Application of synthetic route for inhibiting purine nucleotide in prevention or treatment of orthopoxvirus infection

Abstract

The invention relates to the fields of biology, medicine and clinic. In particular to application of PAICS as a target spot in developing or screening reagents for preventing and / or treating orthopoxvirus infection, and application of inhibiting a synthetic pathway of purine nucleotide and / or inhibiting a metabolic pathway of folic acid in preparing reagents for treating and / or preventing orthopoxvirus infection diseases. In order to restrain the infection of orthopoxvirus, the inventor team uses a CRISPR / Cas9 technology to carry out generic genome screening on host cells infected by orthopoxvirus, determines a host-dependent gene PAICS gene in the orthopoxvirus infection process, and verifies and clarifies the virus-dependent mechanism of the gene; in order to deal with the global continuous orthopoxvirus infection problem, the invention plays an important role in the development and screening of future related prevention or treatment drugs, and also expands the new clinical application of existing drugs including methotrexate, mercaptopurine, mizoribine and the like.

Description

Technical Field

[0001] The present invention relates to the fields of biology, medicine, and clinical medicine; in particular, it relates to the use of PAICS as a target in the development or screening of reagents for preventing and / or treating orthopoxvirus infections, and the use of inhibiting the purine nucleotide synthesis pathway and / or the folate metabolic pathway in the preparation of reagents for treating and / or preventing orthopoxvirus infections. Background Art

[0002] Poxviruses are cytoplasmic DNA viruses that infect a wide range of mammalian species. The most notorious members of the poxvirus family include variola virus, the causative agent of smallpox, and monkeypox virus (MPXV), the cause of the recent global monkeypox (MPOX) outbreak.

[0003] Since the first case of monkeypox was reported in the UK in May 2022, 110 countries and regions have confirmed cases of monkeypox worldwide. This outbreak has been characterized by widespread, rapid spread, an unclear source, predominantly human transmission, community transmission, and accelerated viral mutation, drawing significant global attention. On July 23, 2022, the WHO declared the monkeypox outbreak a Public Health Emergency of International Concern.

[0004] Monkeypox virus, smallpox virus and vaccinia virus (VACV) are all members of the genus Orthopoxvirus in the family Poxviridae.

[0005] Among them, monkeypox virus is a double-stranded DNA enveloped virus with a sequence identity of up to 96.3% with the smallpox virus. Monkeypox virus can be divided into Clade I (Central African clade) and "Clade IIa" and "Clade IIb" (West African clade) evolutionary branches. Among them, the clinical symptoms caused by the Clade I branch are more severe, with a mortality rate of up to 10%; while the Clade II branch is relatively mild, with a mortality rate of approximately 3.6%. The monkeypox virus currently prevalent worldwide is the West African branch Clade IIb. Compared with the original endemic strain Clade IIa in West Africa, it has 50 single nucleotide polymorphism sites deviated, and its evolutionary rate has increased by 6-12 times. This may be one of the reasons for the rapid spread of monkeypox worldwide since May 2022.

[0006] Monkeypox virus mainly infects the human body through the respiratory tract and skin mucosa, and spreads to other parts of the body by infecting macrophages and dendritic cells in the draining lymph nodes.

[0007] The fundamental approach to antiviral drug development is to block any stage in the viral proliferation cycle. Viruses are characterized by their inability to survive independently in the natural environment and their ability to proliferate within living host cells. Generally speaking, a virus's proliferation cycle within a host cell consists of five stages: adsorption, invasion, replication, maturation, and release. Blocking any one of these five stages can inhibit viral spread within the body and ultimately treat viral infections.

[0008] Vaccination is also a common method to resist viral infection, but there is currently no specific vaccine to prevent monkeypox, nor is there any specific drug for monkeypox virus on the market.

[0009] There is an urgent need to develop antiviral drugs against orthopoxviruses including monkeypox virus. Summary of the Invention

[0010] In order to curb the infection of orthopoxvirus, the inventor team of this application used CRISPR / Cas9 technology to perform pan-genome screening of host cells infected by orthopoxvirus, identified the host-dependent gene PAICS gene in the process of orthopoxvirus infection, and verified and clarified the virus-dependence mechanism of this gene, that is, by inhibiting the folic acid metabolism pathway and / or inhibiting the biosynthesis of purine nucleotides (de novo synthesis pathway and / or salvage synthesis pathway), the proliferation of orthopoxvirus in host cells can be effectively inhibited, and the infection of host cells by orthopoxvirus can be effectively inhibited.

[0011] In a first aspect, the present application provides the use of the PAICS gene and / or PAICS protein as a target in any of the following:

[0012] (A1) Application in developing or screening agents for preventing and / or treating orthopoxvirus infection;

[0013] (A2) Application in regulating the susceptibility of host cells or animals to orthopoxviruses;

[0014] (A3) Application of orthopoxviruses in regulating their replication ability in host cells or animals;

[0015] (A4) Application in the preparation of reagents for regulating the susceptibility of host cells or animals to orthopoxviruses;

[0016] (A5) Use in the preparation of reagents for regulating the replication ability of orthopoxvirus in host cells or animals.

[0017] The second aspect of the present application provides the use of a reagent targeting PAICS in any of the following:

[0018] (B1) Use in the preparation of agents for treating and / or preventing orthopoxvirus-infected diseases;

[0019] (B2) Application in the preparation of a reagent for inhibiting orthopoxvirus replication in a host cell:

[0020] (B3) Application in preparing cell models or animal models with enhanced resistance to orthopoxvirus.

[0021] In some embodiments of the present application, the agent targeting PAICS is an agent that inhibits, reduces, knocks out, knocks down or knocks down the expression level of the PAICS gene, or inhibits, reduces or inactivates the activity of the expression product of the PAICS gene.

[0022] In some embodiments of the present application, the PAICS-targeting agent refers to an agent that can regulate the expression level or activity of the PAICS gene or its expression product at the nucleic acid level or protein level. The term "regulate" includes inhibiting, reducing, inactivating, knocking out, knocking down, knocking down, or a combination thereof; optionally, in some embodiments of the present application, the PAICS-targeting agent is an agent that inhibits, reduces, knocks out, knocks down, or knocks down the expression level of the PAICS gene, or inhibits, reduces, or inactivates the activity of the expression product of the PAICS gene.

[0023] In some embodiments of the present application, the reagent targeting PAICS can selectively or specifically recognize and act on the PAICS gene or its expression product; alternatively, the reagent targeting PAICS can reduce the level or activity of the PAICS gene or its expression product; alternatively, the reagent targeting PAICS can knock out the level or activity of the PAICS gene or its expression product; alternatively, the reagent targeting PAICS can knock down or knock down the level or activity of the PAICS gene or its expression product; alternatively, the reagent targeting PAICS can inactivate the PAICS gene or its expression product.

[0024] In some embodiments, the expression products of the PAICS gene refer to various forms of molecules of the PAICS gene at various stages, including but not limited to molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the PAICS gene, such as cDNA, mRNA, non-coding RNA, precursor protein, mature protein, and fragments thereof, or protein modification products. In some preferred embodiments, the protein modification products include phosphorylation products, acetylation modifications, ubiquitination modifications, methylation modifications, glycosylation modifications, ADP-ribosylation modifications, and the like.

[0025] In some embodiments, the PAICS-targeting agent is selected from the group consisting of: small molecule inhibitors, carbohydrates, lipids, proteins, polypeptides, nucleic acid molecules, or interfering viruses. The nucleic acid is selected from the group consisting of: antisense oligonucleotides, siRNA, shRNA, dsRNA, microRNA, lncRNA, sgRNA, esiRNA, etc. The protein or polypeptide is selected from the group consisting of: antibodies or antigen-binding fragments thereof. The antibody is selected from the group consisting of: chimeric antibodies, fully human antibodies, and humanized antibodies; and the antigen-binding fragment includes, but is not limited to: Fab, Fab', F(ab')2, Fv fragments, ScFv, single-chain antibodies, or domain antibodies.

[0026] In some embodiments, an agent targeting "PAICS" or an agent targeting PAICS refers to a PAICS gene or its expression product as a target, and regulating the level or activity of the PAICS gene or its expression product in the cell through the above-mentioned "regulation" method.

[0027] In a third aspect of the present application, there is provided a use of an agent for inhibiting folic acid metabolic pathway in any of the following:

[0028] (C1) Use in the preparation of agents for treating and / or preventing orthopoxvirus-infected diseases;

[0029] (C2) Application in the preparation of a reagent for inhibiting orthopoxvirus replication in a host cell:

[0030] (C3) Application in preparing cell models or animal models with enhanced resistance to orthopoxvirus.

[0031] In some embodiments, the agent that inhibits the folate metabolic pathway comprises an agent that inhibits 10-formyl-tetrahydrofolate (N 10 -Formyl-THF) is a reagent for the synthesis of

[0032] 10-Formyl-tetrahydrofolate (N 10 10-Formyl-THF) is a folic acid derivative and a form of tetrahydrofolate; 10-Formyl-tetrahydrofolate is both a product of the folic acid metabolic pathway and participates in the de novo synthesis of purine nucleotides.

[0033] In some embodiments, the agent that inhibits the folate metabolic pathway comprises a folate antagonist.

[0034] Preferably, the folic acid antagonist comprises methotrexate or a chemically modified derivative thereof, wherein the chemically modified derivative of methotrexate still has the function of inhibiting the folic acid metabolic pathway, and the chemical modification does not affect or substantially does not affect its primary function of inhibiting folic acid metabolism.

[0035] In a fourth aspect, the present application provides the use of an agent for inhibiting the de novo synthesis pathway and / or salvage synthesis pathway of purine nucleotides in any of the following:

[0036] (D1) Use in the preparation of agents for treating and / or preventing orthopoxvirus-infected diseases;

[0037] (D2) Application in the preparation of a reagent for inhibiting orthopoxvirus replication in a host cell:

[0038] (D3) Application in preparing cell models or animal models with enhanced resistance to orthopoxvirus.

[0039] In some embodiments, the agent that inhibits the de novo synthesis pathway and / or the salvage synthesis pathway of purine nucleotides comprises an agent that inhibits purine nucleotide anabolism.

[0040] In some embodiments, the agent that inhibits purine nucleotide synthesis metabolism includes mercaptopurine, mizoribine or methotrexate or their chemically modified derivatives.

[0041] In some embodiments, the orthopoxvirus is selected from any one or combination of the genus Orthopoxvirus of the family Poxviridae.

[0042] Preferably, the orthopoxvirus is selected from any one or a combination of monkeypox virus, vaccinia virus, smallpox virus, or cowpox virus. In some specific embodiments, the orthopoxvirus is monkeypox virus or vaccinia virus.

[0043] In some embodiments, the above-mentioned agent is a pharmaceutical composition; in some specific embodiments, the active ingredient having the above-mentioned function is mixed with an excipient, or diluted with an excipient, or encapsulated in a pharmaceutical carrier. The pharmaceutical composition can be in any existing pharmaceutical dosage form, such as an injection, tablet, pill, powder, capsule, oral liquid (such as syrup), or spray (such as nasal spray). Preferably, an injection is used, such as an intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous drip.

[0044] The above-mentioned pharmaceutical composition can be used to treat orthopoxvirus infection, or to prevent orthopoxvirus infection, comprising administering an effective dose of the pharmaceutical composition to a subject.

[0045] The subject is selected from the group consisting of: humans, mice, rats, guinea pigs, rabbits, horses, monkeys, dogs, and pigs.

[0046] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following embodiments can be combined with each other to form new or preferred technical solutions. Due to space limitations, they are not listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A complete process for whole-genome knockout screening of THP-1 host cells against VTT infection, where a single sgRNA knocks out only one gene in one host cell.

[0048] Figure 2 Results of CRISPR genome-wide knockout screening (sequencing analysis of sgRNA enrichment in surviving cells after VTT infection. The more surviving cells, the more sgRNA sequences they contain are enriched, and thus more likely to be detected. The red marker in the upper right corner of the figure indicates the host gene targeted for knockout by the enriched sgRNA).

[0049] Figure 3 The effect of knocking out the host-dependent candidate genes of VTT infection on viral infection is shown in Figure 2. The numbers represent different sgRNA sequences for the same knockout gene (the same targeted gene can be knocked out using sgRNAs with different sequences, Luc: luciferase expression, KO: gene knockout, **: P < 0.01);

[0050] Figure 4 It is the folate metabolic cycle and the de novo synthesis pathway of purine nucleotides;

[0051] Figure 5 and Figure 6 The results show that MTX inhibits VTT infection of host cell lines and its cytotoxicity; Figure 5 The figure shows the viral inhibition rate and cytotoxicity of different concentrations of MTX in inhibiting VTT infection of Vero-E6 cell line. 50 =1.18uM, CC 50 >11uM; Figure 6 The figure shows the viral inhibition rate and cytotoxicity of different concentrations of MTX in inhibiting VTT infection of BHK-21 cell line. 50 =0.40uM, CC 50 >11uM.

[0052] Figure 7 and Figure 8 This is the result of folinic acid antagonizing the effect of MTX on VTT infection. Figure 7 It shows that different concentrations of folinic acid increased the EC of MTX on VTT-infected Vero-E6 cells. 50 ; Figure 8 It shows that different concentrations of folinic acid increased the EC of MTX on VTT-infected BHK-21 cells 50 . Leucovorin: folinic acid.

[0053] Figure 9 The results of MTX inhibiting MPXV infection of host cell lines and its cytotoxicity. Different concentrations of MTX inhibited the virus inhibition rate and cytotoxicity of MPXV infection in Vero-E6 cell lines, and its EC 50 =275.42nM, CC 50 >11000nM.

[0054] Figure 10 Purine nucleotide salvage synthesis pathway.

[0055] Figure 11 The results showed that 6-MP inhibited VTT infection and cytotoxicity in Vero-E6 cell lines. 50 =2.49uM, CC 50 >410uM.

[0056] Figure 12 and Figure 13 The results show that Miz inhibits VTT infection of Vero-E6 and BHK-21 cell lines and its cytotoxicity. Figure 12 The viral inhibition rate and cytotoxicity of different concentrations of Miz against VTT infection of Vero-E6 cell line are shown. 50 =14.2uM, CC 50 >240uM; Figure 13 The viral inhibition rate and cytotoxicity of different concentrations of Miz against VTT infection in BHK-21 cell lines are shown. 50 =4.56uM, CC 50 >240uM. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and examples, but the present invention is not limited to the scope of the examples. The process parameters not specified in the examples of this application can be carried out according to conventional methods, and the raw materials used can be obtained through commercial channels.

[0058] In order to curb the infection of orthopoxvirus, the inventor team of this application used CRISPR / Cas9 technology to perform pan-genome screening of host cells infected by orthopoxvirus, identified the host-dependent gene PAICS gene in the process of orthopoxvirus infection, and verified and clarified the virus-dependence mechanism of this gene, that is, by inhibiting the folic acid metabolism pathway and / or inhibiting the biosynthesis of purine nucleotides (de novo synthesis pathway and / or salvage synthesis pathway), the proliferation of orthopoxvirus in host cells can be effectively inhibited, and the infection of host cells by orthopoxvirus can be effectively inhibited.

[0059] In response to the ongoing global problem of orthopoxvirus infection, the present invention plays a significant role in the development and screening of related preventive or therapeutic drugs in the future, while also expanding the new clinical applications of existing drugs including methotrexate, mercaptopurine and mizoribine.

[0060] Example 1: Screening and identification of host-dependent genes in orthopoxvirus-infected host cells

[0061] The inventor designed a set of experiments, such as Figure 1 The experimental process shown is to screen and identify host-dependent genes of orthopoxvirus-infected host cells, so as to find relevant targets and antiviral drugs to block the proliferation of orthopoxvirus in host cells.

[0062] The specific operations are as follows:

[0063] 1. Construction of a monoclonal cell line stably expressing Cas9 protein

[0064] The inventors artificially constructed a stable cell line that stably expresses the Cas9 protein. The lentiviral packaging plasmid transfection method was used to transfer a plasmid carrying the Cas9 gene (purchased from Addgene, catalog number Plasmid #52961) into human THP-1 cells (human peripheral blood mononuclear cells, from Wuhan Pronocell Life Sciences Co., Ltd., China). The plasmid also carries the BSD resistance gene (Blasticidin). Through BSD resistance screening, a THP-1-Cas9 monoclonal cell line that stably expresses the Cas9 protein was obtained.

[0065] 2. Construction and purification of VTT strains carrying reporter genes

[0066] The inventors used the plasmid transfection method to transfer a plasmid carrying green fluorescent protein (GFP) and luciferase (luc) reporter genes (synthesized at GenScript Biotech) into the wild VTT strain (Vaccinia virus Tiantan strain, VTT), a representative model strain of the orthopoxvirus genus (donated by Professor Lu Lu's research group at the School of Basic Medical Sciences, Fudan University; Note: The original strains of the orthopoxvirus genus are highly contagious viruses and are not allowed to be operated in laboratories below biosafety level 3. Currently, the attenuated live vaccine VTT strain of vaccinia virus is suitable for easy operation in laboratories with lower safety levels and is also the preventive vaccine for smallpox virus in China.). The plasmid was stably expressed in the VTT strain through plaque gel purification and flow cytometry, and a VTT-GFP-luc monoclonal strain with high virulence and high expression of the reporter gene was obtained.

[0067] 3. Screening of candidate host cell genes knocked out using CRISPR / Cas9 technology

[0068] The inventors ordered the Bassik Human CRISPR Knockout Library (Pooled Libraries #101926, #101927, #101928, #101929, #101930, #101931, #101932, #101933, #101934) from Addgene. This lentiviral genome-scale CRISPR library targets approximately 20,500 protein-coding genes in the human genome. The library is divided into nine sublibraries based on the biological categories of the genes, with the knockout proteins encompassing apoptosis, kinase activity, gene expression, membrane proteins, and the proteasome.

[0069] Based on theoretical speculation, the knockout of these protein-coding genes may interfere with the process of orthopoxvirus infection of host cells (such as adsorption, invasion, replication, maturation and release), thereby making the host cells more resistant to orthopoxvirus infection and able to survive for a relatively long time.

[0070] Therefore, the inventors hope to use the above-mentioned CRISPR library to knock out these important protein-coding genes (a single sgRNA only knocks out one gene in a host cell), and then infect the knocked-out host cells with the VTT-GFP-luc monoclonal strain obtained above, and flow-sort the surviving host cells; finally, extract the genomic DNA from the surviving host cells and enrich the sgRNA; the more surviving cells, the higher the enrichment detection results of the sgRNA sequences they contain, thereby finding the target gene corresponding to the sgRNA, which may be a host-dependent gene related to orthopoxvirus infection of host cells.

[0071] For the specific experimental process, see Figure 1 And the following steps:

[0072] 1) Use a transfection reagent to simultaneously transfer the CRISPR library plasmid and packaging plasmid (psPAX2, pMD2.G, pREV) into the HEK-293T tool cell line to amplify the expression of the CRISPR lentiviral library. The library plasmid also synchronously expresses the mCherry reporter gene and the puromycin resistance gene.

[0073] 2) Set the lentiviral MOI (multiplicity of infection) to 0.3 and infect the THP-1-Cas9 cell line with the CRISPR lentiviral library using the centrifugation method. Polybrene was added to promote infection and the cells were cultured in a 37°C / 5% CO2 incubator for 2 days. After 2 days, puromycin was added to the lentiviral-infected cells for drug selection to kill cells that were not infected with the lentivirus.

[0074] 3) After puromycin selection, a portion of the drug-selected host cells was frozen as a control group. The VTT-GFP-luc virus strain was then added to the drug-selected host cell line for infection. The virus infection amount per cell plate was approximately 10,000 PFU (plaque forming units, PFU) and cultured in a 37°C / 5% CO2 incubator for 2 days.

[0075] 4) After infection, surviving mCherry single-positive cell populations (infected with CRISPR lentivirus but not VTT virus) and mCherry+GFP double-positive cell populations (infected with CRISPR lentivirus and VTT virus) were flow-sorted and cryopreserved.

[0076] 5) After freeze-thaw, genomic DNA is extracted and sgRNA gene sequences are amplified by PCR. The gene fragments are recovered and sent for sequencing.

[0077] 6) Through differential analysis of sgRNAs in the control group, mCherry single-positive cell population, and mCherry+GFP double-positive cell population, the host-dependent candidate genes of VTT-infected human THP-1 cells were confirmed.

[0078] See also Figure 2 , is to analyze the enrichment of sgRNA in surviving cells after VTT strain infection after sequencing; Figure 2 It can be seen that after some genes were knocked out, their corresponding sgRNAs were enriched in surviving cells and thus detected more frequently; specifically, Figure 2 The host genes targeted for knockout by the enriched sgRNAs are highlighted in red in the upper right corner. The inventors speculate that knocking out these genes may enhance THP-1 cell resistance to infection with the VTT strain, thereby extending their survival. However, this remains speculation and requires further verification experiments, as detailed below.

[0079] 4. Verification of candidate host cell genes knocked out using CRISPR / Cas9 technology

[0080] The inventors further determined which host genes, among the enriched candidate genes for VTT infection host dependence, could be knocked out to interfere with and inhibit the life cycle of VTT infection.

[0081] The experimental method is as follows: Using homologous recombination, the sgRNA sequence of the candidate gene was introduced into the pMCB320 plasmid (ordered from Addgene, catalog number Plasmid #89359). In addition, the non-targeting knockout control sgRNA sequence (Nontargeting controls) from the corresponding library was introduced. After obtaining candidate gene knockout cell lines and non-targeting knockout control cell lines using the same method as described above for candidate gene screening, the cells were infected with the VTT-GFP-luc virus, with the virus infection amount per well being approximately 400 PFU. The difference in luc expression between the experimental and control groups was detected. The greater the amount of VTT infection and proliferation, the higher the luc expression, thus confirming whether knockout of the candidate gene inhibits VTT infection in host cells.

[0082] See also Figure 3 , the effect of knocking out the host-dependent candidate genes of VTT infection on viral infection; the numbers represent different sgRNA sequences for the same knockout gene (the same target gene can be knocked out using sgRNAs with different sequences, Luc: luciferase expression, KO: gene knockout, **: P < 0.01). Figure 3 The results clearly show that knocking out the PAICS gene in human THP-1 host cells can significantly inhibit VTT infection.

[0083] 5. Analysis of PAICS gene and its function

[0084] PAICS gene, full name: Bifunctional phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazole succinocarboxamide synthetase (Bifunctional phosphoribosylaminoimidazole carboxylase / phosphoribosylaminoimidazole succinocarboxamide synthetase).

[0085] The above-mentioned phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazole succinylformamide synthetase encoded by the PAICS gene are bifunctional enzymes. The N-terminal region has phosphoribosylaminoimidazole carboxylase activity, and the C-terminal region has phosphoribosylaminoimidazole succinylformamide synthetase activity. The most basic function of this bifunctional enzyme is to participate in the de novo synthesis pathway of purine nucleotides. By regulating the de novo synthesis of purines, it plays a key role in maintaining the balance of purine metabolism in cells.

[0086] See also Figure 4Schematic diagram of the folate metabolic cycle and the de novo purine nucleotide synthesis pathway; the translation of the English characters in the figure is as follows. Folate Cycle: Folate cycle; De novo purine synthesis: Purine de novo synthesis; DHF: dihydrofolate; DHFR: dihydrofolate reductase; THF: tetrahydrofolate; N 5 -Formyl-THF: 5-Formyl-tetrahydrofolate; N 10 -Formyl-THF: 10-formyl-tetrahydrofolate; PRPP: phosphoribosyl pyrophosphate; PRA: 5-phosphoribosamine; GAR: glycinamide nucleotide; FGAR: formylglycinamide nucleotide; FGAM: formylglycinamido nucleotide; AIR: 5-aminoimidazole nucleotide; CAIR: 5-aminoimidazole-4-carboxylic acid nucleotide; SAICAR: 5-aminoimidazole-4-succinyl nucleotide; AICAR: 5-aminoimidazole-4-carboxamide nucleotide; FAICAR: 5-formamidoimidazole-4-carboxamide nucleotide; IMP: inosine nucleotide; PAICS: bifunctional phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazole succinamide synthetase.

[0087] The de novo synthesis of purine nucleotides ultimately leads to the synthesis of IMP (inosine monophosphate), which can be converted into AMP and GMP. The synthesis of IMP begins with PRPP (phosphoribosylpyrophosphate) and proceeds through 10 basic reactions to produce IMP.

[0088] The protein encoded by the PAICS gene (phosphoribosylaminoimidazole carboxylase / phospho-boronaminoimidazole succinylcarboxamide synthetase) is a key catalytic molecule in the de novo synthesis pathway of purine nucleotides in cells. It can catalyze the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxylaminoimidazole ribonucleotide (CAIR) to N-succinylformamide-5-aminoimidazole ribonucleotide (SAICAR), which are the 6th and 7th basic reactions of this pathway.

[0089] In addition, 10-formyl-tetrahydrofolate (N 10 -Formyl-THF) participates in the 3rd and 9th steps of the de novo synthesis pathway of purine nucleotides.

[0090] Based on the experimental results obtained in parts 3-4 of Example 1 above, and the de novo synthesis pathway of purine nucleotides in which the synthetase encoded by the PAICS gene participates, combined with the theoretical basis of the existing technology, purine nucleotides (including AMP and GMP) play an important role in cell metabolism and biosynthesis; after infection, orthopoxviruses need to utilize purine nucleotides produced by the host metabolic system for their own proliferation.

[0091] The inventors speculate that blocking the synthesis pathway of purine nucleotides may inhibit the infection of orthopoxviruses to host cells. The synthesis pathway of purine nucleotides includes Figure 4 The de novo synthesis pathway shown and Figure 10 The salvage synthetic pathway is shown.

[0092] For further verification, the inventors of the present application selected three approved anti-purine nucleotide anabolic drugs in the prior art: methotrexate, mercaptopurine, and mizoribine, based on multiple experiments and theoretical analysis, to verify the mechanism of inhibiting the infection of orthopoxvirus to host cells.

[0093] Example 2 Methotrexate (MTX) inhibits VTT virus infection of host cells

[0094] 1. MTX inhibits VTT virus infection in Vero-E6 and BHK-21 cell lines.

[0095] See also Figure 4 The folic acid metabolic pathway can be blocked by MTX interference, which in turn leads to the obstruction of the de novo synthesis of purine nucleotides.

[0096] MTX, as a synthetic dihydrofolate analogue, mainly competitively inhibits dihydrofolate reductase, resulting in the inability of physiological dihydrofolate to be reduced to physiologically active tetrahydrofolate (an important coenzyme for the synthesis of purine nucleotides in the body), thereby hindering the transfer of one-carbon groups in the biosynthesis of purine nucleotides and inhibiting the biosynthesis of DNA.

[0097] MTX can interfere with and block the folic acid metabolic pathway, thereby inhibiting the biosynthesis of purine nucleotides; combined with the experimental results of Example 1 of the present application and the above-mentioned inventors' speculation, MTX may further affect the infection of orthopoxvirus.

[0098] For verification, the inventors used luciferase luminescence assay to detect the viral inhibition rate of MTX (ordered from MedChemExpress, catalog number HY-14519) after inhibiting VTT strain infection of Vero-E6 and BHK-21 cell lines.

[0099] The detection method is as follows: Vero-E6 and BHK-21 cells were seeded into 96-well plates and incubated in a 37°C / 5% CO2 incubator overnight. After the incubation, different concentrations of MTX dilutions (3-fold gradient dilution, the highest concentration is 11uM) were mixed with an equal volume of VTT-GFP-luc strain, each containing approximately 400 PFU of virus. The mixture was added to Vero-E6 and BHK-21 cells and incubated in a 37°C / 5% CO2 incubator for 48 hours. The control group was dimethyl sulfoxide (DMSO, ordered by MedChemExpress, product number HY-Y0320). The luciferase expression level of the VTT-GFP-luc strain in Vero-E6 and BHK-21 cells was detected according to the instructions of the luciferase detection reagent (ordered by Promega, product number E1501). The half-effective concentration (EC50) of MTX to inhibit the virus was calculated by the Spearman–Karber method. 50 ).

[0100] See also Figure 5 and 6 The black curves in the two figures are the results of MTX inhibiting the virus inhibition rate of VTT infected host cell lines (Vero-E6, BHK-21 cell lines), EC 50 The results showed that methotrexate can effectively inhibit the proliferation of VTT in Vero-E6 and BHK-21 cells.

[0101] 2. Effect of MTX on the proliferation activity of Vero-E6 and BHK-21 cell lines.

[0102] MTX may affect the proliferation activity of host cells. To evaluate the toxicity of the drug, the inventors used a CCK-8 cell proliferation assay to detect the effect of MTX on the proliferation activity of Vero-E6 and BHK-21 cell lines.

[0103] The detection method is as follows: Vero-E6 and BHK-21 cells were seeded into 96-well plates and incubated in a 37°C / 5% CO2 incubator overnight. After the incubation, different concentrations of MTX dilutions (3-fold gradient dilution, the highest concentration is 11uM) were mixed with an equal volume of DMEM (Dulbecco's Modified Eagle Medium, DMEM, ordered by Gibco, product number C11995500BT) cell culture medium, and the mixture was added to Vero-E6 and BHK-21 cells and incubated in a 37°C / 5% CO2 incubator for 48 hours. The control group was DMSO. The cell proliferation activity of Vero-E6 and BHK-21 cells after treatment with MTX was detected according to the instructions of the CCK-8 cell proliferation reagent (ordered by Shanghai Life-iLab Biotech, product number AC11L054). The half-toxic concentration (CC50) of MTX that affects the proliferation of Vero-E6 and BHK-21 cells was calculated by the Spearman–Karber method. 50 ).

[0104] See also Figure 5 and 6 The yellow curves in the two figures are the results of MTX affecting the proliferation activity of host cell lines (Vero-E6 and BHK-21 cell lines); the results show that methotrexate does not produce obvious toxicity to Vero-E6 and BHK-21 cells when it reaches the half effective concentration of virus inhibition. 50 All greater than 11uM.

[0105] 3. Folinic acid can antagonize the inhibitory effect of MTX on VTT-infected host cells.

[0106] Based on existing theories, see Figure 4 , folinic acid (Leucovorin, N 5 -Formyl-THF) can be used as a derivative of tetrahydrofolic acid to convert into N 10 -Formyl-THF, thereby antagonizing the inhibitory effect of MTX on folic acid metabolism and is usually used together with MTX as a rescue agent.

[0107] To further verify that "the inhibitory effect of MTX on VTT-infected host cells is based on the blocking of the folic acid metabolic pathway by MTX", the inventors of the present application detected the changes in the viral inhibition rate of MTX inhibiting VTT strain infection of Vero-E6 and BHK-21 cell lines after supplementation of folinic acid through luciferase luminescence experiments.

[0108] The detection method is as follows: Vero-E6 and BHK-21 cells were seeded into 96-well plates and placed in a 37°C / 5% CO2 incubator for overnight incubation. After the incubation, different concentrations of MTX dilutions (3-fold gradient dilution) were mixed with an equal volume of VTT-GFP-luc strains. Each portion contained approximately 400 PFU of virus. The mixture was added to Vero-E6 and BHK-21 cells, and different concentrations of folinic acid (ordered from MedChemExpress, product number HY-17556) dilutions were supplemented and placed in a 37°C / 5% CO2 incubator for incubation for 48 hours. The control group was DMSO. The luciferase expression level of the VTT-GFP-luc strain in the proliferation of Vero-E6 and BHK-21 cells was detected according to the instructions of the luciferase detection reagent. The half-effective concentration (EC50) of MTX to inhibit the virus was calculated by the Spearman–Karber method. 50 ).

[0109] See also Figure 7 and 8 , which is the result of folinic acid antagonizing MTX to inhibit the viral inhibition rate of VTT infected host cells. It can be seen that different concentrations of folinic acid (10uM, 100uM) increased the EC 50 The results showed that folinic acid could significantly antagonize the inhibitory effect of methotrexate on VTT-infected host cells and increase its EC value for VTT-infected Vero-E6 and BHK-21 cells. 50 , which further verified that the inhibitory effect of MTX on VTT-infected host cells is based on the blocking of the folic acid metabolic pathway by MTX.

[0110] Example 3: Methotrexate inhibits MPXV infection of host cells

[0111] The above experimental results prove that the inhibitory effect of MTX on VTT-infected host cells is based on MTX's blocking of the folate metabolic pathway and the synthesis of purine nucleotides; the mechanism of this inhibitory effect is also applicable to other orthopoxvirus-infected host cells.

[0112] To further verify, the inventors detected the inhibitory effect of MTX on MPXV (monkeypox virus) infection of host cells.

[0113] The inventors used the 50% plaque reduction neutralization test (PRNT) 50) To detect the viral inhibition rate of methotrexate after inhibiting MPXV infection of Vero-E6 cell line.

[0114] The detection method is as follows: Vero-E6 cells were seeded in a 12-well plate and incubated in a 37°C / 5% CO2 incubator overnight. After the incubation, different concentrations of methotrexate dilutions (final concentrations from high to low are 10000nM, 3333nM, 1111nM, 370nM, 123nM, 41nM, 14nM, 5nM) were mixed with an equal volume of MPXV (MPXV-B.1-China-C-Tan-CQ01). Each portion contained approximately 250 PFU of virus and was placed in a 37°C / 5% CO2 incubator for 90 minutes. The control group was Tecovir. After the incubation, the mixture was added to the Vero-E6 cells and incubated in a 37°C / 5% CO2 incubator for 90 minutes. Then, the mixture was discarded and Vero-E6 cells were covered with Dulbecco's minimal essential medium (DMEM, Gibco, Cat. No. 11965092) containing 0.5% methylcellulose and 2.5% inactivated fetal bovine serum, and incubated in a 37°C / 5% CO2 incubator for 2 days. The number of virus plaques formed was counted, and the half-maximal effective concentration (EC50) of MTX to inhibit virus was calculated by the Spearman–Karber method. 50 ).

[0115] See also Figure 9 , is the viral inhibition rate and cytotoxicity results of MTX inhibiting MPXV infection in host cell lines, EC 50 275.42nM, CC 50 Greater than 10000nM, the results indicate that the drug methotrexate can effectively inhibit the proliferation of MPXV in the host Vero-E6 cells.

[0116] Example 4: Mercaptopurine (6-MP) inhibits VTT infection of host cells Figure 4 The de novo synthesis pathway of purine nucleotides. When the host cell's metabolic needs increase (such as orthopoxvirus infection), the purine nucleotide salvage synthesis pathway (such as Figure 10 can reduce the pressure of de novo synthesis and increase the production of purine nucleotides.

[0117] like Figure 10As shown, the salvage synthesis pathway of purine nucleotides primarily involves three enzymes: adenosine kinase (ADK), adenine phosphoribosyltransferase (APRT), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The English characters in the figure are translated as follows: Purine salvage synthesis; IMP: inosine monophosphate; ADSS: adenyloylsuccinate synthetase; AMP: adenine monophosphate; IMPDH: inosine monophosphate dehydrogenase; GMP: guanine monophosphate; APRT: adenine phosphoribosyltransferase; PRPP: phosphoribosyl pyrophosphate; Adenosine: adenosine; ADA: adenosine deaminase; Adenine: adenine; Inosine: inosine; Hypoxanthine: hypoxanthine; HGPRT: hypoxanthine-guanine phosphoribosyltransferase; Guanosine: guanosine; Guanine: guanine.

[0118] from Figure 10 It can be seen from the salvage synthesis pathway of purine nucleotides that mercaptopurine (6-MP), as a hypoxanthine analogue, can inhibit HGPRT activity, thereby partially blocking the salvage synthesis pathway of purine nucleotides, resulting in a decrease in the synthesis of purine nucleotides in host cells.

[0119] Combined with the experimental results of Example 1 of the present application, and the inventors' speculation, 6-MP may inhibit the infection of orthopoxvirus by inhibiting the salvage synthesis pathway of purine nucleotides.

[0120] 1. 6-MP inhibits VTT infection of Vero-E6 cell line.

[0121] The inventors used a luciferase luminescence assay to detect the viral inhibition rate of 6-MP (ordered from MedChemExpress, catalog number HY-13677) after inhibiting the infection of the Vero-E6 cell line with the VTT strain.

[0122] The detection method is as follows: Vero-E6 cells were inoculated into 96-well plates and placed in a 37°C / 5% CO2 incubator for overnight incubation. After the incubation, different concentrations of 6-MP dilutions (3-fold gradient dilution, the highest concentration is 410uM) were mixed with an equal volume of VTT-GFP-luc strain. Each portion contained approximately 400PFU of virus. The mixture was added to Vero-E6 cells and placed in a 37°C / 5% CO2 incubator for incubation for 48 hours. The control group was DMSO. The luciferase expression level of the VTT-GFP-luc strain in the proliferation of Vero-E6 cells was detected according to the instructions of the luciferase detection reagent. The half effective concentration (EC50) of 6-MP to inhibit the virus was calculated by the Spearman–Karber method. 50 ).

[0123] Test results see Figure 11 The black curve is the result of 6-MP inhibiting the virus inhibition rate of VTT infected host cell lines, EC 50 The results showed that the drug mercaptopurine can effectively inhibit the proliferation of VTT in Vero-E6 cells.

[0124] 2. Effect of 6-MP on the proliferation activity of Vero-E6 cell line.

[0125] 6-MP may affect the proliferation activity of host cells. To evaluate the toxicity of the drug, the inventors used a CCK-8 cell proliferation assay to detect the effect of 6-MP on the proliferation activity of the Vero-E6 cell line.

[0126] The detection method is as follows: Vero-E6 cells were seeded into 96-well plates and incubated in a 37°C / 5% CO2 incubator overnight. After the incubation, different concentrations of 6-MP dilutions (3-fold gradient dilution, the highest concentration is 410uM) were mixed with an equal volume of DMEM cell culture medium, and the mixture was added to the Vero-E6 cells and incubated in a 37°C / 5% CO2 incubator for 48 hours. The control group was DMSO. The cell proliferation activity of Vero-E6 cells after treatment with 6-MP was detected according to the instructions of the CCK-8 cell proliferation reagent. The half-toxic concentration (CC) of 6-MP that affects the proliferation of Vero-E6 cells was calculated by the Spearman–Karber method. 50 ).

[0127] Test results see Figure 11 ,,The yellow curve is the result of 6-MP affecting the proliferation activity of host cell lines, CC 50 Greater than 410uM, the result shows that the drug mercaptopurine does not cause obvious toxicity to Vero-E6 cells when it reaches the half effective concentration for inhibiting the virus.

[0128] Example 5: Mizoribine (Miz) inhibits VTT infection of host cells

[0129] Figure 10 In the salvage synthesis pathway of purine nucleotides, IMP can be oxidized by inosine monophosphate dehydrogenase (IMPDH) to generate xanthine, which is then catalyzed by guanylate synthetase (GMPS) to accept the amino group of glutamine to generate GMP.

[0130] Mizoribine (Miz), as an imidazole nucleoside antimetabolite, can inhibit IMPDH activity, thereby partially blocking the GMP synthesis pathway and leading to a decrease in the synthesis of guanine nucleotides in host cells.

[0131] Combined with the experimental results of Example 1 of the present application, and the inventors' speculation, Miz may inhibit the infection of orthopoxvirus by inhibiting the salvage synthesis pathway of purine nucleotides.

[0132] 1. Miz inhibits VTT infection of Vero-E6 and BHK-21 cell lines.

[0133] The inventors used a luciferase luminescence assay to detect the viral inhibition rate of Miz (ordered from MedChemExpress, catalog number HY-17470) after inhibiting VTT strain infection of Vero-E6 and BHK-21 cell lines.

[0134] The detection method is as follows: Vero-E6 and BHK-21 cells were seeded in 96-well plates and placed in a 37°C / 5% CO2 incubator for overnight incubation. After the incubation, different concentrations of Miz dilutions (3-fold gradient dilution, the highest concentration is 240uM) were mixed with an equal volume of VTT-GFP-luc strain. Each portion contained approximately 400PFU of virus. The mixture was added to Vero-E6 and BHK-21 cells and placed in a 37°C / 5% CO2 incubator for incubation for 48 hours. The control group was DMSO. The luciferase expression level of the VTT-GFP-luc strain in the proliferation of Vero-E6 and BHK-21 cells was detected according to the instructions of the luciferase detection reagent. The half-effective concentration (EC50) of Miz to inhibit the virus was calculated by the Spearman–Karber method. 50 ).

[0135] Test results see Figure 12 and 13 The black curves in the two figures are the results of Miz inhibiting the viral inhibition rate of VTT infection in host cell lines (Vero-E6 and BHK-21 cell lines), EC 50 The results showed that mizoribine can effectively inhibit the proliferation of VTT in Vero-E6 and BHK-21 cells.

[0136] 2. Effect of Miz on the proliferation activity of Vero-E6 and BHK-21 cell lines.

[0137] Miz may affect the proliferation activity of host cells. To evaluate the toxicity of the drug, the inventors used a CCK-8 cell proliferation assay to detect the effect of Miz on the proliferation activity of Vero-E6 and BHK-21 cell lines.

[0138] The detection method is as follows: Vero-E6 and BHK-21 cells were seeded in 96-well plates and placed in a 37°C / 5% CO2 incubator for overnight incubation. After the incubation, different concentrations of Miz dilutions (3-fold gradient dilution, the highest concentration is 240uM) were mixed with an equal volume of DMEM cell culture medium, and the mixture was added to Vero-E6 and BHK-21 cells, and placed in a 37°C / 5% CO2 incubator for incubation for 48 hours. The control group was DMSO. The cell proliferation activity of Vero-E6 and BHK-21 cells after Miz treatment was detected according to the instructions of the CCK-8 cell proliferation reagent. The half-toxic concentration (CC) of Miz that affects the proliferation of Vero-E6 and BHK-21 cells was calculated by the Spearman–Karber method. 50 ).

[0139] See also Figure 12 and 13 The yellow curves in the two figures are the results of Miz affecting the proliferation activity of host cell lines (Vero-E6 and BHK-21 cell lines), CC 50 Both were greater than 240uM; the results showed that the drug mizoribine did not produce obvious toxicity to host cells (Vero-E6 and BHK-21 cells) when it reached the half-effective concentration for inhibiting the virus.

[0140] The above-mentioned methotrexate (MTX), mercaptopurine (6-MP) and mizoribine (Miz) are three approved anti-purine nucleotide anabolic drugs in the prior art.

[0141] Methotrexate inhibits folic acid metabolism, leading to a decrease in the production of 10-formyl-tetrahydrofolate, a component of the de novo purine nucleotide synthesis pathway, thereby reducing the biosynthesis of purine nucleotides. Methotrexate was first approved for marketing in the United States in 1947 and was originally used to treat various types of acute leukemia, head and neck cancer, lung cancer, various soft tissue sarcomas, psoriasis, and autoimmune diseases such as breast cancer, ovarian cancer, cervical cancer, malignant hydatidiform mole, choriocarcinoma, testicular cancer, and rheumatoid arthritis.

[0142] Mercaptopurine, as a hypoxanthine analogue, can inhibit HGPRT activity, thereby partially blocking the purine nucleotide salvage synthesis pathway and leading to a decrease in the synthesis of purine nucleotides in cells.

[0143] Mercaptopurine was first approved for marketing in the United States in 1953 and was originally used to treat choriocarcinoma, malignant hydatidiform mole, acute lymphocytic leukemia, acute non-lymphocytic leukemia, and the blast crisis of chronic myeloid leukemia.

[0144] Mizoribine is an imidazole nucleoside antimetabolite that can inhibit IMPDH activity, thereby partially blocking the purine nucleotide salvage synthesis pathway and leading to a decrease in the synthesis of purine nucleotides in cells.

[0145] Mizoribine was first used in clinical treatment in Japan in 1991. It was originally used to inhibit renal transplant rejection, autoimmune diseases such as lupus nephritis, rheumatoid arthritis and nephrotic syndrome.

[0146] In recent years, although some nucleotide and nucleoside analog inhibitors have been tried for the treatment of certain acute and chronic viral infections, such as herpes simplex virus, human immunodeficiency virus, cytomegalovirus, varicella-zoster virus, hepatitis B and hepatitis C virus infections.

[0147] However, only cytarabine has been shown to be active against orthopoxvirus infections. Note: Common nucleotide analogs approved for clinical treatment include lamivudine, adenosine, stavudine, abacavir, zidovudine, and cytarabine.

[0148] These common nucleotide analogs approved for clinical treatment are, first of all, safe and tolerable drugs that are used by viruses rather than by human polymerases for DNA replication; secondly, they are also the basic preparations for a variety of anti-cancer drugs. Nucleotide and nucleoside analogs are important drugs that inhibit the DNA replication process of tumor cells and have definite clinical efficacy.

[0149] Based on the virus-dependent mechanisms of host-dependent genes during orthopoxvirus infection revealed by the inventors of this application, as well as the relevant knowledge gained from other metabolic pathways, the design of antiviral prodrugs will be more inclusive in the future. These can be applied as a single drug or in combination with multiple existing drugs, such as other antiviral drugs, or cell-targeted drugs combined with one or more prodrugs to combat one or more viral infections.

[0150] The present invention is not limited to the above-described embodiments. The scope of protection of the present invention is defined by the appended claims. Those skilled in the art may make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of the present invention. All of these changes, modifications, substitutions, combinations, and simplifications are equivalent and fall within the scope of protection of the present invention.

Claims

1. Use of the PAICS gene and / or PAICS protein as a target in any of the following: (A1) Application in developing or screening agents for preventing and / or treating orthopoxvirus infection; (A2) Application in regulating the susceptibility of host cells or animals to orthopoxviruses; (A3) Application of orthopoxviruses in regulating their replication ability in host cells or animals; (A4) Application in the preparation of reagents for regulating the susceptibility of host cells or animals to orthopoxviruses; (A5) Use in the preparation of reagents for regulating the replication ability of orthopoxvirus in host cells or animals.

2. Use of PAICS-targeting agents in any of the following: (B1) Use in the preparation of agents for treating and / or preventing orthopoxvirus-infected diseases; (B2) Application in the preparation of a reagent for inhibiting orthopoxvirus replication in a host cell: (B3) Application in preparing cell models or animal models with enhanced resistance to orthopoxvirus.

3. The use according to claim 2, characterized in that: The PAICS-targeting agent is an agent that inhibits, reduces, knocks out, knocks down or knocks down the expression level of the PAICS gene, or inhibits, reduces or inactivates the activity of the expression product of the PAICS gene.

4. The use according to claim 3, characterized in that: The agent is selected from small molecule inhibitors, carbohydrates, lipids, proteins, polypeptides, nucleic acid molecules or interfering viruses; Preferably, the agent is selected from any one of the following: small molecule inhibitors, antisense oligonucleotides, siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA, esiRNA, antibodies or antigen-binding fragments thereof.

5. Use of agents that inhibit folic acid metabolic pathways in any of the following: (C1) Use in the preparation of agents for treating and / or preventing orthopoxvirus-infected diseases; (C2) Application in the preparation of a reagent for inhibiting orthopoxvirus replication in a host cell: (C3) Application in preparing cell models or animal models with enhanced resistance to orthopoxvirus.

6. The use according to claim 5, characterized in that: The agents that inhibit the folic acid metabolic pathway include those that inhibit 10-formyl-tetrahydrofolate (N 10 -Formyl-THF) synthesis reagent; Preferably, the agent that inhibits the folate metabolic pathway includes a folate antagonist; more preferably, the folate antagonist includes methotrexate or a chemically modified derivative thereof.

7. Use of an agent that inhibits the de novo synthesis pathway and / or salvage synthesis pathway of purine nucleotides in any of the following; (D1) Use in the preparation of agents for treating and / or preventing orthopoxvirus-infected diseases; (D2) Application in the preparation of a reagent for inhibiting orthopoxvirus replication in a host cell: (D3) Application in preparing cell models or animal models with enhanced resistance to orthopoxvirus.

8. The use according to claim 7, characterized in that: The agents that inhibit the de novo synthesis pathway and / or salvage synthesis pathway of purine nucleotides include agents that inhibit purine nucleotide anabolism; Preferably, the agent that inhibits purine nucleotide synthesis and metabolism includes mercaptopurine, mizoribine or methotrexate or their chemically modified derivatives.

9. The use according to any one of claims 1 to 8, characterized in that: The orthopoxvirus is selected from any one or combination of the genus Orthopoxvirus of the family Poxviridae; preferably, the orthopoxvirus is selected from any one or combination of monkeypox virus, vaccinia virus, smallpox virus or cowpox virus.

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