CN115400121B

CN115400121B - Application of SNX-2112 in preparation of medicines for resisting adenovirus infection

Info

Publication number: CN115400121B
Application number: CN202210503360.8A
Authority: CN
Inventors: 吴建国; 陈绪林; 刘敏丽; 万品; 杨戈
Original assignee: Foshan Institute Of Pathogenic Microorganisms
Current assignee: Foshan Institute Of Pathogenic Microorganisms
Priority date: 2022-04-29
Filing date: 2022-05-09
Publication date: 2023-05-23
Anticipated expiration: 2042-05-09
Also published as: CN115400121A

Abstract

The invention belongs to the technical field of biomedicine, and discloses an application of SNX-2112 in preparing a medicament for resisting adenovirus infection, wherein the medicament for relieving and/or preventing and/or treating adenovirus infection acts by inhibiting the activity of adenovirus replication. Adenoviruses are various types of adenoviruses including, but not limited to, the AdV3 subtype and the AdV5 subtype. Medicaments for alleviating and/or preventing and/or treating an adenovirus infection include SNX-2112 and a pharmaceutically acceptable carrier. The invention provides an application of a small molecular compound SNX-2112 in preparing a medicine for treating adenovirus infection, and provides a safe and effective small molecular compound for treating adenovirus clinically. SNX-2112 can effectively inhibit adenovirus replication in a non-toxic range, can be further developed into a medicament for treating diseases caused by adenovirus infection, and has wide application prospect.

Description

Application of SNX-2112 in preparation of medicines for resisting adenovirus infection

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an application of SNX-2112 in preparation of a medicament for resisting adenovirus infection.

Background

Currently, human adenoviruses (HAdV) belong to the genus mammalian adenoviruses of the family adenoviridae. Adenovirus is a non-enveloped icosahedral nucleocapsid DNA virus whose genome is cored by a linear double stranded DNA molecule, approximately 36kb, and whose genome is packaged in high concentration and organized into chromatin by hundreds of histone-like proteins VII and protamine-like proteins Mu (also known as protein X). The capsid of adenovirus has 240 hexon proteins (hexon) and 12 peaks of the icosahedral capsid are complexes of pentameric (penton) and trimeric (fiber) proteins. The 12 fiber proteins protrude from the capsid surface on the basis of the penton protein, and the fiber tips form the knob region (knob). The knob region of the penton and fiber proteins can bind to viral receptors on the cell surface and play a very important role in the viral infection of cells. Smaller capsid proteins IIIa, VI, VIII and IX are embedded in the capsid, wherein capsid protein VI is located on the inner surface of the capsid and the capsid is linked to the core comprising the viral genome by capsid protein V. There is also a small number of capsid proteins IVa2 in the capsid involved in genome packaging and adenovirus protease (AVP) formation.

Adenovirus infection can occur in any season, but often winter and early spring are peak seasons of viral infection. Human adenoviruses are classified into seven of a, B, C, D, E, F and G, 57 serotypes in total, based on serum neutralization, hemagglutination epitopes, genomic sequences and function. The types of adenovirus which are popular in China mainly comprise type 1, type 3, type 4, type 5, type 7, type 11, type 14, type 40, type 41 and type 55, wherein the types of adenovirus flow mainly comprise type 3 adenovirus flow and type 5 adenovirus flow. The adenovirus types were found to be highly conserved in the genome except for the hexon, fiber, penton genes, and rarely undergo intraspecies recombination. Therefore, the method is favorable for overcoming the limitation of the specificity of the medicaments on adenovirus types, and antiviral medicaments which are usually aimed at adenovirus entering the later stage have broad-spectrum anti-adenovirus activity in adenovirus seeds.

Adenovirus infection has a variety of clinical symptoms and disease manifestations, which depend largely on the adenovirus type, the host's immune status and the site of infection. Common sites of infection for adenoviruses include the respiratory tract, corneal epithelium, and intestinal tract. 90% of cases of viral conjunctivitis are caused by adenovirus infection, usually associated with B, D or type E adenovirus infection, often in the form of conjunctivitis pharyngeal or Epidemic Keratoconjunctivitis (EKC) in new soldiers on the line. B. Adenovirus type D or E also often causes acute respiratory illness and viral pneumonia. Two more serious consequences of respiratory adenovirus infection are pneumonia, which can be fatal in children. And acute respiratory distress syndrome, are more common among the symptomatic panelists. Adenoviruses of types a, F and G are associated with gastrointestinal infections. Adenovirus infection is the main cause of gastroenteritis in children, second only to norovirus and rotavirus. In general, adenoviruses are susceptible to all groups of people, where more severe diseases and even death can often occur in infected newborns, children, and immunocompromised groups (hematopoietic stem cells and organ transplant patients).

There is currently no antiviral drug approved for the treatment of adenovirus infection. Certain DNA/RNA synthesis inhibiting antiviral drugs (e.g., cidofovir, ganciclovir Wei Heli bavin) approved for the treatment of other viral infections have been used clinically to treat severe adenovirus infections as trial drugs by expanding the indications. However, most of these drugs have limited efficacy and serious adverse effects. Thus, there is a need to develop other safer and more effective anti-adenovirus drugs.

SNX-2112 (PF-04928473) is a novel and potent inhibitor of HSP90, which inhibits proliferation of a variety of human tumor cell lines. SNX-2112 inhibits the growth of cancer cells by inducing degradation of related oncogenic client proteins in tumor cells, resulting in the blockade of many critical oncogenic pathways. However, no report has been found concerning the treatment of adenovirus infection by SNX-2112.

Through the above analysis, the problems and defects existing in the prior art are as follows:

(1) There is no report on the treatment of adenovirus infection by SNX-2112.

(2) The prior art has low drug effect and poor effect on the medicaments for treating adenovirus infection.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides an application of SNX-2112 in preparing a medicament for resisting adenovirus infection.

The invention is achieved by the use of SNX-2112 for the preparation of a medicament for the alleviation and/or prevention and/or treatment of an adenovirus infection.

Further, the structural formula of the SNX-2112 is as follows:

further, the adenovirus includes adenovirus type B, subtype AdV3, adenovirus type C, subtype AdV5.

Further, the adenovirus is one or more of human adenovirus type 1, type 3, type 4, type 5, type 7, type 11, type 14, type 40, type 41 and type 55.

Further, the agent for alleviating and/or preventing and/or treating adenovirus infection acts by inhibiting the activity of adenovirus replication.

Further, the medicament for alleviating and/or preventing and/or treating adenovirus infection comprises SNX-2112 and a pharmaceutically acceptable carrier.

Further, the dosage form of the medicament for relieving and/or preventing and/or treating adenovirus infection is any clinically acceptable oral administration dosage form, injection administration dosage form or external administration dosage form.

Further, the medicine for relieving and/or preventing and/or treating adenovirus infection is tablets, capsules, granules, oral liquid and injection.

It is another object of the present invention to provide the use of SNX-2112 for the preparation of an adenovirus inhibitor.

Aiming at the current situation that no antiviral drug for adenovirus infection exists in the prior art, the invention provides a novel anti-adenovirus drug-SNX-2112 in combination with experimental data of low cytotoxicity of SNX-2112 in Vero cells, high antiviral activity on two adenoviruses and the like in the research and development process, provides a novel technology of the antiviral drug for adenovirus infection, and provides a brand new therapy and more treatment options for treating diseases caused by adenovirus infection.

The invention provides an application of a small molecular compound SNX-2112 in preparing a medicine for treating adenovirus infection, and provides a safe and effective small molecular compound for treating adenovirus clinically. SNX-2112 can effectively inhibit adenovirus replication in a non-toxic range, can be further developed into a medicament for treating diseases caused by adenovirus infection, and has wide application prospect.

SNX-2112 of the invention is a small molecule compound that does not yet see any cytotoxicity at 5. Mu.M in Vero cells. Thus, CC ₅₀ (half lethal concentration) greater than 5. Mu.M. SNX-2112 is able to dose-dependently inhibit viral replication for both adenoviruses (AdV 3 and AdV 5). Its IC for AdV3 in Vero cells ₅₀ (half inhibitory concentration) was only 0.17. Mu.M, IC for AdV5 ₅₀ 0.33. Mu.M. By calculation, the Selection Index (SI) of SNX-2112 was greater than 10 on both adenoviruses, indicating that SNX-2112 has a broad spectrum of activity against adenovirus replication.

Regarding the technical scheme as a whole or from the perspective of products, the technical scheme to be protected has the technical effects and advantages as follows:

the invention provides a novel anti-adenovirus drug-SNX-2112, which overcomes the technical defect that the prior art does not have an antiviral drug for adenovirus infection.

As inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:

(1) The expected benefits and commercial values after the technical scheme of the invention is converted are as follows: the invention takes the clinical requirement as the guide, focuses on the core treatment field, is based on clinical value, pharmaceutical economy value and commercial value of products, and has huge commercial value and expected benefit after transformation

(2) The technical scheme of the invention fills the technical blank in the domestic and foreign industries: the invention provides a novel technology of antiviral drugs for adenovirus infection.

Drawings

FIG. 1 shows that Vero cells according to the examples of the invention contain 5% CO at 37℃with different concentration gradients of SNX-2112 ₂ After 48 hours incubation in an incubator, the measured cell viability of Vero cells relative to untreated cells;

FIG. 2 is a graph showing the percentage of inhibition of the Vero cells treated with different drug concentration gradients to AdV3 after incubation at 37℃for 48 hours by adding SNX-2112 with different concentration gradients to the Vero cells provided in the examples of the present invention, to AdV3 infected with MOI 0.55, relative to non-drug-added cells after viral infection;

FIG. 3 is a graph showing the percentage of inhibition of AdV5 in Vero cells treated with different drug concentration gradients relative to non-drug-loaded cells after viral infection, after addition of SNX-2112 at different concentration gradients to Vero cells, infection of AdV5 with MOI 1.1, incubation at 37℃for 48 hours, according to the examples of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

1. The embodiments are explained. In order to fully understand how the invention may be embodied by those skilled in the art, this section is an illustrative embodiment in which the claims are presented for purposes of illustration.

The SNX-2112 provided by the embodiment of the invention is used for preparing the medicine for relieving and/or preventing and/or treating the adenovirus infection and the adenovirus inhibitor.

The chemical structural formula of the SNX-2112 provided by the embodiment of the invention is as follows:

the adenovirus provided by the embodiment of the invention can be any one or more of human adenovirus type 1, type 3, type 4, type 5, type 7, type 11, type 14, type 40, type 41 and type 55.

The drugs for alleviating and/or preventing and/or treating adenovirus infection provided by the embodiments of the present invention act by inhibiting the activity of adenovirus replication.

The medicine for relieving and/or preventing and/or treating adenovirus infection provided by the embodiment of the invention comprises SNX-2112 and a pharmaceutically acceptable carrier.

The dosage form of the medicine for relieving and/or preventing and/or treating adenovirus infection provided by the embodiment of the invention is any clinically acceptable oral administration dosage form, injection administration dosage form or external administration dosage form.

The medicine for relieving and/or preventing and/or treating the adenovirus infection provided by the embodiment of the invention is tablets, capsules, granules, oral liquid and injection.

2. Application example. In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.

The SNX-2112 provided by the embodiment of the invention is applied to the preparation of medicaments for relieving and/or preventing and/or treating adenovirus infection and adenovirus inhibitors. SNX-2112 can effectively inhibit adenovirus replication in a non-toxic range, can be further developed into a medicament for treating diseases caused by adenovirus infection, and has wide application prospect. In view of the fact that no drug for the treatment of adenovirus infection is currently approved for the market, the invention provides a new technology for the treatment of adenovirus infection.

3. Evidence of the effect of the examples. The embodiment of the invention has a great advantage in the research and development or use process, and has the following description in combination with data, charts and the like of the test process.

The invention detects the toxicity of SNX-2112 to cells on an African green monkey kidney cell line Vero, and simultaneously determines the inhibition effect of SNX-2112 to adenovirus 3 (AdV 3) and adenovirus 5 (AdV 5) in the Vero cell line. The results show that the small molecule compound SNX-2112 has significant dose-dependent anti-adenovirus activity on both AdV3 and AdV5. Therefore, the SNX-2112 is a novel antiviral drug for adenovirus, has the advantages of good safety, high selection index, broad-spectrum adenovirus resistance and the like, can be used for developing drugs for treating adenovirus infection, and has wide application prospect.

1. Experimental materials:

(1) Cell lines, laboratory animals and viruses for laboratory purposes

Vero cells were purchased from American Type Culture Collection (ATCC);

strains used: adenovirus type B AdV3, adenovirus type C AdV5.

The required drugs for the experiment: SNX-2112 is available from Selleck Chemicals; in cell experiments, the drug was dissolved in DMSO.

Reagents required for the experiment:

DMEM medium, fetal Bovine Serum (FBS) were purchased from GIBCO;

CellTiter-Glo cell proliferation assay kit was purchased from Promega corporation.

(2) Instrument for experiment

Enspire multifunctional microplate reader available from Perkinelmer company;

CO ₂ cell incubator was purchased from Thermo company;

the operaetta CLS high content imaging analysis system was purchased from PerkinElmer corporation.

2. Experimental method

2.1 Cytotoxicity assays for SNX-2112 were as follows:

(1) Vero was used as a 2X 10 Vero cell line ⁴ Each well was plated into 96-well plates and incubated for 24h.

(2) Adding SNX-2112 diluted with culture medium to different gradient concentrations, and containing 5% CO at 37deg.C ₂ The culture was continued for 48 hours in the incubator of (C).

(3) Cell viability following treatment with different concentrations of drug was tested to detect SNX-2112 cytotoxicity.

(4) The median Cytotoxicity Concentration (CC) of the drug was calculated by plotting Graphpad against the log of Cell relative activity (Cell viability) versus drug concentration ₅₀ )。

2.2 evaluation of antiviral activity of SNX-2112 against adenovirus AdV3 in a cell model includes:

(1) Vero was used as a 2X 10 Vero cell line ⁴ Each well was plated into 96-well plates and incubated for 24h. To test for antiviral efficacy, 0.5 is usedAdenovirus AdV3 at 5MOI (multiplicity of infection) infects Vero cells.

(2) Simultaneously adding SNX-2112 diluted with culture medium to different gradient concentrations, and adding 5% CO at 37deg.C ₂ The culture was continued for 48 hours in the incubator of (C).

(3) The number of virus-infected positive cells (Hexon positive cells) in the drug-treated and untreated cell wells was examined by indirect immunofluorescence experiments (Indirect immunofluorescence assay, IFA) to assess the replication level of adenovirus AdV3 following treatment at different concentrations of SNX-2112.

(4) The median Inhibitory Concentration (IC) of the drug against adenovirus AdV3 was calculated by plotting Graphpad against the log of the Inhibition rate (Inhibition rate) against the drug concentration ₅₀ ). The selection index of SNX-2112 on adenovirus AdV3 on Vero cell line was calculated.

2.3 evaluation of antiviral Activity of SNX-2112 against adenovirus AdV5 in cell model the following was evaluated:

1) Vero was used as a 2X 10 Vero cell line ⁴ Each well was plated into 96-well plates and incubated for 24h. To examine its antiviral effect, vero cells were infected with adenovirus AdV5 at 1.1MOI (multiplicity of infection).

2) Simultaneously adding SNX-2112 diluted with culture medium to different gradient concentrations, and adding 5% CO at 37deg.C ₂ The culture was continued for 48 hours in the incubator of (C).

3) The number of virus-infected positive cells (Hexon positive cells) in the drug-treated and untreated cell wells was examined by indirect immunofluorescence experiments (Indirect immunofluorescence assay, IFA) to assess the replication level of adenovirus AdV5 following treatment at different concentrations of SNX-2112.

4) The median Inhibitory Concentration (IC) of the drug against adenovirus AdV5 was calculated by plotting Graphpad against the log of the Inhibition rate (Inhibition rate) against the drug concentration ₅₀ ). The selection index of SNX-2112 on adenovirus AdV5 on Vero cell line was calculated.

2.4 Evaluation of cytotoxicity of SNX-2112 in Vero cell line

(1) Cell culture

The cells recovered by freezing were passaged 2 times and then treated with a kit comprising 10% fetal bovine serum and a double antibody (penicillin 100U/ml)Streptomycin 100 μg/ml) DMEM medium amplification culture, inoculation density not lower than 1x10 ⁴ cell/ml, passage density not higher than 5x10 ⁴ cell/ml。

(2) Drug-treated cells

Vero cells at 1X10 ⁴ Cells/well (volume 100 μl) were inoculated into 96-well cell culture plates and cultured for 24h until cell well confluence reached 80%; 200 mu L of culture medium solution (DMEM culture medium+2% serum+double antibody) is used for preparing medicines per well, and the medicines are added into corresponding cell holes for uniform mixing. The drug was set to 6 concentration gradients, each gradient was set to 2 multiplex wells at final concentrations of 0.02. Mu.M, 0.06. Mu.M, 0.19. Mu.M, 0.56. Mu.M, 1.67. Mu.M and 5. Mu.M, containing 5% CO at 37 ℃C ₂ The culture was continued for 48 hours in the incubator of (C).

(3) Calculating cytotoxicity of the drug in each assay well

The supernatant was removed and 100. Mu.L was added to each well

Reagents, plates were incubated for 10 min at room temperature to stabilize the luminescence signal. Chemiluminescent readings were measured using an EnSpire microplate reader and cell viability was calculated.

Cell viability (%) = drug treated/untreated control group 100%

As shown in FIG. 1, after the SNX-2112 was treated with 5. Mu.M at the highest concentration for 48 hours, the cell viability was slightly different from that of the control group, indicating that SNX-2112 was slightly toxic to cells at this concentration, and half of the toxic concentration was CC ₅₀ Greater than 5 μm.

2.5 Evaluation of anti-AdV 3 adenovirus Activity of SNX-2112 in Vero cell line

(1) Cell culture

Subjecting the cells after cryopreservation and resuscitation to 2 passages, and culturing with DMEM medium containing 10% foetal calf serum and double antibody (penicillin 100U/ml, streptomycin 100 μg/ml) at inoculation density of not less than 1x10 ⁴ cell/ml, passage density not higher than 5x10 ⁴ cell/ml。

(2) Drug-treated cells

Vero cells at 1X10 ⁴ Cells/well (volume 100. Mu.L)Inoculating the cells into a 96-hole cell culture plate, and culturing for 24 hours until the confluence degree of the cell holes reaches 80%; the infectious group was added with 0.55MOI (multiplicity of infection) of AdV3 virus, and simultaneously with each gradient of drug (5. Mu.M as starting concentration, 6 gradients were serially diluted 3-fold in a gradient, two duplicate wells per gradient) to a total volume of 200. Mu.L of culture medium (DMEM medium+2% serum+diabodies) and incubated in a cell incubator at 37℃for 48h.

(3) Indirect immunofluorescence method for detecting specific fluorescent-labeled virus

Cell culture plates were washed twice with PBS solution and fixed with 4% paraformaldehyde (4% PFA in PBS solution) for 20 min at room temperature. The fixed samples were washed 3 times with PBST (0.05% tween 20 in PBS) and then incubated in blocking buffer (3% BSA,0.3% Triton X-100 and 10% FBS in PBS) for 1 hour at room temperature. Then incubated overnight at 4℃in a binding buffer (3% BSA,0.3% Triton X-100 in PBS) against mouse monoclonal antibodies to adenovirus hexon protein (diluted 1:200). After rinsing 3 times with PBST, the samples were incubated in goat FITC-conjugated anti-mouse secondary antibody (dilution 1:1000) and DAPI (dilution 1:10000) in binding buffer for 1 hour in the dark at room temperature. After rinsing 3 times with PBST, use

CLS ^TM The high content analysis system observes the sample and then captures and analyzes the image.

(4) Calculating the inhibition rate of the drug in each detection hole to viruses

Cells were labeled by DAPI staining, FITC staining intensity representing the level of viral replication. FITC background fluorescence values were measured in uninfected control cells. Cells with FITC intensities three times higher than control cells were defined as adenovirus-infected positive cells. The ratio of adenovirus-infected positive cells in total cells was calculated.

Inhibition (%) = 100% - (drug treated well-blank)/(virus control well-blank) ×100%

As shown in FIG. 2, SNX-2112 significantly inhibited replication of adenovirus AdV3 and was dose dependent, with half of the effective concentration of IC ₅₀ 0.17. Mu.M.

(5) Drug selection index calculation

The drug Selection Index (SI) is used to judge the safety range of the drug effect, and a selection index of more than 3 is effective, and the safety range is larger as the index is larger. The calculation formula is as follows: si=cc ₅₀ /IC ₅₀

In combination with the data, SNX-2112 has a selection index of greater than 10 on adenovirus AdV3 on Vero and has potent anti-adenovirus AdV3 activity.

2.6 Evaluation of anti-AdV 5 adenovirus Activity of SNX-2112 in Vero cell line

(1) Cell culture

(2) Drug-treated cells

Vero cells at 1X10 ⁴ Cells/well (volume 100 μl) were inoculated into 96-well cell culture plates and cultured for 24h until cell well confluence reached 80%; the infectious group was added with 1.1MOI (multiplicity of infection) of AdV5 virus, and simultaneously with each gradient of drug (5. Mu.M as starting concentration, 6 gradients were serially diluted 3-fold in a gradient, two duplicate wells per gradient) to a total volume of 200. Mu.L of culture medium (DMEM medium+2% serum+diabodies) and incubated in a cell incubator at 37℃for 48h.

Cell culture plates were washed twice with PBS solution and fixed with 4% paraformaldehyde (4% pfa in PBS) for 20 min at room temperature. The fixed samples were washed 3 times with PBST (0.05% tween 20 in PBS) and then incubated in blocking buffer (3% BSA,0.3% Triton X-100 and 10% FBS in PBS) for 1 hour at room temperature. Then incubated overnight at 4℃in a binding buffer (3% BSA,0.3% Triton X-100 in PBS) against mouse monoclonal antibodies to adenovirus hexon protein (diluted 1:200). After rinsing 3 times with PBST, the samples were subjected to FITC coupling with goatsThe combined anti-mouse secondary antibody (dilution 1:1000) and DAPI (dilution 1:10000) in binding buffer were incubated in the dark at room temperature for 1 hour. After rinsing 3 times with PBST, use

As shown in FIG. 3, SNX-2112 significantly inhibited replication of adenovirus AdV5 and was dose dependent with half the effective concentration of IC ₅₀ 0.33. Mu.M.

(5) Drug selection index calculation

In combination with the data, SNX-2112 has a selection index of greater than 10 on adenovirus AdV5 on Vero and has potent anti-adenovirus AdV5 activity.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

Claims

1. SNX-2112 in preparation of drugs for relieving and/or preventing and/or treating adenovirus infectionIs used for the purposes of (1); the structural formula of the SNX-2112 is as follows:

；

the adenovirus is the AdV3 subtype of the B group adenovirus or the AdV5 subtype of the C group adenovirus.

2. The use according to claim 1, wherein the medicament for alleviating and/or preventing and/or treating an adenovirus infection comprises SNX-2112 and a pharmaceutically acceptable carrier.

3. The use of claim 1, wherein the adenovirus infection is one or more of a respiratory disease, an intestinal disease, and a corneal disease.

4. The use according to claim 1, wherein the dosage form of the medicament for alleviating and/or preventing and/or treating an adenovirus infection is any one of a clinically acceptable oral dosage form or an injectable dosage form.

5. The use according to claim 1, wherein the dosage form of the medicament for alleviating and/or preventing and/or treating an adenovirus infection is any one of the clinically acceptable topical dosage forms.

6. The use according to claim 1, wherein the medicament for alleviating and/or preventing and/or treating adenovirus infection is a tablet, capsule, granule, oral liquid or injection.