CN120694999A - Application of a small molecule compound in the preparation of drugs for preventing and treating dengue virus - Google Patents

Abstract

The present invention discloses the use of a small molecule compound in the preparation of a drug for treating and/or preventing dengue virus infection. The small molecule compound ZZL-208 has a CC ₅₀ of greater than 1000 μM on HepG2 cells and a half-maximal inhibitory concentration (EC ₅₀ ) of 1.50 μM in HepG2 cells infected with DENV-2; a CC ₅₀ of greater than 500 μM on BHK-21 cells and an EC ₅₀ of 9.19 μM in BHK-21 cells infected with DENV-2; a CC ₅₀ of greater than 500 μM on Vero cells and an EC ₅₀ of 4.78 μM and 1.91 μM in Vero cells infected with DENV-2 and DENV-3, respectively. This small molecule compound can inhibit the replication of DENV-2 and DENV-3 at the nucleic acid level, and the inhibitory effect is dose-dependent.

Description

Application of small molecular compound in preparation of dengue virus prevention and treatment drugs

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a small molecular compound in preparation of an anti-dengue virus medicine.

Background

Dengue fever (Dengue fever, DF) is an acute insect-borne infectious disease caused by Dengue virus (DENV) infection, and is transmitted by aedes aegypti and aedes albopictus, which pose a serious threat to public health in tropical and subtropical areas of the world. Studies have estimated that 39 million people in 128 countries worldwide are at risk of dengue virus infection, about 3.9 million people infected annually, 9600 tens of thousands of clinical symptoms.

The different antigenicity of the DENV envelope protein E is divided into four serotypes (DENV 1-4), and different strains in the same type have certain antigen differences. At present, the lack of specific antiviral drugs for dengue viruses clinically, due to the lack of effective cross protection among serotypes, the existence of antibody dependence enhancement and other characteristics, the stable and effective vaccine for DENV is difficult to obtain, and the dengue fever can only be mainly improved by symptoms such as fever, hemorrhage and the like, which brings great impediment to the clinical treatment of dengue fever. Therefore, the discovery of active molecules against dengue virus is of great biological research and practical significance.

Disclosure of Invention

The invention provides a novel application of a small molecular compound and an application thereof in preparing medicines for resisting dengue virus infection, and the provided small molecular compound has anti-DENV-2 and DENV-3 activities.

The structural formula of the small molecule compound ZZL-208 is as follows:

;

The components (or active ingredients) of the medicine are the micromolecule compounds, and one or more auxiliary materials acceptable in production of products can be added to improve the absorption effect of the active ingredients or facilitate the use, such as capsules or pills, powder, tablets, granules, oral liquid, injection and the like, so that the medicine can be prepared into proper dosage forms.

Viruses DENV-2 and DENV-3 are selected for research. The cytotoxicity of the small molecule compound ZZZL-208 in HepG2, BHK-21 and vero cells was evaluated based on the cell viability assay, the antiviral effect of ZZL-208 in DENV-2-infected BHK-21 cells was examined using the MTT method, the inhibitory effect of this small molecule compound on viral replication in DENV-2-infected HepG2 and vero cells was evaluated based on the viral nucleic acid assay, and the inhibitory effect on viral replication in DENV-3-infected vero cells was evaluated.

The results showed that ZZL-208 had a half-cytotoxicity (CC ₅₀) of greater than 1000. Mu.M on HepG2 cells, a half-inhibitory concentration (EC ₅₀) of 1.50. Mu.M in DENV-2 infected HepG2 cells, a SI value of greater than 666.67, a half-cytotoxicity (CC ₅₀) of greater than 500. Mu.M on BHK-21 cells, a half-inhibitory concentration (EC ₅₀) of 9.19. Mu.M in DENV-2 infected BHK-21 cells, a SI value of greater than 108.81, a half-cytotoxicity (CC ₅₀) of greater than 500. Mu.M on vero cells, a half-inhibitory concentration (EC ₅₀) of 4.78. Mu.M and 1.91. Mu.M in DENV-2 and DENV-3 infected vero cells, respectively, and a SI value of greater than 104.6 and 261.78, respectively. The small molecular compound is shown to be low in toxicity and high in efficiency in the cell model. At the nucleic acid level, the small molecule compound can inhibit the replication of DENV-2 and DENV-3, and the inhibition has dose dependency.

The invention has the advantages and technical effects that:

1. The small molecule compound ZZL-208 has low cytotoxicity, and can inhibit the replication of DENV-2 and DENV-3 in a dose-dependent manner;

2. The small molecular compound has the advantages of relatively simple chemical structure, strong tissue permeability, high bioavailability, better stability and the like, and has wide application prospect in clinic and market.

Drawings

FIG. 1 shows the results of a DENV-2 virus titer assay;

FIG. 2 shows the results of a DENV-3 virus titer assay;

FIG. 3 is a schematic diagram showing the cytotoxicity test results of small molecule compound ZZL-208 in vero cells and the replication inhibition results of DENV-2 and DENV-3, wherein the blue line is the cytotoxicity result of small molecule compound, and the red line is the inhibition result of small molecule compound on DENV-2;

FIG. 4 is a schematic diagram showing experimental results of inhibition of DENV-2 replication by small molecule compound ZZZL-208 in virus-infected HepG2, BHK-21 and vero cells, blue line as result of cytotoxicity of small molecule compound, red line as result of inhibition of DENV-2 by small molecule compound;

FIG. 5 is a schematic representation of experimental results of inhibition of replication of DENV-2 by small molecule compound ZZZL-208 in supernatants of virus infected HepG2, BHK-21 and vero cells;

The experimental results were expressed as mean ± standard error, and were analyzed using One-way ANOVA (or mixed model), and p <0.05, p <0.01, p <0.001, and p <0.0001 were shown in comparison with DENV-2 group.

Detailed Description

In order to more clearly illustrate the technical scheme of the invention, the invention will be described in detail below with reference to specific embodiments. However, the protection scope of the invention is not limited to the above, the experimental methods used in the present embodiment are all conventional methods unless otherwise specified, and the materials and reagents used are all available from commercial sources unless otherwise specified, and the dengue viruses are DENV-2 and DENV-3.

The preparation of the small molecular compound ZZL-208 in the examples below was carried out according to the method described in reference Pan Zhou,Biao Hu,Lingling Lu,Rong Huang&Fuchao Yu.(2017).One-Pot Synthesis of Functionalised 4-Spiro-1,4-Dihydropyridines Via [1+2+1+2]-Cyclisation.Journal of Chemical Research(9),513-516.;

The small molecule compound ZZL-208 powder in the following examples was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution having a storage concentration of 50 mM. Storing the completely dissolved drug in a 4 ℃ refrigerator for subsequent experiments;

EXAMPLE 1 culture and titer determination of DENV-2 and DENV-3

1. Virus amplification Using C6/36 cells

The vigorous C6/36 cells were passaged into T75 flasks for conventional proliferation culture for 24h, after cell confluency of about 90%, the supernatant was discarded, the surface of the C6/36 cells was gently rinsed with PBS, DENV-2 was inoculated with virus at MOI of 0.1, incubated for 2h at 28℃under 3% CO ₂, and fresh 2% FBS-containing RPMI-1640 to 25mL was supplemented. Culturing for 5-7 days at 28 ℃ under the condition of 3% CO ₂, observing the change of C6/36 cells and a culture medium, turning yellow from powder when the diseased cells account for about 50% of the total cell amount (5-6 days), taking out a small amount of supernatant, carrying out qRT-PCR detection, collecting the supernatant in a cell culture bottle if the virus load is higher, centrifuging for 10min at 4000r/min in a centrifuge, sub-packaging the supernatant into sterile 2mL freezing pipes, storing each pipe for 0.5mL, and storing in a refrigerator at-80 ℃, wherein the culture mode of DENV-3 is the same as that of DENV-2.

2. DENV titer assay

And selecting the BHK-21 cells which grow vigorously, washing the inoculation surface in two 6-hole plates by using a culture medium, uniformly inoculating the cells, and culturing overnight for 12 hours, wherein the cell coverage rate reaches 90% -100%. The DENV-2 strain was subjected to 10-fold gradient dilution. Cells were washed with PBS, added to a gradient of 800. Mu.L/well of diluted virus, 2 replicates per gradient, and incubated for 2h in a 37℃5% CO ₂ incubator. 2 XDMEM medium containing 2% FBS was prepared and stored in a 37℃incubator, and the 2% low melting point agar gel was heated and cooled to 37℃to mix the medium with the agar solution in equal volumes. And removing virus inoculation liquid after virus adsorption, adding 4mL of low-temperature agar culture liquid into each hole, and after agar is cooled and solidified, inversely culturing for 5-7 days in a 37 ℃ and 5% CO ₂ incubator. The increase or decrease in the number of plaques caused by cytopathy was recorded daily until the number of plaques no longer increased. About 8 mL/well of 4% paraformaldehyde solution was added for fixation, and after 24 hours, the agar layer was carefully removed to prevent the agar from sliding on the cell surface and scratching the bottom cells. 1 mL/hole crystal violet dye liquor is added for dyeing for 20min, the dye liquor is recovered, the redundant dye liquor is rinsed clean with clear water and is inverted for airing, the number of plaques is photographed and recorded under a white background, and the virus titer of DENV-3 is measured in the same way as that of DENV-2.

3. Experimental results

The DENV-2 and DENV-3 titer results are shown in the following figures 1 and 2, the number of plaques is clear, the number of plaques shows 10-fold bench change, the plaque experiment is considered to be effective, and the number of plaques appearing in the gradient range of 10-100 is checked. DENV-2 was counted at a dilution of 10 ^-7 to obtain 25 and 22 plaques, and according to the result of the counting, the virus titer was calculated using the formula, (25+22)/(2X 0.8X10. 10 ^-7）=2.9375×10⁸ PFU/mL), and DENV-3 was counted at a dilution of 10 ^-5 to obtain 64 and 61 plaques, according to the result of the counting, the virus titer was calculated using the formula, (64+61)/(2X 0.8X10. 10 ^-5）=7.8125×10⁶ PFU/mL. The titers of DENV-2 and DENV-3 were 2.9375X 10 ⁸ PFU/mL and 7.8125X 10 ⁶ PFU/mL, respectively, after which the titer of the newly produced virus was determined for each virus culture.

Example 2 cytotoxicity assay of Small molecule Compound ZZL-208 on HepG2, BHK-21 and vero cells

1. Experimental method

HepG2 cells, which were well grown, were seeded in 96-well cell culture plates (flat bottom), 100. Mu.L of cell suspension was added to each well, and the cell density was adjusted to 2.0X10 ⁴ cells/well. And adding medicine when the cell confluency in the hole reaches 90%. The supernatant was discarded, washed once with PBS, the small molecule compound ZZZL-208 was diluted to 1000. Mu.M with DMEM medium containing 2% FBS, and diluted in sequential multiple ratios with 1000. Mu.M as the initial concentration for 8 concentration gradients (1000. Mu.M, 500. Mu.M, 250. Mu.M, 125. Mu.M, 62.5. Mu.M, 31.25. Mu.M, 15.625. Mu.M, 7.8125. Mu.M), 100. Mu.L of drug-containing medium was added to each well, DMEM medium containing 2% FBS was used as a blank control, DMEM medium containing 2% FBS and 0.5% DMSO was used as a solvent control, and 6 wells were multiplexed. After further incubation for 48h in a 37 ℃ 5% CO ₂ incubator, CPE was observed and recorded. The MTT is used for detecting the activity of cells, the MTT solution with the storage concentration of 5mg/mL is diluted to 1mg/mL by PBS, the MTT working solution is added for incubation for 4 hours after the supernatant of the original culture medium is discarded, the MTT solution is discarded after the reaction, 100 mu L of DMSO is added into each hole, the reaction is carried out for 5 minutes in a dark place, and the formazan crystals in the cells are completely dissolved and uniformly mixed. The absorbance (OD) of the sample at 490nm was measured using a microplate reader. Inhibition of cell viability by the drug to be selected = (control OD value-drug OD value)/(control OD value-blank OD value) ×100% inhibition ratio. Calculating the half-toxicity concentration (CC ₅₀) of the drug by Prism non-regression analysis, and detecting cytotoxicity of ZZL-208 on BHK-21 and vero cells by the same method;

2. Experimental results

The results are shown in Table 1, and the small molecular compounds ZZZL-208 have CC ₅₀ in HepG2, BHK-21 and vero cells of > 1000. Mu.M, > 500. Mu.M and > 500. Mu.M respectively, which indicates that the ZZL-208 has smaller toxicity to the cells, meaning that the ZZL-208 has better safety;

CC ₅₀、IC₅₀ and SI values of tables 1 ZZL-208

。

EXAMPLE 3 inhibition of DENV-2 and DENV-3 replication by the small molecule Compound ZZL-208

1. Experimental method

Well-grown vero cells were seeded into 24-well cell culture plates with 500 μl of cell suspension per well at a cell density of 3.0X10 ⁵/mL. A virus group and a blank group were set, and each group was repeated three times. DENV-2 was diluted to moi=1 virus liquid with DMEM medium containing 2% FBS. After the culture is completed for 12 hours, the virus is inoculated, the original culture medium is discarded, PBS is used for washing once, each hole of the virus group is inoculated with 500 mu L of virus liquid, a blank control group is added with an equal volume of DMEM culture medium containing 2% FBS, the blank control group is placed in a 37 ℃ and 5% CO ₂ constant temperature incubator for incubation for 2 hours, after the incubation is completed, the DMEM culture medium containing 2% FBS is replaced for continuous culture, cytopathic Condition (CPE) is observed and recorded every day, and after 72 hours of infection, the culture supernatant is collected for detecting virus RNA.

The viral 3' UTR gene copy number in the cell culture supernatant was detected by absolute quantitative PCR. Sequentially sucking the cell culture supernatant into a 1.5mL enzyme-free EP tube, centrifuging at 8000rpm/min for 5min, taking 140 mu L to a new enzyme-free 1.5mL centrifuge tube, extracting viral RNA by using an Omega E.Z.N.A. viral RNA extraction R6874-02 kit, preparing a reaction system (shown in table 2) according to Ai Kerui AG11713 kit instruction, detecting the copy number of a viral 3' UTR gene by qRT-PCR, calculating the inhibition rate of a small molecular compound on the virus and EC ₅₀, wherein the inhibition rate (%) = 1- (drug treatment group 3' UTR gene copy number/viral group 3' UTR gene copy number) ×100%, and the primer and probe sequences are shown in table 3, wherein the qRT-PCR reaction program is 42 ℃ reverse transcription for 5min, 95 ℃ pre-denaturation for 30s, 95 ℃ denaturation for 5s, annealing and extension for 39 cycles;

TABLE 2 Ai Kerui (AG 11713) reaction system

TABLE 3 primers and probe sequences for detecting 3' UTR genes of DENV-2, 3

The level of viral replication was measured by measuring the expression of the E protein mRNA in the cells by relative quantitative PCR. Cells in 24 well plates were washed 2 times with pre-chilled PBS, cells were lysed on ice for 10min with 500. Mu.L of Trizol lysate added to each well, and the cells were blown down until complete shedding, and sequentially received into enzyme-free 1.5mL enzyme-free EP tubes, and total RNA of cells was extracted according to Trizol reagent instructions, as follows:

Adding 100 μl of chloroform into each tube sample, shaking vigorously for 15s, incubating at room temperature for 15min, centrifuging at 4 ℃ and 13000rpm/min for 15min, transferring the supernatant to a new enzyme-free EP tube, adding 300 μl of isopropanol, mixing upside down, incubating at room temperature for 10min, centrifuging at 4 ℃ and 13000rpm/min for 15min, discarding the supernatant, retaining white precipitate, adding 1ml of 75% ethanol (absolute ethanol+DEPC water, which is used for preparation), washing the precipitate twice, centrifuging at 4 ℃ and 13000rpm/min for 5min, discarding the supernatant, retaining the precipitate, opening the cover, airing to dryness, adding 20 μl of RNase water, dissolving the precipitate on ice for 15min, measuring RNA concentration by an ultraviolet spectrophotometer, configuring a reaction system according to Ai Kerui AG11728 kit instructions (as shown in Table 5), and performing reverse transcription on the sample into cDNA. The reaction system (as in table 6) was configured according to Vazyme Q kit instructions, the mRNA expression level of the viral E protein gene was detected by qRT-PCR, GAPDH was used as an internal gene, and the inhibition rate of the small molecule compound against the virus and EC ₅₀ were calculated by the 2 ^{-ΔΔ Ct} method, inhibition rate (%) =1- (the viral gene mRNA expression level/the viral gene mRNA expression level of the treatment group administered) ×100%. Reverse transcription reaction procedure was 37℃15min,85℃5s,4℃infinity, qRT-PCR reaction procedure was 95℃pre-denaturation 30s, 95℃denaturation 10s, annealing and extension 60℃20s, denaturation, annealing and extension 39 cycles total, melting sections 95℃15s,60℃1min,95℃15s, primer sequences are shown in Table 4;

TABLE 4 primer sequences for detection of DENV-2, 3E protein mRNA

TABLE 5 Ai Kerui AG11728 reaction system

;

TABLE 6 Vazyme Q311 reaction System

;

2. Experimental results

As shown in Table 7 and FIG. 3, the small molecule compounds ZZL-208 in the DENV-2 and DENV-3 infected vero cells had EC ₅₀ values of 4.78. Mu.M and 1.91. Mu.M, respectively, and SI values of greater than 104.6 and 261.78, respectively. The SI value of the small molecular compound in the vero cells infected by DENV-2 and DENV-3 is more than 100, which indicates that the small molecular compound has broad-spectrum anti-dengue virus activity;

CC ₅₀、IC₅₀ and SI values of tables 7 ZZL-208

。

EXAMPLE 4 inhibition of DENV-2 replication by the small molecule Compound ZZL-208 on different cells (HepG 2, BHK-21, vero cells)

The experimental procedure of this example is the same as that of example 3, and the results are shown in FIGS. 4-5, and the effect of the small molecule compound on viral replication is evaluated based on qRT-PCR detection of viral RNA expression in cells, and the use of the drug treatment after viral infection can significantly inhibit viral replication in cells. Treatment with ZZL-208 after infection of HepG2, BHK-21 and vero cells with DENV-2 significantly inhibited viral release from the cell culture supernatant at half inhibition concentrations (EC ₅₀) of 1.50. Mu.M, 9.17. Mu.M and 4.78. Mu.M, SI values of greater than 666.67, 108.81 and 104.6, respectively, and the inhibition was dose dependent (Table 1).

Claims (2)

1. Use of a small molecule compound having the structural formula shown in Formula I in the preparation of a drug for treating and/or preventing dengue virus infection; . 2. The method according to claim 1, wherein the germline virus is DENV-2 or DENV-3.