Use of phA-767491 as an inhibitor of replication of h3n2 subtype swine influenza virus

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to application of PHA-767491 or a pharmaceutically acceptable salt thereof as a replication inhibitor of H3N2 subtype swine influenza virus. Through in-vitro cell experiments, it is found that the GPCR small-molecule compound PHA-767491 has a significant inhibitory effect on the H3N2 subtype swine influenza virus, with a half-inhibitory concentration (IC 50 ) of 6.07 µM and a half-cell toxicity concentration (CC 50 ) of greater than 200 µM. When administered simultaneously with or after infection of the H3N2 subtype swine influenza virus, the inhibition rate is more than 97%. The above results show that PHA-767491 can be used as a new drug for application to the replication inhibitor of the H3N2 subtype swine influenza virus, has high inhibitory activity, high safety to normal cells, is easy to popularize, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to the application of PHA-767491 as an inhibitor of H3N2 subtype swine influenza virus replication. Background Technology

[0002] Swine influenza virus (SIV) is a representative member of the Orthomyxoviridae family, and the H3N2 subtype is one of the major circulating strains in pig populations worldwide. This virus not only causes respiratory diseases in pigs, leading to economic losses in the swine industry, but also poses a significant public health threat due to its zoonotic characteristics. Pig respiratory epithelial cells contain receptors for both human and avian influenza viruses, and are considered "mixing agents" for the generation of novel reassortant influenza viruses, increasing the risk of cross-species transmission and triggering human influenza pandemics.

[0003] Currently, influenza prevention and control mainly rely on vaccination and antiviral drug treatment. However, the highly variable nature of the influenza virus genome often leads to a mismatch between vaccine strains and circulating strains, resulting in reduced or even failed immune protection. In terms of drugs, the variety of antiviral drugs used clinically is limited, with the main targets being the viral M2 ion channel and neuraminidase (NA). Among these, M2 ion channel inhibitors such as amantadine are no longer recommended due to widespread resistance (currently, the resistance rate of circulating strains exceeds 90%) and neurological side effects. While neuraminidase inhibitors such as oseltamivir remain mainstream drugs, reports of resistant strains are increasing annually, and they are ineffective against certain subtypes of viruses (such as some influenza B viruses). Therefore, developing antiviral drugs that target novel targets and possess entirely new chemical structures and mechanisms of action is a crucial issue that urgently needs to be addressed in the field of zoonotic diseases.

[0004] G protein-coupled receptors (GPCRs) are the largest family of membrane protein receptors in the human body, participating in the regulation of numerous physiological and pathological processes and serving as important targets for drug development. In recent years, studies have revealed that some GPCRs play crucial roles in the progression of viral infection, potentially becoming novel targets for antiviral therapy. PHA-767491 HCl belongs to the GPCR small molecule family. PHA-767491 HCl is an effective, ATP-competitive, dual Cdc7 / CDK9 inhibitor, with the following chemical structure:

[0005]

[0006] PHA-767491 HCL exhibits 20-fold selectivity towards Cdk1, Cdk2, and GSK3-β, 50-fold selectivity towards MK2 and Cdk5, and 100-fold selectivity towards PLK1 and CHK2. PHA-767491 HCL inhibits the proliferation of various cell lines. Due to the specific inhibition of Cdc7 kinase and Mcm2 phosphorylation at the Cdc7-dependent Ser40 site, low-dose PHA-767491 HCL can block DNA replication initiation. Treatment of anti-ABT-737 OCI-LY1 and SU-DHL-4 cells with PHA-767491 HCL significantly reduces upregulated Mcl-1 levels. A mitochondrial-dependent pro-apoptotic effect was observed in quiescent chronic lymphocytic leukemia (CLL) cells treated with 1 μM PHA-767491 HCL. PHA-767491 HCL treatment of CD154 and IL-4 stimulated proliferating CLL cells can abolish DNA synthesis by inhibiting Cdc7 rather than triggering cell death.

[0007] However, to date, there have been no reports of PHA-767491 or its pharmaceutically acceptable salts having activity against the H3N2 subtype of swine influenza virus. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and to provide the application of PHA-767491 as a replication inhibitor of H3N2 subtype swine influenza virus.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention claims protection for the use of PHA-767491 or a pharmaceutically acceptable salt thereof in the preparation of drugs against H3N2 subtype swine influenza virus.

[0011] This invention also claims protection for the use of PHA-767491 or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of H3N2 subtype swine influenza.

[0012] Furthermore, the PHA-767491 is applied to the infection and replication stages of the H3N2 subtype swine influenza virus.

[0013] Furthermore, the drug also includes pharmaceutically acceptable adjuvants in preparation of the drug formulation.

[0014] Furthermore, the pharmaceutical preparations include injections, powders, granules, tablets, or oral liquids.

[0015] The pharmaceutically acceptable salts described in this invention refer to salts that are pharmaceutically considered safe, effective, and suitable for use in pharmaceutical formulations, such as hydrochlorides, sulfates, nitrates, acetates, oxalates, citrates, fumarates, and parabensylsulfonates. Specifically, this invention uses PHA-767491 HCl (CAS No. 942425-68-5), an ATP-competitive dual Cdc7 / CDK9 inhibitor with PHA-767491 as its sole active ingredient. However, there are currently no reports of PHA-767491 or its pharmaceutically acceptable salts exhibiting activity against the H3N2 subtype of swine influenza virus.

[0016] The pharmaceutical excipients may be adhesives, diluents, fillers, preservatives, absorption enhancers, etc.

[0017] This invention evaluates the cytotoxicity of PHA-767491 HCL to MDCK cells using an in vitro cytotoxicity assay (CCK-8 assay) to determine its safe concentration range; and determines the inhibitory effect and half-maximal inhibitory concentration (IC50) of PHA-767491 HCL on H3N2 virus using an in vitro viral inhibition assay. 50 The role of PHA-767491 HCL in the viral infection process was investigated through administration trials at different time points.

[0018] This invention, through in vitro cell experiments, found that PHA-767491 HCl has a significant inhibitory effect on H3N2 subtype swine influenza virus, with an IC50 value of [missing information]. 50 The concentration was 6.07 µM. This invention, through in vitro cytotoxicity assays, found that PHA-767491 HCL induced CC in MDCK cells. 50 The concentration was >200µM, indicating that it had low cytotoxicity at the effective concentration.

[0019] This invention, through administration experiments at different time points, found that PHA-767491 HCL, when administered during and after viral infection, exhibited inhibition rates of up to 98.3% and 97.6% against H3N2 virus, respectively, indicating that it primarily acts on the infection and replication phases of the virus.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention, through in vitro experiments, verified that PHA-767491 or its pharmaceutically acceptable salt has a clear inhibitory effect on H3N2 subtype swine influenza virus. Furthermore, PHA-767491 exhibits low cytotoxicity and good safety at effective antiviral concentrations. In addition, as a small molecule chemical drug, PHA-767491 has a well-defined composition and stable source, providing an important candidate compound for the development of novel anti-H3N2 swine influenza drugs. Attached Figure Description

[0022] Figure 1 To investigate the toxic effects of different concentrations of PHA-767491 HCl on MDCK cells (cell viability assay).

[0023] Figure 2 The effects of different concentrations of PHA-767491 HCl on the infectivity and replication capacity of H3N2 subtype swine influenza virus. A: Effect of different concentrations of PHA-767491 HCl on the replication capacity of H3N2 subtype swine influenza virus. B: Effect of different concentrations of PHA-767491 HCl on the infectivity of H3N2 subtype swine influenza virus.

[0024] Figure 3 The effect of different concentrations of PHA-767491 HCl on the nucleic acid replication of H3N2 subtype swine influenza virus (IC50 determination).

[0025] Figure 4 The inhibitory effect of PHA-767491 HCl on H3N2 subtype swine influenza virus at different administration times was investigated. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these embodiments are commercially available.

[0027] Example 1: Assay of the toxicity of PHA-767491 HCl to MDCK cells (CC) 50 (Measurement)

[0028] MDCK cells in good growth condition were placed at 1×10⁶ cells per well. 4Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator until the cell density reached 70%-80%. The culture medium was discarded, and the cells were gently washed once with PBS. The PHA-767491 HCl stock solution was serially diluted using serum-free DMEM medium, with a concentration gradient of 100 μL / well and three replicates for each concentration. 100 μL of maintenance medium containing the corresponding drug concentration was added to each well. A cell control group (drug-free cell control wells) and a blank control group (cell-free control wells) were also set up. The cell plates were incubated at 37°C in a 5% CO2 incubator for another 24 h. DMEM culture medium and CCK8 solution were mixed at a 9:1 ratio and added to the cell plates (after washing the cell plates once with PBS) at a concentration of 100 μL / well. The cells were incubated at 37°C in the dark for 4 h. The optical density (OD) at 450 nm was measured using a microplate reader.

[0029] Cell viability is calculated using the following formula:

[0030] A (Drug+): Absorbance of the pores containing cells, CCK-8 solution, and drug solution;

[0031] A (Drug-): Absorbance of the pore containing cells and CCK-8 solution but not drug solution;

[0032] A (blank): Absorbance of pores containing culture medium and CCK-8 solution but without cells;

[0033] The results are as follows Figure 1 As shown, PHA-767491 HCL affects CC in MDCK cells. 50 >200µM.

[0034] Example 2: PHA-767491 HCl affects the replication and infectivity of H3N2 subtype swine influenza virus

[0035] The drug was serially diluted 10-fold from its highest concentration and applied to MDCK cells infected with H3N2 subtype swine influenza virus. The effect of different drug concentrations on the ability to inhibit viral replication was studied by measuring viral titer. The viral fluid was serially diluted 10-fold with viral diluent (10... -1 ~10 -8 ) Discard the supernatant from MDCK cell plates (96 wells) that have grown to 70%-80% cell monolayer, wash the cells 3 times with PBS, and incubate each well with 100 µL of virus dilution. Repeat each dilution 3 times.

[0036] After inoculation, 96-well cell culture plates were placed in a cell culture incubator for 48 hours. Cell cytopathic effects (CPE) were observed periodically. When obvious CPE appeared, indirect immunofluorescence staining was performed, and fluorescence was observed and recorded using an inverted fluorescence microscope. Different concentrations of PHA-767491 HCl were applied to MDCK cells infected with H3N2 subtype influenza virus. Cells were fixed as soon as CPE appeared, followed by IFA staining. 100 µL of fixative was added to each well for 30 min, then the fixative was discarded; the cells were gently washed three times with PBS buffer. 3000-fold diluted Anti-Influenza A Virus Nucleoprotein antibody was added, and the cells were incubated at 37°C for 1 h. The liquid in the wells was discarded, and the cells were washed three times with PBS buffer. 500-fold diluted Anti-Mouse IgG-FITC was added, and the cells were incubated at 37°C for 60 min in the dark. The liquid in the wells was discarded, and the cells were washed three times with PBS buffer. The fluorescence staining was observed using an inverted fluorescence microscope.

[0037] The results are as follows Figure 2 As shown, with the increase of drug concentration, the viral replication ability gradually weakened, and the number of viruses infected in cells decreased significantly, indicating that PHA-767491 HCL has a significant inhibitory effect on the infection and replication of H3N2 subtype influenza virus.

[0038] Example 3: Inhibitory effect of PHA-767491 HCl on H3N2 subtype swine influenza virus (IC50) 50 (Measurement)

[0039] Seed MDCK cells into 96-well plates and cultured to 70%-80% confluence. Discard the culture medium and wash once with PBS. Add 100 µL of virus dilution buffer to each well to a final concentration of 1:1. 4.25 TCID 50 H3N2 virus solution was prepared at 37°C for 1 h. After adsorption, serially diluted drug (10 μL / well) was added to each well, with three replicates for each concentration. A virus control group (infected but not treated) and a cell control group (uninfected and not treated) were also included. The cell plates were incubated at 37°C with 5% CO2 for 24 h. When significant cytopathic effect (CPE) appeared in the virus control group, the cell plates were frozen at -80°C and then thawed at 37°C, repeated three times to release the virus. The virus solution was collected, total RNA was extracted, and reverse transcribed into cDNA. Viral nucleic acid copy number was quantified using quantitative real-time PCR (qPCR). Based on the copy number in the virus control group (set as 100% viral replication), nucleic acid copy number was determined after treatment with different drug concentrations, and the half-maximal inhibitory concentration (HMC) for virus inhibition was calculated.

[0040] The results are as follows Figure 3 As shown, the half-maximal inhibitory concentration (IC50) of PHA-767491 HCl against H3N2 subtype swine influenza virus is... 50 The value is 6.07µM.

[0041] Example 4: Study on the inhibitory effect of PHA-767491 HCL on the virus.

[0042] Drug administration experiments were conducted at different time points on MDCK cell plates, employing three methods: drug and virus incubation followed by cell infection (Pre), drug and virus co-infection (During), and drug administration after virus infection (Post) to further explore the stages of drug-induced viral inhibition. The working concentration of PHA-767491 HCl was 10 µM. After culturing all groups for another 24 h, samples were collected and viral nucleic acid copy numbers were measured. The viral inhibition rate of each drug-treated group was calculated with the viral control group (infected only, without drug administration) as 100%.

[0043] Pre (pre-inoculation administration): Mix PHA-767491 HCl with H3N2 virus solution (MOI=0.1) at a volume ratio of 1:9, incubate at 4℃ for 1 h, then inoculate 100 µL of the mixture into cells, adsorb at 37℃ for 1 h, discard the mixture, and replace it with maintenance solution.

[0044] During (drug administration simultaneously with inoculation): PHA-767491 HCl and H3N2 virus solution (MOI=0.1) were mixed at a volume ratio of 1:9, and 100 µL was directly inoculated into cells. After adsorption at 37°C for 1 h, the mixture was discarded and replaced with maintenance solution.

[0045] Post-vaccination administration: 100 µL of H3N2 virus solution (MOI=0.1) was first inoculated, allowed to adsorb at 37℃ for 1 h, then discarded. 100 µL of maintenance solution containing 10 µM PHA-767491 HCl was then added. Results are as follows: Figure 4 As shown, the inhibition rates of the Pre, During, and Post groups against H3N2 virus were 23.1%, 98.3%, and 97.6%, respectively. This indicates that PHA-767491 HCL mainly acts on the intracellular infection and replication stages of the virus.

[0046] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of PHA-767491 or a pharmaceutically acceptable salt thereof in the preparation of drugs against H3N2 subtype swine influenza virus.

2. The application according to claim 1, characterized in that, The PHA-767491 is used in the infection and replication stages of the H3N2 subtype swine influenza virus.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable adjuvants in its formulation.

4. The application according to claim 3, characterized in that, The pharmaceutical preparations mentioned include injections, powders, granules, tablets, or oral liquids.

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