CN115252605B - Application of compound in preparation of medicine for resisting virus infection of cover tower - Google Patents

Application of compound in preparation of medicine for resisting virus infection of cover tower Download PDF

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CN115252605B
CN115252605B CN202210810432.3A CN202210810432A CN115252605B CN 115252605 B CN115252605 B CN 115252605B CN 202210810432 A CN202210810432 A CN 202210810432A CN 115252605 B CN115252605 B CN 115252605B
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acid
virus
formula
compounds
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CN115252605A (en
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刘铮
王川庆
车兴
王傲杰
陈陆
甘世杰
闫安
刘聪聪
高冬生
王永生
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Southern University of Science and Technology
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/30—Prediction of properties of chemical compounds, compositions or mixtures
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    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
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Abstract

The invention discloses application of a compound in preparing a medicine for resisting the infection of a gehead tower virus, wherein the medicine comprises a therapeutically effective amount of the compound shown in a formula I or pharmaceutically acceptable salts, isomers and solvates thereof. In addition, the compound of the formula I in the invention damages E1-E2 interaction of the Gauss virus by combining E2 structural protein in the Gauss virus, and blocks assembly of virus particles, thereby interfering virus infection and replication, and has obvious curative effect and difficult generation of drug resistance in prevention or treatment of the Gauss virus infection.

Description

Application of compound in preparation of medicine for resisting virus infection of cover tower
Technical Field
The invention relates to the field of medicines, in particular to application of a compound in preparation of medicines for resisting infections of getavirus.
Background
Getah virus (GETV) belongs to one of the group of Chimliki forest viruses (Semliki Forest virus, SFV) belonging to the genus Emamoviridae of the family Emamoviridae, the group of SFV also comprises chikungunya virus (Chikungunya virus, chiKV), chimliki forest virus (Semliki Forest virus, SFV), mayaro virus (MAYV), una virus (unaV), brua virus (Bebaru virus, BEBV) and Ounien virus (O' nyong-nyong viruses, ONNV), wherein chiKV, ONNV and MAYV have extremely high morbidity and mortality to susceptible people.
GETV was originally derived from mosquitoes and limited to equine and porcine cases during 40 years 1960-2000, but had been expanded to multiple animals such as cows, sheep, dogs, kangaroos, foxes, wild birds, rabbits, guinea pigs, rats, hamsters, etc. only 20 years into this century, and the frequency of clinical case reports has been increasing. Even more alarming is that anti-GETV antibodies were detected in febrile patients and even healthy people. Although there has been no report on the onset of human infection so far, various epidemiological studies of serum at home and abroad show that there are GETV antibodies in human bodies, suggesting that the virus can infect humans. Because of the occurrence of various animal infections and the common toxic effects of mosquitoes, livestock such as pigs and the like in vast rural areas of China are closely contacted with the crowd, the GETV is very likely to be popular among the crowd, and a great potential risk can exist in future public health events.
Scientists use a frozen electron microscope to study the steps of various viral structures in the genus alphaviruses from unsteady, resolution fromTo->The viruses studied contained Barmah forest virus (BFV), EEEV, WEEV, VEEV, CHIKV, SINV, MAYV, etc. The cryoelectron microscopy density map of the alphaviruses analyzed at present shows that: alphaviruses have the same structural composition. Alphavirus RNAs are hidden in a disordered state in the icosahedral core consisting of 240 copies of capsid (capsid). The outwardly protruding E1 and E2 structural proteins form heterodimers (80 of which form the icosahedral viral envelope) that are linked to the capsid across the phospholipid membrane. And GETV is a single-stranded positive strand RNA virus with a membrane envelope. Mature getavirus is a spherical particle of about 70nm, the genome of about 11kb consists of 2 Open Reading Frames (ORFs) and contains a code and a polyprotein, wherein the polyprotein at the N-terminus comprises 4 nonstructural proteins (non-structural proteins, i.e. nsP1, nsP2, nsP3 and nsP 4) and the polyprotein at the C-terminus consists of 5 structural proteins (structural proteins, capsid-E3-E2-6K-E1). In the prior art, research on the Gaota virus is mainly focused on strengthening the transmission medium of GETV, detection and monitoring of animal infection of pigs, horses, cattle and the like, quarantine of imported livestock and finished products thereof and other works, and the research on the aspects of detection of the Gaota virus, vaccine prevention and the like, and the aspects of medicines for treating the Gaota virus infection are almost absent in the reported literature, so that medicines capable of effectively treating the Gaota virus infection are not available. In addition, in the prior art, a method for screening medicines by using molecular docking software and scoring software also exists, but the screening result of the existing screening method is inaccurate, the screening cannot be performed accurately, and the existing screening method lacks a verification step.
Disclosure of Invention
In order to overcome the problems of the prior art, one of the purposes of the present invention is to provide the application of the compound shown in the formula I or the pharmaceutically acceptable salt, isomer and solvate thereof in preparing the medicine for preventing and/or treating the infection of the gecko virus;
it is a second object of the present invention to provide a pharmaceutical composition for preventing and/or treating infections with katavirus.
It is a further object of the present invention to provide a method for screening compounds for the prevention and/or treatment of infections with katavirus.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention is to provide the application of the compound shown in the formula I or pharmaceutically acceptable salt, isomer and solvate thereof in preparing medicaments for preventing and/or treating the infection of the cover tower virus,
wherein R is 1 Selected from Cl, F, br, H, C 1～5 Alkyl, C 1～5 An alkoxy group;
R 2 selected from H, cl, F, br, C 1～5 An alkyl group;
R 3 selected from F, cl, br, C 1～5 Alkyl or C 1～5 An alkoxycarbonyl group;
n is 0,1,2,3,4 or 5;
m is 0,1,2,3,4 or 5.
Preferably, the C 1～5 Alkyl is selected from-CH 3 、-CH 2 CH 3 、-CH 2 CH 2 CH 3 、-CH 2 CH 2 CH 2 CH 3 、-CH 2 CH 2 CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 、-C(CH 3 ) 3 、-CH 2 CH(CH 3 ) 2 、-CH(CH 3 )CH 2 CH 3 、-CH(CH 3 )CH 2 CH 2 CH 3 、-CH 2 CH(CH 3 )CH 2 CH 3 、-CH 2 CH 2 CH(CH 3 ) 2 、-CH(CH 2 CH 3 ) 2 、-CH 2 C(CH 3 ) 3 、-C(CH 3 ) 2 CH 2 CH 3 、-CH(CH 3 )CH(CH 3 ) 2 。
Preferably, the C 1～5 Alkoxy is selected from-OCH 3 、-OCH 2 CH 3 、-OCH 2 CH 2 CH 3 、-OCH 2 CH 2 CH 2 CH 3 、-OCH 2 CH 2 CH 2 CH 2 CH 3 、-OCH(CH 3 ) 2 、-OC(CH 3 ) 3 、-OCH 2 CH(CH 3 ) 2 、-OCH(CH 3 )CH 2 CH 3 、-OCH(CH 3 )CH 2 CH 2 CH 3 、-OCH 2 CH(CH 3 )CH 2 CH 3 、-OCH 2 CH 2 CH(CH 3 ) 2 、-OCH(CH 2 CH 3 ) 2 、-OCH 2 C(CH 3 ) 3 、-OC(CH 3 ) 2 CH 2 CH 3 、-OCH(CH 3 )CH(CH 3 ) 2 。
Preferably, the C 1～5 Alkoxycarbonyl is selected from-COOCH 3 、-COOCH 2 CH 3 、-COOCH 2 CH 2 CH 3 、-COOCH 2 CH 2 CH 2 CH 3 、-COOCH 2 CH 2 CH 2 CH 2 CH 3 、-COOCH(CH 3 ) 2 、-COOC(CH 3 ) 3 、-COOCH 2 CH(CH 3 ) 2 、-COOCH(CH 3 )CH 2 CH 3 、-COOCH(CH 3 )CH 2 CH 2 CH 3 、-COOCH 2 CH(CH 3 )CH 2 CH 3 、-COOCH 2 CH 2 CH(CH 3 ) 2 、-COOCH(CH 2 CH 3 ) 2 、-COOCH 2 C(CH 3 ) 3 、-COOC(CH 3 ) 2 CH 2 CH 3 、-COOCH(CH 3 )CH(CH 3 ) 2 。
Preferably, in formula I, R 1 Selected from Cl, F, br, H, C 1～3 Alkyl, C 1～3 An alkoxy group; further preferably, R 1 Selected from Cl, F,H. Methyl, methoxy.
Preferably, in formula I, R 2 Selected from H, cl, F, br, C 1～3 An alkyl group; further preferably, R 2 Selected from H, cl, F, methyl.
Preferably, in formula I, R 3 Selected from F, cl, C 1～3 Alkyl or C 1～3 An alkoxycarbonyl group; further preferably, R 3 Selected from Cl, C 1～3 Alkyl or ethoxycarbonyl.
Preferably, in formula I, n is 0,1 or 2; further preferably, n is 0 or 1.
Preferably, in formula I, m is 0,1 or 2.
Preferably, the C 1～3 Alkyl is selected from-CH 3 、-CH 2 CH 3 、-CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 。
Preferably, the C 1～3 Alkoxy is selected from-OCH 3 、-OCH 2 CH 3 、-OCH 2 CH 2 CH 3 、-OCH(CH 3 ) 2 。
Preferably, the C 1～3 Alkoxycarbonyl is selected from-COOCH 3 、-COOCH 2 CH 3 、-COOCH 2 CH 2 CH 3 、-COOCH(CH 3 ) 2 。
Preferably, the compound of formula I is selected from:
preferably, the compound of formula I is
Preferably, the compound of formula I above may be prepared by the following preparation method: the method comprises the following steps:
s1: make the following stepsWith CS 2 、ClCH 2 CO 2 Me reaction to obtain->Make the following stepsAnd ClCH (channel group) 2 COCl reaction to give->Then with R 2 Group-substituted indolinones are reacted to give +.>
S2: make the following stepsAnd (3) mixing and reacting to obtain the compound shown in the formula I.
Preferably, the medicament comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, isomer, solvate thereof.
Preferably, the medicament further comprises pharmaceutically acceptable excipients.
Preferably, the dosage form of the medicament is a pill, a tablet, a granule, a capsule, a syrup or an injection.
The anti-virus infection of the present invention means that a series of diseases caused by the virus infection of the virus can be prevented or treated.
The solvate in the present invention means that the drug crystals contain solvent molecules.
Preferably, the katavirus is derived from bovine, ovine, canine, kangaroo, fox, bird, rabbit, guinea pig, rat or hamster.
In a second aspect, the present invention provides a pharmaceutical composition for preventing and/or treating a cover-tower virus infection, comprising a therapeutically effective amount of a compound shown in formula I or pharmaceutically acceptable salts, isomers and solvates thereof,
wherein R is 1 、R 2 、R 3 N, m are as defined above.
Preferably, the compound shown in formula I is
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
Preferably, the dosage form of the pharmaceutical composition is a pill, a tablet, a granule, a capsule, a syrup or an injection.
The term "therapeutically effective amount" means an amount sufficient to produce a beneficial or desired effect when administered to a subject suffering from a katavirus infection; the effect may be prophylaxis of infection by the rotavirus and/or treatment of clinical symptoms or indications associated with rotavirus infection. It will be appreciated that the total daily dosage of the medicament of the present invention must be determined within the scope of sound medical judgment. For any particular infected person, the particular therapeutically effective dose level will depend on a variety of factors, including the severity of the infection in the infected person being treated; the activity of the particular drug employed; the specific drug or dosage form employed; weight, general health status, and food intake of the infected person; the administration time, administration route and excretion rate of the employed drug; duration of treatment; a drug to be used in combination with or simultaneously with the specific drug to be used; and similar factors well known in the medical arts. For example, it is common in the art to start doses of the drug below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.
The term "pharmaceutically acceptable adjuvant" is a substance that is non-toxic, compatible with the active ingredient and otherwise biologically compatible with the organism. The choice of a particular adjuvant will depend on the mode of administration or the type and state of the disease used to treat a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, binders, lubricants, stabilizers, hydration agents, emulsifying accelerators, buffers, absorbents, colorants, ion exchangers, mold release agents, coating agents, flavoring agents, antioxidants and the like which are conventional in the pharmaceutical arts. Flavoring agent, antiseptic, sweetener, etc. can be added into the pharmaceutical composition if necessary.
The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts including acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, or methanesulfonic acid and the like; also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid (see Berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the invention contain basic and acidic functionalities that can be converted to either base or acid addition salts.
Preferably, the salt is contacted with a base or acid in a conventional manner to isolate the parent compound, thereby regenerating the neutral form of the compound. The parent form of a compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.
As used herein, "pharmaceutically acceptable salts" are derivatives of the compounds of the invention wherein the parent compound is modified by salt formation with an acid or by salt formation with a base. Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, such as salts formed with non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic or organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxy, hydroxynaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalactural, propionic acid, salicylic acid, stearic acid, sulfurous acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, or p-toluenesulfonic acid.
Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
"isomers" as used herein include geometric isomers as well as stereoisomers, such as cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic and other mixtures thereof, all of which are within the scope of the present invention. The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to one of the functional group isomers that has a different point of attachment of hydrogen through displacement of one or more double bonds, for example, the ketone and its enol form are keto-enol tautomers. The term "diastereoisomer" refers to a stereoisomer of a molecule having two or more chiral centers and having a non-mirror image relationship between the molecules. The term "cis-trans isomer" refers to different spatial configurations in which either a double bond or a single bond of a ring-forming carbon atom in the molecule cannot rotate freely.
In the present invention, the substituent R 3 The definitions in each case are independent and the substituents may be the same or different; in general, the variables may be selected from the same or different substituents of the same embodiment; for example, when m is 2 in formula (I), then the benzene ring is substituted with two R 3 Group substitution, wherein each R 3 Are defined independently of each other.
In a third aspect, the present invention provides a method of screening for compounds for the prevention and/or treatment of a cover virus infection comprising the steps of:
s1: obtaining the mapping relation between the antiviral molecules and the antiviral characteristics, and constructing a model;
s2: screening compounds conforming to the model from the database to obtain primary screening compounds;
s3: using protein of the Gatta virus as a substrate, using the antiviral drug Rede Siwei as a molecular probe, using scoring software to evaluate, and screening binding sites from the protein of the Gatta virus;
s4: molecular docking is carried out on the primary screening compound and a binding site in the protein of the cover tower virus, and screening is carried out on the basis of binding parameters, so that the target compound is obtained.
Preferably, the binding parameters include binding energy, conformation.
The conformation in the present invention refers to the spatial arrangement generated by the placement of atoms around only a single bond in one molecule without changing the covalent bond structure. The cleavage and reformation of covalent bonds is not required when one conformation is changed to another. Conformational changes do not alter the optical activity of the molecule.
The calculation formula of the binding energy is as follows:where the sum is for all pairs of atoms that can move relative to each other, typically not including 1-4 interactions, i.e., atoms separated by consecutive 3 covalent bonds. Here, each atom i is assigned a type t i And->Is to define interatomic distance (r ij ) Is a set of symmetric interaction functions.
The beneficial effects of the invention are as follows: the compound of the formula I has excellent effect of preventing or treating the infection of the Gaota virus, has no obvious toxic or side effect on normal cells, and can achieve the effect of inhibiting the Gaota virus at a lower concentration. In addition, the compound of the formula I in the invention damages E1-E2 interaction of the Gauss virus by combining E2 structural protein in the Gauss virus, blocks assembly of virus particles, further interferes with virus infection and replication, has obvious curative effect in preventing or treating the Gauss virus infection and is not easy to generate drug resistance. Specifically, the compound of the formula I has obvious antiviral activity at the concentration of about 12.5 mu mol/L, and has no cytotoxicity in the concentration range of 3.125-50 mu mol/L.
The screening method is simple, easy to operate and high in screening accuracy and efficiency. In addition, the screening method of the invention carries out verification steps on the primary screening compound, specifically uses the structure of Capsed-E2-E1 as a target, selects antiviral drug Rede-Sivir as a small molecular probe, screens a series of binding sites from the Gaitavirus, screens out compounds for preventing and/or treating the Gaitavirus by the binding energy and molecular conformation of the primary screening compound and the binding sites, and has good curative effect and accurate result.
Drawings
FIG. 1 is a Cryo-EM block diagram of the head tower virus of the present invention.
FIG. 2 is a graph showing the prediction of binding of Compound 1 of the present invention to a Kappaphycus virus protein.
FIG. 3 is an enlarged view of a portion of the binding site of Compound 1 and the Katavirus protein in FIG. 2.
FIG. 4 is a chart showing cytotoxicity test of Compound 1 of the present invention.
FIG. 5 is a graph showing the antiviral activity test of Compound 1 of the present invention.
Detailed Description
Specific embodiments of the present invention will be described in further detail below with reference to the drawings and examples, but the practice and protection of the present invention are not limited thereto. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available.
The invention utilizes Cryo-EM (frozen electron microscope) to carry out structural analysis on a strain GETV-V1 which can cause reproductive dysfunction of pregnant female mice, and is specifically shown in figure 1, wherein figure 1 (A) is an outer surface diagram of the Katavirus, and as can be seen in figure 1 (A), the outer surface of the Katavirus has designated symmetry axes of 5 times, 3 times and 2 times; FIG. 1 (B) is a cross-sectional view of a cover tower virus; FIG. 1 (C) is an electron density diagram of the Kappaphycus alvarezier virus; FIG. 1 (D) is an atomic model diagram of a Kappaphycus virus; FIG. 1 (E) is an atomic model of an E1-E2-capsid heterotrimer. From the slaveAtomic separationIn resolution map, new identification of 19 interactions (hydrogen bonds or salt bridges) plays an important role in maintaining the overall structural stability of the virus, 8 glycosylation sites found and built on the particle surface of an atomic model are involved in receptor antibody recognition and viral immune escape, 5S-acylation sites (S-acylation sites) of the atomic model are built on E1/E2 near the inner phospholipid membrane to contribute to viral assembly and transmembrane region stability, and 1 dioleoyl phosphatidylcholine (DOPC) and 3 cholesterol identified in and near the hydrophobic pocket formed by TM helix of E1, TM helix of E2 and domain D (D region) are extremely valuable for maintaining E1/E2 structural stability, especially DOPC and cholesterol in the pocket.
The GETV virus consists of Capid, E3, E2, 6K and E1:
amino acid sequence of Capsid (Capsid):
MNYIPTQTFYGRRWRPRPAYRPWRVPMQPAPPMVIPELQTPIVQAQQMQQLISAVSALTTKQNGKAPKKPKKKPQKAKAKKNEQQKKNENKKPPPKQKNPAKKKKPGKRERMCMKIENDCIFEVKLDGKVTGYACLVGDKVMKPAHVKGVIDNPDLAKLTYKKSSKYDLECAQIPVHMKSDASKYTHEKPEGHYNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWTKDMVTRYTPEGTEEW
amino acid sequence of E2:
SVTEHFNVYKATKPYLAYCADCGDGQFCYSPVAIEKIRDEASDGMIKIQVAAQIGINKGGTHEHNKIRYIAGHDMKEANRDSLQVYTSGVCAIRGTMGHFIVAYCPPGDELKVQFQDAESHTQACKVQYKHAPAPVGREKFTVRPHFGIEVPCTTYQLTTAPTEEEIDMHTPPDIPDITLLSQQSGNVKITAGGKTIRYNCTCGSGNVGTTSSDKTINSCKIAQCHAAVTNHDKWQYTSSFVPRADQLSRKGKVHVPFPLTNSTCRVPVARAPGVTYGKRELTVKLHPDHPTLLTYRSLGADPRPYEEWIDRYVERTIPVTEDGIEYRWGNNPPVRLWAQLTTEGKPHGWPHEIILYYYGLYPAATIAAVSAAGLAVVLSLLASCYMFATARRKCLTPYALTPGAVVPVTLGVLCCAPRAHA
amino acid sequence of E1:
YEHTATIPNVVGFPYKAHIERNGFSPMTLQLEVLGTSLEPTLNLEYITCEYKTVVPSPYIKCCGTSECRSMERPDYQCQVYTGVYPFMWGGAYCFCDTENTQLSEAYVDRSDVCKHDHAAAYKAHTAAMKATIRISYGNLNQTTTAFVNGEHTVTVGGSRFTFGPISTAWTPFDNKIVVYKNDVYNQDFPPYGSGQPGRFGDIQSRTVESKDLYANTALKLSRPSSGTVHVPYTQTPSGFKYWLKERGTSLNDKAPFGCVIKTNPVRAENCAVGNIPVSMDIPDTAFTRVIDAPAVTNLECQVAVCTHSSDFGGIATLTFKTDKPGKCAVHSHSNVATIQEAAVDIKTDGKITLHFSTASASPAFKVSVCSAKTTCTAACEPPKDHIVPYGASHNNQVFPDMSGTAMTWVQRVAGGLGGLTLAAVAVLILVTCVTMRR
the invention utilizes Cryo-EM (frozen electron microscope) to analyze the structure of the GETV-V1 strain, thereby defining the structure of the Katavirus.
The invention is further elaborated in connection with the following specific embodiments:
the invention provides a method for screening compounds for preventing and/or treating infection of a cover tower virus, which comprises the following specific steps:
the method is based on geometric deep learning (Geometric Deep Learning) (developed by Souzhou cloud advanced medical science and technology Co., ltd.), some antiviral molecules which meet commercial availability and chemical diversity are selected, the mapping relation between the existing antiviral molecules and antiviral characteristics is obtained, a screening model is built based on the mapping relation between the existing antiviral molecules and antiviral characteristics, the model is based on a deep learning network (Graph-based deep learning) of Graph learning, and then the screening model is used for predicting the antiviral characteristics of compounds in a large-scale molecular library so as to find potential Miao compounds. And finally, molecular ordering is carried out according to the predicted fraction, then, the compound at the seedling emergence is selected and screened according to the predicted physicochemical property, and finally, the screened compound is cross-verified through molecular docking.
The method for molecular butt-joint cross-validation of the initially screened compound comprises the following steps:
firstly, dividing three-dimensional space grids of proteins (sub 1, sub2, sub3, monomers and tetramers) in the head tower virus, wherein sub1, sub2, sub3 are three subdomains of E2 structural proteins in the head tower virus proteins, the monomers are E1-E2-capsids, and the tetramers are assembled by four monomers, and the space between the monomers is 20 angstroms. Then, the grids are used as centers, 15 angstroms is used as radius, antiviral drug Ruidexivir is selected as a small molecular probe, and docking software is utilized to score, so that a series of binding sites are screened out. For each grid point, a score of the best binding pose was chosen as the assessment of the binding pocket for that site. These binding sites are then examined one by one to select the final binding site which is the substrate binding groove that can be used for the next large-scale small molecule library screening.
The biochemical and LC antiviral small molecule library containing 10157 small molecules was then screened. For more than 700 ten thousand molecules of a comprehensive small molecule library ZINC, 100 ten thousand molecules are randomly selected for molecular docking, 50 small molecules with highest scores are screened out from the LC antiviral small molecule library and the ZINC library, and then similarity inspection is carried out on the small molecules in the rest 600 ten thousand ZINC libraries, wherein in the inspection, the small molecules with the similarity of more than 0.6 with any one of the 50 small molecules can be selected for docking with the binding sites in the protein of the Katavirus. In these molecular docking, 5 conformations were examined for each small molecule at the time of docking, scoring was performed using scoring software, and screening was performed according to binding energy and molecular conformation, thereby screening compound 1 for preventing and/or treating a cover virus infection in the present invention.
Compound 1, having the formula:the chemical name is: 4- (2- {3- [ (5Z) -3- [ (2-chlorophenyl) methyl)]-4-oxo-2-sulfanyl-1, 3-thiazolidine-5-ylidene]-ethyl 2-oxo-2, 3-dihydro-1H-indol-1-yl } acetamide) benzoate of the formula: c (C) 29 H 22 ClN 3 O 5 S 2 The CAS number for this compound is: 617696-38-5.
Further optimizing the molecular structure of the compound 1 to obtain the compounds 2 to 59, wherein the molecular formulas of the compounds 2 to 59 are as follows:
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then, the binding prediction was performed on the above-mentioned compounds 1 to 59 and the protein of the Kappavirus, and the binding energies of the compounds 1 to 59 were calculated as shown in Table 1 below:
TABLE 1 binding energy of Compounds 1 to 59
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As shown in Table 1, the binding energy of the compounds 1 to 59 was between-7.9 and-9.8 kcal/mol, and it was found that the compounds 1 to 59 all had a good binding force with the protein of the Katavirus.
Compounds 1 to 59 are all binding pockets to the D-domain of the E2 protein (subdomain D) of the getavirus, similar to the hydrophobic pocket formed by TM helix and D-domain of the E2 protein, TM helix of the E1 protein. Studies have shown that the D subregion plays an important role in maintaining the stability of the hydrophobic pocket, and also affects lipid release and large conformational changes in the overall structure of E1-E2-Capid in the hydrophobic pocket. It is assumed that the above-mentioned compounds 1 to 59 interfere with viral infection and replication by disrupting E1-E2 interactions, thereby blocking viral particle assembly.
In the invention, compounds 1-59 are combined with the protein of the Katta virus, the combination position is predicted, and the analysis of the predicted optimal combination conformation is performed to determine the action site on the protein, namely the specific position of a combination pocket. Taking compound 1 as an example, the optimal binding conformation of the protein binding prediction of compound 1 and the cover virus is shown in fig. 2 and 3. Specific different kinds of interactions are shown in tables 2 to 4 below:
TABLE 2 proteins create hydrophobic interaction sites with compounds
Sequence number Protein residues AA (amino acid abbreviation)
1 277 TYR
2 279 LYS
3 320 VAL
4 337 LEU
5 338 TRP
6 340 GLN
TABLE 3 sites where proteins and compounds interact hydrogen-bonding
Sequence number Protein residues AA (amino acid abbreviation)
1 338 TRP
TABLE 4 sites where proteins interact with Compounds to produce pi-pi stacks
Sequence number Protein residues AA (amino acid abbreviation)
1 277 TYR
As can be seen from fig. 2-3 and tables 2-4, compound 1 has a hydrophobic interaction with 277tyr,279lys,320val, 307 leu,338trp and 340GLN, respectively; compounds 1 and 338TRP have hydrogen bond interactions; compounds 1 and 277TYR form a pi-pi stacking interaction; therefore, the compound 1 has better binding performance with the binding site in the protein of the cover tower virus.
Compound 1 was then tested for cytotoxicity and anti-kappaphycus activity as follows:
the toxicity of the above compounds on cells cultured in vitro was first evaluated using the CCK-8 kit. The CCK-8 kit was purchased from Donjindo and cell viability of BHK21 cells treated with the above compounds was measured according to the protocol provided. BHK21 cells were inoculated into 96-well plates and cultured to 80% density of monolayer cells, then different concentrations of the above-mentioned compounds (concentrations of the compounds: 50. Mu. Mol/L, 25. Mu. Mol/L, 12.5. Mu. Mol/L, 6.25. Mu. Mol/L, 3.125. Mu. Mol/L, respectively) were added to the wells as experimental groups, 0.1% DMSO was added to the control group, the cells of the experimental groups, the control group and the blank group were treated with no compound or solvent, respectively, for 24 hours, and CCK-8 solution was added to each well and further cultured at 37℃for 2 hours. Absorbance at 450nm was measured using a SPARK 10M (multi-function microplate detector) and the test results are shown in fig. 4. As can be seen from FIG. 4, C was added at various concentrations (3.125 to 50. Mu. Mol/L) 29 H 22 ClN 3 O 5 S 2 The absorbance difference between the drug group and the non-drug group at 450nm is very small, indicating that the compound C in the invention 29 H 22 ClN 3 O 5 S 2 The concentration is 3.125-50 mu mol/L, and the cell-free recombinant DNA is not obvious in toxicity.
Subsequently, the antiviral activity of the above-mentioned compounds was examined at the cellular level by mixing a fixed amount of virus with an equivalent serial double diluted compound. BHK21 cells were seeded into 96-well plates and cultured to 80% density of monolayer cells. According to 100TCID 50 Inoculated Getah virus was added to each well of cells and after incubation for 0.5h the virus solution was discarded, 100. Mu.L of the above compound was rapidly added to each well of cells at a concentration of 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M (where. Mu.M refers to. Mu. Mol/L) at 5% CO 2 After incubation at 37℃for 48h, the Reed-Muench calculation formula (Reed-Muench calculation formula: distance ratio = (percentage of disease rate higher than 50%)/(percentage of disease rate higher than 50% low)Percent at 50% disease rate); logTCID 50 Log of dilution of =difference between log of distance ratio x dilution + greater than 50% disease rate, dilution means that compound concentrations are 50 μm,25 μm,12.5 μm,6.25 μm,3.125 μm) respectively to measure TCID of GETV after effect of different concentrations of the above compound 50 Relative quantification, the test results are shown in FIG. 5. As can be seen from FIG. 5, compound C of the present invention 29 H 22 ClN 3 O 5 S 2 Has effect in inhibiting virus activity, meets basic use requirement of medicine, and can be used for calculating 100TCID of GETV after the above compounds act 50 Value, C can be determined 29 H 22 ClN 3 O 5 S 2 The molecule has obvious antiviral activity at the concentration of 12.5 mu mol/L. Furthermore, one-way ANOVA in SPSS software was used to analyze data for treatment, P<0.01 represents a very significant difference, specifically: the marked horizontal lines and the marked horizontal lines refer to significant differences, i.e., significant differences of 50 μm,25 μm and 12.5 μm, compared to the control group 0.1% dmso bar graph.
Binding force of Compound 2 to Compound 59 screened by the screening method of the present invention to the binding site on the protein of the Gattavirus and Compound 1 (C) 29 H 22 ClN 3 O 5 S 2 ) And thus, the effect of compound 2 to compound 59 on cytotoxicity and anti-kappaphycus activity is also at substantially the same level as that of compound 1.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes may be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (6)

1. The use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a cover-tower virus infection, characterized in that: the compounds of formula I are:
。
2. the use according to claim 1, characterized in that: the medicament comprises a therapeutically effective amount of a compound represented by formula I or a pharmaceutically acceptable salt thereof.
3. The use according to claim 2, characterized in that: the medicine also comprises pharmaceutically acceptable auxiliary materials.
4. The use according to claim 2, characterized in that: the dosage forms of the medicine are pills, tablets, granules, capsules, syrups or injections.
5. The use according to claim 1, characterized in that: the Katavirus is derived from bovine, ovine, canine, kangaroo, fox, bird, rabbit, guinea pig, rat, or hamster.
6. The use according to claim 1, characterized in that: the compound shown in the formula I is combined with E2 structural protein in the cover tower virus.
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