Application of ABHD12 inhibitor in inhibiting and killing viruses

Abstract

The invention belongs to the technical field of medicines, and discloses application of an ABHD12 inhibitor in inhibiting and killing viruses. The inventor finds that ABHD12 is a potential broad-spectrum antiviral target through experiments, an inhibitor (such as DO264 or a derivative thereof) of ABHD12 can effectively inhibit proliferation of viruses (such as Zika virus, mumps virus, coronavirus and the like), and the ABHD12 has very good application prospects and research values in the field of antiviral medicines.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of ABHD12 inhibitors (such as DO264 and its derivatives) in inhibiting and killing viruses. BACKGROUND

[0002] For a long time, human beings have been in an unremitting struggle with viruses, and a large number of scientists and pharmaceutical companies have been committed to the research and development of antiviral vaccines and drugs. Although vaccines are the most effective means for human beings to resist viruses, such as hepatitis B vaccine for newborns, polio and smallpox virus vaccine, etc., which have greatly killed viruses in the cradle. However, some viruses, such as HIV, NV, ZIKV, etc., are difficult to overcome by vaccines due to high variability, multiple types and potential ADE effects of viruses and other factors. Therefore, antiviral drugs are still an important means to resist viral infection. Among them, the broad-spectrum antiviral drugs have more broad-spectrum applicability to multiple viruses or different types of the same virus, which provides important support for preventing and controlling the large-scale outbreak of emerging viruses. According to the different target points, the antiviral drugs are divided into two categories of directly targeting viral proteins and targeting host proteins. Compared with targeting viral proteins, host proteins are usually highly conserved for most strains, and thus have more broad-spectrum antiviral potential and higher drug resistance barrier advantages.

[0003] Lysophosphatidylserine lipase (ABHD12) is widely distributed in the brain, mainly located on the endoplasmic reticulum membrane, and mediates the hydrolysis of lyso-PS, ox-lyso PS and 2-AG. Lyso PS is a class of signal lipids that regulate immune and neural processes, and plays a crucial role in the central nervous system. There is no report on the research of ABHD12 in antiviral aspect. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the present application provides an application of ABHD12 inhibitor in inhibiting and killing viruses.

[0005] In the first aspect of the present application, an application of ABHD12 inhibitor in preparing a product for inhibiting and / or killing viruses is provided.

[0006] Specifically, the ABHD12 inhibitor can be an antibody or an antigen-binding fragment thereof, an interfering RNA or a small molecule compound.

[0007] Specifically, the antigen-binding fragment can be selected from the group consisting of: Fab, Fab', F(ab)2, Fv, dsFv, scFv, Fd and Fd' fragments, and the like.

[0008] Specifically, the interfering RNA can be selected from the group consisting of: siRNA, dsRNA, shRNA, aiRNA, miRNA, and combinations thereof.

[0009] In some embodiments of the present application, the ABHD12 inhibitor is an siRNA; in some embodiments of the present application, the siRNA comprises or consists of a nucleotide sequence selected from the group consisting of: GCCACCCUAUCAUUCUGUATT, GGAAUCUCCAUUCACUAAUTT, GGUUCUUCCUUGAUCCUAUTT, AGGUGACACGGUCAUCGAATT.

[0010] In some embodiments of the present application, the ABHD12 inhibitor is a small molecule compound, for example, the ABHD12 inhibitors described in Chinese patent applications CN112105355A, CN113939290A; Ogasawara D, Ichu TA, Jing H, et al. Discovery and Optimization of Selective and in Vivo Active Inhibitors of the Lysophosphatidylserine Lipase α / β-Hydrolase Domain-Containing 12 (ABHD12). J Med Chem. 2019 Feb 14; 62(3): 1643-1656.; Parkkari T, Haavikko R, Laitinen T, et al. Discovery of triterpenoids as reversible inhibitors of α / β-hydrolase domain containing 12 (ABHD12). PLoS One. 2014 May 30; 9(5): e98286.

[0011] In some embodiments of the present application, the small molecule compound has the following structure:

[0012]

[0013] wherein,

[0014] X is S or O;

[0015] Y is N or CH;

[0016] R 0 Selected from: H, C 1-6 alkyl;

[0017] L is selected from:

[0018] R 1 is one or more independent substituents on the ring, each of which is independently selected from: H, halogen, -CN, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, -C(O)OR 2 、-C(O)R 2 、-OR 3 , phenyl optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy;

[0019] Each R 2 Independently selected from C 1-6 alkyl;

[0020] Each R 3 Independently selected from: H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl, -C 1-6 Alkyl-C 3-6 Cycloalkyl, 4-10 membered heterocycloalkyl, phenyl, -C 1-6 Alkyl-phenyl, where C 3-6 Cycloalkyl, -C 1-6 Alkyl-C 3-6 Cycloalkyl, 4-10 membered heterocycloalkyl, phenyl, -C 1-6 Alkyl-phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -C(O)OR 4 ;

[0021] Each R 4 Independently selected from C 1-6 alkyl;

[0022] n is 0, 1, 2, or 3;

[0023] or a pharmaceutically acceptable salt, stereoisomer, prodrug, solvate thereof.

[0024] In particular, n is 1, 2 or 3, in particular 1 or 2.

[0025] In some preferred embodiments of the application, L is The small molecule compound has the following structure:

[0026]

[0027] In some preferred embodiments of the application, X is S.

[0028] In some preferred embodiments of the application, Y is N.

[0029] More particularly, the small molecule compound has the following structure:

[0030]

[0031] In particular, R 0 is selected from the group consisting of H, C 1-3 alkyl, such as methyl, ethyl, n-propyl, i-propyl.

[0032] In particular, each R 2 is independently selected from the group consisting of C 1-3 alkyl, such as methyl, ethyl, n-propyl, i-propyl.

[0033] In particular, each R 4 is independently selected from the group consisting of C 1-3 alkyl, such as methyl, ethyl, n-propyl, i-propyl.

[0034] In particular, each R 3 is independently selected from the group consisting of C 1-6 alkyl, C 1-6 haloalkyl, phenyl, -C 1-6 alkyl-C 3-6 cycloalkyl, 4-7 membered heterocycloalkyl, -C 1-6 alkyl-phenyl, wherein the phenyl and -C 1-6 alkyl-phenyl is optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, C 1-6 alkyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, -C(O)OR 4 ; in particular, each R 3 is independently selected from the group consisting of C 1-6 haloalkyl, phenyl, -C 1-6alkyl-phenyl, wherein the phenyl group is optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, C 1-6 alkyl-phenyl is optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, C 1-6 haloalkyl, C 1-6 haloalkoxy.

[0035] In some embodiments of the application, each R 3 is independently selected from the group consisting of:

[0036]

[0037] In some embodiments of the application, each R 1 is independently selected from the group consisting of: H, halogen, -CN, C 1-6 alkyl, C 2-6 alkynyl, C 1-6 haloalkyl, -C(O)R 2 , -OR 3 , phenyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy; more specifically, each R 1 is independently selected from the group consisting of: H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -OR 3 , phenyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy.

[0038] In some embodiments of the application, each R 1 is independently selected from the group consisting of: H, F, Cl, cyano, methyl, trifluoromethyl, trifluoromethoxy, -C(O)CH3,

[0039]

[0040] In some embodiments of the application, the small molecule compound has the following structure:

[0041]

[0042] wherein,

[0043] R 1 , R 3 each independently has the above definition.

[0044] In particular, in formula IV, R1 selected from: H, halogen, -CN, C 1-6 alkyl, C 2-6 alkynyl, C 1-6 haloalkyl, -C(O)R 2 , phenyl optionally substituted with 1 or 2 substituents independently selected from: halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy; more specifically, each R 1 is independently selected from: H, halogen, -CN, C 1-6 alkyl, C 1-6 haloalkyl, phenyl optionally substituted with 1 or 2 substituents independently selected from: halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy; more specifically, R 1 is selected from: H, halogen, -CN, C 1-3 alkyl, C 1-3 haloalkyl.

[0045] In some embodiments of the application, the ABHD12 inhibitor is DO264, which has the following structure:

[0046]

[0047] In some embodiments of the application, the ABHD12 inhibitor is a derivative of DO264, which has the following structure:

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] In other embodiments of the application, the ABHD12 inhibitor has the following structure:

[0054]

[0055] Specifically, the above-mentioned ABHD12 inhibitor compound can be prepared, for example, by the method described in Chinese Patent Application CN112105355A, CN113939290A, Discovery and Optimization of Selective and in Vivo Active Inhibitors of the Lysophosphatidylserine Lipase α / β-Hydrolase Domain-Containing 12 (ABHD12), and its ABHD12 inhibitory activity has been verified in the aforementioned documents.

[0056] Specifically, the virus can be Adenoviridae, Herpesviridae (such as EBV), Papovaviridae, Picornaviridae, Poxviridae (such as smallpox virus), Hepadnaviridae (such as hepatitis B virus), Coronaviridae (such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.), Bornaviridae, Filoviridae (such as Ebola virus, Marburg virus), Orthomyxoviridae (such as influenza virus), Paramyxoviridae (such as human parainfluenza virus type 1 (HPV), HPV type 2, HPV type 3, HPV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.), Retroviridae (such as HIV), Reoviridae, Rhabdoviridae (such as rabies virus), Flaviviridae (such as dengue virus, Zika virus, hepatitis B virus, Kyasanur virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.), etc.

[0057] In some embodiments of the present application, the virus is a flavivirus, such as Zika virus, dengue virus, hepatitis B virus, Kyasanur virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.

[0058] In other embodiments of the present application, the virus is an orthomyxovirus, such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.).

[0059] In other embodiments of the present application, the virus is a paramyxovirus, such as human parainfluenza virus type 1 (HPV), HPV type 2, HPV type 3, HPV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.

[0060] In some embodiments of the application, the virus is a herpesvirus, such as herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), human herpesvirus 8 (HHV-8), or a combination thereof.

[0061] In particular, the product can be used for diagnostic or therapeutic purposes, but also for non-diagnostic or non-therapeutic purposes.

[0062] In particular, the inhibition and / or killing of the above-mentioned viruses can be carried out in vivo, but also in vitro.

[0063] In some embodiments of the application, the product is a pharmaceutical composition.

[0064] In particular, in the pharmaceutical composition, the ABHD12 inhibitor can be used as the only active ingredient, but also in combination with one or more other active ingredients for the same indication or for a different indication, wherein the ABHD12 inhibitor and the other active ingredient(s) can be formulated for simultaneous, separate or sequential administration.

[0065] In some embodiments of the application, the pharmaceutical composition further comprises a second antiviral agent.

[0066] In particular, the second antiviral agent is selected from one or more of the following: remdesivir, idoxuridine, trifluridine, brivudine, vidarabine, entecavir, telbivudine, foscarnet, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, nevirapine, delavirdine, efavirenz, etravirine, rilpivirine, saquinavir, lopinavir, indinavir, nelfinavir, amprineravir, lopinavir-ritonavir, atazanavir, fosamprenavir, tipranavir, darunavir, telaprevir, boceprevir, simeprevir, asunaprevir, paritaprevir, glecaprevir, ritonavir, elbasvir, elvitegravir, dolutegravir, palivizumab, docosanol, enfuvirtide, maraviroc, zoster immune globulin, acyclovir, ganciclovir, famciclovir, valacyclovir, penciclovir, valganciclovir, cidofovir, tenofovir disoproxil fumarate, adefovir dipivoxil.

[0067] In some embodiments of the present application, the pharmaceutical composition further comprises one or more of an anti-inflammatory agent, a corticosteroid, an antihistamine, a bronchodilator, a short-acting beta agonist, a long-acting beta agonist, a short-acting muscarinic antagonist, a long-acting muscarinic antagonist, an immunosuppressant, an antibiotic, an antiviral agent, an antifungal agent, an anti-infective agent.

[0068] In particular, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0069] In particular, the pharmaceutically acceptable excipient can include one or more of a sweetening agent (in particular, sucrose, xylitol, fructooligosaccharide, acesulfame, stevia, aspartame, etc.), an aromatic agent (e.g., a flavoring agent, a flavoring essence, etc.), a gum base (in particular, sodium alginate, acacia gum, gelatin, methylcellulose, sodium carboxymethylcellulose, etc.), a clarifying agent (in particular, chitosan, gelatin, etc.), a preservative (in particular, benzoic acid and its salts, sorbic acid and its salts, the nipagin series, etc.), a disintegrant (in particular, low-substituted hydroxypropylcellulose, crospovidone, sodium starch glycolate, crosscarmellose sodium, starch, etc.), a binder (in particular, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone, copovidone, pregelatinized starch, etc.), a lubricant (in particular, stearic acid, magnesium stearate, sodium fumarate stearate, etc.), a wetting agent (in particular, polyoxyethylene sorbitan fatty acid ester, poloxamer, polyoxyethylene castor oil derivative, etc.), a suspending agent (in particular, hydroxypropylmethylcellulose, hydroxypropylcellulose, povidone, copovidone, sodium carboxymethylcellulose, methylcellulose, etc.), a stabilizer (in particular, citric acid, fumaric acid, succinic acid, etc.), a filler (in particular, starch, sucrose, lactose, microcrystalline cellulose, etc.), a binder (in particular, cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone, etc.), and the like.

[0070] In particular, the pharmaceutical composition can take any dosage form or administration form, particularly an oral dosage form, which can be selected by a person skilled in the art according to the circumstances, including, but not limited to, a tablet (including a sugar-coated tablet, a film-coated tablet, a sublingual tablet, an oral disintegrating tablet, an oral tablet, etc.), a pill, a powder, a granule, a capsule (including a soft capsule, a microcapsule), a lozenge, a syrup, a solution, an emulsion, a suspension, a controlled release preparation (e.g., an instant release preparation, a sustained release preparation, a sustained release microcapsule), an aerosol, a film (e.g., an oral disintegrating film, an oral mucosa-adhesive film), an injection (e.g., a subcutaneous injection, an intravenous injection, an intramuscular injection, an intraperitoneal injection), an intravenous drip, a transdermal absorption preparation, an ointment, a lotion, an adhesive preparation, a suppository (e.g., a rectal suppository, a vaginal suppository), a nasal preparation, a pulmonary preparation (an inhalant), an eye drop, and the like.

[0071] In other embodiments of the present application, the product is a functional food composition.

[0072] In particular, the ABHD12 inhibitor can be used as the sole active ingredient in the functional food composition, or in combination with one or more other active ingredients.

[0073] In particular, the functional food composition can further comprise food adjuvants.

[0074] In particular, the functional food composition can be in any form, such as tablets, pills, capsules (e.g. soft capsules, microcapsules), candies (e.g. compressed candies, gummy candies, gum candies, etc.), solid beverages (e.g. powders, granules, etc.), liquid beverages, etc.

[0075] In particular, the functional food composition can be prepared according to conventional methods in the field of functional foods.

[0076] In other embodiments of the present application, the product described above is a disinfectant product, which can be a medical disinfectant product, or a daily household disinfectant product.

[0077] In a second aspect of the present application, there is provided use of an ABHD12 inhibitor in the preparation of a medicament for preventing and / or treating a disease caused by or associated with a viral infection.

[0078] In particular, the ABHD12 inhibitor is as described in the first aspect of the present application, in particular DO264.

[0079] In particular, the virus is as described in the first aspect of the present application, in particular a flavivirus (e.g. Zika virus, dengue virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus), orthomyxovirus (e.g. influenza A virus, influenza B virus, influenza C virus), paramyxovirus (e.g. human parainfluenza virus type 1 (HPV), HPV type 2, HPV type 3, HPV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus), coronavirus (e.g. HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2 (including variants thereof, such as the Omicron strain (B.1.1.529), the Delta strain (B.1.617.2), the Alpha strain (B.1.1.7), the Gamma strain (P.1), the Beta strain (B.1.351)).

[0080] In particular, the disease is selected from the group consisting of a pulmonary disease, a sinus disease, an airway disease, an ear disease, a heart disease, hypertension, diabetes, a kidney disease, a liver disease, a gastrointestinal disease, a central nervous system disease, dementia, Alzheimer’s disease, stroke, an immunocompromised state, cancer, or obesity.

[0081] More specifically, the disease is selected from one or more of acute bronchitis, chronic bronchitis, rhinitis, sinusitis, croup, acute bronchiolitis, pharyngitis, tonsillitis, laryngitis, tracheitis, asthma, pneumonia, influenza, and the like.

[0082] In some embodiments of the application, the disease is a disease caused by Zika virus infection, such as Zika fever, Guillain-Barre Syndrome (GBS), microcephaly, meningoencephalitis, myelitis, retinopathy, and the like.

[0083] In some embodiments of the application, the disease is a disease caused by Dengue virus infection, such as Dengue fever, encephalitis, meningoencephalitis, myelitis, and the like.

[0084] In some embodiments of the application, the disease is a disease caused by Mumps virus infection, such as mumps, orchitis, oophoritis, pancreatitis, meningoencephalitis, Mumps virus ocular disease, and the like.

[0085] In some embodiments of the application, the disease is a disease caused by Coronavirus (such as SARS-CoV-2) infection, such as COVID-19, Severe Acute Respiratory Syndrome (SARS), pneumonia, and the like.

[0086] In particular, the medicament can be in any suitable dosage form, such as a dosage form for administration via the gastrointestinal tract, for example, including, but not limited to, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, oral disintegrating tablets, oral tablets, and the like), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, solutions, emulsions, suspensions, controlled release preparations (e.g., instant release preparations, sustained release preparations, sustained release microcapsules), aerosols, films (e.g., oral disintegrating films, oral mucosal-adhesive films), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), intravenous drip preparations, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories (e.g., rectal suppositories, vaginal suppositories), nasal preparations, lung preparations (inhalants), eye drops, and the like.

[0087] In a third aspect of the application, there is provided a method of inhibiting and / or killing a virus, comprising the step of inhibiting ABHD12.

[0088] In particular, the virus is as described in the first aspect of the application, in particular a flavivirus (such as Zika virus, Dengue virus, Japanese encephalitis virus, Chikungunya virus, Yellow fever virus, Hepatitis C virus, West Nile virus), orthomyxovirus (such as Influenza A virus, Influenza B virus, Influenza C virus), paramyxovirus (such as Human parainfluenza virus type 1 (HPV), HPV type 2, HPV type 3, HPV type 4, Sendai virus, Mumps virus, Measles virus, Respiratory syncytial virus, Newcastle disease virus), coronavirus (such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2 (including variants thereof, such as the Omicron strain (B.1.1.529), the Delta strain (B.1.617.2), the Alpha strain (B.1.1.7), the Gamma strain (P.1), the Beta strain (B.1.351)).

[0089] In particular, the inhibition of ABHD12 can be achieved by a method of gene editing (e.g. knocking out or knocking down the gene disrupting ABHD12 expression), or by administering an ABHD12 inhibitor.

[0090] In particular, the method can be used for diagnostic or therapeutic purposes, but also for non-diagnostic or non-therapeutic purposes.

[0091] In particular, the method can be performed in vivo, but also in vitro.

[0092] In a fourth aspect of the application, a method of preventing and / or treating a disease caused by or associated with a viral infection is provided, comprising the step of administering an ABHD12 inhibitor to a subject in need thereof.

[0093] In particular, the ABHD12 inhibitor is as described in the first aspect of the application, in particular DO264.

[0094] In particular, the virus is as described in the first aspect of the present application, in particular, a flavivirus (such as Zika virus, dengue virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus), orthomyxovirus (such as influenza A virus, influenza B virus, influenza C virus), paramyxovirus (such as human parainfluenza virus type 1 (HPV), HPV type 2, HPV type 3, HPV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus), coronavirus (such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2 (including its variants, such as the Omicron strain (B.1.1.529), the Delta strain (B.1.617.2), the Alpha strain (B.1.1.7), the Gamma strain (P.1), the Beta strain (B.1.351)).

[0095] In particular, the disease is as described in the second aspect of the present application, in particular, a disease caused by Zika virus infection (such as Zika fever, Guillain-Barre syndrome, microcephaly, meningoencephalitis, myelitis, retinopathy), a disease caused by dengue virus infection (such as dengue fever, encephalitis, meningoencephalitis, myelitis), a disease caused by mumps virus infection (such as mumps, orchitis, oophoritis, pancreatitis, meningoencephalitis, mumps virus eye disease), a disease caused by coronavirus infection (such as COVID-19, SARS, pneumonia).

[0096] In particular, the subject is an animal; in some embodiments of the present application, the subject is a mammal, such as a human, a monkey, an ape, a cow, a horse, a pig, a seal, etc.; in other embodiments of the present application, the subject is an avian, such as a chicken.

[0097] In particular, the administration route can be any suitable route, for example, a gastrointestinal administration (such as oral, sublingual, rectal administration) or a non-gastrointestinal administration (such as intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, respiratory tract administration, etc.). In some embodiments of the present application, the administration route is oral. In other embodiments of the present application, the administration route is injection (such as subcutaneous injection, intravenous injection, intramuscular injection).

[0098] The inventors of the present application have found through experiments that ABHD12 is a potential broad-spectrum antiviral target, and its inhibitors (such as DO264 or its derivatives) can effectively inhibit the proliferation of viruses (such as Zika virus, mumps virus, coronavirus, etc.), which can have very good application prospects and research value in the field of antiviral medicine. BRIEF DESCRIPTION OF DRAWINGS

[0099] Figure 1 The results of U87MG cells with knock-out of ABHD12 and siRNA interference inhibiting ZIKV infection are shown. Among them, sg1 to sg4 correspond to sgABHD12-1 to sgABHD12-4 respectively, si1 to si3 correspond to siRNA-1 to siRNA-3 respectively, and si4 corresponds to siRNA-3’UTR.

[0100] Figure 2 The results of specific small molecule inhibitor DO264 of ABHD12 inhibiting ZIKV infection in 293T, U87MG, Paki and Huh7 cells are shown.

[0101] Figure 3 The results of DO264 inhibiting flavivirus, mumps virus and novel coronavirus infection are shown. The left Y-axis represents the inhibition level of the inhibitor on the virus, and the right Y-axis represents the cytotoxicity of the inhibitor.

[0102] Figure 4 The results of overexpression of ABHD12 weakening the inhibitory effect of DO264 on ZIKV infection are shown.

[0103] Figure 5 The results of exogenous supplement of Lyso PS inhibiting ZIKV infection at the cellular level are shown.

[0104] Figure 6 The results of ABHD12 gene knockout MEF cells limiting ZIKV infection are shown.

[0105] Figure 7 The results of ABHD12 gene knockout AG129 mice inhibiting ZIKV infection in vivo are shown.

[0106] Figure 8 The results of DO264 administration effectively inhibiting ZIKV infection in vivo are shown. DETAILED DESCRIPTION

[0107] Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0108] In the present application, the term “ABHD12 inhibitor” refers to a molecule having an inhibitory effect on ABHD12, which includes but is not limited to: inhibiting the activity of ABHD12, inhibiting the transcription or expression of ABHD12 gene. The ABHD12 inhibitor includes but is not limited to an antibody or an antigen-binding fragment thereof, an interfering RNA, a small molecule compound, etc.

[0109] Inhibiting ABHD12 activity means to reduce the activity of ABHD12; specifically, the activity of ABHD12 is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% compared to before the inhibition.

[0110] Inhibiting ABHD12 gene transcription or expression means to not transcribe the gene of ABHD12, or to reduce the transcription activity of the gene of ABHD12, or to not express the gene of ABHD12, or to reduce the expression activity of the gene of ABHD12.

[0111] The skilled person can use conventional methods to modulate the gene transcription or expression of ABHD12, such as gene knockout, homologous recombination, interfering RNA, etc.

[0112] The inhibition of the gene transcription or expression of ABHD12 can be verified by detecting the expression amount through experiments such as PCR and Western Blot.

[0113] Preferably, the gene transcription or expression of ABHD12 is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% compared to the wild type, and in particular, the gene of ABHD12 is completely not expressed.

[0114] In the present application, the term "alkyl" refers to a straight-chain or branched-chain hydrocarbon radical that is not unsaturated and that is attached to the rest of the molecule by a single bond. Typical alkyl groups contain 1 to 12, 1 to 8, 1 to 6, or 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, n-hexyl, i-hexyl, n-heptyl, i-heptyl, and the like. Alkyl groups can be substituted, for example, if an alkyl group is substituted with a cycloalkyl group, it corresponds to a "cycloalkylalkyl", such as cyclopropylmethyl; if an alkyl group is substituted with a halogen, it corresponds to a "haloalkyl"; if an alkyl group is substituted with an aryl group, it corresponds to an "aralkyl", such as benzyl, diphenylmethyl, or phenethyl; if an alkyl group is substituted with a heterocyclyl group, it corresponds to a "heterocyclylalkyl"; and the like.

[0115] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon radical that contains at least two carbon atoms, at least one unsaturated bond, and that is attached to the rest of the molecule by a single bond. Typical alkenyl groups contain 2 to 12, 2 to 8, 2 to 6, or 2 to 3 carbon atoms, such as ethenyl, 1-methyl-ethenyl, 1-propenyl, 2-propenyl, or butenyl.

[0116] The term "alkynyl" refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms, at least one carbon-carbon triple bond, and which is attached to the rest of the molecule by a single bond. Typical alkynyl groups contain 2 to 12, 2 to 8, 2 to 6, or 2 to 3 carbon atoms, such as ethynyl, propynyl (e.g., 1-propynyl, 2-propynyl), or butynyl (e.g., 1-butynyl, 2-butynyl, 3-butynyl).

[0117] The term "alkoxy" refers to an alkyl group, as previously defined, appended to the parent molecular moiety through an oxygen atom. Typical alkoxy groups contain 1 to 12 carbon atoms, such as methoxy, ethoxy, propyloxy, butyloxy, and the like.

[0118] The term "cycloalkyl" refers to an alicyclic hydrocarbon. Typical cycloalkyl groups contain 1 to 4 rings and / or fused rings and contain 3 to 18 carbon atoms, such as 3 to 10 carbon atoms or 3 to about 6 carbon atoms, such as cyclopropyl, cyclohexyl.

[0119] The term "aryl" refers to a monocyclic or polycyclic radical, including both single and / or fused ring radicals. Typical aryl groups contain 1 to 3 rings and 6 to 18 carbon ring atoms, preferably 6 to 14 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indenyl, phenanthryl, or anthryl radicals.

[0120] The term "heterocyclyl" includes both heteroaromatic and heteroalicyclic radicals containing 1 to 3 rings and / or fused rings and 3 to 18 ring atoms. Specifically, the heteroaromatic and heteroalicyclic radicals contain 5 to about 10 ring atoms. Suitable heteroaryl groups can contain 1, 2, or 3 heteroatoms selected from N, O, or S atoms. "Heterocycloalkyl" refers to saturated heterocyclyl groups.

[0121] The term "halogen" refers to bromine, chlorine, iodine, or fluorine. Haloalkyl refers to an alkyl group in which one or more hydrogen atoms have been replaced with a halogen atom (F, Cl, Br, I), such as -CH2Rh, -CHRh2, -CRh3, where Rh is F, Cl, Br, or I; such as -CF3.

[0122] The term "pharmaceutically acceptable salt" refers to a salt of an acid or base that is theoretically non-toxic, irritating, and allergenic, and that is capable of achieving or providing the clinically acceptable pharmacokinetic, absorption, distribution, and metabolic properties of the drug molecule to achieve the intended purpose. The salts described herein include pharmaceutically acceptable acid or base salts of the acidic, basic, or amphoteric groups of the compounds. A list of suitable salts can be found in S. M. Birge, et al., J. Pharm. Sci., 66, 1-19 (1977).

[0123] The pharmaceutically acceptable salts referred to in the present invention include acid addition salts and base addition salts.

[0124] The acid addition salts include, but are not limited to, those derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, and phosphonic acids, as well as organic acids, such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Such salts thus include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, and methanesulfonate, as well as salts of amino acids such as arginate, gluconate, galacturonate, and the like. The acid addition salts can be prepared by contacting the free base form with a sufficient amount of the desired acid to form the salt. The free base form can be regenerated by contacting the salt form with a base.

[0125] The base addition salts referred to in the present invention are those formed between the free acid form of the invention and a suitable base such as an alkali or alkaline earth metal. Examples of metals which can be formed into salts include sodium, potassium, magnesium, and calcium. Examples of suitable amines include, but are not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane- 1,2-diamine), N-methylglucamine, and procaine. The base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base to form the salt. The free acid form can be regenerated by contacting the salt form with a suitable acid. The term "solvate" is to be understood as meaning any form of the compounds of the present invention in which the compound is associated with another molecule, typically by non-covalent bonding, such as a polar solvent, and in particular includes hydrates and alcoholates, e.g. methanolate. In particular, the solvate is a hydrate.

[0126] The term "solvate" is to be understood as meaning any form of the compounds of the present invention in which the compound is associated with another molecule, typically by non-covalent bonding, such as a polar solvent, and in particular includes hydrates and alcoholates, e.g. methanolate. In particular, the solvate is a hydrate.

[0127] The term "prodrug" is used in its broadest sense and encompasses derivatives of the compounds of the application that are convertible in vivo to the compounds of the application. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds, including biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable acyloxymethyl esters, and biohydrolyzable phosphate analogs. Preferably, the prodrugs with carboxyl functionality are lower alkyl esters of carboxylic acids. The carboxylate esters are readily formed from any carboxylic acid moiety present in the molecule by esterification. Prodrugs can generally be prepared using known methods, such as those described in Burger's "Medicinal Chemistry and Drug Discovery" Sixth Edition (Donald J. Abraham ed., 2001, Wiley) and "Design and Applications of Prodrugs" (H. Bundgaard ed., 1985, Harwood Academic Publishers).

[0128] Any compound referred to herein is intended to represent a particular compound and some of its modifications or forms. In particular, the compounds referred to herein can have asymmetric centers and therefore exist in different enantiomeric or diastereomeric forms. Thus, any given compound referred to herein is intended to represent any one of the racemates, one or more of the enantiomeric forms, one or more of the diastereomeric forms, and mixtures thereof. Likewise, stereoisomers or geometric isomers of double bonds can exist, and in some cases the molecule can exist as an (E)-isomer or a (Z)-isomer (trans and cis isomers). If the molecule contains multiple double bonds, each double bond can have its own stereochemistry, which can be the same or different from the stereochemistry of the other double bonds in the molecule. In addition, the compounds referred to herein can exist as atropisomers. All stereoisomers, including enantiomers, diastereomers, geometric isomers, and atropisomers, and mixtures of stereoisomers, of the compounds described herein are within the scope of the application.

[0129] In addition, any compound referred to herein can exist in tautomeric forms. In particular, the term tautomer refers to one of two or more structural isomers of a compound that are in equilibrium with one another and can be interconverted by a tautomerization. Common tautomeric pairs are enol-keto, amide-imidic acid, ketone-enol, lactam-lactim, and the like.

[0130] Unless otherwise stated, the compounds of the application also include isotopically-labeled forms of the compounds. For example, compounds having a hydrogen atom replaced by a deuterium or tritium atom, or a carbon atom replaced by a 13C- or 14C-enriched carbon atom, are within the scope of this application. Such compounds are useful in metabolic studies, as reversible inactivators of enzymes involved with the metabolism of the compounds, and the like. Further, deuterium substitution can be used to modify the metabolic profile of a compound in vivo or in vitro. In particular, deuterium 13C or 14 C of carbon to replace at least one carbon, or using a compound of an existing structure that is enriched in 15 N of nitrogen to replace at least one nitrogen of an existing structure are all included in the scope of the present application.

[0131] The compound described in the present application or its salt, solvate, stereoisomer, ether, ester is preferably in a pharmaceutically acceptable form. Among them, "pharmaceutically acceptable" means that when the molecular body and the composition containing it are properly administered to a subject, they will not produce adverse, allergic or other adverse reactions.

[0132] The disclosures of various publications, patents and published patent specifications referred to herein are hereby incorporated by reference in their entireties.

[0133] The technical solutions of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0134] The DO264 described in the embodiments is an ABHD12 specific small molecule inhibitor (Ogasawara D, Ichu TA, Vartabedian VF, Benthuysen J, Jing H, Reed A, Ulanovskaya OA, Hulce JJ, Roberts A, Brown S, Rosen H, Teijaro JR, Cravatt BF. Selective blockade of the lyso-PSlipase ABHD12 stimulates immune responses in vivo. Nat Chem Biol. 2018 Dec; 14(12): 1099-1108.), which has the following structure:

[0135]

[0136] Example 1: Anti-viral function verification of ABHD12 gene knockout or knockdown cells

[0137] In this embodiment, CRISPR Cas9 technology is used to knock out ABHD12 gene and siRNA is used to interfere with the expression of ABHD12, then Zika virus is used to infect U87MG cell lines with ABHD12 knockdown or knockout, and then western blot is used to detect the amount of viral protein.

[0138] 1. Experimental method

[0139] 1.1, sgRNA expression vector construction

[0140] sgRNA primer sequences:

[0141] sgABHD12-1 forward primer: 5'-caccg GCACACCGTCCCTGCAGTC-3';

[0142] sgABHD12-1 reverse primer: 5'-aaac GCACACCGTCCCTGCAGTC c-3';

[0143] sgABHD12-2 forward primer: 5'-caccg AGTGGCCCCAGATGTACAC-3';

[0144] sgABHD12-2 reverse primer: 5'-aaac GTGTACATCTGGGGCCACT c-3';

[0145] sgABHD12-3 forward primer: 5'-caccg GTGGCCCCAGATGTACACG-3';

[0146] sgABHD12-3 reverse primer: 5'-aaac CGTGTACATCTGGGGCCAC c-3';

[0147] sgABHD12-4 forward primer: 5'-caccg CTGCTCATCCTGCACGCTG-3';

[0148] sgABHD12-4 reverse primer: 5'-aaac CAGCGTGCAGGATGAGCAG c-3'.

[0149] The annealing system for the synthesized sgRNA sequence is as follows:

[0150]

[0151] The mixed system is phosphorylated at 37°C for 30 min, denatured at 95°C for 5 min, and annealed at 25°C by cooling at a rate of 1.5°C per minute. The double-stranded DNA fragment with BsmBI sticky ends is obtained.

[0152] The double-stranded DNA fragment is then diluted 1:20 and ligated with the CRISPR V2 vector digested with BsmbI at 37°C for 2 h according to the following system.

[0153]

[0154] Finally, the ligation product was transformed into stbl3 competent cells, which were plated on LB plates with ampicillin resistance. After incubation at 37°C overnight, positive colonies were identified by sequencing and the plasmid CRISPR V2-sgABHD12 was extracted.

[0155] 1.2, Construction of ABHD12 knockout cell line

[0156] 293T cells were plated at 4x10 5 cells per well in a 6-well plate and incubated overnight. Then, CRISPR V2-sgABHD12 plasmid and lentivirus packaging plasmids were transfected. After 48 hours, the lentivirus supernatant was collected, filtered through a 0.22 uM filter, mixed with polybrene, and added to U87 cells cultured in a 6-well plate. The final concentration of polybrene was 8 ug / mL. After 48 hours of virus infection, the cells were passaged and screened for successful expression of CRISPR V2-sgABHD12 using 2 ug / mL puromycin. After about a week of screening, the successfully knocked-out cell line was selected for subsequent virus infection experiments.

[0157] 1.3, Verification of the antiviral function of the gene knockout cell line

[0158] The next day after successfully knocking out the ABHD12 gene in the cells, an appropriate amount of ZIKV was added to infect the cells. The cell culture plate was incubated at 37°C for another 48 hours. Then, the culture medium was discarded and the cells were washed twice with PBS. The cells were collected and the virus load was determined by Western blot.

[0159] 1.4, Verification of the antiviral function of ABHD12 knockdown

[0160] The following siRNA sequences were synthesized by Jintuobio Technology Co., Ltd.:

[0161] siRNA-1: GCCACCCUAUCAUUCUGUATT;

[0162] siRNA-2: GGAAUCUCCAUUCACUAAUTT;

[0163] siRNA-3: GGUUCUUCCUUGAUCCUAUTT;

[0164] siRNA-3'UTR: AGGUGACACGGUCAUCGAATT.

[0165] The synthesized siRNA sequences were diluted to 20 uM stock solution with DEPC water according to the instructions and stored in aliquots.

[0166] On the second day after cell plating, the cell confluence reached about 70%, and siRNA transfection was performed. Taking a 24-well plate as an example: 25 uL of Opti-MEM was used to dilute 0.6 uL of 20 uM siRNA storage solution, and the mixture was gently mixed. Another 25 uL of Opti-MEM was used to dilute 1.2 uL of transfection reagent Lipofectamine RNAiMAX, and the mixture was gently mixed. Then the diluted siRNA was added to the diluted transfection reagent, gently mixed, incubated at room temperature for 3-5 min, and then added to the cells to interfere with the expression of ABHD12. Then the culture plate was incubated at 37°C for 24-36 h, and then the subsequent ZIKV infection experiment was performed. Finally, the cells were collected, and the virus amount was determined by Western blot.

[0167] 1.5, Western blot

[0168] After the cells were collected, an appropriate amount of RIPA (medium) lysis buffer was added to each well, and the cells to be tested were lysed on ice for 15 min, then centrifuged at 4°C at 12000 rpm for 15 min, and the supernatant was collected. After adding loading buffer, it was boiled in a 95°C metal bath for 10 minutes. After 10% SDS-PAGE electrophoresis, it was transferred to a PVDF (0.45 uM) membrane. Then 5% skim milk prepared with PBST was used to block at room temperature for 1 h, and the primary antibody corresponding to the protein was incubated at 4°C, 20 rpm overnight. anti-ABHD12 (abcam, ab180944), anti-GAPDH (ZSGB-Bio, TA-08), anti-ZIKV E (Genetex, GTX133314), anti-GFP (Proteintech, PABG1). The next day, the primary antibody was recovered, and the membrane was washed with TBST at room temperature 3 times, 50 rpm, 10 min. Then the HRP-conjugated secondary antibody was incubated at room temperature for 1-2 h, and then the membrane was washed with TBST at room temperature, 50 rpm, 3 times, 50 rpm, 10 min. The developer was configured, and the target band was detected by an exposure instrument.

[0169] 2, Experimental results

[0170] The results of inhibiting Zika virus replication after ABHD12 knockout or knockdown are shown in Figure 1 ABHD12 expression level was greatly inhibited in ABHD12 knockout or knockdown cell lines, and the expression level of Zika virus E protein was also much lower than that of the control group.

[0171] The above experimental results show that ABHD12 gene is required for normal infection of Zika virus.

[0172] Example 2: Anti-viral function verification of ABHD12 specific small molecule inhibitor (DO264)

[0173] The following experiments are included in this example:

[0174] (1) After treating different cell lines with different concentrations of DO264, ZIKV was added to infect the cells, and the effect of the drug on viral infection was detected by RT-qPCR, and the effect of the drug on cell activity was detected by CCK-8.

[0175] (2) Dengue virus (DENV) and ZIKV were added to U87MG cells treated with different concentrations of DO264, and the cells were collected 48 hours after infection. The effect of the drug on DENV and ZIKV infection was quantitatively analyzed by qPCR.

[0176] (3) Mumps was added to 293T cells treated with different concentrations of DO264, and the cell supernatant was collected 48 hours after infection. The effect of the drug on Mumps infection was detected by RT-qPCR.

[0177] (4) SARS-CoV2 Omicron BA.2 strain was added to Vero cells treated with different concentrations of DO264, and the cell supernatant was collected 48 hours after infection. The effect of the drug on Omicron BA.2 replication was detected by RT-qPCR.

[0178] 1. Experimental methods

[0179] 1.1, The cells to be tested were plated in 24-well plates and cultured overnight until the cell confluence reached about 70%. 0, 0.5uM, 1uM, 2uM, 5uM, and 10uM of DO264 were added, respectively. After 2-4h of treatment, an appropriate amount of virus was added, and the cells were collected 48h after infection. The culture medium was discarded, and the cells were washed twice with PBS. The RNA was extracted from the cells, and the viral load was determined by qPCR.

[0180] 1.2, CCK8 was used to determine the cytotoxicity of DO264. The cells to be tested were plated in 96-well plates and cultured overnight until the cell confluence reached about 70%. 0, 0.5uM, 1uM, 2uM, 5uM, and 10uM of DO264 were added, respectively. After 48h of continuous culture, fresh culture medium was added to each well, and 10ul of CCK-8 (Vazyme, A311) was added. The OD450nm absorbance was measured.

[0181] 1.3, Real time qPCR

[0182] After the collected cell RNA was extracted using the kit HiPure Total RNA Mini Kit (Magen, R4111), cDNA was obtained by reverse transcription using 5x All-In-One MasterMix (ABM, G490). The reverse transcription system was 4uL 5x All-In-One MasterMix and 16uL RNA (total amount less than 2ug). After mixing evenly, reverse transcription was performed according to the program 25℃ 10min, 42℃ 15min, 85℃ 5min. Finally, qPCR was performed using SYBR Green qPCR master mix (Vazyme, Q311-02), and each reaction system was 10ul. Two technical repeats were set for each RNA sample. The reaction system was as follows:

[0183]

[0184]

[0185] Quantitative detection was performed using Bio-Rad CFX96 fluorescent quantitative PCR instrument according to the program. The program was set as: 95℃ pre-denaturation 3min, denaturation 10s, 60℃ annealing 30s, repeating denaturation and annealing 40 cycles.

[0186] The primer sequences were as follows:

[0187] Human GAPDH forward primer: 5'-AGATCCCTCCAAAATCAAGTGG-3';

[0188] Human GAPDH reverse primer: 5'-GGCAGAGATGATGACCCTTTT-3';

[0189] ZIKV NS5 forward primer: 5'-GGTCAGCGTCCTCTCTAATAAAGG-3';

[0190] ZIKV NS5 reverse primer: 5'-GCACCCTAGTGTCCACTTTTTCC-3'

[0191] DENV forward primer: 5'-CATGCCATCCAT GAAAAGATTCAGA-3';

[0192] DENV reverse primer: 5'-TTGTTGCTGCGATTTGTAAGG GAG-3';

[0193] Mumps-GFP forward primer: 5'-AGTCCGCCCTGAGCAAAGA-3';

[0194] Mumps-GFP reverse primer: 5′-TCCAGCAGGACCATGTGATC-3′;

[0195] Omicron N forward primer: 5′-GACCCCAAAATCAGCGAAAT-3′;

[0196] Omicron N reverse primer: 5′-TCTGGTTACTGCCAGTTGAATCTG-3′;

[0197] Mouse GAPDH forward primer: 5′-AGGTCGGTGTGAACGGATTTG-3′;

[0198] Mouse GAPDH reverse primer: 5'-TGTAGACCATGTAGTTGAGGTCA-3'.

[0199] 2. Experimental results

[0200] The results showed that the ABHD12-specific small molecule inhibitor DO264 can effectively inhibit the infection of ZIKV, DENV, Mumps-GFP, and SARS-CoV2 (Omicron). Figure 2 As shown in the figure, different concentrations of DO264 were added to 293T, U87MG, Paki and Huh7 cells, and after 2-4 hours of treatment, ZIKV was added. It was found that DO264 could significantly inhibit ZIKV infection. Moreover, the half-maximal inhibitory concentration of DO264 was much lower than the concentration that caused half of its toxicity to cells. Figure 3 As shown, DENV and ZIKV were added to DO264-treated U87MG cells for infection. qPCR results showed that DO264 effectively inhibited dengue virus and Zika virus infection. Similarly, when Mumps-GFP virus was added to DO264-treated 293T cells, DO264 also significantly inhibited mumps virus infection. Furthermore, DO264 also effectively reduced the level of viral RNA secreted by the Omicron BA.2 strain in the supernatant of Vero cells.

[0201] The above experimental results show that specific inhibitors of ABHD12 can effectively inhibit the infection of multiple viruses.

[0202] Example 3: Effect of ABHD12 overexpression on DO264 antiviral infection

[0203] In this example, the AAV system was used to increase the expression level of ABHD12 in cells, and then drug treatment was added to conduct infection experiments. The inhibitory effect of DO264 on ZIKV infection was detected by western blot.

[0204] 1. Experimental method

[0205] The cells to be tested were plated in 24-well plates, and 5 x 10 10 GC ABHD12-AAV and GFP-AAV, after 36 h of culture, 0, 0.125 uM, 0.25 uM, 0.5 uM, 1 uM, and 2 uM of DO264 were added, respectively. After 2-4 h of treatment, Zika virus was added. After 48 h of continuous culture, the culture medium was discarded, and the cells were washed twice with PBS, and the cells were collected to extract RNA, and the level of ZIKV infection was detected by Western blot (see Example 1 for experimental procedures).

[0206] 2. Experimental results

[0207] The results show that overexpression of ABHD12 weakens the inhibitory effect of DO264 on ZIKV infection. As shown in FIG. 2, after 36 h of AAV infection in U87MG cells, drug treatment and virus infection experiments were performed, and the cells were collected after 48 h. Western blot detection showed that the expression level of ABHD12 in U87MG cells was significantly improved by using the AAV overexpression system, and the partial inhibitory effect of DO264 on ZIKV infection was eliminated. Figure 4 The above experimental results show that increasing the expression level of ABHD12 in cells can weaken the inhibitory effect of DO264 on ZIKV infection.

[0208] Example 4: Verification of the antiviral function of Lyso PS

[0209] In this example, lyso PS and DO264 were added before, at the same time of, and 4 h after virus infection, respectively, and their inhibitory effect on ZIKV infection was detected by Western blot.

[0210] 1. Experimental method

[0211] U87MG cells were plated in 24-well plates at 5 x 10 4 After 37°C culture overnight, 0, 50 nM, 100 uM, and 200 nM of lyso PS or 0, 0.25 uM, 0.5 uM, and 1 uM of DO264 were added before, at the same time of, and 4 h after virus infection, respectively, and the level of cell infection was detected by qPCR after 48 h of virus infection.

[0212] 2. Experimental results

[0213]

[0214] ​The results show that exogenous addition of Lyso PS inhibits ZIKV infection. ABHD12 is an important enzyme in the phospholipid metabolism pathway, mediating the hydrolysis of lysophosphatidylserine (lyso-PS), oxidized lysophosphatidylserine (ox-lyso PS), and 2-arachidonoylglycerol (2-AG). As shown in Figure 5 Figure 1, exogenous supplementation of lyso PS and DO264 treatment can both significantly inhibit ZIKV infection, indicating that ABHD12 can mediate the viral infection process by regulating the level of lyso PS. Moreover, lyso PS or DO264 added before, simultaneously with, and after viral infection all show good antiviral activity, indicating that ABHD12 can be involved in multiple stages of viral infection.

[0215] The above experimental results show that the inhibition of virus by DO264 is due to the blockage of lyso PS metabolism.

[0216] Example 5: In vivo antiviral activity verification of ABHD12

[0217] This example includes the following experiments:

[0218] (1) ABHD12 + / - After mating (Heter)C57BL / 6 mice, MEF cells were obtained by dissecting 14-day pregnant mice, and ABHD12 + / + (WT) and ABHD12 - / - (KO) type MEF cells were selected for Zika virus infection experiments, and the viral level was detected by RT-qPCR.

[0219] (2) After mating ABHD12 + / - type AG129 mice, 5-6-week-old mice of similar body weight were selected from the offspring, and 10 3 PFU of virus was injected intraperitoneally for infection experiments, and blood was collected from the mouse tail on days 3-4 after infection, and the viral level in the mouse blood was detected by RT-qPCR, and the survival of the mice was recorded daily.

[0220] (3) After mating wild-type AG129 mice, 5-6-week-old mice of similar body weight were selected from the offspring, and 10 3 PFU of virus was injected intraperitoneally, and drug treatment was performed continuously at 12 h before viral infection and 3 days after viral infection, with 2 doses of 8 mg / kg per mouse per day. Blood was collected from the mouse tail on days 3-4 after infection, and the viral level in the mouse blood was detected by RT-qPCR, and the survival of the mice was observed.

[0221] 1. Experimental methods

[0222] 1.1, compare the difference of viral infection level between wild type and gene knockout type B6 mouse embryonic fibroblast (MEF) cells. First, ABHD12 heterozygous mice were mated, and the next day the mice were separated by gender. The pregnant mice were dissected in the morning on the 14th day after mating to obtain MEF cells. After genotyping by PCR, ABHD12 + / + and ABHD12 - / - type MEF cells were selected for viral infection experiments.

[0223] 1.2, compare the infection of ABHD12 + / + and ABHD12 - / - type AG129 mice. Wild type and knockout type mice were obtained after mating ABHD12 + / - mice. After 5-6 weeks, several mice with similar body weights were selected, each injected with 10 3 PFU of virus suspension for infection. The viral load in the blood on the 3rd-4th day after infection was determined, and the survival of the mice was observed.

[0224] 1.3, compare the infection of AG129 mice in the treatment group and the control group. Several 5-6 week old mice with similar body weights were selected, and each was injected with 10 3 PFU of virus suspension for infection. Drug treatment was performed by intraperitoneal injection 12 hours before viral infection and three days after viral infection. The drug was administered twice a day, and the dose was 8 mg / kg per mouse. The viral load in the blood on the 3rd-4th day after infection was determined, and the survival of the mice was observed.

[0225] 2, experimental results

[0226] The results show that knocking out ABHD12 or DO264 drug treatment can effectively inhibit ZIKV infection in mice. First, mouse embryonic fibroblast cells MEF, which are close to in vivo cell infection, were isolated, and the infection of wild type and knockout type MEF cells to ZIKV was compared. As Figure 6 shown, the infection level of ZIKV in WT, KO type MEF cells was significantly reduced. Subsequently, several WT and KO type 5-6 week old AG129 mice with similar body weights were selected for infection experiments. As Figure 7 shown, the viral titer of KO mice was more than ten times lower than that of WT mice, and the survival time was prolonged by one day. In addition, several 5-6 week old AG129 mice were selected for drug administration experiments. As Figure 8 shown, the viral titer of mice in the drug administration group was ten times lower than that of mice in the control group, and the survival time was prolonged by two days. This shows that knocking out ABHD12 or inhibiting its enzyme activity can effectively inhibit the infection of Zika virus in vivo.

[0227] Summary: ABHD12 is a potential broad-spectrum antiviral target, and its inhibitors such as DO264 have good application prospects in antiviral.

[0228] The above merely describes preferred embodiments of the present application but should not be used to restrict the present application, and any modification, equivalent replacement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

[0229] The foregoing embodiments and methods described in the present application can be different based on the ability, experience and preference of the person skilled in the art.

[0230] The steps of the method described in the present application are only listed in a certain order and do not constitute any limitation on the order of the steps of the method.

Claims

1. Use of an ABHD12 inhibitor in the preparation of a product for inhibiting and / or killing viruses; Preferably, the ABHD12 inhibitor is selected from: an antibody or an antigen-binding fragment thereof, an interfering RNA or a small molecule compound.

2. The use according to claim 1, characterized in that The small molecule compound has the following structure: in, X is S or O; Y is N or CH; R 0 Selected from: H, C 1-6 alkyl; L is selected from: R 1 is one or more independent substituents on the ring, each of which is independently selected from: H, halogen, -CN, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, -C(O)OR 2 、-C(O)R 2 、-OR 3 , phenyl optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy; Each R 2 Independently selected from C 1-6 alkyl; Each R 3 Independently selected from: H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl, -C 1-6 Alkyl-C 3-6 Cycloalkyl, 4-10 membered heterocycloalkyl, phenyl, -C 1-6 Alkyl-phenyl, where C 3-6 Cycloalkyl, -C 1-6 Alkyl-C 3-6 Cycloalkyl, 4-10 membered heterocycloalkyl, phenyl, -C 1-6 Alkyl-phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -C(O)OR 4 ; Each R 4 Independently selected from C 1-6 alkyl; n is 0, 1, 2, or 3; or a pharmaceutically acceptable salt, stereoisomer, prodrug, or solvate thereof.

3. The use according to claim 2, characterized in that The small molecule compound has the following structure: Preferably, X is S; Preferably, Y is N; Preferably, R 0 Selected from: H, C 1-3 alkyl.

4. The use according to claim 2 or 3, characterized in that Each R 2 Independently selected from C 1-3 Alkyl; and / or, each R 4 Independently selected from C 1-3 alkyl; Preferably, each R 3 Independently selected from: C 1-6 Alkyl, C 1-6 Halogenated alkyl, phenyl, -C 1-6 Alkyl-C 3-6 Cycloalkyl, 4-7 membered heterocycloalkyl, -C 1-6 Alkyl-phenyl, wherein phenyl and -C 1-6 Alkyl-phenyl is optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -C(O)OR 4 ; More preferably, each R 3 Independently selected from:

5. The use according to claim 4, characterized in that Each R 1 Independently selected from: H, halogen, -CN, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, -C(O)R 2 、-OR 3 , phenyl optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy; Preferably, each R 1 Independently selected from: H, F, Cl, cyano, methyl, trifluoromethyl, trifluoromethoxy, -C(O)CH3, 6. The use according to claim 1, wherein The small molecule compound is selected from: or a pharmaceutically acceptable salt, stereoisomer, prodrug, or solvate thereof; Preferably 7. The use according to any one of claims 1 to 6, characterized in that The virus is Adenoviridae, Herpesviridae, Papovaviridae, Picornaviridae, Poxviridae, Hepadnaviridae, Coronaviridae, Bornaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Retroviridae, Reoviridae, Rhabdoviridae, Flaviviridae; Preferably, the virus is a flavivirus, preferably one or more selected from: Zika virus, dengue virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus; Preferably, the virus is a paramyxovirus, preferably one or more selected from: HPV type 1, HPV type 2, HPV type 3, HPV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus; Preferably, the virus is a coronavirus, preferably one or more selected from: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2.

8. The use according to any one of claims 1 to 7, characterized in that The product is a pharmaceutical composition or a disinfectant product.

9. The use according to any one of claims 1 to 7, characterized in that The application is the use of ABHD12 inhibitors in the preparation of drugs for preventing and / or treating diseases caused by viral infection or related to viral infection.

10. The use according to claim 9, characterized in that The medicine is in oral dosage form or injection form.

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