CN111374983B - Application of CH5138303 in preparation of anti-rotavirus drugs - Google Patents

Abstract

Experiments prove that the CH5138303 can inhibit rotavirus from establishing infection in cells and prevent infected cells from generating new viruses with infection capacity, namely inhibit infection and replication of rotavirus, so that the effect of resisting the rotavirus 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 CH5138303 in preparation of anti-rotavirus drugs

Technical Field

The invention belongs to the technical field of anti-rotavirus medicaments, and relates to application of CH5138303 in preparing 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.

CH5138303(MW:415.9) is a highly orally bioavailable Hsp90 inhibitor, structurally distinct from currently available HSP90 inhibitors such as Geldanamycin (GA, MW:560.64) and its derivative, Taneslimycin (17-AAG, MW:585.69) (see FIGS. 1 a-1 c). CH5138303Kd was 0.48 nM. CH5138303 acts primarily on HSP90 α subunit, with a high binding affinity for N-terminal HSP90 α (Kd ═ 0.52 nM). In vitro CH5138303 has strong inhibition capability on human cancer cell strains HCT116 and NCI-N87, and IC50 is 0.098 μ M and 0.066 μ M respectively. CH5138303 has high oral bioavailability (F ═ 44.0%) in mice, and has strong antitumor effect (tumor growth inhibition rate ═ 136%) in human NCI-N87 gastric cancer xenograft model. However, no report on the rotavirus resistance of CH5138303 is found at home and abroad so far.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of CH5138303 in preparing an anti-rotavirus medicament.

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

1. application of CH5138303 in preparing anti-rotavirus medicines.

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 CH5138303 as effective component.

Preferably, the medicament is a single or compound preparation of CH 5138303.

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:

the invention uses a human intestinal epithelial cell Caco-2 infected rotavirus Wa strain model to evaluate the effect of CH5138303 on rotavirus infection, and flow cytometry finds that the CH5138303 obviously inhibits the rotavirus Wa strain from infecting the intestinal epithelial cell Caco-2, so that the infection efficiency is reduced by over 50 percent; meanwhile, the detection of the titer of the nascent virus shows that CH5138303 can strongly inhibit the generation of active rotavirus, and the inhibition efficiencies of CH5138303 at different concentrations (10 mu M and 1 mu M) are respectively 90 percent and 80 percent; further, in an in vivo experiment of a suckling mouse, oral administration of CH5138303 can obviously inhibit diarrhea caused by rotavirus infection and obviously reduce the death rate of severe lethal rotavirus infection. The experimental results show that CH5138303 can inhibit rotavirus from establishing infection in cells and prevent infected cells from generating new viruses with infection capacity, namely inhibit infection and replication of rotavirus, so that the effect of resisting the rotavirus is achieved, and adverse effects such as diarrhea and death 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.

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 formulas of CH5138303, GA and 17-AAG, respectively.

Figure 2 shows that CH5138303 significantly inhibits the establishment of rotavirus infection.

In vitro rotavirus Wa strain infects human intestinal epithelial cells Caco-2(moi is 1), the infection is divided into 3 groups, namely a simulated infection group (mockinfection), an infection + DMSO group (RV + DMSO) and an infection + CH5138303 group (RV +10 mu m CH5138303), and the frequency of the infected cells is detected by flow cytometry 20h after infection.

Figure 3 shows that CH5138303 strongly inhibits the production of active rotavirus.

In vitro rotavirus Wa strain infects human intestinal epithelial cells Caco-2(moi is 1), the infection is divided into 5 groups, namely a simulated infection group (Mockinfectcion), an infection + DMSO group (RV + DMSO), an infection + CH5138303 group (RV +1 mu M CH5138303), an infection + Geldamamycin group (RV +1 mu M Geldamamycin) and an infection +17-AAG group (RV +10 mu M17-AAG), and the rotavirus titer in culture supernatant is detected by an immunofluorescence colony assay (FFA) after 24h of infection.

FIG. 4 shows the inhibitory effect of CH5138303 at various concentrations on rotavirus infection.

Human intestinal epithelial cells Caco-2(moi ═ 1) were infected with rotavirus Wa strain in vitro, and various concentrations (10 μ M, 1 μ M, 100nM, 10nM, 1nM) of CH5138303 (dissolved in DMSO) were added to the virus solution, with the same volume of DMSO 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 CH5138303 with corresponding concentration) is added into each well 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 showing the effect of CH5138303 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 CH5138303, Geldamamycin and 17-AAG (all 10mg/kg) were administered 1 time each at 0 and 8 hours of infection in suckling mice, and the control group was gavaged with an equal volume of DMSO dissolved in an equal volume of 0.01M PBS.

FIG. 6 is a graph showing the effect of CH5138303 on the mortality rate of suckling mice in severe 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. CH5138303, Geldamamycin and 17-AAG (all 10mg/kg) were gavaged 1 time each at 0, 8 and 16 hours after suckling mice were infected, and the control group was gavaged with an equal volume of DMSO dissolved in an equal volume of 0.01M PBS.

FIG. 7 shows that CCK-8 detects CH5138303 cytotoxicity.

5000 Caco-2 cells are inoculated in a 96-well plate, the cells adhere to the wall after 12h of culture, then CH5138303, Geldanamycin and 17-AAG with the final concentration of 10 MuM are respectively added, DMSO with the same volume is used as a control, and a group of cell-free controls are simultaneously arranged. 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.

Examples of the compounds of formula (I) include CH5138303, Geldanamycin (GA) and 17-AAG, the chemical structures of which are shown in FIG. 1a, FIG. 1b and FIG. 1c, respectively.

1.CH5138303 obviously inhibits 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, infection was performed with rotavirus Wa strain (purchased from ATCC, VR-2018) (MOI ═ 1), and the infection was divided into 3 groups, a Mock infection group (Mock infectcion, using an equal volume of DMEM medium instead of the virus solution), an infection + DMSO group (RV + DMSO, rotavirus Wa strain was infected and added in an equal volume (same volume as added CH5138303 (purchased from seleck corporation, s 7340)) DMSO), and an infection + CH5138303 group (RV + CH5138303, rotavirus Wa strain was infected and added with 10 μ M CH5138303 (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 1% for 7min, cells were washed with 0.01M PBS (pH7.3) 2 times, and goat anti-rotavirus polyclonal antibody (purchased from virostat, USA) labeled with FITC (fluorescein isothiocyanate) was used to stain RV antigen(1:100 dilution) the frequency of infected cells was determined by flow cytometry (FIG. 2). The result shows that CH5138303 can obviously inhibit rotavirus from infecting intestinal epithelial cells Caco-2, so that the infection capacity of the intestinal epithelial cells is reduced by more than 50%.

2. CH5138303 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, rotavirus Wa strain was used for infection (MOI 1), and the infection was divided into 3 groups, a Mock infection group (Mock infecticion, using an equal volume (1ml) of DMEM medium instead of the virus solution), an infection + DMSO group (RV + DMSO, rotavirus Wa strain was infected and an equal volume (same volume of CH5138303 solution added) of DMSO was added), an infection + CH5138303 group (RV + CH5138303, rotavirus Wa strain was infected and CH5138303 (dissolved in DMSO) was added at a final concentration of 1. mu.M), an infection + Geldamycin group (RV + 1. mu.MGeldanamycin) and an infection +17-AAG group (RV + 10. mu.M 17-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 ul of culture supernatant (containing newly generated rotavirus), adding trypsin with the final concentration of 10 ug/ml to digest for 30 minutes at 37 ℃, adding 100 ul 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 put into a cell culture box for further culture for 20 hours, the cells are fixed for 15 minutes by paraformaldehyde with the mass concentration of 4 percent, 7 minutes by Triton-x-100 permeabilized cells with the mass concentration of 1 percent, 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) and is washed for one time by 0.01M PBS, then liquid is sucked up, the cells are observed and infected under an Olympus inverted fluorescence microscope, and the calculation is carried outVirus content in culture supernatant. The results showed that CH5138303 strongly inhibited the production of active rotavirus, and 10 μ M CH5138303 inhibited the production of 90% of active rotavirus in vitro (fig. 3).

3. Shows the inhibition effect of CH5138303 on rotavirus infection at different concentrations

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 different concentrations (10 μ M, 1 μ M, 100nM, 10nM, 1nM) of CH5138303 (dissolved in DMSO) were added to the virus solution, as was the volume of DMSO for 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 CH5138303 with corresponding concentration) is added into each well 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 and 1 mu M CH5138303 can obviously inhibit the infection of the rotavirus to the intestinal epithelial cells (the inhibition efficiency is about 50 percent), and 100nM, 10nM and 1nM CH5138303 have no obvious inhibition effect on the infection of the rotavirus to the intestinal epithelial cells. 10 mu M and 1 mu M CH5138303 can effectively inhibit the generation of active rotavirus, and the inhibition efficiency is respectively 90% and 80%; 100nM, 10nM, 1nM CH5138303 was not effective in inhibiting the production of active rotaviruses. Therefore, 1-10 μ M CH5138303 was effective in inhibiting rotavirus infection and active virus production in vitro (FIG. 4).

4. CH5138303 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 CH5138303, Geldanamycin and 17-AAG (all 10mg/kg) were administered 1 time each at 0 and 8 hours of infection in suckling mice. The drugs were all 50mM stock solutions in DMSO. The administration mode is that the medicine will contain CH513 with corresponding mass8303 stock solution was diluted to 50. mu.l with 0.01MPBS and gavaged. The control group was gavaged with an equal volume of DMSO diluted to 50. mu.l with an equal volume 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 20% of the administration groups showed diarrhea symptoms at 24h of infection; thus, CH5138303 can obviously inhibit the occurrence of diarrhea caused by intestinal rotavirus infection (figure 5).

5. CH5138303 obviously reduces the mortality 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. The drugs CH5138303, Geldamamycin and 17-AAG (all 10mg/kg) were each administered 1 time by gavage at 0, 8 and 16 hours of infection in suckling mice. The drugs were all 50mM stock solutions in DMSO. The administration was performed by gavage of a stock solution containing the corresponding mass of CH5138303 diluted to 50. mu.l with 0.01 MPBS. 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 significantly reduced the mortality rate of rotavirus infection, and only 20% of suckling mice died 72h after CH5138303 infection, and no longer died thereafter, indicating that CH5138303 significantly reduced the mortality rate of severe rotavirus infection (fig. 6).

6. CH5138303 was significantly less cytotoxic.

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 CH5138303, Geldanamycin and 17-AAG with a final concentration of 10 μ M, respectively, and DMSO with the same volume was used as a control, and a group of no-cell controls was set. 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 the cytotoxicity of CH5138303 is significantly lower than that of the currently used CH5138303 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 a relatively obvious inhibitory effect on rotavirus replication in vitro experiments, but in vivo experiments in suckling mice show that the ability of resisting rotavirus diarrhea and the ability of preventing 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.

The results can determine that the CH5138303 can obviously inhibit rotavirus from infecting intestinal epithelial cells and strongly inhibit the generation of active rotavirus, and the CH5138303 has lower cytotoxicity, effectively inhibits the generation of rotavirus diarrhea in a suckling mouse and obviously reduces the death rate of the suckling mouse 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 severe lethal rotavirus prevention 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 CH5138303 in preparing anti-rotavirus medicaments; the chemical structural formula of CH5138303 is as follows:

   
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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