CN115260183A - Application of small molecular compound in preparation of medicine for resisting Getta virus infection - Google Patents

Application of small molecular compound in preparation of medicine for resisting Getta virus infection Download PDF

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CN115260183A
CN115260183A CN202210809558.9A CN202210809558A CN115260183A CN 115260183 A CN115260183 A CN 115260183A CN 202210809558 A CN202210809558 A CN 202210809558A CN 115260183 A CN115260183 A CN 115260183A
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CN115260183B (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
高东生
王永生
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Southwest University of Science and Technology
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Abstract

The invention discloses an application of a small molecular compound in preparing a medicine for resisting a Getavirus infection, wherein the medicine comprises a therapeutically effective amount of a compound shown as a formula I or pharmaceutically acceptable salts, esters, isomers and solvates thereof. In addition, the compound of the formula I in the invention breaks the E1-E2 interaction of the Getavirus by combining with the E2 structural protein in the Getavirus, blocks the assembly of virus particles, further interferes with virus infection and replication, has obvious curative effect on preventing or treating the Getavirus infection and is not easy to generate drug resistance.

Description

Application of small molecular compound in preparation of medicine for resisting Getta virus infection
Technical Field
The invention relates to the field of medicines, in particular to application of a small molecular compound in preparing a medicine for resisting a Getavirus infection.
Background
Getah virus (GETV) belongs to an insect-borne virus in the Semliki Forest virus group (SFV) of the genus alphavirus of the family togaviridae, and the SFV group further comprises Chikungunya virus (CHIKV), semliki Forest Virus (SFV), malay virus (Mayaro virus, MAYV), una virus (UNAV), babaru virus (Bebaru virus, BEBV) and onien virus (O' nyong-nyong virus, ONNV), wherein CHIKV, ONNV and MAYV have a very high incidence and mortality in susceptible people.
GETV was originally derived from mosquitoes, and was limited to horse and pig cases in 40 years of 1960-2000, but only 20 years of this century, and has been expanded to many animals such as cattle, sheep, dogs, kangaroos, foxes, wild birds, and rabbits, guinea pigs, rats, hamsters, and the frequency of clinical cases is also increasing. Even more alarming is the detection of anti-GETV antibodies in febrile patients and even in healthy people. Although no report about human infection onset exists so far, a plurality of serum epidemiological surveys at home and abroad show that a GETV antibody exists in a human body, and the virus is predicted to infect the human. Because various animals are infected and attacked and mosquitoes are generally toxic, the livestock such as pigs and the like in vast rural areas of China are in close contact with people, GETV is likely to be popular among people, and a great potential risk is likely to exist in future public health events.
Scientists have used cryoelectron microscopy to investigate the steps and resolution of viral structures of various classes in the alphavirus genus never stopped
Figure BDA0003740090460000011
To
Figure BDA0003740090460000012
The viruses studied include Barmah Forest Virus (BFV), EEEV, WEEV, VEEV, CHIKV, SINV, MAYV, and the like. Cryo-electron microscopy density charts of the currently resolved alphaviruses show that the alphaviruses have the same structural composition, and that the alphavirus RNA is hidden in a disordered state in an icosahedral core consisting of 240 copies of capsid (capsid). The exogenously protruding E1 and E2 structural proteins form heterodimers (80 copies of which form an icosahedral viral coat) that are attached to the capsid across the phospholipid membrane. And GETV is a single-stranded positive-strand RNA virus with an envelope. Mature Galtavirus is a spherical particle of about 70nm, and the genome of about 11kb is composed of 2 Open Reading Frames (ORFs), and contains a code and a polyprotein, wherein the polyprotein at the N-terminal comprises 4 non-structural proteins (nsP 1, nsP2, nsP3 and nsP 4), and the polyprotein at the C-terminal consists of 5 structural proteins (capsid-E3-E2-6K-E1). The research on the Galta virus in the prior art mainly focuses on enhancing the transmission vector of GETV, the detection and monitoring of animal infection of pigs, horses, cattle and the like, the quarantine of imported livestock and finished products thereof and the like, and the currently reported literature also focuses on the detection of the Galta virus, the vaccine prevention and the like, and the research on the medicine for treating the Galta virus infection is almost not available, so that the medicine capable of effectively treating the Galta virus infection is still lacked. In addition, methods for screening drugs by using molecular docking software and scoring software exist in the prior art, but the screening results of the existing screening methods are inaccurate, the screening cannot be accurately performed, and the existing screening methods lack verification steps.
Disclosure of Invention
In order to overcome the problems of the prior art, the invention provides the application of the compound of formula I or pharmaceutically acceptable salts, esters, isomers and solvates thereof in preparing a medicament for preventing and/or treating the Getavirus infection;
Figure BDA0003740090460000021
the invention also aims to provide a pharmaceutical composition for preventing and/or treating the Getavirus infection.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the present invention is the use of a compound of formula I or a pharmaceutically acceptable salt, ester, isomer, solvate thereof, for the manufacture of a medicament for the prevention and/or treatment of a Getavirus infection,
Figure BDA0003740090460000022
wherein R is1Selected from H, C1～10Alkyl radical, C1～10An alkoxy group;
R2selected from H, cl, F, br, C1～5An alkyl group;
n is 1, 2,3, 4, 5, 6, 7, 8, 9 or 10.
Preferably, said C1～5Alkyl is selected from-CH3、-CH2CH3、-CH2CH2CH3、-CH2CH2CH2CH3、-CH2CH2CH2CH2CH3、-CH(CH3)2、-C(CH3)3、-CH2CH(CH3)2、-CH(CH3)CH2CH3、-CH(CH3)CH2CH2CH3、-CH2CH(CH3)CH2CH3、-CH2CH2CH(CH3)2、-CH(CH2CH3)2、-CH2C(CH3)3、-C(CH3)2CH2CH3or-CH (CH)3)CH(CH3)2。
Preferably, it isSaid C is1～10The alkyl group being selected from C1～10Straight chain alkyl or C3～10A branched alkyl group.
Preferably, C1～10The straight chain alkyl is selected from: -CH3、-CH2CH3、-CH2CH2CH3、-CH2CH2CH2CH3、-CH2CH2CH2CH2CH3、-CH2CH2CH2CH2CH2CH3、-CH2CH2CH2CH2CH2CH2CH3、-CH2CH2CH2CH2CH2CH2CH2CH3、-CH2CH2CH2CH2CH2CH2CH2CH2CH3or-CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3。
Preferably, C3～10The branched alkyl group is selected from: c3Branched alkyl radical, C4Branched alkyl radical, C5Branched alkyl radical, C6Branched alkyl, C7Branched alkyl radical, C8Branched alkyl, C9Branched alkyl, C10A branched alkyl group.
Preferably, said C1～10Alkoxy is selected from C1～10Straight-chain alkoxy or C3～10A branched alkoxy group.
Preferably, said C1～10The linear alkoxy group is selected from the group consisting of-OCH3、-OCH2CH3、-OCH2CH2CH3、-OCH2CH2CH2CH3、-OCH2CH2CH2CH2CH3、-OCH2CH2CH2CH2CH2CH3、-OCH2CH2CH2CH2CH2CH2CH3、-OCH2CH2CH2CH2CH2CH2CH2CH3、-OCH2CH2CH2CH2CH2CH2CH2CH2CH3or-OCH2CH2CH2CH2CH2CH2CH2CH2CH2CH3。
Preferably, said C3～10The branched alkoxy is selected from C3Branched alkoxy, C4Branched alkoxy radical, C5Branched alkoxy, C6Branched alkoxy, C7Branched alkoxy, C8Branched alkoxy, C9Branched alkoxy or C10A branched alkoxy group.
Preferably, in formula I, R1Selected from H, C1～8Alkyl radical, C1～8An alkoxy group; further preferably, R1Selected from H, C1～5Alkyl radical, C1～5An alkoxy group.
Preferably, in formula I, R2Selected from H, cl, F, br, C1～5An alkyl group; further preferably, R2Selected from H, cl, F, br, C1～3An alkyl group.
Preferably, in formula I, n is 2,3, 4, 5, 6, 7 or 8; further preferably, n is 3, 4, 5 or 6; still further preferably, in formula I, n is 3, 4 or 5.
Preferably, the compound of formula I is selected from:
Figure BDA0003740090460000031
Figure BDA0003740090460000032
Figure BDA0003740090460000041
preferably, the compound of formula I is
Figure BDA0003740090460000042
Preferably, the reaction scheme for the preparation of the compounds of formula I above is:
Figure BDA0003740090460000051
preferably, the compound of formula I binds to the E2 structural protein in the gabotavirus.
Preferably, the medicament comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, ester, isomer, solvate thereof.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the medicament is in the form of pills, tablets, granules, capsules, syrups and injections.
The ester of the invention is a compound of formula I with C1～10The alkyl alcohol is prepared by esterification.
Preferably, said C1～10The alkyl alcohol comprises C1～10Straight chain alkyl alcohol or C3～10A branched alkyl alcohol.
Preferably, said C1～10The linear alkyl alcohol is selected from: CH (CH)3OH、CH3CH2OH、CH2CH3CH2OH、CH3CH2CH2CH2OH、CH3CH2CH2CH2CH2OH、CH3CH2CH2CH2CH2CH2OH、CH3CH2CH2CH2CH2CH2CH2OH、CH3CH2CH2CH2CH2CH2CH2CH2OH、CH3CH2CH2CH2CH2CH2CH2CH2CH2OH or CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2OH。
Preferably, the first and second electrodes are formed of a metal,said C is3～10The branched alkyl alcohol is selected from C3Branched alkyl alcohol, C4Branched alkyl alcohol, C5Branched alkyl alcohol, C6Branched alkyl alcohol, C7Branched alkyl alcohol, C8Branched alkyl alcohol, C9Branched alkyl alcohol or C10A branched alkyl alcohol.
Solvate in the present invention means that the drug crystal contains solvent molecules.
Preferably, the gata virus is of bovine, ovine, canine, kangaroo, fox, avian, rabbit, guinea pig, rat or hamster origin.
The second aspect of the present invention provides a pharmaceutical composition for preventing and/or treating a Getavirus infection, comprising a therapeutically effective amount of a compound represented by formula I or a pharmaceutically acceptable salt, ester, isomer, solvate thereof,
Figure BDA0003740090460000061
wherein R is1、R2N is as defined above.
Preferably, the compound of formula I is
Figure BDA0003740090460000062
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
Preferably, the pharmaceutical composition is in the form of pills, tablets, granules, capsules, syrups or injections.
The term "therapeutically effective amount" means an amount of the agent of the invention sufficient to produce a beneficial or desired effect when administered to a human infected with a Galtavirus; the effect may be prevention of infection by the gacovirus and/or treatment of clinical symptoms or indications associated with infection by the gacovirus. It will be appreciated, however, that the total daily amount of the medicament of the invention must be determined within the scope of sound medical judgment. For any particular infected subject, the particular therapeutically effective dose level will depend upon a variety of factors including the severity of the infection in the subject being treated; the activity of the particular drug employed; the specific drug or dosage form employed; body weight, general health, diet of the infected; the time of administration, route of administration and rate of excretion of the drug employed; the duration of treatment; drugs used in combination or concomitantly with the specific drug employed; and similar factors well known in the medical arts. For example, it is common in the art to start doses of drug at levels below those required to achieve the desired therapeutic effect and to 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 applicable to 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 the pharmaceutically acceptable adjuvants include, but are not limited to, solvents, diluents, dispersing agents, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, antioxidants, and the like, which are conventional in the pharmaceutical field. If necessary, a flavor, a preservative, a sweetener and the like may be further added to the pharmaceutical composition.
The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present 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 a base in neat 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 compounds of the present 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 neat 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 salts of organic acids including such acids as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, 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 (1977). Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.
Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms by certain physical properties, such as solubility in polar solvents.
As used herein, "pharmaceutically acceptable salts" belong to derivatives of the compounds of the present invention, wherein the parent compound is modified by forming a salt with an acid or a salt 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 the conventional non-toxic salts or the quaternary ammonium salts of the parent compound, for example, 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 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, hydroxyl, 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, glycolic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannin, tartaric acid, or p-toluenesulfonic acid.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting 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 the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
The "isomers" of the present invention 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 one or more double bond shifts, e.g., a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to a stereoisomer in which the molecules have two or more chiral centers and a non-mirror image relationship between the molecules. The term "cis-trans isomers" refers to different spatial configurations in which a double bond or a single bond of a ring-forming carbon atom in a molecule does not rotate freely.
The invention has the beneficial effects that: the compound of the formula I has excellent effect of preventing or treating the Getaavirus infection, has no obvious toxic or side effect on normal cells, and can achieve the effect of inhibiting the Getaavirus at lower concentration. In addition, the compound of the formula I in the invention breaks the E1-E2 interaction of the Getavirus by combining with the E2 structural protein in the Getavirus, blocks the assembly of virus particles, further interferes with the virus infection and replication, has obvious curative effect on preventing or treating the Getavirus infection and is difficult to generate drug resistance. Specifically, the compound of formula I in the invention has obvious antiviral activity at a concentration of 25 mu mol/L, and has no cytotoxicity in a concentration range of 3.125-50 mu mol/L.
Drawings
FIG. 1 shows a structural diagram of Cryo-EM of Getavirus of the present invention.
FIG. 2 is a graph showing the prediction of binding of Compound 1 of the present invention to a Galavirus protein.
FIG. 3 is a partially enlarged view of the binding site of Compound 1 to the Galavirus protein in FIG. 2.
FIG. 4 is a graph 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 are described in further detail below with reference to the figures and examples, but the practice and protection of the present invention is not limited thereto. It is noted that the following processes, if not described in particular detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
The invention provides a method for screening a compound for preventing and/or treating a Getaavirus infection, which comprises the following steps:
s1: acquiring a mapping relation between antiviral molecules and antiviral characteristics, and constructing a model;
s2: screening a compound conforming to the model from the database to obtain a primary screened compound;
s3: taking the protein of the Getavirus as a substrate, taking the antiviral drug of the Reed-Sivir as a molecular probe, evaluating by using scoring software, and screening out a binding site from the protein of the Getavirus;
s4: and carrying out molecular docking on the primary screening compound and a binding site in the protein of the Getavirus, and screening based on the binding parameters to obtain the target compound.
Preferably, the binding parameters include binding energy, conformation.
The conformation in the present invention refers to a spatial arrangement resulting from the placement of atoms around a single bond in one molecule without changing the covalent bond structure. No cleavage and reformation of covalent bonds is required to change from one conformation to another. The conformational change does not alter the optical activity of the molecule.
The calculation formula of the binding energy is:
Figure BDA0003740090460000091
where the sum is for all pairs of atoms that can move relative to each other, it is generally excluded that the atoms are 1-4 interactions, i.e. separated by 3 consecutive covalent bonds. Here, each atom i is assigned a type tiAnd is and
Figure BDA0003740090460000093
is to define the distance (r) between atomsij) A set of symmetric interaction functions.
Firstly, the invention carries out structural analysis on a GETV-V1 strain which can cause the pregnant mother mouse to generate breeding obstacle by utilizing Cryo-EM (frozen electron microscope), and concretely refers to figure 1, wherein figure 1 (A) is an external surface diagram of the Getavirus, and as can be seen from figure 1 (A), the external surface of the Getavirus has specified symmetry axes of 5 times, 3 times and 2 times; FIG. 1 (B) is a sectional view of a Getavirus; FIG. 1 (C) is an electron density diagram of Getavirus; FIG. 1 (D) is an atomic model diagram of a Getavirus; FIG. 1 (E) is an atomic model of E1-E2-capsid heterotrimer. From
Figure BDA0003740090460000092
In the atomic resolution map, 19 newly identified interaction forces (hydrogen bonds or salt bridges) play an important role in maintaining the overall structure stability of the virus, 8 glycosylation sites which are found and built up on the surface of particles and participate in receptor antibody recognition and virus immune escape, 5S-acylation sites (S-acylation sites) which are built up on an E1/E2 near an inner phospholipid membrane of an atomic model contribute to virus assembly and transmembrane region stabilization, and 1 Dioleoylphosphatidylcholine (DOPC) and 3 cholesterol which are identified in and near a hydrophobic pocket formed by a TM helix (transmembrane helical region) of E1, a TM helix (transmembrane helical region) of E2 and a domain D (D region) of E2 have great value in maintaining the structure stability of E1/E2, particularly the pocket has great valueDOPC and cholesterol.
GETV virus consists of Capsid, 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 (freezing electron microscope) to carry out structural analysis on GETV-V1 strains, thereby defining the structure of the Getavirus.
The invention is described in further detail below with reference to specific examples:
the invention provides a method for screening a compound for preventing and/or treating a Getaavirus infection, which comprises the following specific steps:
a method based on Geometric Deep Learning (geological Deep Learning) (developed by Suzhou cloud medicine science and technology Co., ltd.) is adopted, antiviral molecules which meet commercial availability and chemical diversity are selected, a mapping relation between the existing antiviral molecules and antiviral properties is obtained, a screening model is constructed based on the mapping relation between the existing antiviral molecules and the antiviral properties, the model is a Graph-based Deep Learning network (Graph-based Deep Learning), and then the screening model is used for predicting the antiviral properties of the compounds in a large-scale molecular library so as to discover potential seedling-end compounds. And finally, performing molecular sequencing according to the predicted scores, selecting and screening the seedling-end compounds according to the predicted physicochemical properties, and finally performing molecular docking cross validation on the screened compounds.
The method for molecular docking cross-validation of the primarily screened compounds specifically comprises the following steps:
firstly, dividing three-dimensional space grids of proteins (sub 1, sub2, sub3, monomers and tetramers) in the Getavirus, wherein the sub1, sub2 and sub3 are three sub domain names of E2 structural proteins in the Getavirus proteins, the monomers are E1-E2-capsids, and the tetramers are assembled by four monomers, and the intervals are 20 angstroms. Then, by taking the grids as the center, taking 15 angstroms as the radius, selecting an antiviral drug namely the Rudeciclovir as a small molecular probe, and scoring by using docking software, thereby screening a series of binding sites. For each grid point, the score of the best binding posture among them was chosen as the assessment of the binding pocket for that site. And then, the binding sites are examined one by one, so that the final binding site is selected, and the binding site is a substrate binding groove and can be used for next large-scale small molecule library screening.
Then, a life chemistry and LC antiviral small molecule library containing 10157 small molecules is screened. For more than 700 ten thousand molecules of a more comprehensive small molecule library ZINC, 100 ten thousand molecules are randomly selected for molecular docking, 50 molecules with the highest score are screened out from an LC antiviral small molecule library and a ZINC library in total, similarity inspection is carried out on the remaining 600 molecules in the ZINC library, and in the inspection, the molecule with any similarity of 50 molecules larger than 0.6 can be selected for docking with a binding site in a protein of a togavirus. In these molecular docking, in order to increase computational efficiency, for each small molecule, 5 conformation images were examined at the time of docking, scored using scoring software, and screened for binding energy and binding conformation, thereby screening compound 1 for preventing and/or treating a togavirus infection in the present invention.
Compound 1 of the formula
Figure BDA0003740090460000111
The chemical name is: 2,6-bis ({ 5- [ (3Z) -1-methyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfonyl sulfoxide-1,3-thiazolidin-3-yl }) hexanoic acid of the formula: c30H24N4O6S4The CAS number of the compound is: 306322-08-7.
Further optimizing the molecular structure of the compound 1 to obtain compounds 2 to 31, wherein the molecular formulas of the compounds 2 to 31 are as follows:
Figure BDA0003740090460000112
Figure BDA0003740090460000121
Figure BDA0003740090460000131
then, the binding energies of the compounds 1 to 31 were calculated while predicting the binding of the above-mentioned compounds 1 to 31 to the proteins of the Getavirus, respectively, and the calculated binding energies of the compounds 1 to 31 are shown in Table 1 below:
TABLE 1 binding energy of Compounds 1 to 31
Figure BDA0003740090460000132
As is clear from Table 1, the binding energy of the compounds 1 to 31 was in the range of-7.4 to-9.3 kcal/mol, and it was found that the compounds 1 to 31 all had a good binding force to the proteins of the Getavirus.
Compounds 1 to 31 are binding pockets to the D domain (subdomain D) of the E2 protein of the Galtavirus, and are close to the hydrophobic pockets formed by the TM helix (transmembrane helix) and D domains of the E2 protein, and the TM helix of the E1 protein. Studies indicate that the D region 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 presumed that the compounds 1 to 31 interfere with virus infection and replication by disrupting the E1-E2 interaction, inhibiting the assembly of virus particles.
According to the invention, the compounds 1-31 are combined with the proteins of the Galavirus, the combination position is predicted, and the analysis of the interaction of the predicted optimal combination conformation defines the action site on the protein, namely the specific position of the combination pocket. Taking compound 1 as an example, the predicted optimal binding conformation of protein binding of compound 1 to the gata virus is shown in fig. 2 and 3, and specific different types of interactions are shown in the following tables 2 to 5:
TABLE 2 sites of hydrophobic interaction of proteins with compounds
Serial number Protein residues AA (amino acid abbreviation)
1 277 TYR
2 320 VAL
TABLE 3 sites of hydrogen bonding interaction of proteins with compounds
Serial number Protein residues AA
1 277 TYR
TABLE 4 sites of pi-pi stacking interaction of proteins with compounds
Serial number Protein residues AA
1 338 TRP
TABLE 5 sites of pi-Cation interaction of proteins with Compounds
Serial number Protein residues AA
1 338 TRP
As can be seen from FIGS. 2 to 3 and tables 2 to 5, compound 1 and 277 TYR,320 VAL have hydrophobic interactions; compound 1 and 277 TYR have hydrogen bond interactions; compound 1 and 338 TRP formed pi-pi stacking and pi-cation interactions; therefore, the compound 1 of the present invention has a good binding property to the binding site in the proteins of the Getavirus.
Compound 1 was then tested for cytotoxicity and anti-togavirus activity as follows:
the CCK-8 kit was first used to evaluate the toxicity of the above compounds on cells cultured in vitro. The CCK-8 kit was purchased from Donjindo, and the cell viability of BHK21 cells treated with the above-described compound was measured according to the protocol provided. BHK21 cells were seeded in a 96-well plate and cultured to a monolayer of cells at 80% density, then the above-mentioned compounds (the concentrations of the compounds were 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M, respectively, where. Mu.M means. Mu. Mol/L) were added to different wells as an experimental group, 0.1. Mu.L of DMSO was added to a control group, no compound or solvent was added to a blank group, and the experimental group, the control group, and the blank group were treated separatelyAfter 24h for cells from both the group and blank, CCK-8 solution was added to each well and further incubated at 37 ℃ for 2h. The absorbance at 450nm was measured using SPARK 10M (multifunctional microplate detector) and the results are shown in FIG. 4. As can be seen from FIG. 4, C was added at various concentrations (3.125. Mu. Mol/L to 50. Mu. Mol/L)30H24N4O6S4The difference between the absorbance at 450nm of the drug group and the absorbance at 450nm of the drug-free group is very small, which indicates that the compound C in the invention30H24N4O6S4The concentration of the compound is 3.125-50 mu mol/L, and the compound has no obvious toxicity to cells.
Subsequently, the antiviral activity of the above compounds was examined at the cellular level by mixing a fixed viral load with an equivalent series of dilutions of the compounds. BHK21 cells were seeded into 96-well plates and cultured to 80% density monolayers. According to 100TCID50Getah virus inoculated into each well cell, incubated for 0.5h, then virus solution was discarded, and then 100. Mu.L of the above-mentioned compound was rapidly added to each well cell at concentrations of 50. Mu. Mol/L, 25. Mu. Mol/L, 12.5. Mu. Mol/L, 6.25. Mu. Mol/L, and 3.125. Mu. Mol/L, at 5% CO2After incubation at 37 ℃ for 48h, the equation for Reed-Muench calculation (Reed-Muench calculation: distance ratio = (percentage higher than 50% disease rate-50%)/(percentage higher than 50% disease rate-percentage lower than 50%); "logTCID50= distance ratio x difference between the logarithm of dilutions + the logarithm of dilutions above 50% disease rate, dilution means concentration of compound 50 μ M,25 μ M,12.5 μ M,6.25 μ M,3.125 μ M) respectively) to measure TCID of GETV after effect of different concentrations of the above-mentioned compound50The relative quantification was determined, and the results are shown in FIG. 5, and it is clear from FIG. 5 that Compound C of the present invention30H24N4O6S4Has effect in inhibiting viral activity, meets basic application requirement as medicine, and is calculated by calculating the 100TCID of GETV50Value of can determine C30H24N4O6S4The molecule has obvious antiviral activity at the concentration of 25 mu mol/L. In addition, data was entered using one-way ANOVA in SPSS softwareLine process analysis, P<0.01 represents a very significant difference, specifically: the marked horizontal and horizontal lines are significantly different, i.e. both 50 μ M and 25 μ M are significantly different, compared to the control group of 0.1% dmso histograms.
The binding ability of compounds 2 to 31 to the binding site on the protein of the gavirus screened by the screening method of the present invention is comparable to that of compound 1, and therefore, the effects of compounds 2 to 31 in cytotoxicity and anti-gavirus activity are also at substantially the same level as that of compound 1.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.

Claims (15)

1. The application of the compound of the formula I or the pharmaceutically acceptable salt, ester, isomer and solvate thereof in preparing the medicine for preventing and/or treating the Getavirus infection,
Figure FDA0003740090450000011
wherein R is1Selected from H, C1～10Alkyl radical, C1～10An alkoxy group;
R2selected from H, cl, F, br, C1～5An alkyl group;
n is an integer of 1 to 10.
2. Use according to claim 1, characterized in that:
in the formula I, R1Selected from H, C1～8Alkyl radical, C1～8An alkoxy group; preferably, R1Selected from H, C1～5Alkyl radical, C1～5An alkoxy group.
3. Use according to claim 1, characterized in that:
in the formula I, R2Selected from H, cl, F, br, C1～5An alkyl group; preferably, R2Selected from H, cl, F, br, C1～3An alkyl group.
4. Use according to claim 1, characterized in that: in the formula I, n is an integer of 2-8; preferably, n is an integer of 3 to 6.
5. Use according to claim 1, characterized in that: the compound of formula I is selected from:
Figure FDA0003740090450000012
Figure FDA0003740090450000013
Figure FDA0003740090450000021
6. use according to claim 5, characterized in that: the compounds of the formula I are
Figure FDA0003740090450000022
7. Use according to any one of claims 1 to 6, characterized in that: the compounds of formula I bind to E2 structural proteins in the togavirus.
8. Use according to any one of claims 1 to 6, characterized in that: the medicament comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, ester, isomer, solvate thereof.
9. Use according to claim 8, characterized in that: the medicine also comprises pharmaceutically acceptable auxiliary materials.
10. Use according to any one of claims 1 to 6, characterized in that: the ester is a compound of formula I with C1～10The alkyl alcohol is prepared by esterification.
11. Use according to any one of claims 1 to 6, characterized in that: the dosage form of the medicine is pills, tablets, granules, capsules, syrup or injection.
12. Use according to claim 1, characterized in that: the Galtavirus is derived from cattle, sheep, dog, kangaroo, fox, bird, rabbit, guinea pig, rat, or hamster.
13. A pharmaceutical composition for the prevention and/or treatment of a togavirus infection, characterized in that: comprises a therapeutically effective amount of a compound shown as a formula I or pharmaceutically acceptable salts, esters, isomers and solvates thereof,
Figure FDA0003740090450000031
wherein R is1、R2N is as defined in any one of claims 1 to 4.
14. The pharmaceutical composition for the prevention and/or treatment of a Getavirus infection according to claim 13, wherein: the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.
15. The pharmaceutical composition for the prevention and/or treatment of a Getaavirus infection according to claim 13, characterized in that: the dosage form of the pharmaceutical composition is pills, tablets, granules, capsules, syrups or injections.
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