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

Abstract

The invention discloses the use of compounds in the preparation of medicines against Geta virus infection. The medicines include a therapeutically effective amount of the compound represented by formula I or its pharmaceutically acceptable salt, its isomer, and its solvate, The compound of formula I of the present invention has excellent effect in preventing or treating Getta virus infection, and at the same time has no obvious toxic side effects on normal cells, and can achieve the effect of inhibiting Getta virus at a lower concentration. In addition, the compound of Formula I of the present invention binds to the E2 structural protein in Geta virus, destroys the E1-E2 interaction of Geta virus, hinders the assembly of virus particles, and thereby interferes with viral infection and replication. It is useful in preventing or treating Geta virus. It has obvious efficacy in infection and is not prone to drug resistance.

Description

Application of compound in preparation of medicine for resisting Geta virus infection

Technical Field

The present invention relates to the field of medicine, and in particular to application of a compound in preparing a medicine for resisting Geta virus infection.

Background Art

Getah virus (GETV) is an arbovirus belonging to the Semliki Forest virus group (SFV) of the Togaviridae family. The SFV group also includes Chikungunyavirus (CHIKV), Semliki Forest virus (SFV), Mayarovirus (MAYV), Una virus (UNAV), Bebaru virus (BEBV) and O’nyong-nyong virus (ONNV). Among them, CHIKV, ONNV and MAYV have extremely high morbidity and mortality rates in susceptible populations.

GETV originally originated from mosquitoes. In the 40 years from 1960 to 2000, it was limited to cases in horses and pigs. However, in just 20 years since the beginning of this century, it has expanded to cattle, sheep, dogs, kangaroos, foxes, wild birds, rabbits, guinea pigs, rats, hamsters and other animals. At the same time, the frequency of clinical case reports is also accelerating. What is more worrying is that anti-GETV antibodies have been detected in patients with fever and even in healthy people. Although there have been no reports of human infection so far, many serological epidemiological surveys at home and abroad have shown the presence of GETV antibodies in the human body, indicating that the virus can infect humans. Due to the infection of multiple animals and the widespread infection of mosquitoes, and the close contact between pigs and other livestock and people in the vast rural areas of my country, GETV is very likely to be prevalent in the human population, and there may be a large potential risk in future public health events.

Scientists have been using cryo-electron microscopy to study the structure of various viruses in the alphavirus genus, with resolution ranging from arrive The viruses studied include Barmah forest virus (BFV), EEEV, WEEV, VEEV, CHIKV, SINV, MAYV, etc. The cryo-electron microscopy density map of the alphavirus currently analyzed shows that the alphavirus has the same structural composition. The alphavirus RNA is hidden in an icosahedral core composed of 240 copies of capsid (capsid) in a disordered state. The outward protruding E1 and E2 structural proteins form heterodimers (80 copies of which form an icosahedral virus shell), and the heterodimers are connected to the capsid across the phospholipid membrane. GETV is a single-stranded positive-strand RNA virus with an envelope. The mature Geta virus is a spherical particle of about 70nm, and the genome of about 11kb is composed of 2 open reading frames (ORFs), and contains a coding and a polyprotein, wherein the polyprotein at the N end contains 4 non-structural proteins (non-structural proteins, i.e., nsP1, nsP2, nsP3 and nsP4), and the polyprotein at the C end is composed of 5 structural proteins (structural proteins, capsid-E3-E2-6K-E1). The research of Geta virus in the prior art mainly focuses on strengthening the detection and monitoring of infection of animals such as GETV, pigs, horses and cattle, and the quarantine of imported livestock and their finished products. The literature reported at present also focuses on the detection and vaccine prevention of Geta virus, and there is almost no research on drugs for treating Geta virus infection, so there is still a lack of drugs that can effectively treat Geta virus infection. In addition, there is also a method for screening drugs using molecular docking software and scoring software in the prior art, but the screening results of the existing screening method are inaccurate, and it is impossible to accurately screen, and the existing screening method lacks a verification step.

Summary of the invention

In order to overcome the problems existing in the above-mentioned prior art, one of the objects of the present invention is to provide a compound represented by Formula I or a pharmaceutically acceptable salt, isomer, solvate thereof for use in the preparation of a drug for preventing and/or treating Geta virus infection;

A second object of the present invention is to provide a pharmaceutical composition for preventing and/or treating Geta virus infection.

A third object of the present invention is to provide a method for screening compounds for preventing and/or treating Geta virus infection.

In order to achieve the above object, the technical solution adopted by the present invention is:

The first aspect of the present invention is to provide a compound represented by formula I or a pharmaceutically acceptable salt, isomer, solvate thereof for use in the preparation of a drug for preventing and/or treating Geta virus infection.

Wherein, R ₁ is selected from Cl, F, Br, H, C _1-5 alkyl, C _1-5 alkoxy;

R ₂ is selected from H, Cl, F, Br, C _1-5 alkyl;

R ₃ is selected from F, Cl, Br, C _1-5 alkyl or C _1-5 alkoxycarbonyl;

n is 0, 1, 2, 3, 4 or 5;

m is 0, 1, 2, 3, 4 or 5.

Preferably, the C _{1 to 5} alkyl group 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 to 5} alkoxy group 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 to 5} alkoxycarbonyl group 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 is selected from Cl, F, Br, H, C _1-3 alkyl, C _1-3 alkoxy; further preferably, R ₁ is selected from Cl, F, H, methyl, methoxy.

Preferably, in Formula I, R ₂ is selected from H, Cl, F, Br, C _1-3 alkyl; further preferably, R ₂ is selected from H, Cl, F, methyl.

Preferably, in Formula I, R ₃ is selected from F, Cl, C _1-3 alkyl or C _1-3 alkoxycarbonyl; further preferably, R ₃ is 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 group is selected from -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , and -CH(CH ₃ ) ₂ .

Preferably, the C _1-3 alkoxy group is selected from -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₃ , and -OCH(CH ₃ ) ₂ .

Preferably, the C _1-3 alkoxycarbonyl group is selected from -COOCH ₃ , -COOCH ₂ CH ₃ , -COOCH ₂ CH ₂ CH ₃ , and -COOCH(CH ₃ ) ₂ .

Preferably, the compound represented by formula I is selected from:

Preferably, the compound shown in formula I is

Preferably, the compound of formula I above can be prepared by the following preparation method: comprising the following steps:

S1: Make Reaction with CS ₂ , ClCH ₂ CO ₂ Me to obtain make Reaction with ClCH ₂ COCl to obtain Then react with indole dione substituted with _R2 group to obtain

S2: Make The mixture is mixed and reacted to obtain the compound represented by formula I.

Preferably, the drug comprises a therapeutically effective amount of the compound represented by Formula I or a pharmaceutically acceptable salt, isomer, or solvate thereof.

Preferably, the drug further comprises a pharmaceutically acceptable excipient.

Preferably, the drug is in the form of pills, tablets, granules, capsules, syrups or injections.

The anti-Geta virus infection in the present invention refers to the ability to prevent or treat a series of diseases caused by Geta virus infection.

The solvate in the present invention refers to drug crystals containing solvent molecules.

Preferably, the Getavirus is derived from cattle, sheep, dogs, kangaroos, foxes, birds, rabbits, guinea pigs, rats or hamsters.

The second aspect of the present invention is to provide a pharmaceutical composition for preventing and/or treating Geta virus infection, comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, isomer, or solvate thereof,

wherein R ₁ , R ₂ , R ₃ , n and m are as defined above.

Preferably, the compound represented by formula I is

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

Preferably, the pharmaceutical composition is in the form of pills, tablets, granules, capsules, syrups or injections.

The term "therapeutically effective amount" means an amount sufficient to produce a beneficial or desired effect when the drug of the present invention is administered to a Geta virus infected person; the effect may be the prevention of Geta virus infection, and/or the treatment of clinical symptoms or indicators associated with Geta virus infection. However, it should be recognized that the total daily dose of the drug of the present invention must be determined within the scope of sound medical judgment. For any specific infected person, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the severity of the infection of the infected person being treated; the activity of the specific drug used; the specific drug or dosage form used; the weight, general health, and diet of the infected person; the administration time, route of administration, and excretion rate of the drug used; the duration of treatment; drugs used in combination or simultaneously with the specific drug used; and similar factors known in the medical field. For example, the practice in the art is to start the dosage of the drug at a level lower than that required to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is obtained.

The term "pharmaceutically acceptable excipient" is a substance that is non-toxic, compatible with the active ingredient and otherwise biologically suitable for use in an organism. The selection of a particular excipient will depend on the mode of administration or the type and state of disease used to treat a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, conventional solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, adhesives, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, etc., in the pharmaceutical composition. If necessary, flavoring agents, preservatives, and sweeteners, etc., may also be added to the pharmaceutical composition.

The term "pharmaceutically acceptable salt" refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, 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 include salts of amino acids (such as arginine, etc.), 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 present invention contain basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid in a conventional manner and isolating the parent compound. The parent form of the 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 salification with an acid or salification 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 or organic salts of acid radicals 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 and organic acids selected from 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, hydroiodide, 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, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin, tartaric acid, or p-toluenesulfonic acid.

The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

The "isomer" described in the present invention includes geometric isomers and stereoisomers, such as cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic mixtures 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 a type of functional group isomer that has different hydrogen attachment points through one or more double bond displacements, for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to a stereoisomer in which a molecule has two or more chiral centers and is a non-mirror image between molecules. The term "cis-trans isomer" refers to different spatial configurations in which double bonds or single bonds of ring carbon atoms in a molecule cannot rotate freely.

In the present invention, the definition of the substituent _R3 in each case is independent, and the substituents may be the same or different; generally, the variable may be selected from the same or different substituent groups of the same technical solution; for example, when m in the general formula (I) is 2, the benzene ring is substituted by two _R3 groups, wherein the definition of each _R3 is independent of each other.

The third aspect of the present invention is to provide a method for screening compounds for preventing and/or treating Geta virus infection, comprising the following steps:

S1: Obtain the mapping relationship between antiviral molecules and antiviral properties and build a model;

S2: Screen the compounds that meet the model from the database to obtain the primary screening compounds;

S3: Using the Geta virus protein as a substrate and the antiviral drug remdesivir as a molecular probe, scoring software was used for evaluation to screen out binding sites from the Geta virus protein;

S4: Molecular docking of the primary screening compounds with the binding sites in the Geta virus protein was performed, and screening was performed based on the binding parameters to obtain the target compounds.

Preferably, the binding parameters include binding energy and conformation.

The conformation in the present invention refers to the spatial arrangement of atoms placed around single bonds in a molecule without changing the covalent bond structure. When one conformation changes to another, it does not require the breaking and reformation of covalent bonds. Conformational changes will not change the optical activity of the molecule.

The formula for calculating the binding energy is: where the sum is over all pairs of atoms that can move relative to each other, typically excluding 1-4 interactions, i.e. atoms separated by three consecutive covalent bonds. Here, each atom i is assigned a type t _i , and is a set of symmetric interaction functions that define the interatomic distances ( _rij ).

The beneficial effects of the present invention are as follows: the compound of formula I of the present invention has an excellent effect of preventing or treating Geta virus infection, and has no obvious toxic side effects on normal cells, and can achieve the effect of inhibiting Geta virus at a relatively low concentration. In addition, the compound of formula I of the present invention destroys the E1-E2 interaction of Geta virus by binding to the E2 structural protein in Geta virus, hinders the assembly of virus particles, and then interferes with virus infection and replication. It has obvious efficacy in preventing or treating Geta virus infection and is not easy to produce drug resistance. Specifically, the compound of formula I of the present invention has obvious antiviral activity at a concentration of about 12.5 μmol/L, and has no cytotoxicity in the concentration range of 3.125 to 50 μmol/L.

The screening method of the present invention is simple, easy to operate, and has high screening accuracy and efficiency. In addition, the screening method of the present invention carries out a verification step for the primary screening compound, specifically using the Capsid-E2-E1 structure as a target, selecting the antiviral drug remdesivir as a small molecule probe, and screening a series of binding sites from the Geta virus. Through the binding energy and molecular conformation of the primary screening compound and the binding site, the compound for preventing and/or treating the Geta virus is screened, and the screened compound has good efficacy and accurate results.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a Cryo-EM structure diagram of the Geta virus in the present invention.

FIG. 2 is a diagram showing the predicted binding of compound 1 of the present invention to Geta virus protein.

FIG3 is a partial enlarged view of the binding position between compound 1 and Geta virus protein in FIG2 .

FIG4 is a graph showing the cytotoxicity test of compound 1 of the present invention.

FIG5 is a graph showing the antiviral activity test of compound 1 of the present invention.

DETAILED DESCRIPTION

The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

The present invention utilizes Cryo-EM (cryo-electron microscopy) to perform structural analysis on a GETV-V1 strain that can cause reproductive disorders in pregnant mice, as specifically shown in Figure 1, wherein Figure 1(A) is a diagram of the outer surface of the Getavirus. It can be seen from Figure 1(A) that the outer surface of the Getavirus has designated 5-fold, 3-fold and 2-fold symmetry axes; Figure 1(B) is a cross-sectional diagram of the Getavirus; Figure 1(C) is an electron density diagram of the Getavirus; Figure 1(D) is an atomic model diagram of the Getavirus; and Figure 1(E) is an atomic model of the E1-E2-capsid heterotrimer. In the atomic resolution map, 19 newly identified interaction forces (hydrogen bonds or salt bridges) play an important role in maintaining the overall structural stability of the virus. Eight glycosylation sites discovered and atomically modeled on the particle surface are involved in receptor antibody recognition and viral immune escape. Five S-acylation sites (S-acylation sites) atomically modeled on E1/E2 near the inner phospholipid membrane contribute to viral assembly and transmembrane stability. One dioleoylphosphatidylcholine (DOPC) and three cholesterols identified in and near the hydrophobic pocket formed by the TM helix (transmembrane helix region) of E1, the TM helix and domain D (D region) of E2 are of great value in maintaining the stability of the E1/E2 structure, especially the DOPC and cholesterol in the pocket.

The GETV virus is composed of Capsid, E3, E2, 6K and E1:

Capsid amino acid sequence:

MNYIPTQTFYGRRWRPRPAYRPWRVPMQPAPPMVIPELQTPIVQAQQMQQLISAVSALTTKQNGKAPKKPKKKPQKAKAKKNEQQKKNNKKPPPKQKNPAKKKKPGKRERMCMKIENDCIFEVKLDGKVTGYACLVGDKVMKPAHVKGVIDNPDLAKLTYKKSSKYDLECAQIPVHMKSDASKYTHEKPEGHYNWH HGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWTKDMVTRYTPEGTEEW

Amino acid sequence of E2:

SVTEHFNVYKATKPYLAYCADCGDGQFCYSPVAIEKIRDEASDGMIKIQVAAQIGINKGGTHEHNKIRYIAGHDMKEANRDSLQVYTSGVCAIRGTMGHFIVAYCPPGDELKVQFQDAESHTQACKVQYKHAPAPVGREKFTVRPHFGIEVPCTTYQLTTAPTEEEIDMHTPPDIPDITLLSQQSGNVKITAGGKTIRYNCTCGSGNV GTT SSDKTINSCKIAQCHAAVTNHDKWQYTSSFVPRADQLSRKGKVHVPFPLTNSTCRVPVARAPGVTYGKRELTVKLHPDHPTLLTYRSLGADPRPYEEWIDRYVERTIPVTEDGIEYRWGNNPPVRLWAQLTTEGKPHGWPHEIILYYYGLYPAATIAAVSAAGLAVVLSLLASCYMFATARRKCLTPYALTPGAVVPVTLGVLCCAPRAHA

Amino acid sequence of E1:

YEHTATIPNVVGFPYKAHIERNGFSPMTLQLEVLGTSLEPTLNLEYITCEYKTVVPSPYIKCCGTSECRSMERPDYQCQVYTGVYPFMWGGAYCFCDTENTQLSEAYVDRSDVCKHDHAAAYKAHTAAMKATIRISYGNLNQTTTAFVNGEHTVTVGGSRFTFGPISTAWTPFDNKIVVYKNDVYNQDFPPYGSGQPGRFGDI QSRTVESKDLYANTAL KLSRPSSGTVHVPYTQTPSGFKYWLKERGTSLNDKAPFGCVIKTNPVRAENCAVGNIPVSMDIPDTAFTRVIDAPAVTNLECQVAVCTHSSDFGGIATLTFKTDKPGKCAVHSHSNVATIQEAAVDIKTDGKITLHFSTASASPAFKVSVCSAKTTCTAACEPPKDHIVPYGASHNNQVFPDMSGTAMTWVQRVAGGLGGLTLAAVAVLILVTCVTMRR

The present invention utilizes Cryo-EM (cryo-electron microscopy) to perform structural analysis on the GETV-V1 strain, thereby clarifying the structure of the GETA virus.

The present invention is further described in detail below with reference to specific embodiments:

The present invention provides a method for screening compounds for preventing and/or treating Geta virus infection, and the specific steps are as follows:

We used a method based on geometric deep learning (developed by Suzhou Yunsheng Pharmaceutical Technology Co., Ltd.) to select some antiviral molecules that are commercially available and chemically diverse, obtain the mapping relationship between existing antiviral molecules and antiviral properties, and build a screening model based on the mapping relationship between existing antiviral molecules and antiviral properties. The model is a graph-based deep learning network, and then the screening model is used to predict the antiviral properties of compounds in a large-scale molecular library to discover potential lead compounds. Finally, the molecules are ranked according to the predicted scores, and the lead compounds are selected and screened according to the predicted physical and chemical properties. Finally, the screened compounds are cross-validated through molecular docking.

The specific method of molecular docking cross-validation of the initially screened compounds is as follows:

First, the proteins in the Getavirus (sub1, sub2, sub3, monomers and tetramers; among them, sub1, sub2, and sub3 are the three subdomains of the E2 structural protein in the Getavirus protein, the monomer is E1-E2-capsid, and the tetramer is assembled from four monomers) were divided into three-dimensional space grids with a spacing of 20 angstroms. Then, with these grids as the center and 15 angstroms as the radius, the antiviral drug Remdesivir was selected as a small molecule probe, and the docking software was used to score it, thereby screening out a series of binding sites. For each grid point, the score of the best binding posture was selected as the evaluation of the site as a binding pocket. Next, these binding sites were examined one by one to select the final binding site, which is a substrate binding groove, which can be used for the next large-scale small molecule library screening.

Then, the life chemistry and LC antiviral small molecule libraries containing 10,157 small molecules were screened. For the more comprehensive small molecule library ZINC, which has more than 7 million molecules, we first randomly selected 1 million molecules for molecular docking, screened a total of 50 small molecules with the highest scores from the LC antiviral small molecule library and the ZINC library, and then performed a similarity check on the remaining 6 million small molecules in the ZINC library. In this check, those with a similarity greater than 0.6 with any of the 50 small molecules can be selected to dock with the binding site in the protein of the Geta virus. In these molecular dockings, in order to increase the computational efficiency, for each small molecule, 5 conformations were examined during docking, scored using scoring software, and screened according to binding energy and molecular conformation, thereby screening out compound 1 for preventing and/or treating Geta virus infection in the present invention.

Compound 1, molecular formula: The chemical name is: ethyl 4-(2-{3-[(5Z)-3-[(2-chlorophenyl)methyl]-4-oxo-2-sulfonyl-1,3-thiazolidin-5-ylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}acetamide)benzoate, the molecular formula is: C ₂₉ H ₂₂ ClN ₃ O ₅ S ₂ , and the CAS number of the compound is: 617696-38-5.

The molecular structure of compound 1 was further optimized to obtain compounds 2 to 59. The molecular formulas of compounds 2 to 59 are as follows:

Then, the above compounds 1 to 59 were respectively predicted to bind to the proteins of Geta virus, and the binding energies of compounds 1 to 59 were calculated. The calculated binding energies of compounds 1 to 59 are shown in Table 1 below:

Table 1 Binding energy of compounds 1 to 59

As shown in Table 1, the binding energies of compounds 1 to 59 are between -7.9 and -9.8 kcal/mol, which indicates that compounds 1 to 59 all have good binding affinity with the protein of Geta virus.

Compounds 1 to 59 all bind to the D subdomain (subdomain D) of the Geta virus E2 protein, which is close to the hydrophobic pocket formed by the TM helix (transmembrane helix) and D subdomain of the E2 protein and the TM helix of the E1 protein. Studies have shown that the D subdomain plays an important role in maintaining the stability of the hydrophobic pocket, and also affects the release of lipids in the hydrophobic pocket and the large-scale conformational changes of the overall structure of E1-E2-Capsid. It is speculated that the above compounds 1 to 59 disrupt the E1-E2 interaction, hinder the assembly of virus particles, and thus interfere with virus infection and replication.

The present invention combines compounds 1 to 59 with the protein of Geta virus and predicts the binding position. We have analyzed the interaction of the predicted optimal binding conformation and have clarified the specific position of the action site on the protein, that is, the binding pocket. Among them, taking compound 1 as an example, the predicted optimal binding conformation of compound 1 binding to the protein of Geta virus is shown in Figures 2 and 3. The specific different types of interactions are shown in Tables 2 to 4 below:

Table 2 Hydrophobic interaction sites between proteins and compounds

Serial number Protein residues AA (short for amino acid) 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 produce hydrogen bond interactions

Serial number Protein residues AA (short for amino acid) 1 338 TRP

Table 4 Sites where proteins and compounds generate π-π stacking interactions

Serial number Protein residues AA (short for amino acid) 1 277 TYR

As shown in Figures 2 to 3 and Tables 2 to 4, compound 1 has hydrophobic interactions with 277TYR, 279LYS, 320VAL, 337LEU, 338TRP and 340GLN, respectively; compound 1 has hydrogen bond interactions with 338TRP; compound 1 and 277TYR form π-π stacking interactions; therefore, compound 1 in the present invention has good binding performance with the binding site in the protein of Geta virus.

Compound 1 was then subjected to cytotoxicity test and anti-Geta virus activity test, as follows:

First, the CCK-8 kit was used to evaluate the toxicity of the above compounds to cells cultured in vitro. The CCK-8 kit was purchased from Donjindo, and the cell viability of BHK21 cells treated with the above compounds was measured according to the provided protocol. BHK21 cells were inoculated into 96-well plates and cultured to a monolayer cell density of 80%, and then different concentrations of the above compounds were added to different wells (the concentrations of the compounds were: 50 μmol/L, 25 μmol/L, 12.5 μmol/L, 6.25 μmol/L, 3.125 μmol/L) as the experimental group, the control group was added with 0.1% DMSO, and the blank group was not added with any compound or solvent. After treating the cells of the experimental group, the control group and the blank group for 24 hours, the CCK-8 solution was added to each well and further cultured at 37°C for 2 hours. The absorbance at 450nm was measured using SPARK 10M (multi-function microplate reader), and the test results are shown in Figure 4. As can be seen from Figure 4, the absorbance at 450nm _between the drug group with different concentrations ( _{3.125-50 μmol} / _{L )} of _{C29H22ClN3O5S2} and the group _{without drug addition is very small, indicating that the compound C29H22ClN3O5S2 in the present} invention has no obvious toxicity to cells when the concentration is 3.125-50 μmol/L.

Subsequently, the antiviral activity of the above compounds was detected at the cellular level by mixing a fixed amount of virus with an equal amount of serially diluted compounds. BHK21 cells were inoculated into 96-well plates and cultured to a monolayer cell density of 80%. Getah virus was inoculated into each well of cells at 100TCID ₅₀ , and after incubation for 0.5h, the virus solution was discarded and 100μL of the above compounds with concentrations of 50μM, 25μM, 12.5μM, 6.25μM, and 3.125μM (μM here refers to μmol/L) were quickly added to each well of cells. After incubation for 48h at 5% CO ₂ and 37°C, the Reed-Muench calculation formula was used (Reed-Muench calculation formula is: distance ratio = (percentage of lesion rate above 50% - 50%) / (percentage of lesion rate above 50% - percentage of lesion rate below 50%); logTCID ₅₀ = distance ratio × difference between the logarithms of dilution + logarithm of the dilution with a lesion rate higher than 50%, where the dilution refers to the compound concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, respectively) to measure the relative quantification of TCID ₅₀ of GETV after the above-mentioned compounds at different concentrations acted on. The test results are shown in FIG5 . As can be seen from FIG5 , the compound C ₂₉ H ₂₂ ClN ₃ O ₅ S ₂ in the present invention has an effect of inhibiting viral activity and meets the basic requirements for use as a drug. By calculating the 100 TCID ₅₀ value of GETV after the above-mentioned compounds acted on, it can be determined that the C ₂₉ H ₂₂ ClN ₃ O ₅ S ₂ molecule has obvious antiviral activity at a concentration of 12.5 μmol/L. In addition, one-way ANOVA in SPSS software was used to process and analyze the data. ***, P<0.01 represents an extremely significant difference. Specifically, compared with the 0.1% DMSO bar graph of the control group, the marked horizontal line and *** indicate a significant difference, that is, 50μM, 25μM and 12.5μM all have significant differences.

The binding forces of compounds 2 to 59 screened by the screening method of the present invention to the binding site on the Geta virus protein are comparable to those of compound 1 (C ₂₉ H ₂₂ ClN ₃ O ₅ S ₂ ). Therefore, the effects of compounds 2 to 59 in terms of cytotoxicity and anti-Geta virus activity are also at substantially the same level as those of compound 1.

The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims (6)

1. The application of a compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating Geta virus infection, characterized in that: the compound represented by Formula I is: . 2. The application according to claim 1, characterized in that the drug includes a therapeutically effective amount of the compound represented by Formula I or a pharmaceutically acceptable salt thereof. 3. The application according to claim 2, characterized in that: the medicine further includes pharmaceutically acceptable excipients. 4. The application according to claim 2, characterized in that: the dosage form of the drug is pills, tablets, granules, capsules, syrups or injections. 5. The application according to claim 1, characterized in that: the Geta virus originates from cattle, sheep, dogs, kangaroos, foxes, birds, rabbits, guinea pigs, rats or hamsters. 6. The application according to claim 1, characterized in that: the compound represented by formula I binds to the E2 structural protein in Geta virus.