CN115381816A - Application of VER50589 in preparing medicine for resisting enterovirus 71 - Google Patents
Application of VER50589 in preparing medicine for resisting enterovirus 71 Download PDFInfo
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Abstract
The invention provides an application of VER50589 in preparation of an anti-enterovirus 71 medicament, wherein an HSP90 beta inhibitor (VER-50589) has certain EV71 inhibition activity, can strongly inhibit RD cytopathic effect caused by EV71 viruses, enhances cell survival rate, inhibits replication of virus RNA level, remarkably reduces death rate of infected mice, and can be applied in preparation of anti-EV 71 virus medicaments.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to application of VER50589 in preparation of an anti-enterovirus 71 medicament.
Background
Enterovirus type 71 (EV 71) belongs to a member of Enterovirus (Enterovirus) of Picornaviridae (Picornaviridae), is one of the most major pathogens causing hand-foot-and-mouth disease in infants and young children, sometimes accompanied by serious central nervous system complications including aseptic meningitis, brainstem encephalitis, autonomic nerve disorders, pulmonary edema, and the like, and even leading to death. Since the first report in 1969, EV71 infectious diseases have been frequently outbreaks and epidemics worldwide, and the situation is severe in Asia-Pacific region, especially China. In the last decade, the HFMD outbreak associated with EV71 has proliferated. In view of the great harm to the life and health of people in China caused by the spread and prevalence of the hand-foot-and-mouth disease, the spread and prevalence of the hand-foot-and-mouth disease are strictly controlled in China. Currently, the prevention and treatment of viral diseases relies primarily on vaccines and drugs, and there is no specific data to support the protection of other serotypes of enteroviruses by marketed vaccines. The treatment methods for EV71 virus infection are quite limited, and the main treatment means are symptomatic support treatment and broad-spectrum antiviral treatment, so that the treatment effects are limited, the individual difference is large, and the popularization is difficult.
Therefore, the research and development of related antiviral drugs are key efforts to overcome the virus, and the development of specific and effective anti-EV 71 drugs is imperative.
Disclosure of Invention
The invention aims to provide application of VER50589 in preparation of an anti-enterovirus 71 type medicament, and the application discovers that VER50589 has a strong inhibiting effect on enteroviruses for the first time, and provides a direction for treating and preventing enterovirus infectious diseases.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of VER50589 in preparing an anti-enterovirus 71 medicament.
Further, the anti-enterovirus 71 medicament also comprises pharmaceutically acceptable auxiliary materials and carriers.
Further, the adjuvant includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener, or a colorant.
Further, the dosage form of the anti-enterovirus 71 medicament comprises at least one of granules, tablets, pills, capsules, injections or dispersing agents.
Further, the anti-viral means of the anti-enterovirus 71 drug comprises: inhibit enterovirus intracellular nucleic acid replication, viral protein expression and infection.
Further, the inhibition rate of the VER-50589 against EV71 virus was 96.3% at a VER-50589 concentration of 20. Mu. Mol/mL, and the inhibition rate of the EV71 virus was 89% at a VER-50589 concentration of 0.16. Mu. Mol/mL.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
according to the application of VER50589 in preparation of an anti-enterovirus 71 medicament, the HSP90 beta inhibitor (VER-50589) has good activity of inhibiting EV71 virus through a large number of biological experiments. The specific expression is that the cell pathological effect caused by the EV71 virus can be inhibited, the survival rate of infected cells is enhanced, and the replication and proliferation of the EV71 virus in the cells are inhibited. Therefore, the compound HSP90 beta inhibitor has potential to be prepared into a specific treatment medicament for resisting EV71 infection and has better clinical application prospect.
Meanwhile, the VER50589 micromolecule compound has a simple synthesis process, and is easy to produce and popularize on a large scale. The anti-EV 71 virus activity of the HSP90 beta inhibitor is not reported, and the HSP90 beta inhibitor has a certain guiding effect on the development of the anti-EV 71 virus activity. An anti-EV 71 drug is searched from a compound with a similar structure, so that an action target of the anti-EV 71 drug can be easily found through structure-activity relationship research, and a certain reference significance is provided for further drug development.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 shows the results of the toxicity test of HSP90 beta inhibitor (VER-50589) on RD cells affected by EV71 in example 1;
FIG. 2 shows the results of the toxicity test of HSP 90. Beta. Inhibitor (VER-50589) on Hela cells affected by EV71 in example 1;
FIG. 3 is the TICD of example 2 50 Measuring antiviral activity test results of HSP90 beta inhibitor (VER-50589) at different concentrations;
FIG. 4 is a graph showing the results of measurement of antiviral activity of HSP 90. Beta. Inhibitor (VER-50589) at various concentrations by the plaque assay in example 2; wherein FIG. 4A is a photograph taken by a plaque method; FIG. 4B is a statistical result of the number of plaques;
FIG. 5 shows the effect of the real-time quantitative fluorescent PCR (qPCR) method of example 3 on the inhibition of viral replication by HSP90 β inhibitor (VER-50589);
FIG. 6 is a graph showing the effect of the western blot assay of example 3 on the inhibition of viral replication by the HSP90 β inhibitor (VER-50589);
FIG. 7 shows the results of the effect of VER-50589 and the control group on the cytopathic effect (CPE) produced in the host cell RD by EV71 in example 4; wherein FIG. 7A is the result of the cytopathic effect of control Rib generation ribavirin on EV71 in host cell RD; FIG. 7B shows the results of the cytopathic effect of VER-50589 on EV71 generation in host cell RD;
FIG. 8 shows that the HSP90 beta inhibitor (VER-50589) significantly inhibited EV71 replication at ICR3d suckling mouse level in example 5, thereby significantly improving survival of infected mice.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
VER50589 is HSP90 beta inhibitor, is a kind of organic small molecule compound with biological activity, and the protein family of the target protein heat shock protein 90 (HSP 90 beta) acted by it is one of the most abundant molecular chaperones, and is highly conserved from bacteria to eukaryotes. VER50589 has the following chemical structure:
it should be noted that VER50589 is referred to as number JYT046 in the present invention.
VER-50589 is a potent HSP90 β inhibitor with an IC50 of 21nM for HSP90 β.
The invention discovers through experiments that: the compound HSP90 beta inhibitor (VER-50589) inhibits the cytopathic effect (CPE) produced by EV71 in host cells RD, enhances cell survival; inhibiting the replication and proliferation of the EV71 virus in cells; protects against death of EV71 virus infected mice. In conclusion, the results of the invention show that the compound VER50589 has the potential to prepare the specific treatment medicine for resisting EV71 infection and has better clinical application prospect.
Therefore, the invention provides an application of VER50589 in preparing an anti-enterovirus 71 medicament, wherein the application refers to the step of adding pharmaceutically acceptable auxiliary materials and carriers into VER50589 to prepare an anti-EV 71 virus preparation, the auxiliary materials comprise at least one of a filling agent, a disintegrating agent, a binding agent, an excipient, a diluent, a lubricant, a sweetening agent or a coloring agent, and different auxiliary materials are selected according to the requirements of medicament forms. The preparation is granule, tablet, pill, capsule, injection or dispersant.
It is understood that the VER50589 is used as a lead compound for further structural optimization, and is used for preparing a medicament for treating enterovirus infectious diseases, and the method also belongs to the protection scope of the invention.
The use of VER50589 in the present application for the preparation of an anti-enterovirus type 71 medicament will be described in detail below with reference to examples and experimental data.
EXAMPLE 1 cytotoxicity assay of HSP90 beta inhibitor of interest (VER-50589)
1. In RD cells, the cytotoxicity of HSP90 β inhibitor (VER-50589) was examined. Plating RD cells in 96-well plates, at 37 ℃ 5% 2 Culturing in incubator for 12-16h, discarding cell culture solution, adding cell maintenance solution containing different concentrations of test compounds, culturing, performing drug action for 48h, MTT staining, and detecting OD 492 nm, cell viability was analyzed. Prism7 software calculates the Median Cytotoxic Concentration (CC) of drug to cells 50 ) As shown in fig. 1.
As can be seen from FIG. 1, the median toxic concentration CC of the drug to the cells in RD cells 50 ＝22123nM。
2. In HeLa cells, the cytotoxicity of HSP 90. Beta. Inhibitor (VER-50589) was examined. Plating RD cells in 96-well plates at 37 ℃ with 5% CO 2 Culturing in incubator for 12-16h, discarding cell culture solution, adding cell maintenance solution containing different concentrations of test compounds, culturing, performing drug action for 48h, MTT staining, and detecting OD 492 nm, cell viability was analyzed. Prism7 software calculates the Median Cytotoxic Concentration (CC) of drug to cells 50 ) As shown in fig. 2.
As can be seen from FIG. 2, the half toxic concentration CC of the drug to the cells in Hela cells 50 ＝29027nM。
Example 2 inhibitory Effect of VER50589 on EV71 Virus replication
1、TICD 50 Method for determining inhibition effect of target compound HSP90 beta inhibitor (VER-50589) on EV71 virus replication at different concentrations
Plating RD cells in 96-well plates5% CO at 37 ℃ 2 Culturing in an incubator until the culture medium is overgrown, and removing the culture medium, wherein MOI =3 × 10 7 Cells were incubated for 2h with CPE/mL of EV71 virus solution to infect cells. Cell maintenance solutions containing different concentrations of test compounds were added separately and the incubation continued for about 24h, and the same volume of PBS was added for negative control. When the virus control hole has CPE lesion of about 90%, repeatedly freezing and thawing the cell plate, and collecting the cell culture medium containing virus (the virus titer is to be TCID) without the action of drug concentration 50 Detection by the method, and detection by the plaque method), using TCID 50 Method of determining the TCID of the virus without drug action 50 The trend of change of (c). Calculating TCID after EV71 replication inhibition at different concentrations by using Reed-Muench formula 50 And TCID of non-drug group virus well 50 . Histogram with Prism7 software Y-axis log 10 (TCID 50 In ml), the X-axis is in nmol/L, without understanding the drug concentration.
As shown in FIG. 3, it was revealed that the inhibition ratio against EV71 virus was 96.3% at the VER-50589 concentration of 20. Mu. Mol/mL and 89% at the VER-50589 concentration of 0.16. Mu. Mol/mL.
2. Plaque assay for determining inhibition effect of target compound HSP90 beta inhibitor (VER-50589) on EV71 virus replication at different concentrations
As is clear from FIG. 4, VER50589 has a good inhibitory effect on EV71 virus replication.
Example 3 detection of antiviral Activity of VER50589 against EV71
1. Inoculation of RD cells on 12-well plate cellsIn the culture plate, 5% CO at 37% 2 The cells were cultured in an incubator for 12-16h to a monolayer, treated with test compounds containing different concentrations, and the negative control was added with the same volume of PBS. After infection with EV71 virus and incubation at 37 ℃ for 2 hours, the medium was changed to DMEM medium containing 2% FBS, and after 24 hours, the effect of HSP 90. Beta. Inhibitor (VER-50589) in inhibiting virus replication was examined by real-time fluorescent quantitative PCR (qPCR).
The results are shown in FIG. 5, which shows that: under different concentration conditions, the HSP90 inhibitor (VER-50589) has obvious inhibition effect on the replication of EV71 virus.
2. The above experiment was repeated, and after 24h, cell pellets were collected and lysed on ice to prepare protein samples, which were then examined in western blots and photographed with Photoshop software.
The results are shown in FIG. 6, which indicates that HSP90 beta inhibitor (VER-50589) has better inhibitory activity against EV 71.
Example 4 detection of the inhibitory Effect of VER-50589 on EV 71-induced RD cell CPE
Plating RD cells in 96-well plates at 37 ℃ with 5% CO 2 After the culture box is used for culturing the full monolayer, the culture solution is discarded, and 100 TCID50 EV71 virus solution infects cells for 2h, and the cells are incubated by (ribavirin as a positive control drug). After the culture is continued for about 48 hours, when the virus control wells show CPE lesions of about 90%, the cytopathic effect (CPE) is observed under a microscope.
The results are shown in fig. 7, and show that the HSP90 beta inhibitor (VER-50589) inhibits the EV 71-induced RD cell CPE effect, the EV 71-infected RD cell becomes round and detached from the cell plate wall, and the treatment of the test compound at different concentrations has a remarkable inhibitory effect on the pathological effect. And compared with the Rib ribavirin substitute of a control group, the HSP90 beta inhibitor has stronger inhibition effect.
Example 5 Effect of the target Compound HSP90 beta inhibitor (VER-50589) on EV 71-infected mice
ICR3d suckling mouse is bred in SPF environment, mouse infection is carried out by intraperitoneal injection method, and inoculation dose is 10 8 PFU/mouse, volume 40. Mu.L. Subsequently, the mice were divided into 3 groups: (1) EV71+ vehicle group; (2) EV71+ JYT046 mg/Kg-d; (3) EV71+ JYT046 mg/Kg/d group. Mock group is an equal volume of placebo. The administration is carried out by intraperitoneal injection.
The dosage of JYT046 (VER-50589) was strictly performed according to the experimental protocol, 1 time per day for a total of 8 days. The overall experimental procedure followed strictly the guidelines of the national institutes of health of the united states as set forth in the guidelines for the operation of the animal care and use committee. The mouse daily monitoring indexes include: motility, stress, weight, limb flexibility, etc.
As shown in fig. 8, the mortality rate of mice in the administration group was significantly reduced and dose-related as compared to the control group. It was suggested that HSP90 β inhibitor (JYT 046) could significantly inhibit EV71 replication at the mouse level, thereby significantly improving survival of infected mice.
In conclusion, the HSP90 beta inhibitor (VER-50589) has certain EV71 inhibition activity, can strongly inhibit RD cytopathic effect caused by EV71 viruses, enhances cell survival rate, inhibits replication of viral RNA level, remarkably reduces death rate of infected mice, and has potential for further developing and preparing a medicament for clinically and effectively resisting EV71 virus infection.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention. It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (6)
- Application of VER50589 in preparing medicine for resisting enterovirus 71 is provided.
- 2. The use of claim 1, wherein the anti-enterovirus type 71 medicament further comprises pharmaceutically acceptable excipients and carriers.
- 3. The use according to claim 2, wherein the adjuvant comprises at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener, or a coloring agent.
- 4. The use of claim 1, wherein the anti-enterovirus type 71 medicament is in a dosage form comprising at least one of granules, tablets, pills, capsules, injections or dispersions.
- 5. The use according to claim 1, wherein the anti-viral means of the anti-enterovirus type 71 drug comprises: inhibit enterovirus intracellular nucleic acid replication, viral protein expression and infection.
- 6. The use according to claim 1, wherein the inhibition of EV71 virus is 96.3% at a VER-50589 concentration of 20 μmol/mL and 89% at a VER-50589 concentration of 0.16 μmol/mL.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116253695A (en) * | 2022-12-19 | 2023-06-13 | 青岛泰博恒生物医药科技有限公司 | HSP90 inhibitor and preparation method and application thereof |
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|---|---|---|---|---|
| US20090305998A1 (en) * | 2008-02-01 | 2009-12-10 | Takeda Pharmaceutical Company Limited | Hsp90 inhibitors |
| WO2020231979A1 (en) * | 2019-05-13 | 2020-11-19 | The Trustees Of Princeton University | Small molecule inhibitors of viral replication |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090305998A1 (en) * | 2008-02-01 | 2009-12-10 | Takeda Pharmaceutical Company Limited | Hsp90 inhibitors |
| WO2020231979A1 (en) * | 2019-05-13 | 2020-11-19 | The Trustees Of Princeton University | Small molecule inhibitors of viral replication |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116253695A (en) * | 2022-12-19 | 2023-06-13 | 青岛泰博恒生物医药科技有限公司 | HSP90 inhibitor and preparation method and application thereof |
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