CN114948918A - Application of protein kinase inhibitor in preparation of anti-hantaan virus medicine - Google Patents

Abstract

The invention discloses application of a protein kinase inhibitor in preparing a hantavirus-resistant medicament, wherein the structural formula of the protein kinase inhibitor is shown as a formula (I). The research of the invention finds that the HTNV virus load in cells is obviously reduced after the protein kinase inhibitor shown in the formula (I) is added; the expression level of nucleic acid is obviously reduced, and the protein kinase inhibitor shown in the formula (I) can effectively inhibit the replication and proliferation of hantaan virus.

Description

Application of protein kinase inhibitor in preparation of anti-hantaan virus medicine

Technical Field

The invention belongs to the field of antiviral drug development, and particularly relates to application of a kinase inhibitor ZM 39923HCl in preparation of an anti-hantaan virus drug.

Background

Hantavirus infectious diseases can mainly cause acute renal failure (nephritis), hemorrhagic fever with renal syndrome or acute non-cardiogenic pulmonary edema syndrome. Among them, Hemorrhagic fever with renal syndrome (HFRS) is a natural epidemic disease transmitted globally, mainly transmitted by rodents such as hyphomus, and the clinical manifestations mainly include acute renal function impairment, fever, hemorrhage, shock, etc. Hantaan virus (HTNV), a major pathogen causing severe HFRS, belongs to Bunyavirales (Bunyavirales), Hantaviridae (Hantaviridae), Orthohantavirus (Orthohantavirus), is a enveloped single negative strand RNA virus (-ssRNA), a spherical viroid with an average diameter of 120nm, the genome of which comprises l (large), and encodes the viral RNA-dependent RNA polymerase (RdRp); m (medium), encodes an envelope Glycoprotein Precursor (GPC); s (small), encodes the viral Nucleocapsid Protein (NP). There is currently no FDA-approved specific therapeutic HFRS drug or vaccine, and the current primary method of clinical treatment of HFRS is symptomatic support therapy. Therefore, the research and development of the medicine with anti-Hantaan virus activity and better safety have important clinical significance.

Disclosure of Invention

Based on the research of the inventor, the invention provides the application of the protein kinase inhibitor in preparing the anti-hantaan virus medicine on one hand, the structural formula of the protein kinase inhibitor is shown as the formula (I),

the invention also provides application of the protein kinase inhibitor in preparing a medicine for treating hantavirus infection diseases, wherein the structural formula of the protein kinase inhibitor is shown as the formula (I).

The invention proves that the kinase inhibitor ZM 39923HCl can effectively inhibit the replication of Hantaan virus, reduce the virus load in infected cells and inhibit the proliferation of the virus; in addition, the compounds have less effect on cell viability at concentrations that exert an antiviral effect.

Drawings

FIG. 1 shows the results of Western blot detection of structural protein NP in HTNV-infected cells of the present invention in example 2, to which ZM 39923HCl was added at various concentrations (1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M), in which NC is a cell group; PC: cell + hantaan virome;

FIG. 2 shows the results of immunofluorescence of NP protein in HTNV-infected cells in example 2 of the present invention with different concentrations of ZM 39923 HCl;

FIG. 3 shows the results of the measurement of the virus S gene expression level in HTNV-infected cells in example 2 of the present invention with different concentrations of ZM 39923HCl added;

FIG. 4 shows the results of the measurement of the activity level of A549 cells by adding ZM 39923HCl at different concentrations in example 3 of the present invention.

Detailed Description

Unless otherwise specified, the terms or methods herein are understood or implemented using established methods of correlation, as recognized by one of ordinary skill in the relevant art.

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The experimental materials and reagents used in the following examples are all commercially available products.

Example 1: screening for kinase inhibitors that inhibit HTNV

The inventor tries to research whether the protein kinase inhibitor has an inhibitory effect on hantaan virus, but based on the fact that protein kinase inhibitor micromolecule compounds have multiple types and large chemical structure difference, even though the protein kinase inhibitors with similar chemical structures have different substitution groups, the selectivity and inhibitory activity on kinase are also obviously different, and the antiviral effect and the inhibitory effect on specific viruses cannot be presumed from simple structure-activity relationship. Through multiple researches of the inventor, a kinase inhibitor with an extremely outstanding HTNV inhibition rate effect is unexpectedly obtained: ZM 39923HCl (Compound 6 in Table 1). A partial representative screening protocol is as follows:

human non-small cell lung cancer cell a549 cells (purchased from wuhan kinuosai life technologies ltd., china) were digested, inoculated into 96-well plates, 1 × 104 cells per well, cultured overnight in 5% CO2 at 37 ℃, and treated with HTNV virus (international standard strain 76-118, given by the chinese center for disease prevention and control) infection and representative 9 protein kinase inhibitor small molecule compounds when the cell density reached 60%, as shown in table 1.

Each compound was added to a virus dilution at a working concentration of 10. mu.M, and the multiplicity of infection (MOI) of the virus was 1, both of which were simultaneously applied to the cells, and after 4 hours of treatment, the supernatant was removed, and the cells were further cultured in DMEM medium containing 2% FBS, three duplicate wells were provided for each compound, and an immunofluorescence assay was performed at 96hours (96hours post-infection,96hpi) after infection.

In an immunofluorescence experiment of HTNV infected A549 cells, the expression level of NP protein is used as an index for screening a compound with anti-HTNV activity, the NP protein expression is in negative correlation with the virus inhibition effect of the compound, namely, the NP protein is less expressed when the compound can obviously inhibit HTNV replication, so that the anti-HTNV effect of the compound can be evaluated through the expression of the NP protein, and the result is shown in Table 1.

TABLE 1 kinase inhibitors and assay results for their anti-HTNV activity

Example 2: identification of inhibition of HTNV replication by ZM 39923HCl cellular levels

(I) Western blot detection

Digesting A549 cells, then passaging to a T25 cell culture bottle, infecting HTNV according to MOI (molar equivalent of identity) 1 when the cell density reaches 70%, simultaneously adding cell culture supernatant according to different compound concentrations, uniformly mixing, then culturing in a CO2 cell culture box at 37 ℃, changing the culture solution after 2 hours to a DMEM culture solution containing ZM 39923HCl with the corresponding concentration and dissolving in 2% FBS, and continuously culturing for 4 days and then collecting the cells;

adding the obtained cell sample into a Western blot sample buffer, boiling for 7min, cooling, performing electrophoresis by 12% SDS-PAGE, and performing electrophoresis on a concentrated gel part by 80V constant voltage electrophoresis for about 30 min; then the sample enters separation gel, constant voltage electrophoresis of 120V is carried out, the time is about 60min, and the electrophoresis is cut off when the front end of the sample reaches the lower edge of the glass plate, so that the electrophoresis is finished; electrically transferring to the PVDF membrane at constant pressure of 100V for 80min, taking out the PVDF membrane after the membrane transfer is finished, putting in 3% BSA (prepared by using TBST as a solvent) as soon as possible, and sealing for 2-3h at room temperature by using a shaking table; then, HTNV-NP specific monoclonal antibody was added, incubated overnight at 4 ℃, excess antibody was washed off with TBST buffer, infrared labeled anti-mouse secondary antibody was added, incubated at room temperature in the dark for 60min, followed by TBST washing three times for 10min each, scanned with Odyssey infrared scanner and the results analyzed.

Results as shown in figure 1, intracellular viral NP protein expression was significantly reduced after addition of ZM 39923HCl compared to Positive Control (PC) results.

(II) immunofluorescence assay

1) Fixing: in a biological safety cabinet, taking a 96-well plate from which a supernatant culture medium is removed, gently adding 200 mu L PBS into each well for washing, then adding 100 mu L of 4% (wt/vol) Paraformaldehyde (PFA) precooled at 4 ℃, and fixing for 20min at room temperature;

2) punching: preparing 0.5% Triton X-100, using PBS as a solvent, taking out the fixed 96-well plate, removing supernatant liquid, rinsing for 3 times by using 200 muL PBS, adding 100 muL 0.5% Triton X-100, and perforating for 20min at room temperature;

3) and (3) sealing: preparing a blocking solution (3% BSA), weighing 3g BSA powder, adding the powder into 100mL PBS, uniformly mixing for later use, taking out a 96-well plate after punching, removing a supernatant liquid, washing the bottom of the well for 2 times by using 200 mu L PBS, removing the punching solution as much as possible, then adding 100 mu L blocking solution, and placing the plate in a 37 ℃ thermostat for treatment for 1 h;

4) primary antibody incubation: the sealed 96-well plate was removed, the supernatant liquid was removed, 40. mu.L of primary antibody (murine anti HTNV NP mAb 1A8, 1:100 dilution) was added, and overnight at 4 ℃;

5) and (3) secondary antibody incubation: the primary antibody was removed from the overnight incubated 96-well plate, recovered, rinsed 2 times with 200. mu.L PBS, 50. mu.L secondary antibody (Cy 3-labeled goat anti-mouse IgG, 1:400 dilution) was added, and treated in a 37 ℃ incubator for 1 h;

6) dyeing the core: the 96-well plate incubated with the secondary antibody was removed, the supernatant liquid was removed, and the plate was rinsed 3 times with 200. mu.L of PBS to remove the background as much as possible, 100. mu.L of stained nuclei was added with DAPI (1:10000 dilution), and the plate was rinsed 1 time with PBS, 100. mu.L of 70% glycerol was added, and the distribution and intensity of red fluorescence were observed under a fluorescence microscope.

The results are shown in fig. 2, and the red fluorescence intensity in each well is significantly reduced after addition of ZM 39923HCl, indicating that hantaan virus replication is significantly inhibited.

(III) qRT-PCR detection

1) Extracting total RNA of cells: taking out a 6-hole plate of the total RNA of the cells to be extracted, removing supernatant of a cell culture medium, extracting the total RNA of the cells according to the instruction flow of the Axygen total RNA preparation kit, finally adding DEPC water, centrifuging to obtain a total RNA sample of the cells, then, spotting the sample on a Take3 micro-detection plate, and measuring the concentration by using a multifunctional enzyme-labeling instrument;

2) and (3) cDNA synthesis: calculating the volume of RNA required by the reverse transcription of 2000ng according to the concentration result measured by the multifunctional microplate reader, preparing a reverse transcription reaction solution according to the specification, and obtaining the required synthesized cDNA after the reaction is finished;

3) qPCR reaction: the preparation of the reaction liquid system was carried out on ice: qPCR SYBR Green premix 10. mu.L, Forward and Reverse primers 1. mu.L each, template DNA 1. mu.L, DEPC water 7. mu.L.

The results are shown in FIG. 3, where nucleic acid expression levels of HTNV were significantly lower in infected cells treated with ZM 39923HCl compared to the positive control group for HTNV infection.

Example 3: ZM 39923HCl cytotoxicity assay

1) A549 cells (104 cells/well) were inoculated in a 96-well plate, cultured in 100. mu.L of a medium, plated at 8X 11, and cultured overnight in A5% CO2 incubator at 37 ℃;

2) dissolving ZM 39923HCl (100 mu M, 50 mu M, 25 mu M, 10 mu M, 5 mu M, 2.5 mu M and 1 mu M) with different concentrations in serum-containing DMEM according to an experimental design scheme, taking out 96-well culture plates with uniformly adhered cells, and removing supernatant to change into 100 mu L of culture medium containing diluted compounds per well; column 12, no cells, 100 μ L of medium per well as a blank control; the first row was inoculated with cells, and 100 μ L of medium was added per well as a cell control;

3) treating a 96-hole cell culture plate in a cell incubator for 48h, sucking CCK-8 solution by using an electric pipette, adjusting the range and adding the solution according to 10 mu L per hole (the operation is gentle in the liquid adding process, so that the introduction of foam is avoided, and otherwise, the reading result of the instrument is influenced);

4) the 96-well culture plate was incubated in an incubator for 2h, then taken out and gently shaken to ensure uniform color distribution in the wells, and absorbance was measured by a microplate reader at 450 nm.

5) Cell viability was calculated according to the formula:

cell survival (%) - (Cs-Cb)/(Cc-Cb) x 100

Cs — experimental absorbance (containing a549 cells, DMEM, CCK-8, and ZM 39923 HCl);

cb ═ blank absorbance (with DMEM and CCK-8);

cc absorbance (containing a549 cells, DMEM and CCK-8);

the results are shown in FIG. 4, and the effect of gradient ZM 39923HCl on the cell viability is obtained, and the half cytotoxic concentration (CC50) is calculated to be 99.88 +/-1.19 mu M, which shows that the compound has better anti-hantaan virus activity and has smaller effect on the viability of host cells.

The ZM 39923HCl is proved to have the effect of obviously inhibiting the replication of hantaan virus and reducing the virus load in infected cells by various experiments from the expression level of protein and nucleic acid of the hantaan virus; in addition, ZM 39923HCl has less effect on cell viability at concentrations that exert antiviral effects. Therefore, ZM 39923HCl can also be used for preparing medicines for treating hantaan virus infection diseases.

Claims (3)

1. The application of the protein kinase inhibitor in preparing the anti-Hantaan virus medicine, the structural formula of the protein kinase inhibitor is shown as the formula (I),

2. the application of a protein kinase inhibitor in preparing a medicine for treating hantavirus infection diseases is disclosed, wherein the structural formula of the protein kinase inhibitor is shown as a formula (I).

3. The use of claim 2, wherein the hantavirus infectious disease is acute renal failure, hemorrhagic fever with renal syndrome, or acute non-cardiogenic pulmonary edema syndrome.

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