercidin inhibits the EC of SARS-CoV-2RdRp₅₀0.75. mu.M, and the EC of the positive compound, Reidesvir₅₀The value was 1.07. mu.M, both EC₅₀The ratio of (5-iodotubercidin/Redoxivir) was 0.69. Experimental results show that the small molecular 5-iodotubercidin is a SARS-CoV-2RdRp inhibitor with great potential.

Example 35 iodotubercidin inhibition of the transcriptional Activity of SARS-CoV-2RdRp at the mRNA level

The effect of 5-iodotubercidin on SARS-CoV-Gluc was examined at the mRNA level to further confirm the effect of 5-iodotubercidin on SARS-CoV-2RdRp transcriptional activity. The invention adopts qRT-PCR method to detect the inhibition of 5-iodotubercidin on SARS-CoV-2RdRp at mRNA level.

Mixing 2.5X 10⁵HEK 293T cell suspension per mL was seeded at 2mL per well in 6-well plates. When the cells grew to 80%, the HEK 293T cell group was co-transfected with 10ng pCoV-Gluc, 200ng eukaryotic codon optimized plasmid pCOVID19-nsp12, 600ng eukaryotic codon optimized plasmid pCOVID19-nsp7 and 600ng eukaryotic codon optimized plasmid pCOVID19-nsp8 per well. The medium was changed to DMEM medium containing 10% Fetal Bovine Serum (FBS) 4 hours after transfection, one group was added with 2. mu.L of 5.00mM or 10.00mM of 5-iodotubercidin per well, the other group was added with DMSO (dimethyl sulfoxide) as a negative control, while the broad spectrum antiviral nucleoside inhibitor Reidsciclovir was used as a positive control, and 2. mu.L of 5.00mM or 10.00mM of Reidsciclovir per well, and incubation was continued for 24 hours. And finally, absorbing the culture medium, adding 1ml of Trizol reagent into each hole, extracting mRNA from the Trizol, obtaining whole genome cDNA by using a reverse transcription method, and then carrying out qRT-PCR to detect the expression quantity of Gluc mRNA. The invention utilizes GAPDH as an internal reference gene. Three replicates were set up and statistically analyzed, where P < 0.01 and P < 0.001 was referenced in the DMSO group. Experimental data on

Expressed and plotted using GraphPad Prism5.0 and statistically analyzed. The experimental results are shown in FIG. 3, wherein A is a graph showing the effect of 5-iodotubercidin on the transcription activity of SARS-CoV-2RdRp at the mRNA level in example 3 of the present invention; b is a graph showing the effect of the positive compound of Reidcisvir in the mRNA level on the transcription activity of SARS-CoV-2RdRp in example 3 of the present invention.

The test result shows that the 5-iodotubercidin can obviously inhibit the Gluc mRNA expression quantity in SARS-CoV-Gluc under the concentration of 2.00 mu M and 10.00 mu M, and the invention respectively illustrates that the 5-iodotubercidin is a novel SARS-CoV-2RdRp small molecule inhibitor from different aspects by combining the test result of the example 2.

Example 45 resistance test of iodotubercidin to New coronavirus exonuclease nsp14

When nucleoside analogs inhibit the new coronavirus RdRp, most nucleoside analogs are less resistant to nsp14 due to the calibrating function of the new coronavirus exonuclease nsp 14. This also results in nucleoside analogs being less effective as RdRp inhibitors in the treatment of new coronaviruses. Therefore, the invention co-expresses nsp14 and nsp10 on the basis of SARS-CoV-Gluc system, and detects the resistance of the compound to the new coronavirus exonuclease.

The specific experimental procedures were as described in example 1, except that: in HEK 293T cells, CoV-Gluc (10ng) was expressed separately per well, CoV-Gluc (10ng), nsp12(200ng), nsp7(600ng) and nsp8(600ng) were co-expressed, CoV-Gluc (10ng), nsp12(200ng), nsp7(600ng), nsp8(600ng), eukaryotic codon optimized plasmid pCOVID19-nsp14(nsp14) (500ng) and eukaryotic codon optimized plasmid pCOVID19-nsp10(nsp10) (500ng) were co-expressed, and the other operations were the same. In order to show the position and expression of nsp10 and nsp14 proteins more clearly, two proteins were also separately detected in Western Blot of A in FIG. 4. Three parallel sets were set up and statistically analyzed, where P < 0.001, with reference to the individual CoV-Gluc expressing set. Experimental data on

Expressed and plotted using GraphPad Prism5.0 and statistically analyzed. Specific results are shown in FIG. 4, wherein A is the expression results of nsp12, nsp7, nsp8, nsp14 and nsp10 proteins in a report system for detecting CoV-RdRp-Gluc exonuclease nsp14 by using Western Blot in example 4 of the invention; b is the microplate reader Centro XS used in example 4 of the invention³LB 960 detects the result of Gluc expression in CoV-RdRp-Gluc exonuclease nsp14 reporter system.

This example further tests the resistance of 5-iodotubercidin and Reidesvir to SARS-CoV-2 exonuclease nsp 14. The specific method comprises the following steps:

specific experimental procedures refer to example 2, except that CoV-Gluc (10ng), nsp12(200ng), nsp7(600ng), nsp8(600ng), nsp14(500ng) and nsp10(500ng) were co-expressed in HEK 293T cells, and the other operations were the same.

The detection result is shown as C, D, E in FIG. 4, C is the result of the resistance of 5-iodotubercidin to SARS-CoV-2 exonuclease nsp14 in example 4 of the present invention; d is a graph showing the results of the resistance of the positive compound of Reidcisvir to SARS-CoV-2 exonuclease nsp14 in example 4 of the present invention, and E is a graph showing the results of the resistance of the negative compound of ribavirin to SARS-CoV-2 exonuclease nsp14 in example 4 of the present invention.

The test result shows that 5-iodotubercidin has EC against SARS-CoV-2 exonuclease₅₀1.42. mu.M, EC of positive compound Reidesvir against SARS-CoV-2 exonuclease₅₀The value was 2.08. mu.M, both EC₅₀The ratio of (5-iodotubercidin/Reidesvir) was 0.68. And the negative compound ribavirin has EC on SARS-CoV-2 exonuclease₅₀Has a value of>1000. mu.M. The experimental result shows that the 5-iodotubercidin is insensitive to SARS-CoV-2 exonuclease nsp14 and is slightly superior to the Redexilvir.

Example 55-experiment of iodotubercidin inhibition of replication of coronavirus HCoV-OC43 and HCoV-NL63

HCT-8 or LLC-MK2 cells at 1.0X 10⁴Each cell was inoculated in a 96-well plate at 100. mu.L/well in a DMEM medium containing 10% FBS for 48 hours. The supernatant was discarded and replaced with fresh DMEM medium containing 2% FBS. HCoV-OC43 (ATCC: VR-1558) was infected with HCT-8 cells at MOI of 0.1 or HCoV-NL43(Amsterdam I) virus was infected with LLC-MK2 at MOI of 0.01. Gradient diluted compound 5-iodotubercidin (final concentration 2-fold dilution from 50. mu.M, dilution 9 gradients) was added to each well in 1. mu.L, negative control group was added to each well in 1. mu.L of DMSO (dimethyl sulfoxide), and the broad spectrum antiviral nucleoside inhibitor Reidsivir was used as a positive control (final concentration 2-fold dilution from 50. mu.M, dilution 9 gradients) in 1. mu.L per well, followed by further incubation at 33 ℃ for 60 hours. mu.L of Cell Proliferation Assay (MTS) reagent was added to each well for 3 hours at 37 ℃. Finally, the absorption peak generated at 490nm wavelength is detected. The calculation formula of the inhibition rate is as follows: inhibition rate ═ 100% for (virome-sample group)/(virome-blank group).

FIG. 5 shows the compound 5-iodotubercidin and Redexilvir of example 5 of the present invention against coronavirus HCoV-OC43 andinhibitory Effect of HCoV-NL63 strain. The test results show that 5-iodotubercidin inhibits EC in coronavirus HCoV-OC43₅₀EC inhibition of coronavirus HCoV-NL63 at about 1.56. mu.M (A in FIG. 5)₅₀About 3.62 μ M (B in FIG. 5), which is close to the anti-coronavirus activity of the positive compound Rudexilvir, further shows that 5-iodotubercidin is expected to develop a novel anti-new coronavirus lead compound and provides a theoretical basis for developing COVID-19 effective therapeutic drugs.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of medical and Biotechnology of Chinese academy of medical sciences

Application of adenosine kinase inhibitor in preparation of anti-coronavirus preparation

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Claims (6)

1. Use of an adenosine kinase inhibitor for the preparation of an anti-coronavirus formulation, characterised in that the adenosine kinase inhibitor is 5-iodotubercidin.

2. The use according to claim 1, wherein the coronavirus is SARS-CoV-2.

3. Use according to claim 2, wherein the coronaviruses are HCoV-OC43 and HCoV-NL 63.

4. An anti-coronavirus medicine is characterized in that the active component is 5-iodotubercidin.

5. The medicament of claim 4, further comprising a pharmaceutically acceptable adjuvant.

6. A medicament as claimed in claim 4 or claim 5, wherein said adjuvant comprises one or more of a carrier, excipient or diluent.

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