阅读说明：本技术 Dyngo-4a和/或Dynasore在制备抗轮状病毒腹泻药物中的应用 (Application of Dyngo-4a and/or Dynasore in preparation of anti-rotavirus diarrhea medicine ) 是由 吴叔文 蓝柯 张全志 于 2019-09-03 设计创作，主要内容包括：本发明提供了Dyngo-4a和/或Dynasore在制备抗轮状病毒腹泻药物中的应用。所述抗轮状病毒腹泻药物包括含Dyngo-4a和/或Dynasore的药理或生理上可接受的盐、含Dyngo-4a和/或Dynasore的药学上可接受的载体或赋形剂。本发明提供的Dyngo-4a和Dynasore两种小分子化合物可以有效抑制轮状病毒的感染和复制,其对细胞的毒性小,可用于制备抗轮状病毒腹泻药物,适于临床推广。(The invention provides application of Dyngo-4a and/or Dynasore in preparing medicaments for resisting rotavirus diarrhea. The rotavirus diarrhea resisting medicine comprises pharmacologically or physiologically acceptable salt containing Dyngo-4a and/or Dynasore and pharmaceutically acceptable carrier or excipient containing Dyngo-4a and/or Dynasore. The Dyngo-4a and Dynasore micromolecule compounds provided by the invention can effectively inhibit infection and replication of rotavirus, have low toxicity to cells, can be used for preparing anti-rotavirus diarrhea medicines, and are suitable for clinical popularization.)

Use of Dyngo-4a and/or Dynasore in the manufacture of a medicament for the treatment of rotavirus diarrhoea.

2. The use of claim 1, wherein: the anti-rotavirus diarrhea medicine comprises pharmacologically or physiologically acceptable salts containing Dyngo-4a and/or Dynasore.

3. The use of claim 1, wherein: the anti-rotavirus diarrhea medicine comprises a pharmaceutically acceptable carrier or excipient containing Dyngo-4a and/or Dynasore.

4. The use of claim 1, wherein: the structural formula of Dyngo-4a is as follows,

5. the use of claim 1, wherein: the structural formula of Dynasore is shown as follows,

Technical Field

The invention belongs to the technical field of medicines, and relates to application of Dyngo-4a and/or Dynasore in preparation of a rotavirus diarrhea resistant medicine.

Background

Rotavirus diarrhea (rotavirus diarrheea) is an acute infectious disease caused by rotavirus, mainly infects mammals and birds, including human and domestic animals, poultry, and causes acute gastroenteritis of newborns and young animals of various animals such as cows, pigs, horses, sheep/goats, rabbits, deer, mice, chickens, ducks, pigeons, quails, etc., and is an important cause of death of newborns of human and animals, and 20 ten thousand infants can die worldwide every year; adults and animals can also be infected, but are generally asymptomatic or less symptomatic; about 2400 million patients require hospitalization each year due to rotavirus infection, of which about 230 require hospitalization; in addition to the immediate economic loss of an animal due to its death when it is infected, the stagnation of the animal's growth during the disease can also cause significant economic loss.

The source of rotavirus diarrhea is Rotavirus (RV). RV is a member of the Reoviridae (Reoviridae) family. To date, RV class 10 (a-J) has been discovered, with rotavirus a accounting for substantially 90% of all rotavirus infections and cases of death; based on the differences in rotavirus glycoprotein (VP7, labeled "G") and host protease target (VP4, labeled "P"), rotavirus type A is commonly designated GiP [ j ], such as G1P [8] strain; there are 32 known types of G protein and 47 known types of P protein of rotavirus, but there are G1, G2, G3, G4, G9 and G12 as G protein subtypes of rotavirus A which are more prevalent in the global range, P4, P6 and P8 as P protein subtypes, and G1P [8], G2P [4], G3P [8], G4P [8], G9P [8] and G12P [8] as main prevalent strains. No cross immunity phenomenon exists among serotypes.

At present, no specific treatment means for rotavirus diarrhea exists, symptomatic treatment is mainly adopted clinically, and prevention of excessive dehydration of patients is usually taken as a treatment means; in the aspect of vaccines, by 2017, the inoculation rate of oral rotavirus vaccines in China is usually less than 10% (1723 thousands of people born in China in 2017, the batch issuance amount of monovalent oral rotavirus vaccines in Lanzhou in the current year is 484 thousands, and the oral rotavirus vaccines adopt a three-dose oral immunization program), and the vaccines are adopted to prevent rotavirus diarrhea and have the defect of insufficient coverage rate; it is therefore necessary to develop new antiviral strategies to cope with the threat of the disease. Some researchers have tried to prevent and control rotavirus diarrhea by using red wine extract, small molecule chloride channel inhibitor, etc., and although some progress is made at the cellular or animal level, none of them have been put into use.

At present, few reports of preventing and controlling rotavirus diarrhea by adopting chemical micromolecules exist, so that obtaining some active compounds capable of better inhibiting rotavirus infection is very beneficial to developing rotavirus diarrhea medicines.

Disclosure of Invention

The invention aims to provide application of Dyngo-4a and/or Dynasore in preparation of a drug for resisting rotavirus diarrhea, wherein the Dyngo-4a and/or Dynasore has low toxicity to cells and has a certain application prospect in prevention and treatment of rotavirus diarrhea.

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

application of Dyngo-4a and/or Dynasore in preparing anti-rotavirus diarrhea medicine.

The anti-rotavirus diarrhea medicine comprises pharmacologically or physiologically acceptable salts containing Dyngo-4a and/or Dynasore.

The anti-rotavirus diarrhea medicine comprises a pharmaceutically acceptable carrier or excipient containing Dyngo-4a and/or Dynasore.

The structural formula of Dyngo-4a is as follows,

the structural formula of Dynasore is shown as follows,

the experimental research proves that two micromolecular compounds, Dyngo-4a and Dynasore, can inhibit the cytoplasmic transportation process of rotavirus, thereby inhibiting infection, replication and reinfection of rotavirus.

Compared with the prior art, the invention has the following beneficial effects:

the Dyngo-4a and Dynasore micromolecule compounds provided by the invention can effectively inhibit rotavirus infection from entering cells and new virus particles from exiting cells, have low toxicity to cells, can be used for preparing medicaments for resisting rotavirus diarrhea, and overcomes the defects and shortcomings of the existing means for preventing and controlling rotavirus diarrhea; and can be further widely popularized to prevention and treatment of rotavirus diarrhea of people, livestock, poultry and the like after clinical test verification, and further development of public health and animal husbandry is supported.

Drawings

FIG. 1 is a graph showing the results of the cytotoxicity assay of Dyngo-4a according to the present invention;

FIG. 2 is a graph showing the results of the cytotoxicity assay of Dynasore in the present invention.

Detailed Description

Further features and advantages of the present invention will be understood by reference to the following detailed description and drawings. The examples provided are merely further illustrative of the present invention and do not limit the remainder of the disclosure in any way.

[ example ] Dyngo-4a, Dynasore bioactivity assay

1.Dyngo-4a, Dynasore cytotoxicity assay:

the reagent CCK-8 containing WST-8 (chemical name: 2- (2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt) is a compound similar to MTT, which can be reduced by intramitochondrial dehydrogenase to Formazan product with high water solubility in the presence of an electron coupling reagent 1-Methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS), and the amount of Formazan generated is proportional to the number of living cells. Therefore, the cell proliferation and toxicity analysis can be directly carried out by utilizing the characteristic, and the more the cell proliferation is faster, the darker the color is; the more cytotoxic, the lighter the color.

For the experiments, MA-104 cells were administered at 2X 10 per well⁴The density of (2) was transferred to a 96-well plate, and after 24 hours of incubation at 37 ℃, the medium was aspirated, and cell culture medium containing various concentration gradient compound (Dyngo-4a or Dynasore) drugs was added to each well (DMSO, 0.98. mu.M, 1.95. mu.M, 3.91. mu.M, 7.81. mu.M, 15.63. mu.M, 31.25. mu.M, 62.5. mu.M, 125. mu.M, 250. mu.M, 500. mu.M), respectively, while blank control (absorbance of wells with medium and CCK-8 solution without cells), negative (0-dose) control (absorbance of wells with cells, CCK-8 solution without drug solution) was made; after 24 hours, 10. mu.l of CCK-8 reagent was added to each well and the cell plates were incubated at 37 ℃ CO₂Culturing for 4h in incubator, and measuring 450nm wave with microplate readerOD value in length. Cell viability (%) ([ a (medicated) -a (blank)]/[ A (0 dosing) -A (blank)]X 100, wherein: a (dosing): absorbance of wells with cells, CCK-8 solution and drug solution (dynano-4 a or Dynasore), a (blank): absorbance of wells with medium and CCK-8 solution without cells, a (0 dosing): absorbance of wells with cells, CCK-8 solution, but no drug solution. Median inhibitory concentration (CC) of the Compound₅₀) As an indicator of the cytotoxicity of the compound.

2. Dyngo-4a, Dynasore anti-Rotavirus (RV) activity assay:

antiviral activity of the compounds was assessed by the reduction in the production of supernatant virus following infection of MA-104 cells. 96-well plates confluent MA-104 cells were deinfected with rotavirus containing various concentration gradients, and after 1 hour the virus-containing medium was removed and medium (DMSO, 0.98. mu.M, 1.95. mu.M, 3.91. mu.M, 7.81. mu.M, 15.63. mu.M, 31.25. mu.M, 62.5. mu.M, 125. mu.M, 250. mu.M, 500. mu.M) containing the drug to be tested (Dyngo-4a or Dynasore) at a specific concentration was added, respectively. At 37 deg.C, 5% CO₂After culturing for 48 hours under the condition, taking cell culture fluid to extract the total RNA of the supernatant virus, carrying out qPCR quantification on the total RNA after reverse transcription, wherein the relative Ct value reflects the relative quantity of the virus in the cell culture supernatant after RV infection and can reflect the influence of the medicament on virus infection and replication. EC (EC)₅₀Refers to the concentration of the specific drug required to effectively inhibit the virus production in the cell culture supernatant to 50% of the control wells.

3. Dyngo-4a, Dynasore cytotoxicity assay and anti-RV activity test results:

the results are shown in the following table 1,

TABLE 1

Compounds	EC50(μM)	CC50(μM)	SI(CC50/EC50)
				Dyngo-4a	1.2	213	178
Dynasore	2.1	471	224

The results show that: the compounds Dyngo-4a and Dynasore can inhibit the cytoplasmic transportation process of rotavirus, effectively inhibit infection of rotavirus into cells and new virus particles from coming out of cells, thereby inhibiting infection, replication and reinfection of rotavirus, and showing very good activity and very high selectivity against rotavirus; at the same time, the cell toxicity (CC)₅₀) As shown in the data (figure 1 and figure 2), Dyngo-4a and Dynasore have low cytotoxicity, so that the two compounds have strong application prospects in the aspects of prevention and treatment of rotavirus diarrhea.