Use of ufmylation modulators in the preparation of a medicament for the treatment of HIV infection

Abstract

The application discloses application of a UFMylation regulator in preparation of a medicine for resisting HIV infection. + The application evaluates the inhibiting effect of the UFMylation regulator, namely, the UFSP2 inhibitor Compound-8, on HIV virus through in-vivo and in-vitro pharmacodynamic experiments. The experimental results show that Compound-8 can significantly inhibit the replication of HIV virus in MT-4 cells, primary CD4 T cells and mice, can maintain a low viral load after drug withdrawal, and has no obvious influence on the proliferation of normal cells and the weight of mice, and has good anti-HIV infection effect and high safety. The application firstly discloses that Compound-8 effectively inhibits HIV-1 replication, indicates that Compound-8 can be used as a candidate medicine for HIV-1 treatment medicine, provides a new treatment strategy for AIDS, and has a wide application prospect in the field of anti-HIV infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of UFMylation modulators in the preparation of drugs for treating HIV infection. Background Technology

[0002] AIDS (Acquired immune deficiency syndrome) is an infectious disease caused by the human immunodeficiency virus (HIV), which seriously threatens human health. Currently, the most widely used clinical treatment is antiretroviral therapy (ART), which effectively suppresses HIV replication, rebuilds the immune system of HIV / AIDS patients, improves their quality of life, prolongs survival, and reduces mortality. Although ART can suppress viral replication, viral rebound is inevitable after discontinuation due to the presence of a viral reservoir, posing multiple risks to patient health, treatment regimens, and public health. Identifying key pathways and novel factors regulating HIV-1 replication, and developing HIV-1 prevention and control strategies accordingly, is a crucial breakthrough for ultimately achieving a functional cure for AIDS.

[0003] UFMylation modification is a newly identified ubiquitination-like modification in recent years, conserved in almost all eukaryotes. Through a cascade reaction of three enzymes—E1 (UBA5), E2 (UFC1), and E3 (UFL1)—and with the synergistic action of cofactors DDRGK1 and CDK5RAP3, the ubiquitin-like molecule UFM1 covalently binds to the lysine residues of the substrate, thereby regulating the protein abundance, biological activity, and cellular localization of the substrate, playing a crucial regulatory role in various life processes. Simultaneously, like ubiquitination, UFMylation modification is reversible; the UFM1 bound to the substrate protein undergoing UFMylation modification can be removed by the de-UFMylation enzyme UFSP2, thus forming a cyclical regulatory pattern. Therefore, regulating the activity state of UFSP2 is an effective way to regulate UFMylation modification. The UFMylation regulator Compound-8 is a covalent inhibitor of the de-UFMylation enzyme UFSP2, effectively inhibiting UFSP2 activity and enhancing UFMylation modification. There are currently no reports on the use of UFMylation modulators in the preparation of anti-HIV infection drugs. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide the application of a UFMylation modulator in the preparation of drugs for treating HIV infection.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides the use of a UFMylation modulator in the preparation of a drug for treating HIV infection, wherein the UFMylation modulator is a UFSP2 inhibitor.

[0006] In this invention, HIV refers to Human Immunodeficiency Virus, a retrovirus with two subtypes: HIV-1 and HIV-2. The HIV envelope glycoprotein and CD4... + T cells infect cells after binding to the CD4 receptor molecule on their surface. + T-cell depletion eventually leads to severe immunodeficiency and the development of AIDS, also known as acquired immunodeficiency syndrome (AIDS).

[0007] Furthermore, the UFMylation modifier is Compound-8 or its derivatives, including pharmaceutically acceptable salts, hydrates, isomers, solvates, crystalline forms, or functional compounds obtained by chemically modifying the Compound-8 core structure, wherein the structure of Compound-8 is shown in Formula (I).

[0008]

[0009] Equation (Ⅰ).

[0010] In some embodiments, the derivative refers to a compound whose structure is modified or functionalized by chemical, physical, or biological methods, such as by introducing new functional groups, changing the molecular conformation, or combining with other molecules, thereby obtaining a compound with enhanced or expanded functions while maintaining the core structure and function of the parent compound. Exemplary derivatives include compounds obtained by introducing new functional groups into the parent compound, such as through esterification, acylation, or sulfonation.

[0011] In some embodiments, the pharmaceutically acceptable salt refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali (including inorganic and organic alkalis). Salts derived from inorganic alkalis include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese sulfide salts, potassium salts, sodium salts, zinc salts, etc. Specific embodiments include calcium salts, magnesium salts, zinc salts, cesium salts, potassium salts, sodium salts, and salts of organic amines. Salts derived from pharmaceutically acceptable, non-toxic organic amines include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and salts from basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, heparin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine, leucine, isoleucine, methionine, alanine, meglumine, etc.

[0012] In some embodiments, the solvate refers to an association or complex of one or more solvent molecules with a compound. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide (DMSO), ethyl acetate, acetic acid, acetonitrile, and ethanolamine. The hydrate refers to a complex in which the solvent molecules are water.

[0013] In some implementations, the crystalline form refers to the process and morphology of a substance forming crystals from a gaseous, liquid (solution or melt), or amorphous solid state through ordered arrangement. Common methods include evaporation crystallization, cooling crystallization, recrystallization, and precipitation crystallization. Crystal morphologies vary, mainly including needle-like, polygonal plate-like, dendritic, and spherical shapes, and are often characterized by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), and microscopic observation.

[0014] In some embodiments, isomers refer to compounds with the same molecular formula but different structures (or different spatial arrangements). Due to differences in structure or spatial configuration, isomers typically possess different physical, chemical, or biological properties. Isomerism is classified into two main categories: structural isomers, including carbon chain isomers, positional isomers, functional group isomers, and tautomers, which differ in the order of atomic connection in the molecule; and stereoisomers, including configurational isomers (cis-trans isomers or enantiomers) and conformational isomers, which have the same atomic connection order but different spatial orientations.

[0015] In some embodiments, the drug is administered in a therapeutically effective amount in a manner compatible with the dosage form. The dosage and timing of administration depend on the subject to be treated, the subject's ability to utilize the active ingredient, or the desired degree of therapeutic effect. The precise amount of the active ingredient to be administered, such as the UFMylation modifier provided in the first aspect of the invention, depends on the physician's judgment and varies from individual to individual. Suitable dosing regimens are variable, but are represented by an initial dose followed by repeated administration at intervals of one or more hours via subsequent injections or other administrations, or by considering continuous intravenous infusion sufficient to maintain blood concentrations within the range specified for in vivo treatment.

[0016] In some embodiments, an effective amount or therapeutically effective amount refers to an amount of compound sufficient to provide therapeutic or preventive benefit in the treatment or prevention of a disease, or sufficient to delay or minimize disease-related symptoms. Furthermore, a therapeutically effective amount of a compound used in this invention refers to an amount of the compound, alone or in combination with other therapies that provide therapeutic benefit in the treatment or prevention of a disease. When used in conjunction with compounds used in this invention, the term may include amounts that improve overall treatment, reduce or avoid symptoms or causes of a disease, enhance the therapeutic efficacy of another therapeutic agent, or synergize with another therapeutic agent. The full therapeutic effect does not necessarily occur with the administration of a single dose (or a single administration), but may occur only after a series of doses. Therefore, an effective amount may be administered in one or more administrations.

[0017] Furthermore, the concentration of Compound-8 is not less than 1.25 μg / ml.

[0018] Furthermore, the concentration of Compound-8 is 1.25-10 μg / ml.

[0019] Furthermore, the concentration of Compound-8 is 5 μg / ml.

[0020] In some embodiments, the subject is an animal, preferably a mammal (human and non-human animals), including but not limited to: humans, non-human primates (especially higher primates, such as macaques, cynomolgus monkeys, stump-tailed macaques, rhesus monkeys, shrews, golden snub-nosed monkeys, and tree shrews), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, any livestock or pets, etc. In a preferred embodiment, the subject is a human.

[0021] Furthermore, the anti-HIV infection includes suppressing early asymptomatic infection and / or treating / preventing late-stage AIDS symptoms.

[0022] In some embodiments, the treatment refers to reducing or alleviating, improving, or eradicating a disease or one or more symptoms associated with the disease. In some embodiments, the term refers to minimizing the spread or worsening of the disease due to the administration of one or more preventative or therapeutic agents to a patient suffering from the disease. For the purposes of the various aspects and embodiments provided by the present invention, treatment includes, but is not limited to, reducing, alleviating, or improving one or more clinical manifestations or side effects of the treated disease or condition, improving one or more clinical outcomes, reducing the severity of the disease, delaying or slowing the progression of the disease, improving, alleviating, or stabilizing the disease state, and other beneficial results described in the present invention.

[0023] In some embodiments, prevention refers to preventing the onset, recurrence, or spread of a disease or symptom, or one or more of its symptoms. In some embodiments, the term refers to treatment with or administration of the compounds provided herein prior to the appearance of symptoms, specifically to a patient at risk of the disease or symptom described herein. The term covers the suppression or reduction of symptoms of a particular disease. In some embodiments, this is specifically a candidate for a prevention program in a subject with a family history of the disease. Furthermore, subjects with a history of recurrent symptoms are also potential candidates for prevention. In this respect, the term "prevention" may be used interchangeably with the term "preventive treatment."

[0024] Furthermore, Compound-8 achieves its anti-HIV infection effect by inhibiting HIV viral replication.

[0025] Furthermore, the effect of Compound-8 in inhibiting HIV viral replication showed no significant rebound within at least 4 weeks after discontinuation of the drug.

[0026] Furthermore, the effect of Compound-8 in inhibiting HIV viral replication showed no significant rebound within at least two weeks after discontinuation of the drug.

[0027] The second aspect of the present invention provides for any of the following applications: 1) The use of the UFMylation modulator described in the first aspect of the present invention in the preparation of a pharmaceutical composition for treating HIV infection, wherein the pharmaceutical composition may also include other drugs that can be used to treat HIV infection.

[0028] 2) The use of the UFMylation modifier described in the first aspect of the present invention in the preparation of a pharmaceutical formulation for treating HIV infection, the pharmaceutical formulation further comprising pharmaceutically acceptable excipients.

[0029] In some embodiments, the pharmaceutical composition refers to any mixture of two or more products, substances, or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. As used herein, the term "pharmaceutical composition" refers to a formulation in which the biological activity of one or more active ingredients is effective and which does not contain other components that would have unacceptable toxicity to a subject to which the formulation is administered. Therefore, it is a pharmaceutical composition suitable for medicinal use in a subject.

[0030] In some embodiments, the two drugs in the pharmaceutical composition can be administered simultaneously, separately, or sequentially. Simultaneous administration means that the two drugs are administered concurrently. If not administered simultaneously, they are administered sequentially within a time frame so that both can be therapeutically effective within the same time frame. Therefore, sequential administration allows for the administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours after administering one drug, provided that the circulating half-life of the first administered drug allows for a simultaneously therapeutically effective amount of both. The time delay between administrations of the components will vary depending on the exact nature of the components, their interactions, and their respective half-lives. This differs from simultaneous or sequential administration, which refers to a significant interval between the administration of one drug and another, meaning that when the second drug is administered, the first administered drug may no longer be present in the bloodstream at a therapeutically effective amount.

[0031] Furthermore, the other drugs that can be used to treat HIV infection are antiretroviral drugs, including nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion enzyme inhibitors, and co-receptor antagonists.

[0032] In some embodiments, the other drugs that can be used to treat HIV infection are not limited to the types of drugs listed above in this invention, and any drug that may be used to treat HIV infection will fall within the protection scope of this invention.

[0033] In some embodiments, the excipients include binders, surfactants, flavoring agents, osmotic pressure regulators, antibacterial agents, solubilizers, antioxidants, and stabilizers. The binders include carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol, povidone, glycerin, polyvinyl alcohol, polyvinylpyrrolidone, polyalkyl styrene, polymethacrylate, and / or polyacrylate. The surfactants include hydroxypropyl-β-cyclodextrin, polyoxyethylene alkyl ethers, Tween, sodium lauryl sulfate, sodium lauryl sulfate, poloxamer, polysorbate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, and / or polyethylene glycol alkyl groups. The flavoring agents include sodium saccharin, cyclamate, aspartame, acetylsupan-K, glycerin, sorbitol, mannitol, sucrose, simple syrup, and aromatic syrup. The osmotic pressure regulators include sodium chloride, potassium chloride, mannitol, glycerin, and / or other pharmaceutically or traditional Chinese medicine-acceptable osmotic pressure regulators. The antibacterial agents include BAK, benzalkonium chloride, sorbic acid, oxychloride complex, citric acid, chlorobutanol, thimerosal, phenylmercuric acetate, disodium ethylenediaminetetraacetate, phenylmercuric nitrate, perborate, and / or benzyl alcohol. The cosolvents include hydrochloric acid, phosphoric acid, propionic acid, acetic acid, lactic acid, citric acid, tartaric acid, boric acid, glucuronic acid, gluconic acid, lactobionic acid, malic acid, threonic acid, gluconic acid, 2,5-dihydroxybenzoic acid, and acidic amino acids. The antioxidants and stabilizers include sulfurous acid, sulfites, bisulfites, metabisulfites, dithionite, thiosulfates, thiourea, glutathione, dimercaprol, mercaptoacetic acid and its salts, thiolactic acid and its salts, thiodipropionic acid and its salts, gallic acid and its salts, caffeic acid or its pharmaceutical salts, ferulic acid or its pharmaceutical salts, di-tert-butyl-p-phenol, 2,5-dihydroxybenzoic acid or its salts, salicylic acid or its salts, ascorbic acid and its salts, isoascorbic acid and its salts, nicotinamide, tartaric acid, phosphates, pharmaceutical salts of acetate, citrates, EDTA and its salts.

[0034] In some embodiments, the excipients may also include substances recognized in the art and including, for example, substances involved in carrying or transporting any subject composition from one organ or part of the body to another organ or part of the body, including but not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository wax; (9) Oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; (21) other non-toxic compatible substances used in traditional Chinese medicine preparations.

[0035] In specific implementation schemes, the choice of which pharmaceutical excipients to combine with the drug formulation depends on the formulation to be made. When making ordinary formulations, the choice of which pharmaceutical excipients to use is well known to those skilled in the art.

[0036] Furthermore, the dosage form of the pharmaceutical preparation includes oral dosage form, parenteral dosage form, or topical dosage form.

[0037] In some embodiments, the present invention does not particularly limit the dosage form of the pharmaceutical preparation, which can be any dosage form suitable for oral, parenteral, or topical administration, including but not limited to: injections, tablets, capsules, granules, powders, powders for injection, transdermal patches, ointments, gels, suppositories, oral solutions, oral suspensions, emulsions for injection, oral emulsions, sustained-release tablets, and controlled-release tablets. All of the above-mentioned dosage forms of pharmaceutical preparations can be prepared according to conventional methods in the pharmaceutical field.

[0038] In some embodiments, the pharmaceutical preparation is administered systemically or topically, such as orally (e.g., using capsules, powders, solutions, suspensions, tablets, sublingual tablets, etc.), by inhalation (e.g., using aerosols, gases, inhalers, nebulizers, etc.), to the ear (e.g., using ear drops), topically (e.g., using creams, gels, liniments, lotions, ointments, pastes, transdermal patches, etc.), to the eye (e.g., using eye drops, ophthalmic gels, ophthalmic ointments), rectally (e.g., using enemas or suppositories), to the nose, cheek, vagina (e.g., using irrigators, intrauterine devices, vaginal suppositories, vaginal rings, or tablets, etc.), through implanted receptacles, etc., or parenterally, depending on the severity and type of disease being treated.

[0039] A third aspect of the present invention provides a method for inhibiting HIV viral replication in vitro, the method comprising treating an HIV-infected system with the UFMylation modulator described in the first aspect of the present invention.

[0040] Furthermore, the HIV infection system includes HIV-infected cellular systems, subcellular systems, tissue systems, or organ systems.

[0041] Furthermore, the HIV-infected cell system includes HIV-infected MT-4 cells and CD4+ cells. + T cells.

[0042] Furthermore, the method described is not for therapeutic purposes.

[0043] In some implementation schemes, the specific dosage of the UFMylation modifier can be conventionally selected and adjusted by those skilled in the art based on actual circumstances.

[0044] Advantages and beneficial effects of the present invention: This invention evaluates the inhibitory effect of Compound-8, a UFMylation modulator and UFSP2 inhibitor, on HIV virus through in vitro and in vivo pharmacodynamic experiments. The results show that Compound-8 significantly inhibits MT-4 cells and primary CD4+ cells. + The drug effectively inhibited HIV replication in T cells and mice, maintaining a low viral load even after drug withdrawal. It also had no significant impact on normal cell proliferation or mouse weight, demonstrating good anti-HIV efficacy and high safety. This invention is the first to reveal that Compound-8 effectively inhibits HIV-1 replication, suggesting its potential as a candidate drug for HIV-1 treatment. It provides a new treatment strategy for AIDS and has broad application prospects in the field of anti-HIV infection. Attached Figure Description

[0045] Figure 1The results of Compound-8 inhibiting HIV viral proteins in MT-4 cells are shown in the figure.

[0046] Figure 2 To suppress the original CD4 in Compound-8 + The results of HIV infection on T cells are shown in the diagram. In the diagram, A is the experimental flowchart and B is the viral infection rate.

[0047] Figure 3 This diagram shows the HIV rebound after discontinuation of Compound-8 medication. In the diagram, A is the experimental flowchart and B is the HIV p24 expression level.

[0048] Figure 4 This is a toxicity assay of Compound-8 against normal immune cells, where A represents the relative viability of MT-4 cells, and B represents primary CD4 cells. + The relative activity of T cells.

[0049] Figure 5 The graph shows the results of Compound-8 inhibiting HIV virus in vivo. In the graph, A is the experimental flowchart, B is the change in viral load before and after drug administration, C is the viral rebound after drug withdrawal, and D is the change in mouse body weight after drug administration.

[0050] Figure 6 This is a flowchart of the preparation process for Compound-8. Detailed Implementation

[0051] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0052] The drugs, reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0053] Example 1: Anti-HIV-1 experiment of Compound-8 in MT-4 cells I. Experimental Methods: 1. Experimental materials: MT-4 cells (NIH AIDS Reagent Program, ARP-120), HIV-NL4-3 (GenBank: MN685337.1), anti-HIV-1 antibody (Abcam, ab63913), GAPDH antibody (Rapid Antibody, RM2002), horseradish peroxidase-conjugated mouse secondary antibody (Jackson, 115-035-062), rabbit secondary antibody (Jackson, 115-035-062), ECL chromogenic substrate (Millipore, WBKLS0050).

[0054] Compound-8 (CAS: 850188-43-1) was synthesized by Beijing Realchem ​​Technology Co., Ltd. The synthesis route is as follows: Figure 6 The synthesis steps are as follows: 27 mg of starting material 1, 5 mL of THF, 14 mg of anhydrous potassium carbonate, and 23 mg of starting material 2 were added to a 25 mL round-bottom flask. The mixture was stirred at room temperature for 4 hours. The solvent was removed by rotary evaporation. The residue was separated by preparative chromatography to obtain 35 mg of the target product 3, which is Compound-8.

[0055] 2. Experimental Procedure: MT-4 cells were cultured at 37°C in a 5% CO2 incubator. The next day, they were infected with HIV-NL4-3 (MOI=0.1) and cultured for another 10 hours. After centrifugation at 1000 rpm for 5 minutes, the virus-containing culture medium was discarded. The cells were then resuspended in culture medium containing different concentrations (0, 1.25, 2.5, 5 μg / mL) of Compound-8 and seeded into the corresponding wells. After culturing at 37°C in a 5% CO2 incubator for 24 hours, the cells were harvested and detected by Western blotting.

[0056] II. Experimental Results The results are as follows Figure 1 As shown, Compound-8 dose-dependently inhibits HIV-1 replication in MT-4 cells, indicating that Compound-8 can effectively inhibit HIV-1 replication at the cellular level.

[0057] Example 2 Compound-8 in primary CD4 + Anti-HIV-1 experiment in T cells I. Experimental Methods See the experimental flowchart. Figure 2 A: CD4 cells isolated from peripheral blood of healthy individuals + T cells were seeded in 24-well plates (3 × 10⁻⁶ cells per well). 5Cells were cultured in 37°C, 5% CO2 incubators (cells / well). The next day, cells were infected with HIV-NL4-3 (MOI=0.1) and cultured for another 10 hours. After centrifugation at 1000 rpm for 5 minutes, the virus-containing culture medium was discarded. The cells were then resuspended in culture medium containing different concentrations (0, 1.25, 2.5, 5, 10 μg / mL) of Compound-8 and seeded into the corresponding wells. The cells were then cultured in 37°C, 5% CO2 incubators.

[0058] Forty-eight hours later, the cell culture supernatant was collected and centrifuged at 12,000 rpm for 5 minutes to remove cell debris. 60 μL of the cell culture supernatant was seeded into three independent replicates in each 24-well plate. 1 × 10⁶ cells were seeded into each well. 5 TZM-bl cells (ATCC, PTA-5659) were cultured at 37°C in a 5% CO2 incubator. After 48 hours, the culture medium was discarded, and 500 μL of 0.25% trypsin was added to each well for digestion for 5 minutes. Then, 1 mL of complete culture medium was added to stop the digestion reaction, and the cells were centrifuged at 12,000 rpm for 5 minutes to collect the TZM-bl cell pellet.

[0059] The harvested TZM-bl cell pellet was lysed with 60 μL of cell lysis buffer from the Promega luciferase reporter assay kit at room temperature for 30 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. 4 μL of the supernatant was collected and mixed with 20 μL of substrate reaction solution. The chemiluminescence value was read using a GloMax 20 / 20 luminometer (Promega), and the results were recorded to calculate the virus inhibition rate.

[0060] II. Experimental Results The results are as follows Figure 2 As shown in Figure B, 1.25 μg / mL Compound-8 inhibits CD4. + HIV-1 replication in T cells reached over 50%, and Compound-8 at concentrations of 2.5, 5, and 10 μg / mL inhibited CD4+. + HIV-1 replication in T cells increased several tens of times. This indicates that Compound-8 cells in primary CD4 cells... + T cells can effectively inhibit HIV-1 replication.

[0061] Example 3: Compound-8 inhibits viral rebound in HIV-1-infected MT-4 cells after drug withdrawal. I. Experimental Methods MT-4 cells were cultured at 37°C in a 5% CO2 incubator. The following day, cells were infected with HIV-NL4-3 (MOI=0.1) and cultured for another 10 hours. After centrifugation at 1000 rpm for 5 minutes to discard the virus-containing culture medium, the cells were resuspended in azidothymidine (AZT) containing 20 µM of anti-HIV-1 or 5 μg / mL Compound-8 medium and then seeded into the corresponding wells. Cells were then cultured at 37°C in a 5% CO2 incubator. After 3 days, both groups of cells were divided in half. One half was continued to be treated with 20 µM AZT or 5 μg / mL Compound-8, while the other half was replaced with regular culture medium. After 3 days, the cell culture supernatant was collected, and HIV-1 levels were detected using an HIV-1 p24 ELISA kit. A schematic diagram of the experimental procedure is shown below. Figure 3 A.

[0062] II. Experimental Results The results are as follows Figure 3 As shown in Figure B, treatment with 20 µM AZT and 5 μg / mL Compound-8 significantly inhibited viral replication. However, after drug withdrawal, viral rebound occurred in the AZT treatment group, while MT-4 cells treated with Compound-8 maintained a low level of p24 without significant rebound, indicating that Compound-8 can effectively inhibit viral rebound after antiviral treatment compared to AZT.

[0063] Example 4 Compound-8 on CD4 + Effects of T cell and MT-4 cell proliferation I. Experimental Methods CD4 separated from peripheral blood of healthy individuals + T cells and MT-4 cells were counted and seeded at 100 μL in 96-well plates (2 × 10⁶ cells / well). 4 (Cells / well) Simultaneously, 100 μL of Compound-8 culture medium was added to each well to achieve a final concentration of 5 μg / mL, with three replicates. A control well containing DMSO was also included. Cells were incubated at 37°C in a 5% CO2 incubator. After 24, 48, and 72 hours of incubation, 20 μL of CCK-8 assay reagent (Beyotime) was added, and incubation continued for 1.5–2 hours. The absorbance of each well was measured at 450 nm using an ELISA reader. The results were recorded, and the cell count was calculated.

[0064] II. Experimental Results The results are as follows Figure 4 As shown, after adding Compound-8, CD4 +The number of T cells and MT-4 cells increased, indicating that 5 μg / mL Compound-8 did not significantly affect normal CD4 counts compared to DMSO. + Proliferation of T cells and MT-4 cells.

[0065] Example 5: Inhibitory effect of Compound-8 on HIV-1 in a humanized mouse model. I. Experimental Methods This invention uses 4-6 week old female NPG-RF immunodeficient mice (Vitonda) as a model, and injects 100 μL of a solution containing 1×10⁻⁶ NPG-RF into the tail vein. 5 CD34 + Humanized mice were constructed using stem cell culture medium. Two weeks after infection, approximately 50 μL of blood was collected from the submandibular region via a tail vein injection of 100 μL containing 20 ng of HIV-JRCSF (GenBank: M38429.1) viral stock solution. The blood sample was collected into a blood collection tube containing 0.5 M EDTA as an anticoagulant, and plasma was obtained by centrifugation at 1600 rpm for 8 minutes. The viral load in the plasma was detected using a viral load detection kit (DaAn Gene, DA0331).

[0066] Plasma RNA was extracted. 800 μL of Trizol solution (Invitrogen) was added to mouse plasma, quickly dispersed, and then 200 μL of chloroform (Sinopharm) was added. The mixture was thoroughly mixed and centrifuged at 12000 ×g for 20 minutes at 4°C. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was precipitated at -20°C for 30 minutes. Centrifuged at 12000 ×g for 20 minutes at 4°C, the supernatant was discarded, and 1 mL of 70% ethanol was added. The mixture was then centrifuged at 12000 ×g for 10 minutes at 4°C. The supernatant was discarded, and 30 μL of LEPC water was added to dissolve the precipitate, yielding mouse plasma RNA. Reverse transcription was performed using a reverse transcription kit (Mona) to obtain cDNA. A real-time quantitative PCR system was prepared according to the instructions of the Human Immunodeficiency Virus Type 1 Nucleic Acid Assay Kit, including standards, blank controls, positive controls, and the test sample—mouse plasma cDNA. Three replicates were prepared for each sample. The viral load of each mouse was determined based on the PCR results.

[0067] See the detailed experimental procedure diagram below. Figure 5A. Mice were randomly divided into three groups of six mice each, based on their average viral load. Group 1 was the control group (solvent formulation: 5% DMSO, 40% PEG300, 5% Tween80, 50% (20% SED in saline W / V)). Group 2 was the antiretroviral therapy (ART) group, receiving subcutaneous injections of ART (45 mg / kg CAB, 45 mg / kg RPV, 40 mg / kg 3TC, 40 mg / kg ABC). Group 3 was the Compound-8 group, receiving subcutaneous injections of Compound-8 (3 mg / kg). After two weeks of daily administration, medication was discontinued once the viral load in the blood fell below the detection threshold, and observation continued for another four weeks. Blood samples were collected weekly, and viral load was measured using a viral load assay kit.

[0068] II. Experimental Results The results are as follows Figure 5 As shown in B, the Compound-8 group effectively reduced the viral load in the peripheral blood of humanized mice to near or below the detection limit. After drug withdrawal, viral load was further monitored; the Compound-8 group maintained a stable low viral load for at least 2 weeks, while the AZT group showed a significant viral rebound. Figure 5 C). Monitoring of mouse body weight showed no significant change in mouse body weight. Figure 5 D).

[0069] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of a UFMylation modulator in the preparation of drugs for treating HIV infection, characterized in that, The UFMylation modifier is Compound-8 or a pharmaceutically acceptable salt thereof, and the structure of Compound-8 is shown in formula (I); Equation (Ⅰ).

2. The application according to claim 1, characterized in that, The concentration of Compound-8 is not less than 1.25 μg / ml.

3. The application according to claim 1, characterized in that, The concentration of Compound-8 is 1.25-10 μg / ml.

4. The application according to claim 1, characterized in that, The anti-HIV infection measures include suppressing early asymptomatic infection and / or treating / preventing late-stage AIDS symptoms.

5. The application according to claim 1, characterized in that, The Compound-8 drug achieves its anti-HIV effect by inhibiting HIV viral replication, and the inhibitory effect of Compound-8 on HIV viral replication has no significant rebound effect within at least 2 weeks after discontinuation of the drug.

6. Any of the following applications: 1) The use of the UFMylation modulator of claim 1 in the preparation of a pharmaceutical composition for treating HIV infection, wherein the pharmaceutical composition may further include other drugs that can be used to treat HIV infection; 2) The use of the UFMylation modifier of claim 1 in the preparation of a pharmaceutical formulation for treating HIV infection, wherein the pharmaceutical formulation further comprises pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, Other drugs that can be used to treat HIV infection are antiretroviral drugs, including nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion enzyme inhibitors, and co-receptor antagonists.

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