CN111214473B - Application of HSP990 in preparation of anti-rotavirus drugs - Google Patents

Abstract

Experiments prove that HSP990 can establish infection in cells by inhibiting rotavirus and prevent infected cells from generating new viruses with infection capacity, namely inhibit infection and replication of the rotavirus, so that an anti-rotavirus effect is achieved, and adverse effects such as diarrhea, death and the like caused by rotavirus infection are obviously relieved. Therefore, the rotavirus-resistant medicament is taken as a main active component, is expected to develop a high-efficiency specific rotavirus-resistant medicament, relieves the rotavirus diarrhea occurrence, reduces the death rate of severe lethal rotavirus infection, meets the clinical treatment requirement and ensures the health of people.

Description

Application of HSP990 in preparation of anti-rotavirus drugs

Technical Field

The invention belongs to the technical field of anti-rotavirus medicaments, and relates to application of HSP990 in preparation of anti-rotavirus medicaments.

Background

Rotavirus (RV) infection is the major cause of diarrhea in infants, causing about 1.25 million cases of gastroenteritis in children every year, causing 352,000 and 592,000 cases of death from Rotavirus infection in children under 5 years of age, mainly occurring in developing countries.

The rotavirus belongs to reoviridae and is a cell-free double-stranded RNA (dsRNA) virus, 11 pieces of dsRNA are respectively wound around 1 molecule of RNA-dependent RNA polymerase (VP1) and 1 molecule of guanylic acid and methyltransferase (VP3) and are positioned at the core of a virus particle, and 12 proteins are coded and comprise 6 structural proteins (VP 1-4, 6-7) and 6 non-structural proteins (NSP 1-6). VP2 constitutes the inner capsid of rotavirus encapsulating the viral genome; a middle capsid constructed from VP6 is wrapped around VP2 to form a rotavirus double-layered capsid particle (DLP); the outer capsid of VP4 and VP7 is further wrapped around DLP to form a complete rotavirus triple-capsid particle (TLP). Rotavirus enters cells through a cell membrane under the mediation of a receptor, DLP is formed by removing an outer capsid, a replication process is started, newly generated virus dsRNA and protein are aggregated in cytoplasm to form virus plasmid, the DLP is assembled in the virus plasmid to generate the DLP, the DLP has no infection capacity, the DLP subsequently enters an endoplasmic reticulum, is assembled with newly synthesized VP4 and VP7 to form TLP with infection capacity, and is secreted out of a host cell, and the replication process of the virus is completed.

So far, the research on the mechanism of rotavirus infection is still insufficient, the clinical treatment is mainly symptomatic treatment, and safe and effective medicaments directly aiming at viruses are lacked. The traditional antiviral gamma interferon and metabolic antiviral drugs are less used for resisting rotavirus infection due to toxic and side effects or poor effects and the like. In order to reduce the infection rate and the death rate, the clinical application has very urgent need for specific drugs capable of resisting rotavirus.

HSP990(MW:379.39) is a HSP90 inhibitor based on a 2-amino-4-methyl-7, 8-dihydropyrido [4,3-d ] pyrimidin-5 (6H) -one backbone, which differs structurally from currently common HSP90 inhibitors such as Geldanamycin (Geldamamycin, GA, MW:560.64) and its derivative, Taneslimycin (17-AAG, MW:585.69) (FIGS. 1 a-1 c). HSP990 is combined with the N end of an ATP binding domain of HSP90, and has obvious inhibition effect on 4 subtypes (HSP90 alpha, HSP90 beta, GRP94 and TRAP-1) of HSP 90.

It has now been found that HSP990 frees HSP90-p23 complexes in GTL-16 gastric cancer cells and depletes c-Met and induces HSP70 expression in tumor cells. HSP990 effectively inhibits the growth of human cell lines and various types of primary tumors.

HSP990 inhibits the proliferation of all glioma tumor initiating cells (GICs). In GICs with high expression of Olig2, HSP990 significantly attenuated their proliferation. HSP990 may also promote tumor cell apoptosis by inhibiting the expression of CDK2 and CDK 4. However, no report on the anti-rotavirus aspect of HSP990 at home and abroad is found so far.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of HSP990 in the preparation of anti-rotavirus drugs.

In order to achieve the purpose, the invention provides the following technical scheme:

1.HSP990 is used in preparing medicine for resisting rotavirus.

Preferably, the rotavirus resisting medicine is a rotavirus infection resisting or severe lethal rotavirus infection preventing medicine.

Preferably, the anti-rotavirus medicament is an anti-rotavirus medicament suitable for infants.

2. An anti-rotavirus medicine contains HSP990 as effective component.

Preferably, the medicament is a single or compound preparation of HSP 990.

Preferably, the preparation form of the medicament is selected from any one of tablets, capsules, granules, powder, oral liquid or injection.

The invention has the beneficial effects that:

according to the invention, a human intestinal epithelial cell Caco-2 infected rotavirus Wa strain model is used for evaluating the effect of HSP990 on rotavirus infection, and flow cytometry finds that the HSP990 obviously inhibits the rotavirus Wa strain from infecting the intestinal epithelial cell Caco-2, so that the infection efficiency is reduced by over 60%; meanwhile, the titer of the nascent virus is detected to find that HSP990 can strongly inhibit the generation of active rotavirus, and the inhibition efficiencies of HSP990 at different concentrations (10 mu M, 1 mu M and 100nM) are 100%, 95% and 85% respectively; further, in vivo experiments in suckling mice, oral administration of HSP990 can significantly inhibit diarrhea caused by rotavirus infection and significantly reduce the mortality rate of severe lethal rotavirus infection. The above experimental results show that HSP990 can inhibit rotavirus from establishing infection in cells and prevent infected cells from generating new viruses with infection capacity, i.e. inhibit infection and replication of rotavirus, thereby achieving the effect of resisting rotavirus and obviously reducing adverse effects such as diarrhea and death caused by rotavirus infection. Therefore, the rotavirus-resistant medicament is taken as a main active component, is expected to develop a high-efficiency specific rotavirus-resistant medicament, relieves the rotavirus diarrhea occurrence, reduces the death rate of severe lethal rotavirus infection, meets the clinical treatment requirement and ensures the health of people.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1a, FIG. 1b and FIG. 1c are the chemical structural formulae of HSP990, GA and 17-AAG, respectively.

Figure 2 is that HSP990 significantly inhibited the establishment of rotavirus infection.

In vitro rotavirus Wa strain infects human intestinal epithelial cells Caco-2(moi ═ 1), and the infection is divided into 3 groups: the frequency of infected cells was measured by flow cytometry 20h after infection by Mock infection (Mock infectcion), infection + DMSO (RV + DMSO) and infection + HSP990 (RV +10 μm HSP 990).

FIG. 3 is a graph showing that HSP990 strongly inhibits the production of active rotavirus.

In vitro rotavirus Wa strain infects human intestinal epithelial cells Caco-2(moi ═ 1), and the infection is divided into 5 groups: the rotavirus titer in the culture supernatant was detected by immunofluorescence colony assay (FFA) 24h after infection in a Mock infection group (Mock infectcion), an infection + DMSO group (RV + DMSO), an infection + HSP990 group (RV + 10. mu.M HSP990), an infection + Geldanamycin group (RV + 10. mu.M Geldanamycin) and an infection +17-AAG group (RV + 10. mu.M 17-AAG).

Figure 4 is a graph of the inhibitory effect of HSP990 at various concentrations on rotavirus infection.

Human intestinal epithelial cells Caco-2(moi ═ 1) were infected with rotavirus Wa strain in vitro, and HSP990 (dissolved in DMSO) was added to the virus solution at various concentrations (10 μ M, 1 μ M, 100nM, 10nM, 1nM), and the same volume of DMSO was used as a negative control. After the virus liquid is absorbed for 1h, the virus liquid is discarded. Cells were washed 2 times for 3min in 1ml DMEM medium. Finally, 1ml of DMEM medium (containing HSP990 with corresponding concentration) is added into each hole and placed into a cell culture box for further culture for 24 hours. Flow cytometry and FFA methods were used to detect infection efficiency and culture supernatant virus titer.

FIG. 5 is a graph of the effect of HSP990 on the development of rotavirus suckling mouse diarrhea.

Selecting 5-7 day old Balb/c newborn mouse with weight of 3.5-5.0g, intragastric infection of 5 × 10⁵PFU rotavirus (SA11 strain), then returned to the mother rat cage and fed by the mother rat. The drugs HSP990, Geldanamycin and 17-AAG (all 10mg/kg) were administered 1 time each at 0 and 8 hours of suckling mice infection, and the control group was gavaged with an equal volume of DMSO dissolved in an equal volume of 0.01M PBS.

Figure 6 is a graph of the effect of HSP990 on the mortality of suckling mice in a severely lethal rotavirus infection.

Selecting 5-day-old Balb/c newborn mice with weight of 4.6-5g, intragastric infection of 2 × 10⁶PFU rotavirus (SA11 strain), then returned to the mother rat cage and fed by the mother rat. HSP990, Geldamamycin and 17-AAG (all 10mg/kg) were gavaged 1 time each at 0, 8 and 16 hours after suckling mice were infected, and control groups were gavaged with an equal volume of DMSO dissolved in an equal volume of 0.01M PBS.

FIG. 7 is a diagram of CCK-8 detecting HSP990 cytotoxicity.

5000 Caco-2 cells are inoculated in a 96-well plate, the cells adhere to the wall after being cultured for 12h, then HSP990, Geldanamycin and 17-AAG with the final concentration of 10 mu M are respectively added, DMSO with the same volume is used as a control, and a group of cell-free controls are arranged at the same time. After 1h of drug addition, 10. mu.L of the cytotoxic assay reagent CCK-8 (purchased from Biyuntian) was added to each well, and the OD450 absorbance of the sample was measured after 2h of incubation.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The experimental procedures, in which specific conditions are not specified, in the preferred examples are generally carried out according to conventional conditions, for example, as described in the molecular cloning protocols (third edition, J. SammBruk et al, Huangpetang et al, scientific Press, 2002), or according to the conditions recommended by the manufacturers.

HSP990, Geldamamycin (GA) and 17-AAG mentioned in the examples, the chemical structures of which are shown in FIG. 1a, FIG. 1b and FIG. 1c, respectively.

1.HSP990 for remarkably inhibiting rotavirus infection establishment

Human intestinal epithelial cells Caco-2 (purchased from ATCC, HTB-37) were seeded in 6-well cell culture plates using DMEM high-sugar medium + 10% by volume peptide bovine serum at 37 ℃ with a volume percentage of 5% CO₂Culturing in a cell culture box. When the cells were completely confluent, rotavirus Wa strain (purchased from ATCC, VR-2018) was used for infection (MOI ═ 1), and the infection was divided into 3 groups: mock-infected (Mock in fectcion, using an equal volume of DMEM medium instead of viral fluid), infected + DMSO (RV + DMSO, rotavirus Wa strain infected with an equal volume of DMSO added (same volume of HSP990 solution) and infected + HSP990 (RV + HSP990, rotavirus Wa strain infected with 10 μ M HSP990 (dissolved in DMSO)). After 20h of infection, cells were digested with trypsin-EDTA at a mass concentration of 0.25%, cells were fixed with paraformaldehyde at a mass concentration of 4% for 15min, cells were permeabilized with Triton-x-100 at a mass concentration of 7min, cells were washed 2 times with 0.01M PBS (pH7.3), goat anti-rotavirus polyclonal antibody (purchased from Virostat, USA) labeled with FITC (fluorescein isothiocyanate) stained for RV antigen (1:100 dilution), and the frequency of infected cells was examined by flow cytometry (FIG. 2). The result shows that HSP990 can obviously inhibit rotavirus from infecting intestinal epithelial cells Caco-2, so that the infection capacity of the intestinal epithelial cells is reduced by over 60 percent.

2. HSP990 strongly inhibits the production of active rotaviruses

Human intestinal epithelial cells Caco-2 were seeded in 12-well cell culture plates using DMEM high-sugar medium plus 10% by volume fetal bovine serum at 37 ℃ with 5% by volume CO₂Culturing in a cell culture box. When the cells were completely confluent, infection with rotavirus Wa strain (MOI ═ 1) was used, and the infections were divided into 5 groups: mock-infected group (Mock infectcion, using an equal volume (1ml) of DMEM medium instead of virus fluid), infected + DMSO group (RV + DMSO, infected and added an equal volume (equal volume of HSP990 solution added) DMSO), infected + HSP990 group (RV +10 μ M HSP990, rotavirus strain Wa infected and added HSP990 (dissolved in DMSO) at a final concentration of 10 μ M), infected + Geldamamycin group (RV +10 μ M Geldamamycin), and infected +17-AAG group (RV +10 μ M17-AAG). After the virus liquid is absorbed for 1h, the virus liquid is discarded. Cells were washed 2 times for 3min in 1ml DMEM medium. Finally, adding 1ml of DMEM medium (without fetal calf serum) into each well, placing the DMEM medium into a cell culture box for further culture for 24 hours, sucking culture supernatant into a 1.5ml EP tube, centrifuging for 5min at 2000g to remove cell residues, taking 100 mul of culture supernatant (containing newly generated rotavirus), adding trypsin with the final concentration of 10 mug/ml for digestion at 37 ℃ for 30 minutes, adding 100 mul of trypsin digested culture supernatant into MA104 cells (purchased from ATCC, CRL-2378) cultured in a 96-well plate (the confluence is 100%), and sucking virus liquid after the virus liquid is absorbed for 1 hour. Cells were washed 2 times for 3min in 100. mu.l DMEM medium (without fetal calf serum). Finally, 100 mul of DMEM medium (without fetal calf serum) is added into each hole and is placed into a cell culture box for further culture for 20 hours, cells are fixed for 15 minutes by paraformaldehyde with the mass concentration of 4%, 7 minutes by Triton-x-100 permeabilized cells with the mass concentration of 1%, the cells are washed for 2 times by 0.01M PBS, a goat anti-rotavirus polyclonal antibody (purchased from Virostat, USA) marked by FITC (fluorescein isothiocyanate) is used for staining rotavirus antigen (room temperature for 3 hours) after 1:100 dilution), liquid is sucked up after 0.01M PBS is washed once, the cell is placed under an Olympus inverted fluorescence microscope to observe infected cells, and the virus content in culture supernatant is calculated. The results show that HSP990 strongly inhibits the production of active rotavirus, and that 10 μ M HSP990 inhibits the production of active rotavirus almost 100% in vitro (by 6 orders of magnitude); same concentration (10. mu.M) of other common HSP9The 0 inhibitor geldanamycin and its derivative 17-AAG also inhibited rotavirus replication (by 2 orders of magnitude), but was significantly weaker than HSP990 (fig. 3).

3. Inhibition of rotavirus infection by various concentrations of HSP990

Human intestinal epithelial cells Caco-2 were seeded on 12-well cell culture plates and cultured in DMEM high-sugar medium plus 10% by volume fetal bovine serum in a 5% CO2 cell culture chamber at 37 ℃. When the cells were completely confluent, rotavirus strain Wa was used for infection (MOI ═ 1), and HSP990 (dissolved in DMSO) was added to the virus solution at various concentrations (10 μ M, 1 μ M, 100nM, 10nM, 1nM), and a negative control was made with the same volume of DMSO. After the virus liquid is absorbed for 1h, the virus liquid is discarded. Cells were washed 2 times for 3min in 1ml DMEM medium. Finally, 1ml of DMEM medium (containing HSP990 with corresponding concentration) is added into each hole and placed into a cell culture box for further culture for 24 hours. Sucking culture supernatant into 1.5ml EP tube, and detecting the titer of the new virus by the same method as that of FIG. 3; the cells were digested with trypsin-EDTA at a mass concentration of 0.25% to determine the infection rate of the cells, as shown in FIG. 2. The results show that 10 mu M, 1 mu M and 100nM HSP990 can obviously inhibit the infection of the rotavirus on the intestinal epithelial cells (the inhibition efficiency is about 60 percent), and the 10nM and 1nM HSP990 has no obvious inhibition effect on the infection of the rotavirus on the intestinal epithelial cells. 10 mu M, 1 mu M and 100nM HSP990 can effectively inhibit the generation of active rotavirus, and the inhibition efficiency is 100%, 95% and 85% respectively; HSP990 at 10nM, 1nM, was not effective in inhibiting the production of active rotavirus. Thus, 0.1-10 μ M HSP990 in vitro was able to effectively inhibit rotavirus infection and the production of active virus (fig. 4).

4. HSP990 obviously relieves diarrhea symptoms of rotavirus suckling mice

Selecting 5-7 day old Balb/c newborn mouse (purchased from army and military medical university animal center) with weight of 3.5-5.0g, and gavage infection of 5 × 10⁵PFU rotavirus (SA11 strain, purchased from ATCC VR-1565), then returned to the mother rat cage and fed by the mother rat. The drugs HSP990, Geldanamycin and 17-AAG (all 10mg/kg) were administered 1 time each at 0 and 8 hours of suckling mice infection. The drugs were all 50mM stock solutions in DMSO. The administration was performed by gavage after diluting the stock solution containing the corresponding mass of drug to 50. mu.l with 0.01M PBS. ControlThe administration group was gavaged with an equal amount of DMSO diluted to 50. mu.l with an equal amount of 0.01M PBS. The results show that the control group virus infected 16h partial suckling mice begin to have obvious diarrhea symptoms (watery stool), and 100% of the mice after infection for 24h all have diarrhea symptoms; however, only 10% of HSP990 group showed diarrhea symptoms after 24h infection, which indicates that HSP990 can obviously inhibit diarrhea caused by intestinal rotavirus infection. The oral administration of equal amounts of Geldanamycin and 17-AAG had no obvious inhibitory effect on the occurrence of rotavirus diarrhea in suckling mice, and the protective effect was significantly lower than that of HSP990 (FIG. 5).

5. HSP990 obviously reduces the mortality rate of mice in severe lethal rotavirus infection model

Firstly, establishing a severe lethal rotavirus infection model: selecting 5-day-old Balb/c newborn mouse (purchased from the laboratory animal center of army medical university) with weight of 4.6-5g, and intragastric infection of 2 × 10⁶PFU rotavirus (SA11 strain), then returned to the mother rat cage and fed by the mother rat. HSP990, Geldanamycin and 17-AAG 10mg/kg body weight were administered 1 time each by gavage at 0, 8 and 16 hours of suckling mice infection. The drugs were all 50mM stock solutions in DMSO. The administration was performed by gavage after diluting the stock solution containing the corresponding mass of drug to 50. mu.l with 0.01M PBS. The control group was gavaged with an equal volume of DMSO diluted to 50. mu.l with an equal volume of 0.01M PBS. Part of suckling mice die after the control group virus is infected for 48 hours, and all suckling mice die after the control group virus is infected for 72 hours; however, the administration group can obviously reduce the mortality rate of rotavirus infection, only 20 percent of suckling mice die after HSP990 is infected for 72 hours, and the suckling mice do not die any more after the HSP990 is infected, which shows that HSP990 can obviously reduce the mortality rate of severe rotavirus infection. Oral administration of equal amounts of Geldanamycin and 17-AAG had no significant inhibitory effect on the death of suckling mice, and the protective effect was significantly lower than HSP990 (fig. 6).

6. HSP990 has significantly lower cytotoxicity

Cytotoxicity is an important indicator of compound druggability. We inoculated 5000 Caco-2 cells in a 96-well plate, cultured for 12h, the cells attached to the wall, then added HSP990, Geldanamycin and 17-AAG with the final concentration of 10 μ M respectively, DMSO with the same volume is used as a control, and a group of no-cell controls are arranged at the same time. After the drug is added for 1h, 10 mul of a cytotoxic assay reagent CCK-8 (purchased from Biyuntian) is added into each well, and the OD450 light absorption value of the sample is detected after the sample is cultured for 2h, wherein the higher the OD450 value is, the lower the cytotoxicity is. The results show that HSP990 is significantly less cytotoxic than the currently used HSP90 inhibitors Geldanamycin and 17-AAG (fig. 7).

From the above results, it can be determined that the common HSP90 inhibitor Geldanamycin and its derivative 17-AAG have inhibitory effect on rotavirus replication in vitro experiments, but in vivo experiments in suckling mice show that the ability to resist rotavirus diarrhea and the ability to prevent severe lethal rotavirus infection are poor, so the common HSP90 inhibitor Geldanamycin and its derivative 17-AAG are not suitable for preparing medicines for infants for resisting rotavirus infection.

In contrast, HSP990 can significantly inhibit rotavirus from infecting intestinal epithelial cells, and more strongly inhibit the generation of active rotavirus; HSP990 also has lower cytotoxicity. In vivo, HSP990 is effective in inhibiting the occurrence of rotavirus diarrhea and significantly reducing the mortality of suckling mice caused by severe lethal rotavirus infection. The compound can be used as an active component and is independently applied or combined with other active components to form a compound, and the compound is prepared into various dosage forms of the infant rotavirus infection resisting and severe lethal rotavirus preventing medicines such as tablets, capsules, granules, powder, oral liquid or injection and the like by adopting pharmaceutically acceptable auxiliary materials and a preparation conventional method for clinical use.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (3)

1. The application of HSP990 in preparing anti-rotavirus medicaments; HSP990 has the following structural formula:

   
2. 2. the use of claim 1, wherein the anti-rotavirus drug is an anti-rotavirus infection or a severe lethal rotavirus infection prevention drug.
3. 3. The use of claim 1, wherein the anti-rotavirus medicament is an anti-rotavirus medicament suitable for infants.

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