阅读说明：本技术 N-乙酰-d-甘露糖胺在制备抗病毒药物中的应用 (Application of N-acetyl-D-mannosamine in preparation of antiviral drugs ) 是由 郑海学 薛钊宁 朱紫祥 刘会胜 薛巧 宋影影 于 2021-10-08 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种N-乙酰-D-甘露糖胺在制备抗病毒药物中的应用。本发明意外发现,在培养SVA的培养基中加入N-乙酰-D-甘露糖胺,能够使SVA的复制水平降低,表明N-乙酰-D-甘露糖胺具有抑制SVA复制的作用,可用于制备抗SVA病毒感染的药物或佐剂,用于抑制SVA病毒的复制。但本发明并不局限于SVA,在本发明所述的了N-乙酰-D-甘露糖胺能够抑制SVA病毒的复制的基础上,同样也能抑制小RNA病毒科其他病毒的复制,也可用于抑制其他病毒的复制,制备抗病毒感染的药物,或佐剂。(The invention belongs to the technical field of biology, and particularly relates to application of N-acetyl-D-mannosamine in preparation of an antiviral drug. The invention unexpectedly discovers that the addition of N-acetyl-D-mannosamine into a culture medium for culturing SVA can reduce the replication level of SVA, and shows that the N-acetyl-D-mannosamine has the effect of inhibiting SVA replication, can be used for preparing a medicament or an adjuvant for resisting SVA virus infection and inhibiting SVA virus replication. However, the invention is not limited to SVA, and the N-acetyl-D-mannosamine can inhibit the replication of SVA virus, can also inhibit the replication of other viruses of the picornaviridae family, and can also be used for inhibiting the replication of other viruses and preparing medicaments or adjuvants for resisting virus infection.)

Use of N-acetyl-D-mannosamine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of viral infection.

2. The use of claim 1, wherein the virus is a picornaviridae virus.

3. The use of claim 2, wherein the virus is seneca virus.

4. The use of any one of claims 1 to 3, wherein the N-acetyl-D-mannosamine or a pharmaceutically acceptable salt thereof is formulated with a pharmaceutically acceptable carrier and/or adjuvant into any pharmaceutically acceptable dosage form.

5. The use of claim 4, wherein the dosage form comprises a powder injection, a capsule, a tablet, a suspension.

Use of N-acetyl-D-mannosamine or a pharmaceutically acceptable salt thereof in the preparation of a viral vaccine adjuvant.

7. The use of claim 6, wherein the virus is a picornaviridae virus.

8. The use of claim 7, wherein the virus is seneca virus.

9. The use of any one of claims 6 to 8, wherein the N-acetyl-D-mannosamine or a pharmaceutically acceptable salt thereof is formulated with a pharmaceutically acceptable carrier and/or adjuvant into any pharmaceutically acceptable dosage form.

10. The use of claim 9, wherein the dosage form comprises a powder injection, a capsule, a tablet, a suspension.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of N-acetyl-D-mannosamine in preparation of an antiviral drug.

Background

Senecavirus A (SVA) is a virus newly introduced into China in recent years, is a unique member of a new Senecavirus genus of Picornaviridae (Picornaviridae), has clinical symptoms which are difficult to distinguish from foot-and-mouth disease, and is mainly characterized in that blisters and ulcers are generated on the nose, hooves and the like. Before 2014, SVA happened only sporadically in the United states and Canada, but since 2015, SVA epidemic appeared in Brazil, Vietnam, Columbia, Thailand, China and the like, and spread continuously. In 2015, SVA was introduced into China, and Chinese scholars firstly found cases of pig infection SVA in Guangdong province. Subsequently, epidemic situations occur in provinces such as Hubei, Heilongjiang, Fujian, Henan, Guangxi, Hebei, Liaoning, Shandong, Zhejiang, Anhui, Sichuan, Jiangxi and Shaanxi, which are popular and spread continuously, the pathogenicity is enhanced, and the harm is aggravated. Since SVA is a new infectious disease, no commercial vaccine is available and the pathogenic and immune mechanisms are still not completely understood.

Prior studies showed that RIG-I is a key molecule for SVA to activate innate immunity, but 2C and 3C of SVA^proThe protein can reduce protein expression of RIG-I and has opposite antagonistic function to RIG-I; 2C and 3C of SVA^proCan also activate Caspase-3 to induce apoptosis; 3C of SVA^proThe enzyme activity of the enzyme is utilized to cut key natural immune molecules such as MAVS, TRIF, TANK and the like, and the natural immune response of a host is inhibited; 3C of SVA^proThe protein also inhibits protein expression of IRF3/7, removes ubiquitination of RIG-I, TBK1 and TRAF3 molecules, and antagonizes the antiviral function of a host; therefore, due to the presence of the aforementioned immune-critical molecules and SVA proteins, replication of SVA is inhibited and large-scale replication cannot be achieved. In addition, SVA infection can also activate autophagy of the cell. The above studies have focused mainly on the interaction regulation of SVA and key molecules in the innate immune pathway, and no studies on the inhibition of SVA replication by chemicals have been reported.

N-Acetyl-D-mannosamine (N-Acetyl-D-mannosamine) is white-yellowish crystal powder, can be used for treating neurological diseases, and can be used as synthetic intermediate to produce multiple downstream products. N-acetyl-D-mannosamine is an essential precursor for the synthesis of N-acetylneuraminic acid (NeuAc) and is also a specific monomer for bacterial capsular Polysialic Acid (PA). N-acetyl-D-mannosamine is metabolized by GNE and GlcNAc 2-epimerase (renin-binding protein, RnBP) to MannAC-6-phosphate and GlcNAc, respectively. The N-acetyl-D-mannosamine and the derivatives thereof activate Hypocretin (HCRT) gene expression in orexin neurons, and provide potential possibility for treating neurological diseases. The invention unexpectedly discovers that the addition of N-acetyl-D-mannosamine into a culture medium for culturing SVA can reduce the replication level of SVA, and shows that the N-acetyl-D-mannosamine has the effect of inhibiting SVA replication, can be used for preparing a medicament or an adjuvant for resisting SVA virus infection and inhibiting SVA virus replication.

Disclosure of Invention

The invention provides application of N-acetyl-D-mannosamine in preparing a medicament for resisting virus infection. The method specifically comprises the following steps:

in a first aspect, the present invention provides the use of N-acetyl-D-mannosamine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis or treatment of viral infections.

Preferably, the virus is a picornaviridae virus.

Preferably, the virus is a seneca virus.

Preferably, the N-acetyl-D-mannosamine or pharmaceutically acceptable salts thereof is added with pharmaceutically acceptable carriers and/or excipients to prepare any pharmaceutically acceptable dosage form.

Preferably, the dosage form comprises powder injection, capsules, tablets and suspension.

In a second aspect, the present invention provides the use of N-acetyl-D-mannosamine or a pharmaceutically acceptable salt thereof in the preparation of a viral vaccine adjuvant.

Preferably, the virus is a picornaviridae virus.

Preferably, the virus is a seneca virus.

Preferably, the N-acetyl-D-mannosamine or pharmaceutically acceptable salts thereof is added with pharmaceutically acceptable carriers and/or excipients to prepare any pharmaceutically acceptable dosage form.

Preferably, the dosage form comprises powder injection, capsules, tablets and suspension.

The invention has the beneficial effects that: the invention unexpectedly discovers that N-acetyl-D-mannosamine added into a culture medium for culturing SVA can inhibit the replication of SVA, and the N-acetyl-D-mannosamine has the effect of inhibiting the replication of SVA, can be used for preparing a medicament or an adjuvant for resisting SVA virus infection and inhibiting the replication of SVA virus.

Drawings

FIG. 1 is a graph showing the results of inhibition of SVA replication in IB cells by N-acetyl-D-mannosamine;

FIG. 2 is a graph showing the results of inhibition of SVA replication in IB cells at various concentrations of N-acetyl-D-mannosamine.

Detailed Description

The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.

N-acetyl-D-mannosamine available from sigma;

seneca virus strain SVA/FJ-201 was isolated by this team and maintained in the foot-and-mouth disease and new disease epidemiology team of Lanzhou veterinary institute of Chinese agricultural academy of sciences and the national foot-and-mouth disease reference laboratory.

Example 1N-acetyl-D-mannosamine inhibition of SVA replication

1. Preparation of N-acetyl-D-mannosamine-containing Medium

In DMEM medium, N-acetyl-D-mannosamine was added at different dosages to concentrations of 0mM/L, 0.2mM/L, 0.25mM/L and 0.5mM/L, respectively.

Sample preparation of N-acetyl-D-mannosamine incubated cells

IB cells were incubated with formulated mannosamine-containing medium, followed by SVA/FJ-201(1MOI) inoculation and supernatant harvested 8h after inoculation.

SVA Virus Titer assay

Determination of the TCID of the supernatant obtained in step 2 above₅₀And performing virus titer analysis.

TCID₅₀The measurement steps are as follows: IB cells were seeded into 96-well plates 16h in advance, after a monolayer of cells had formed, the IB cells were washed 3 times with PBS and the supernatant harvested in step 2 (10) was seeded^-1-10^-10) Two additional columns of negative control wells were used as infection wells. Adding 100 μ L virus filtrate or diluted virus diluent in multiple ratio into infected hole, adsorbing at 37 deg.C for 1h, and shaking gently once every 20min to ensure uniform virus adsorption. After 1h of adsorption, the supernatant was aspirated and the plate was washed gently with PBS 1 time. Adding virus maintenance solution, observing cytopathic condition every 12h after 48h, recording pathological change hole number after 72h, and calculating TCID₅₀Triplicate determinations were made and the mean was taken as the final virus titer.

As shown in FIG. 1, TCID of SVA was determined after addition of N-acetyl-D-mannosamine during IB cell culture₅₀The significant reduction indicates that the N-acetyl-D-mannosamine significantly reduces the viral TCID of SVA₅₀And inhibits the replication of SVA virus.

EXAMPLE 2 different concentrations of mannosamine in inhibiting SVA replication

1. Preparation of N-acetyl-D-mannosamine-containing Medium

In DMEM medium, N-acetyl-D-mannosamine was added at different doses to concentrations of 0mM/L, 0.25mM/L, 0.5mM/L, 1.0mM/L and 2.0mM/L, respectively.

Sample preparation of N-acetyl-D-mannosamine incubated cells

IB cells were incubated with formulated mannosamine-containing medium, followed by SVA/FJ-201(1MOI) inoculation and supernatant harvested 8h after inoculation.

SVA Virus Titer assay

Cell supernatants were collected after SVA infection by TCID₅₀And (4) determining and performing virus titer analysis. Measurement of viral titer: IB cells were seeded into 96-well plates 16h in advance, after the cells formed a monolayer, the IB cells were washed 3 times with PBS, and the supernatants harvested in step 2 were separately seeded (10)^-1-10^-10) Two additional columns of negative control wells were provided. Adding 100 μ L of virus filtrate or diluted virus diluent into infected wells, adsorbing at 37 deg.C for 1 hr, and shaking gently every 20minShaking once to ensure the virus to be adsorbed evenly. After 1h of adsorption, the supernatant was aspirated and the plate was washed gently with PBS 1 time. Adding virus maintenance solution, observing cytopathic condition every 12h after 48h, recording pathological change hole number after 72h, and calculating TCID₅₀Triplicate determinations were made and the mean was taken as the final virus titer.

As shown in FIG. 2, the effect of adding N-acetyl-D-mannosamine at various concentrations during IB cell culture on SVA replication was consistent. TCID of SVA when 0.25mM/L mannosamine was added₅₀Decreases, and with increasing N-acetyl-D-mannosamine content, TCID of SVA₅₀The reduction is dose-dependent, and the optimal concentration is 0mM/L-0.5 mM/L. The above results indicate that N-acetyl-D-mannosamine significantly reduced viral TCID of SVA₅₀And inhibits the replication of SVA virus.

In summary, the host cell IB cell is taken as an example in the embodiment of the invention, and research proves that the N-acetyl-D-mannosamine can inhibit the replication of SVA virus in the host cell IB cell, and the N-acetyl-D-mannosamine can inhibit the replication of SVA virus, and can be used for preparing a medicine or an adjuvant for resisting SVA virus infection.

It should be noted that picornaviridae mainly includes the following four genera: enterovirus, rhinovirus, cardiovirus, aphthovirus, and SVA belongs to aphthovirus. Due to the high degree of conservation of viral structural proteins between the four genera of picornaviridae. Although the invention takes SVA of virus of picornaviridae as an example to prove that N-acetyl-D-mannosamine can inhibit the replication of SVA virus, the invention is not limited to SVA, and on the basis that the N-acetyl-D-mannosamine can inhibit the replication of SVA virus, the invention can also inhibit the replication of other virus of picornaviridae, and can also be used for inhibiting the replication of other virus, and preparing drugs for resisting virus infection or adjuvants.