Application of PPM1A inhibitor in resisting RNA virus

Abstract

The invention provides an application of a PPM1A inhibitor in resisting RNA (Ribonucleic Acid) viruses. The disclosure also provides the use of the PPM1A inhibitor in the preparation of a medicament for the treatment and / or prevention of novel coronavirus infection.

Description

Technical Field

[0001] This invention relates to the application of PPM1A inhibitors in the treatment of RNA viruses. Background Technology

[0002] As a single-stranded positive-sense RNA virus, the novel coronavirus exhibits a high degree of genetic variation. Over time, several variants with stronger transmissibility and immune evasion capabilities have emerged (such as Delta, Omicron, and their derived subtypes XBB, EG.5, and JN.1), gradually weakening the protective efficacy of existing vaccines. Therefore, there is an urgent need to develop broad-spectrum antiviral measures against the novel coronavirus to address the continuously emerging mutant strains.

[0003] In the treatment of novel coronavirus infection, several antiviral drugs have been approved, including Remdesivir (PMID: 32020029; PMID: 32445440), Paxlovid (nematvir / ritonavir combination) (PMID: 34726479), Molnupiravir (PMID: 34914868), and Ambasidium / romisvir (BRII-196 / BRII-198). These drugs mostly act on viral proteins or key enzymes in the replication cycle (such as RNA-dependent RNA polymerase, main protease, etc.), exhibiting certain antiviral activity. However, because their target is the virus itself, their efficacy is easily reduced or even drug resistance occurs when facing continuous structural mutations in the virus. Furthermore, these drugs often require use within a short time window in the early stages of infection, and some patients have contraindications or risk of side effects, limiting their widespread clinical application. Therefore, current treatment strategies for the novel coronavirus still face prominent problems such as insufficient effectiveness, limited applicability, and inadequate response to new variant strains. Summary of the Invention

[0004] This disclosure provides a drug that can target the host, restore innate immune homeostasis, effectively inhibit the replication of RNA viruses, especially the novel coronavirus, and reduce inflammatory damage to lung tissue. The drug is a PPM1A inhibitor, particularly SMIP-031, and is suitable for adjunctive treatment or combination therapy for current and future coronavirus mutant infections.

[0005] According to one aspect of this disclosure, the use of PPM1A inhibitors in the preparation of medicaments against RNA viruses is provided.

[0006] In some implementations, the RNA virus includes coronaviruses.

[0007] In some embodiments, the RNA virus is a novel coronavirus.

[0008] In this document, PPM1A inhibitors are compounds that can inhibit the activity of metal-dependent protein phosphatase 1A (PPM1A). In some embodiments, the PPM1A inhibitors include SMIP-30, SMIP-031, or their solvates, hydrates, or pharmaceutically acceptable salts.

[0009] The structure of SMIP-30 is shown below: .

[0010] The structure of SMIP-031 is shown below: .

[0011] In some embodiments, the PPM1A inhibitor is SMIP-031 or its solvate, hydrate, or pharmaceutically acceptable salt.

[0012] In some embodiments, the PPM1A inhibitor is able to restore homeostasis of the antiviral innate immune response.

[0013] In some embodiments, the PPM1A inhibitor is capable of reconstructing the innate immune response.

[0014] In some embodiments, the PPM1A inhibitor can upregulate the expression of interferon-stimulated genes.

[0015] In some embodiments, the PPM1A inhibitor is capable of treating and / or preventing the RNA virus infection or related diseases.

[0016] In some embodiments, the interferon-stimulated gene includes Isg15 , Ifit1 , Ifitm1 One or more of them.

[0017] According to another aspect of this disclosure, the use of PPM1A inhibitors in the preparation of medicaments for the treatment and / or prevention of novel coronavirus infection is provided.

[0018] In some embodiments, the PPM1A inhibitor includes SMIP-30, SMIP-031, or their solvates, hydrates, or pharmaceutically acceptable salts.

[0019] In some embodiments, the PPM1A inhibitor is SMIP-031 or its solvate, hydrate, or pharmaceutically acceptable salt.

[0020] In some implementations, the PPM1A inhibitor is able to inhibit the replication of the novel coronavirus.

[0021] In some embodiments, the PPM1A inhibitor is able to reduce the viral RNA copy number of the novel coronavirus in vivo and in vitro.

[0022] In some embodiments, the PPM1A inhibitor can reduce inflammatory damage to lung tissue.

[0023] In some embodiments, the PPM1A inhibitor is able to restore the lung's innate immune response.

[0024] In some embodiments, the PPM1A inhibitor is able to restore the host's innate immune homeostasis.

[0025] In some implementations, the PPM1A inhibitor is able to rebuild the antiviral barrier.

[0026] In some embodiments, the PPM1A inhibitor can increase the expression of interferon-stimulated genes.

[0027] In some embodiments, the interferon-stimulated gene includes Isg15 , Ifit1 , Ifitm1 One or more of them.

[0028] According to another aspect of this disclosure, a pharmaceutical composition for treating and / or preventing novel coronavirus infection against RNA viruses is provided, said pharmaceutical composition comprising a PPM1A inhibitor.

[0029] In some implementations, the RNA virus includes coronaviruses.

[0030] In some embodiments, the RNA virus is a novel coronavirus.

[0031] In some embodiments, the PPM1A inhibitor includes SMIP-30, SMIP-031, or their solvates, hydrates, or pharmaceutically acceptable salts.

[0032] In some embodiments, the PPM1A inhibitor is SMIP-031 or its solvate, hydrate, or pharmaceutically acceptable salt.

[0033] In some embodiments, the pharmaceutical composition further includes other active ingredients capable of fighting RNA viruses.

[0034] In some embodiments, the pharmaceutical composition further includes other active ingredients that can be used to treat and / or prevent novel coronavirus infection.

[0035] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0036] In some embodiments, the pharmaceutically acceptable carrier includes one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, or synergists.

[0037] In some embodiments, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0038] In some embodiments, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, spray, suppository, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0039] In some embodiments, the non-gastrointestinal dosage form includes at least one of the following: injectable dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0040] In some embodiments, the drug is administered to animals.

[0041] According to another aspect of this disclosure, a method for treating and / or preventing novel coronavirus infection against RNA viruses is provided, the method comprising administering an effective amount of the pharmaceutical composition described in this disclosure to a subject in need.

[0042] In some embodiments, the method uses routes of administration including oral administration, injection administration, intravenous infusion administration, sublingual administration, spray inhalation, or rectal administration.

[0043] In some implementations, the subject is an animal.

[0044] This disclosure presents a host-targeted antiviral therapy strategy based on small molecule drug PPM1A inhibitors, particularly SMIP-031, which can effectively inhibit the replication of the novel coronavirus and reduce viral load in the lungs, achieving dual antiviral effects in vivo and in vitro.

[0045] In this disclosure, by pre-incubating SMIP-031 with the novel coronavirus and treating target cells, effective intervention of the drug in the early stages of viral infection was achieved. Simultaneously, an in vivo infection model was established using K18-hACE2 transgenic mice to systematically verify the drug's in vivo antiviral effect and dose-dependent inhibitory characteristics. Furthermore, this disclosure uses qPCR technology to detect the effect of SMIP-031 administration on novel coronavirus infection. Isg15 , Ifit1 , Ifitm1The study investigated the effects of interferon-stimulated gene expression, finding that SMIP-031 treatment significantly upregulated interferon-stimulated gene expression levels, suggesting its ability to reconstruct the host's innate immune response and thus establish an endogenous immune barrier against viral infection. Furthermore, this disclosure combined H&E staining and IHC techniques to observe the effect of SMIP-031 on improving virus-induced inflammatory damage in mouse lung tissue. The results showed that the drug significantly reduced alveolar structural disorder, decreased inflammatory cell infiltration, and inhibited the expression of the novel coronavirus N protein, exerting a tissue-protective effect.

[0046] This disclosure has the following beneficial effects: (1) SMIP-031 is used for the first time in the treatment of novel coronavirus infection: SMIP-031 was previously used in anti-tuberculosis treatment research. This disclosure is the first time that it has been used for the "repurposing" of an old drug to the field of RNA virus infection, especially the prevention and treatment of coronavirus (especially novel coronavirus) infection, filling the research gap of this drug in viral infectious diseases.

[0047] (2) Targeting the host rather than the virus itself, avoiding the problem of viral drug resistance and mutation: Most current anti-novel coronavirus drugs (such as remdesivir and Paxlovid) target viral proteins and are easily rendered ineffective by viral mutations. SMIP-031 enhances the host's innate immune response by regulating key host factors, thereby improving the host's antiviral ability from the root, without relying on the conservation of viral sequences, thus having a broader spectrum and more durable antiviral efficacy.

[0048] (3) First time revealing that SMIP-031 can enhance the host's antiviral innate immune response: This publication is the first to report that SMIP-031 can upregulate the expression of interferon-stimulated genes and has a significant immune activation effect. This is an important breakthrough in the study of its mechanism and lays the foundation for the application of immune regulation in other disease fields. Attached Figure Description

[0049] Figure 1 The SMIP-031 structure is shown.

[0050] Figure 2 The results of quantitative PCR detection of the novel coronavirus N gene replication at the cellular level using the TaqMan probe method are shown. The results indicate that SMIP-031 significantly inhibited viral replication at both 1.25 μM and 0.625 μM concentrations in a concentration-dependent manner.

[0051] Figure 3 A schematic diagram of the K18-hACE2 transgenic mouse experiment is shown.

[0052] Figure 4The results of viral RNA level detection in lung tissue of K18-hACE2 transgenic mice are shown, indicating that SMIP-031 drug inhibits the replication of the novel coronavirus in mice.

[0053] Figure 5 The results show the expression levels of interferon-stimulated genes (ISGs) in the lung tissue of K18-hACE2 transgenic mice detected by qRT-PCR, indicating that SMIP-031 significantly upregulated the expression of ISGs in the lung tissue of mice after SARS-CoV-2 infection, suggesting that it helps restore innate immune homeostasis.

[0054] Figure 6 The results of H&E staining of lung tissue from K18-hACE2 transgenic mice are shown (scale bar 50 μm), indicating that SMIP-031 can alleviate pathological damage to mouse lung tissue caused by novel coronavirus infection.

[0055] Figure 7 The results of immunohistochemical staining for the expression of SARS-CoV-2 N protein in the lung tissue of K18-hACE2 transgenic mice are shown (scale bar 50 μm), indicating that SMIP-031 effectively inhibits the replication and expression of SARS-CoV-2 in lung tissue. Detailed Implementation

[0056] Most current antiviral drugs focus on strategies that directly inhibit the virus itself, with fewer treatments developed from the host's perspective to regulate the innate immune response. Studies have shown that the novel coronavirus inhibits the host's interferon pathway and evades immune surveillance through multiple mechanisms. Therefore, restoring or enhancing the host's innate immune function has become a crucial direction in combating novel coronavirus infection. In recent years, therapeutic strategies targeting host factors have gradually gained attention, especially those targeting key signaling pathways and regulatory proteins in the viral infection process. Developing immunomodulatory drugs with broad-spectrum activity and low risk of drug resistance has become a significant breakthrough in drug innovation.

[0057] SMIP-031 is a small molecule immunomodulator derived from SMIP-30 through optimization and modification. It was initially developed for the treatment of tuberculosis. Its mechanism of action differs from traditional antibacterial drugs. It mainly enhances anti-infection ability by inhibiting the host protein phosphatase PPM1A and regulating the host cell signaling pathway (rather than directly killing bacteria). This drug has good oral bioavailability and host-targeting activity (PMID: 35320734, PMID: 38958057).

[0058] The small molecule drug SMIP-031 has been found to inhibit Mycobacterium tuberculosis by targeting and inhibiting the host PPM1A protein to activate host cell autophagy. Mycobacterium tuberculosisHowever, there are no literature or patent reports on the effectiveness of SMIP-031 in resisting novel coronavirus infection. Its potential in regulating host innate immunity, interfering with viral replication, and preventing immune dysregulation has not yet been fully explored. The inventors unexpectedly discovered that the small molecule drug SMIP-031, by targeting and inhibiting PPM1A activity, helps the body restore its antiviral response and inhibits novel coronavirus replication, thereby achieving the goal of preventing and treating novel coronavirus infection.

[0059] Therefore, applying SMIP-031 to the prevention and treatment of novel coronavirus infection, by targeting host factors rather than the virus itself, is expected to circumvent the treatment challenges brought about by viral mutations, improve the stability and broad spectrum of efficacy, and demonstrate good industrial transformation potential and public health value.

[0060] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0061] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0062] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0063] The term “restoring homeostasis of antiviral innate immune response” used in this article refers to the regulation of the immune response to return to a “moderate and controllable” balance when the innate immune system is “overactivated” (such as a cytokine storm) or “continuously suppressed” (such as viral hijacking of immune signaling pathways) due to viral infection, so as to ensure the antiviral effect while avoiding damage to the body’s own tissues.

[0064] The term "reconstruction of host innate immune response" used in this article refers to the process of rebuilding a complete and effective innate immune response system when the host's innate immune system suffers from "systemic functional defects" due to long-term viral infection, depletion of immune cells, irreversible damage to signaling pathways, etc., through deeper interventions (such as cell therapy, signaling pathway repair, and immune cell reprogramming).

[0065] The term “treatment” as used herein includes actions that occur when a subject has a specific disease, disorder, or condition, which reduce the severity of the disease, disorder, or condition, or delay or slow its development (“therapeutic treatment”), as well as actions that occur before a subject begins to have a specific disease, disorder, or condition (“preventive treatment”).

[0066] Generally, the “effective amount” of a compound refers to the amount sufficient to elicit a response in the target organism. As will be understood by those skilled in the art, the effective amount of the compounds disclosed herein can vary depending on factors such as the biological target, the pharmacokinetics of the compound, the disease being treated, the administration method, and the age, health status, and symptoms of the subject. Effective amounts include therapeutic effective amounts and prophylactic effective amounts.

[0067] The term "subject" in the administration includes, but is not limited to: humans (i.e., men or women of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, such as mammals, like primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0068] "Pharmaceutically acceptable" means that it is used to prepare a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes that it is acceptable for human pharmaceutical use. "Pharmaceutically acceptable salts" include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, p-toluenesulfonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecyl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, etc.

[0069] Pharmaceutically acceptable excipients used in this disclosure are non-toxic carriers, adjuvants, or mediators that do not impair the pharmacological activity of the compounds formulated together. Pharmaceutically acceptable carriers, adjuvants, or mediators that may be used in the compositions of this disclosure include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates), glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acid metaglycerides, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0070] The compounds or pharmaceutical compositions disclosed herein can be administered via a variety of routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, administration via implantation, or other routes of administration.

[0071] To overcome the limitations of existing antiviral drugs in terms of broad-spectrum activity and single-target efficacy against the novel coronavirus, this invention discloses the development of a drug targeting a broad spectrum of host targets. Through extensive screening, the inventors selected PPM1A, an antiviral innate immune negative regulator, as the host target, and investigated whether the small molecule drug SMIP-031, an anti-Mycobacterium tuberculosis drug, could inhibit novel coronavirus infection and replication by targeting PPM1A. The feasibility of SMIP-031 as an anti-novel coronavirus small molecule drug was evaluated by measuring viral replication capacity and innate immune recovery capacity at the cellular and mouse animal levels.

[0072] Based on this, this disclosure provides the application of the small molecule drug SMIP-031 in the preparation of drugs against the novel coronavirus. The results provided in this disclosure show that SMIP-031 can inhibit viral replication in cells and mice, and can help the body restore its natural immune homeostasis, thus achieving an anti-novel coronavirus effect.

[0073] This disclosure investigates the role of the host factor PPM1A inhibitor SMIP-031 in the treatment of novel coronavirus infection. The molecular formula of SMIP-031 is C0. 17 H 17 BrFNO2, its structural formula is as follows Figure 1 As shown. This small molecule drug has previously been reported to inhibit Mycobacterium tuberculosis infection in mice, and this disclosure further evaluates its potential in inhibiting the replication of the novel coronavirus and restoring the host's innate immune homeostasis.

[0074] This disclosure first describes an in vitro cell experiment in which the small molecule drug SMIP-031 was mixed with the novel coronavirus and co-cultured with host cells. The final concentrations of SMIP-031 used were 1.25 μM and 0.625 μM. After 24 hours of co-culture, cellular RNA was extracted, and the copy number of the novel coronavirus nucleocapsid (N) gene in the cells was detected to assess the effect of SMIP-031 on the replication of the novel coronavirus.

[0075] This disclosure further evaluates the effects of SMIP-031 on SARS-CoV-2 replication and innate immune homeostasis in humanized mice. SMIP-031 was administered intraperitoneally multiple times before and after infection to evaluate its preventive and therapeutic effects. Dosage designs were implemented using 0, 10, and 15 mg / kg concentrations of SMIP-031, based on an average mouse weight of 20 g and an administration volume of 100 μL. Infection was administered via nasal instillation at an infectious dose of 4 × 10⁻⁶ mg / kg. 4 FFU / animal, volume 50 μL. Multiple doses were administered during the viral infection window period after infection, with a total experimental period of 54 h, to simulate the clinical treatment process and examine its intervention effect on viral replication.

[0076] After the experiment, lung tissue from mice was collected, RNA was extracted, and the copy number of the novel coronavirus and the relative expression levels of interferon-stimulated genes (ISGs) were determined. Viral copy number was used to evaluate the drug's effect on viral replication; the expression level of ISGs was used to reflect the recovery of innate immune homeostasis. Considering the significant delay in the innate immune response to novel coronavirus infection, which may lead to rapid viral load growth and affect clinical prognosis, this disclosure focuses on analyzing the relative expression levels of ISGs to evaluate the regulatory effect of the small molecule drug SMIP-031 on the innate immune response in the early stages of viral infection.

[0077] In addition, this disclosure describes the collection of lung tissues subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analysis. H&E staining was used to observe pathological changes and inflammatory cell infiltration in the lung tissues; IHC was performed using a novel coronavirus N protein-specific antibody to detect the distribution and abundance of viral antigens in the tissues, in order to further verify the inhibitory effect of the drug on viral replication.

[0078] Therefore, this disclosure presents experiments on SMIP-031 treatment and novel coronavirus infection in vitro (cell level) and in vivo (animal level). Through novel coronavirus load analysis, interferon-stimulated gene expression analysis, pathological observation, and tissue viral antigen analysis, the application value of the PPM1A inhibitor SMIP-031 small molecule drug in the treatment of novel coronavirus infection is systematically evaluated.

[0079] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0080] The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0081] Example 1: SMIP-031 inhibits novel coronavirus replication at the cellular level HEK293T-hACE2 cells were pre-seeded in 12-well plates and grown to 75%–80% confluence. They were then infected with the novel coronavirus Omicron JN.1 (BA.2.86.1.1) variant and treated with different final concentrations of SMIP-031 (1.25 μM and 0.625 μM), with DMSO as a solvent control. Total RNA was extracted 24 h after infection, and viral N gene expression was detected using the TaqMan probe assay. The specific steps are as follows.

[0082] 1. The HEK293T-hACE2 cell line overexpressing human ACE2 was evenly seeded in a 12-well plate and cultured under the following conditions: DMEM basal medium (containing 10% fetal bovine serum FBS and 1% penicillin and streptomycin) and cultured in a 5% CO2, 37°C incubator until the cell density reached 75%~80%.

[0083] 2. Dissolve SMIP-031 in DMSO to prepare a 20 mM stock solution. Dilute it to 2.5 μM and 1.25 μM respectively using DMEM medium containing 10% fetal bovine serum for later use.

[0084] 3. The novel coronavirus Omicron JN.1 (BA.2.86.1.1) variant was mixed with diluted SMIP-031 solution at a 1:1 volume ratio. The final concentrations of SMIP-031 in the mixture were 1.25 μM and 0.625 μM, respectively. The control group (Mock) was diluted with DMSO and mixed with the virus strain at a 1:1 volume ratio.

[0085] 4. Add the mixture obtained in step 3 to HEK293T-hACE2 cells, with a volume of 1 mL per well. Terminate the culture 24 h after infection, aspirate the supernatant, and wash the cells three times with PBS.

[0086] 5. Add RNA lysis buffer and extract total RNA from cells using a cell RNA extraction kit (EZBioscience). Elute with 50 μL of RNase-free water and determine RNA concentration and purity using NanoDrop.

[0087] 6. Using a 2019-nCoV nucleic acid detection kit (DaAn Gene Co., Ltd.), 10 μL of RNA was taken from each group as a template, and the copy number of the novel coronavirus N gene was detected by TaqMan probe-based quantitative PCR to reflect the effect of drug treatment on the replication of the novel coronavirus. Simultaneously, RNA standards with known copy numbers were used as templates, diluted to different concentrations, and subjected to probe-based qPCR in the same manner to construct a standard curve corresponding to copy number versus Ct value.

[0088] 7. Substitute the detected Ct value into the standard curve formula to obtain the viral copy number of the drug-treated group and the control group.

[0089] The results showed that the viral copy number in the SMIP-031-treated group was lower than that in the DMSO control group. Both 1.25 μM and 0.625 μM treatments showed inhibitory effects, with the 1.25 μM group showing a more significant inhibitory effect, indicating that SMIP-031 can dose-dependently inhibit the replication of the novel coronavirus at the cellular level. Figure 2 ).

[0090] Example 2: SMIP-031 inhibits novel coronavirus replication in humanized mice 1. Four- to five-week-old K18-hACE2 transgenic mice were selected. This model is susceptible to the novel coronavirus and its variants, making it suitable for evaluating the efficacy of antiviral drugs in vivo. The experimental animals were randomly divided into three groups: a high-dose SMIP-031 group (15 mg / kg / dose), a low-dose group (10 mg / kg / dose), and a control group (Mock), with five mice in each group. The drug was dissolved in 10% DMSO, 30% PEG300, 5% Tween-80, and 55% PBS. The control group served as a solvent control.

[0091] 2. Mice were administered and infected in the following manner: a total of five doses were given. The first two doses were administered 4 hours before and after infection, the third dose was administered 24 hours later, and the last two doses were administered 12 hours apart. Six hours after the last dose, mice were sacrificed by cervical dislocation. The administration method was intraperitoneal injection. Mice were infected with the novel coronavirus Omicron JN.1 (BA.2.86.1.1) via intranasal instillation. The infection dose was 4 × 10⁻⁶. 4 FFU / only ( Figure 3 ).

[0092] 3. Mouse lung tissue was collected, and 30 mg of sample was weighed to extract tissue RNA using a tissue RNA extraction kit (EZBioscience). RNA was finally eluted with 50 μL ddH2O, and RNA concentration and purity were determined by NanoDrop.

[0093] 4. Using the 2019-nCoV nucleic acid detection kit (DaAn Gene Co., Ltd.), 10 μL of total RNA from the lungs was used as a template to detect the Ct value of the virus-specific target gene fragment N.

[0094] 5. Substitute the detected Ct values ​​into the standard curve formula to calculate the viral copy number of the novel coronavirus in the lung tissue of mice in the control group and the drug-treated group.

[0095] The results showed that both SMIP-031 groups significantly reduced viral RNA copy number, with the 15 mg / kg group showing a more significant effect than the 10 mg / kg group, in a dose-dependent manner. The viral RNA level in the lung tissue of the control group was significantly higher than that in the drug-treated group. Figure 4 ).

[0096] Example 3: SMIP-031 can help restore natural immune homeostasis. The expression levels of interferon-stimulated genes (ISGs) in mouse lung tissue were detected by qRT-PCR, including... Isg15 , Ifit1 and Ifitm1The SMIP-031 treatment groups (10 mg / kg and 15 mg / kg) were compared with the control group (Mock). Each experimental group contained 5 mice (n=5). The specific steps are as follows.

[0097] 1. Take 1 μg of mouse lung RNA obtained in Example 2 and use a reverse transcription kit (StarScript Pro All-in-one RT Mix with gDNA Remover, GenStar) to perform reverse transcription to synthesize first-strand cDNA for subsequent real-time quantitative PCR (qPCR) analysis.

[0098] 2. Synthesize representative type I interferon downstream response gene, interferon-stimulated gene 15. Isg15 Interferon-induced protein with tetratricopeptide repeats 1, Ifit1 ) and interferon-induced transmembrane protein 1, Ifitm1 ) Specific primers, to Gapdh As an internal control gene, detection was performed using the SYBR Green qPCR method (Taq Pro Universal SYBR qPCR Master Mix, Novizan). Each reaction system contained: SYBR Green reagent (10 μl), cDNA template (2 μl), forward and reverse primers (0.4 μl each), and ddH2O (7.2 μl); the reaction conditions were: 95℃ pre-denaturation for 10 min, followed by 40 cycles (95℃ denaturation for 10 s, 60℃ annealing / extension for 1 min). 2 -ΔΔCt The method calculates the relative expression level.

[0099] Primer sequences are shown in Table 1: Table 1. qPCR primer sequence list

[0100] The results showed that both the 10 mg / kg and 15 mg / kg administration groups increased the concentration of certain substances in the lung tissue of mice. Isg15 , Ifit1、 Ifitm1 The expression level suggests that SMIP-031 can restore the lung's innate immune response, promote the transcriptional expression of ISGs, and ultimately help rebuild the antiviral barrier. Figure 5 ).

[0101] Example 4: Detection of lung damage in mice by SMIP-031 1. Take the mouse lung tissue obtained in Example 2, fix it in 4% paraformaldehyde solution for 7 days, embed it in paraffin, section it, and perform routine H&E staining to observe changes in alveolar structure and inflammatory cell infiltration.

[0102] The results showed that the alveolar structure of mice in the Mock group was disordered, with significant septal thickening and extensive inflammatory cell infiltration. In contrast, the lesions in the SMIP-031 treatment group were significantly alleviated, especially in the 15 mg / kg group where the alveolar structure was relatively intact and the number of inflammatory cells was significantly reduced. Figure 6 ).

[0103] 2. Further immunohistochemical analysis was performed using a novel coronavirus N protein-specific monoclonal antibody (catalog number: 40143-R001; Sinocare) to detect viral replication and expression in lung tissue. After dewaxing and hydration, sections underwent antigen retrieval, non-specific blocking, and incubation with primary antibody (N protein antibody, 1:5000 dilution) overnight at 4°C. After incubation with secondary antibody (goat anti-rabbit IgG H&L (HRP)) (catalog number: ab6721; abcam), colorimetric detection was performed. Positive staining was mainly characterized by brownish-yellow cytoplasmic deposition.

[0104] The results showed that widespread N protein-positive cells were observed in the lung tissue of mice infected with the virus, indicating high viral replication in the lung tissue. In contrast, the number of N protein-positive cells was significantly reduced in the SMIP-031 treatment group, showing a dose-dependent decreasing trend. The 15 mg / kg dose group had the smallest N protein-positive area, almost invisible, suggesting that viral replication was significantly inhibited at this dose. Image analysis results indicate that SMIP-031 can effectively reduce the viral load of the novel coronavirus in lung tissue, consistent with its histopathological improvement effect. Figure 7 ).

[0105] In summary, this disclosure demonstrates the successful application of the small molecule drug SMIP-031 in the treatment of novel coronavirus infection. This drug can inhibit the replication of the novel coronavirus, reduce the viral RNA copy number of the novel coronavirus in vivo and in vitro, restore the homeostasis of the antiviral innate immune response, and increase the expression of interferon-stimulated genes.

[0106] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Application of PPM1A inhibitors in the preparation of drugs against RNA viruses.

2. The application according to claim 1, characterized in that, The RNA virus includes coronaviruses, preferably novel coronaviruses; and / or The PPM1A inhibitors include SMIP-30, SMIP-031, or their solvates, hydrates, or pharmaceutically acceptable salts, preferably SMIP-031 or their solvates, hydrates, or pharmaceutically acceptable salts.

3. The application according to claim 1 or 2, characterized in that, The PPM1A inhibitor can restore homeostasis of the antiviral innate immune response; and / or The PPM1A inhibitor can reconstruct the innate immune response; and / or The PPM1A inhibitor can upregulate interferon-stimulated gene expression; and / or The PPM1A inhibitor can treat and / or prevent the RNA virus infection or related diseases; Preferably, the interferon-stimulated gene includes Isg15 , Ifit1 , Ifitm1 One or more of them.

4. The use of PPM1A inhibitors in the preparation of drugs for the treatment and / or prevention of novel coronavirus infection.

5. The application according to claim 4, characterized in that, The PPM1A inhibitors include SMIP-30, SMIP-031, or their solvates, hydrates, or pharmaceutically acceptable salts, preferably SMIP-031 or their solvates, hydrates, or pharmaceutically acceptable salts.

6. The application according to claim 4 or 5, characterized in that, The PPM1A inhibitor can inhibit the replication of the novel coronavirus; and / or The PPM1A inhibitor can reduce the viral RNA copy number of the novel coronavirus in vivo and in vitro; and / or The PPM1A inhibitor can reduce inflammatory damage to lung tissue; and / or The PPM1A inhibitor can restore the lung's innate immune response; and / or The PPM1A inhibitor is capable of restoring the host's innate immune homeostasis; and / or The PPM1A inhibitor can rebuild the antiviral barrier; and / or The PPM1A inhibitor can increase the expression of interferon-stimulated genes. Preferably, the interferon-stimulated gene includes Isg15 , Ifit1 , Ifitm1 One or more of them.

7. A pharmaceutical composition for use against RNA viruses or for the treatment and / or prevention of novel coronavirus infection, said pharmaceutical composition comprising a PPM1A inhibitor.

8. The pharmaceutical composition according to claim 7, characterized in that, The RNA virus includes coronaviruses, preferably novel coronaviruses; and / or The PPM1A inhibitors include SMIP-30, SMIP-031, or their solvates, hydrates, or pharmaceutically acceptable salts, preferably SMIP-031 or their solvates, hydrates, or pharmaceutically acceptable salts.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that, The pharmaceutical composition also includes other active ingredients that can be used against RNA viruses or can be used to treat and / or prevent novel coronavirus infection; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier; More preferably, the pharmaceutically acceptable carrier includes one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorbent, lubricant, or synergist; More preferably, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a non-gastrointestinal dosage form; More preferably, the gastrointestinal dosage form includes at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, spray, suppository, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet. More preferably, the non-gastrointestinal dosage form includes at least one of the following: injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form; More preferably, the drug is administered to animals.

10. A method for treating and / or preventing novel coronavirus infection against RNA viruses, the method comprising administering to a subject in need an effective amount of the pharmaceutical composition of any one of claims 7-9; Preferably, the administration route used in the method includes oral administration, injection administration, intravenous infusion administration, sublingual administration, spray inhalation, or rectal administration; Preferably, the subject is an animal.

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