Use of salinomycin in the manufacture of a medicament for treating or preventing intracellular MRSA infection

Abstract

The application discloses application of salinomycin (SAL) in preparation of a medicine for treating or preventing intracellular methicillin-resistant Staphylococcus aureus (MRSA) infection, and belongs to the field of biological medicine. Specifically, SAL directly targets host cell RAB7A protein, acts as an agonist or function enhancer of RAB7A, and significantly enhances the ability of the host to remove intracellular MRSA. Experiments prove that both therapeutic administration and prophylactic administration of SAL can effectively reduce the intracellular bacterial load, and are suitable for chronic or recurrent infections mediated by intracellular MRSA retentate. The application provides a brand-new host-oriented drug strategy for resisting intracellular MRSA infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of salinomycin (SAL) in the preparation of drugs for the treatment or prevention of intracellular methicillin-resistant Staphylococcus aureus (MRSA) infection. Background Technology

[0002] Staphylococcus aureus S. aureus Methicillin-resistant Staphylococcus aureus (MRSA) is a facultative intracellular pathogen and one of the most common Gram-positive bacteria causing acquired infections. It can cause a range of diseases, from minor skin and soft tissue infections to fatal necrotizing pneumonia, endocarditis, toxic shock syndrome, and sepsis. S. aureus The emergence of MRSA (metastatic leukemia) has exacerbated this problem, as MRSA exhibits resistance to all penicillin-type β-lactam antibiotics. Currently, vancomycin remains one of the first-line drugs for treating systemic MRSA infections, but even with standard antibiotic treatment, the recurrence rate of MRSA infections remains quite high.

[0003] Traditionally, S. aureus It was considered an extracellular pathogen. However, research over the past two decades has accumulated a wealth of evidence indicating that... S. aureus Its hidden intracellular infection cycle is the core reason why it is difficult to eradicate. [1] The clinical challenges of intracellular MRSA infection mainly lie in the following aspects: (1) Intracellular inaccessibility of antibiotics: Most traditional antibiotics (including vancomycin, β-lactams, aminoglycosides, etc.) have poor cell membrane penetration and are difficult to reach effective therapeutic concentrations within host cells. Even if some drugs can enter cells, their activity is often significantly reduced in the acidic environment of lysosomes and under the action of hydrolytic enzymes. Bacteria hide inside cells, which is equivalent to obtaining a natural "refuge" that allows them to evade the killing effect of antibiotics for a long time.

[0004] (2) Formation of Persisters: After entering the host cell, some MRSA can enter a metabolic dormant state, forming so-called "persisters". Persisters have natural resistance to lethal concentrations of antibiotics. Although antibiotics can eliminate actively proliferating bacteria, they are difficult to eradicate these dormant bacteria. Once antibiotic treatment is stopped, persisters can be reactivated and resume proliferation, leading to recurrence of infection.

[0005] (3) Intracellular immune escape and intercellular transmission: S. aureusMultiple strategies have evolved to defend against the host cell's bactericidal mechanisms. After invading specialized phagocytes such as macrophages, approximately 90% of bacteria can be successfully eliminated, but the remainder can tolerate the harsh environment of the phagocytosolic lysosome and survive intracellularly for extended periods. These surviving bacteria can use the host cell as a "Trojan horse" to evade immune surveillance and even induce macrophages to polarize towards the immunosuppressive M2 phenotype, creating a microenvironment conducive to bacterial persistence. Ultimately, the host cell is lysed by the overproliferating bacteria, and the released bacteria can continue to infect neighboring cells or cause systemic dissemination.

[0006] These characteristics make intracellular MRSA infection closely associated with a variety of chronic, refractory infectious diseases, including recurrent mastitis, osteomyelitis, endocarditis, necrotizing pneumonia, and sepsis. [2] Furthermore, the presence of an intracellular bacterial reservoir is also considered an important reason for infection recurrence after discontinuation of antibiotic treatment. Taking mastitis as an example, S. aureus It can remain dormant in mammary epithelial cells and macrophages for a long time, leading to chronic mastitis or recurrent attacks, which brings great difficulties to clinical treatment.

[0007] Currently, clinical treatment for MRSA still primarily relies on traditional antibiotics. However, these antibacterial drugs, which directly target pathogens, have two inherent drawbacks: First, the strong selective pressure accelerates the development of resistance. The use of antibiotics directly exerts evolutionary selection pressure on bacteria, easily selecting for drug-resistant mutants and fostering "superbugs." In recent years, vancomycin-resistant... S. aureus (VRSA) has already been reported, further limiting the options for clinical treatment. Secondly, it severely disrupts the host's symbiotic microbiota. While broad-spectrum antibiotics kill pathogens, they also severely disrupt the balance of the host's symbiotic microbiota, inducing dysbiosis and related complications. Most importantly, traditional antibiotic strategies cannot effectively eliminate intracellular persistent bacteria, which is the fundamental reason for the high relapse rate after discontinuation of MRSA infection. Therefore, it is urgent to develop new anti-infective strategies to fundamentally overcome the treatment bottleneck of intracellular bacterial infections.

[0008] Given the inherent limitations of traditional antibiotic strategies, host-directed therapy has attracted widespread attention in recent years. This strategy does not directly target pathogens, but rather enhances the host's ability to clear pathogens by modulating the immune function or key signaling pathways of host cells.

[0009] Existing host-guided strategies mainly include: regulating macrophage polarization (e.g., reprogramming M2 macrophages to M1 macrophages using IFN-γ), targeting intracellular membrane transport pathways, and utilizing nanodelivery systems to achieve precise intracellular drug delivery. [3]However, the number of clearly identified host targets and corresponding small molecule drugs is still limited. Developing host-guided anti-intracellular MRSA drugs with promising clinical applications is an important direction in this field.

[0010] The Rab GTPase family are key molecular switches for intramembrane transport in eukaryotic cells, with RAB7A being a core protein regulating the endocytosis-lysosome pathway. RAB7A is primarily located in late endosomes and lysosomes, and is a key regulator of the maturation of early endosomes into late endosomes / lysosomes, the transport of endosomes to the negative microtubule end, and endosome-lysosome fusion. Through its interactions with effector proteins (such as RILP and FYCO1), it precisely regulates multiple steps of membrane transport. [4] Participating in mediating phagocytosis S. aureus The process of phagosome maturation and acidification [5] Therefore, maintaining or enhancing the activity of RAB7A may help promote S. aureus It is delivered to lysosomes and eventually cleared.

[0011] However, whether RAB7A can serve as a direct drug target for host-directed therapy against MRSA, and whether there are small molecule compounds that can enhance intracellular MRSA clearance by targeting RAB7A, have not been previously reported.

[0012] SAL is a polyether ionocarrier antibiotic that has long been widely used in veterinary medicine as an anticoccidial agent and growth promoter. Recent studies have revealed that SAL possesses multiple biological activities, including antibacterial, antitumor, autophagy-inducing, and cell death-regulating activities. As a multi-target drug, SAL can exert broad regulatory effects on host cells, but its specific molecular targets and mechanisms of action against intracellular MRSA infection remain unclear.

[0013] References: [1]Le C, et al. Intracellular Habitation of Staphylococcus aureus: Molecular Mechanisms and Prospects for Antimicrobial Therapy. Biomedicines.2022. [2]Wang X, et al. Cascade-targeting Poly(amino acid) NanoparticlesEliminate Intracellular Bacteria via on-site Antibiotic Delivery. AdvancedMaterials. 2022. [3]Sun XR, et al. Intracellular MRSA-targeted lipid-polymer hybridnanoparticles for macrophage reprogramming and intracellular MRSAeradication. Chemical Engineering Journal. 2025. [4] Chavan I, et al. Lysosome heterogeneity and diversity mapped through its distinct cellular functions. Cellular and Molecular LifeSciences. 2025 [5]Ma L, et al. Mechanism and role of mitophagy in the development of severe infection. Cell Death Discovery. 2024. Summary of the Invention The present invention aims to address the shortcomings of existing drugs for treating intracellular methicillin-resistant Staphylococcus aureus (MRSA) infections by providing a drug use of salinomycin in treating intracellular MRSA infections.

[0014] To achieve the above objectives, this invention establishes a co-infection model of RAW264.7 macrophages and MRSA USA300 to evaluate the effects of therapeutic (post-infection) and prophylactic (pre-infection) administration of SAL on intracellular bacterial load. Molecular docking and isothermal titration (ITC) were used to detect the direct binding of SAL to the RAB7A protein. RAB7A expression was bidirectionally regulated using overexpression and RNA interference techniques to verify its function in host clearance of intracellular MRSA.

[0015] The results showed that after SAL treatment following bacterial challenge, the intracellular MRSA load was significantly reduced compared to the control group. p<0.001); 6 h or 12 h after prophylactic administration, challenge with bacteria resulted in a significant decrease in intracellular bacterial load ( p <0.05 and p <0.01 indicates that SAL exerts its anti-intracellular bacterial effect by regulating host cells. Molecular docking showed that SAL forms hydrogen bonds with key residues (GLY-66, GLN-71) in the Switch I / II region of RAB7A, and ITC confirmed that the two bind directly (ΔH ≈ -80 kcal / mol, ΔG ≈ -6.6 kcal / mol). Functional experiments showed that overexpression of RAB7A significantly reduced intracellular MRSA load ( p <0.01), while knocking down RAB7A significantly increased the bacterial load ( p <0.01).

[0016] Therefore, SAL can effectively promote the clearance of intracellular MRSA by directly targeting and binding to the host RAB7A protein, thereby enhancing its function. This invention reveals for the first time a novel mechanism by which SAL exerts its host-directed anti-infective effect as a RAB7A agonist, providing a new drug strategy and target for the treatment of chronic and recurrent infections mediated by MRSA-residual intracellular bacteria (such as mastitis, osteomyelitis, endocarditis, etc.).

[0017] Based on this, the present invention provides the following technical solution: The use of salinomycin in the preparation of drugs for the treatment or prevention of intracellular MRSA infection.

[0018] The drug exerts its anti-intracellular MRSA infection effect by targeting the RAB7A protein in host cells.

[0019] Furthermore, the drug is an agonist or functional enhancer of the RAB7A protein.

[0020] The intracellular MRSA infection includes chronic or recurrent infections mediated by MRSA intracellular persistent bacteria.

[0021] Furthermore, the chronic or recurrent infection is selected from one or more of the following diseases: mastitis, osteomyelitis, endocarditis, necrotizing pneumonia, and sepsis.

[0022] The drug is a pharmaceutical preparation made by combining SAL as the active ingredient with a pharmaceutically acceptable carrier or excipient.

[0023] Furthermore, the formulation is selected from injections, powder for injection, tablets, capsules, granules, oral liquids, external applications, or inhalants.

[0024] Furthermore, the drug is used for preventative or therapeutic administration.

[0025] The beneficial effects of this invention are: This invention reveals for the first time that SAL exerts its host-directed anti-intramural MRSA effect by directly targeting the host RAB7A protein and enhancing its function, overcoming the shortcomings of traditional antibiotics, such as inaccessibility to intracellular structures, easy induction of drug resistance, and disruption of symbiotic flora. Experiments have demonstrated that SAL significantly clears intracellular MRSA and effectively targets persistent bacteria, reducing the risk of chronic and recurrent infections, regardless of whether administered therapeutically or prophylactically. This mechanism is clearly defined and the target is well-identified, providing a novel treatment strategy for refractory intracellular MRSA infections such as mastitis, osteomyelitis, and endocarditis, and has significant potential for clinical translation. Attached Figure Description

[0026] Figure 1 The therapeutic effect of SAL on the clearance of intracellular MRSA.

[0027] Figure 2 The protective effect of SAL prophylactic administration against intracellular MRSA infection.

[0028] Figure 3 Results of molecular docking between SAL and RAB7A protein.

[0029] Figure 4 The thermodynamic curves of the binding between SAL and RAB7A were determined by isothermal titration calorimetry (ITC). The left graph shows the heat change (exothermic peak) as SAL is successively added to the RAB7A protein solution, and the right graph shows the integrated binding isotherm. Fitting analysis yielded a binding enthalpy change (ΔH) of approximately -80 kcal / mol and a Gibbs free energy change (ΔG) of approximately -6.6 kcal / mol, indicating a direct binding interaction between SAL and RAB7A.

[0030] Figure 5 Effects of RAB7A overexpression on intracellular MRSA infection.

[0031] Figure 6 The effect of interfering with RAB7A expression on intracellular MRSA infection. Detailed Implementation

[0032] The technical solution of the present invention will be described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the feasibility and technical effects of the present invention and do not constitute any limitation on the scope of protection of the present invention. Equivalent substitutions or simple modifications that can be reasonably foreseen by those skilled in the art based on their understanding of the technical solution of the present invention all fall within the scope of protection of the present invention.

[0033] Unless otherwise specified, all reagents, materials, and instruments used in the following examples are commercially available; all experimental methods used are conventional methods in the field, and specific conditions may be adjusted appropriately according to actual operation. Statistical analysis was performed using GraphPad Prism 9.0 software. Data are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using t-tests or one-way ANOVA. p <0.05 indicates a statistically significant difference.

[0034] Example 1: Evaluation of the preventive and therapeutic effects of salinomycin on intracellular MRSA infection 1. Experimental Materials RAW264.7 cells, MRSA USA300, fetal bovine serum, high glucose DMEM medium, PBS, lysozyme, gentamicin, DMSO, SAL, Triton, 6-well cell culture plates, TSA plates and TSB medium.

[0035] 2. Experimental Methods 2.1 Bacterial Culture MRSA USA300 strain was streaked onto TSA plates and incubated at 37°C for 18 h. A single colony was picked and inoculated into 5 mL of TSB medium, and incubated overnight at 37°C with shaking at 220 rpm. The next day, the colony was transferred to fresh TSB medium at a 1:100 ratio and cultured until the logarithmic growth phase (OD200). 600 ≈ 0.4~0.6). The bacterial culture was centrifuged (4,000 rpm, 10 min) to collect the bacterial cells, washed twice with sterile PBS and resuspended. After serial dilution, the cells were plated and counted to determine the bacterial concentration for later use.

[0036] 2.2 Cell Culture RAW264.7 cells were cultured in high-glucose DMEM medium containing 10% FBS at 37°C in a 5% CO2 incubator. Cells were passaged daily, gently detached by pipetting, and passaged at a 1:2 ratio. All experiments were performed when the cells were in the logarithmic growth phase.

[0037] 2.3 Therapeutic drug administration experiments (post-infection administration) (1) Cell seeding: Take RAW264.7 cells in logarithmic growth phase, gently pipette them off, resuspend them in DMEM medium containing 10% FBS, count them, and adjust the density to 1.0 × 10⁶ cells / year. 6 Cells / mL. Cells were seeded in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h until the cells were fully adhered.

[0038] (2) Bacterial infection: Discard the culture supernatant, add USA300 bacterial solution at a ratio of multiple of infection (MOI) = 10, add culture medium to each well to make up to 2 mL, mix gently and incubate for 6 h in an incubator.

[0039] (3) Removal of extracellular bacteria: After infection, discard the culture medium and carefully wash the cells three times (2 mL each time) with pre-warmed PBS. Add 2 mL of DMEM medium containing 20 μg / mL lysozyme to each well and incubate at 37°C for 15 min to destroy bacteria that may adhere to the cell surface. Afterward, aspirate the culture medium containing lysozyme solution and replace it with DMEM medium containing 200 μg / mL gentamicin, and continue treatment for 1 h to completely kill any remaining extracellular bacteria.

[0040] (4) Drug treatment: After treatment with gentamicin, the culture medium was discarded, and DMEM medium containing DMSO (solvent control group) and 2 μM SAL was added to each well. The wells were then incubated for 12 h. Each treatment group was set up with 3 replicates.

[0041] (5) Intracellular bacterial count: After drug treatment, discard the culture supernatant and wash three times with pre-cooled PBS to completely remove residual drugs. Add 500 μL of 0.1% Triton X-100 lysis buffer to each well and lyse on ice for 10 min, repeatedly pipetting during the process to ensure complete cell lysis and release of intracellular bacteria. Dilute the lysis buffer 10-fold serially with PBS, and spread 100 μL of each dilution evenly onto TSA plates, with three parallel plates for each dilution. Incubate the plates upside down at 37°C for 18–24 h, count the colony count (CFU), and calculate the intracellular bacterial load (CFU / mL) per well. Experimental results are expressed as CFU / mL.

[0042] (6) Data analysis: CFU count results are expressed as mean ± standard deviation, and differences between groups are analyzed by unpaired t-test.

[0043] 2.4 Prophylactic drug administration experiment (pre-infection administration) To clarify whether the effect of SAL stems from direct regulation of host cells rather than direct killing of bacteria, an experiment was conducted involving drug pretreatment followed by bacterial infection.

[0044] (1) Cell seeding and drug pretreatment: RAW264.7 cells were seeded in 6-well plates as described above and cultured for 24 h. The culture medium was then discarded, and DMEM medium containing DMSO (control group) or 2 μM SAL was added respectively. The cells were cultured for 6 h or 12 h for pretreatment.

[0045] (2) Bacterial infection: After pretreatment, discard the drug-containing culture medium and wash the cells three times with PBS to completely remove residual drugs (avoid direct contact between drugs and bacteria). Add USA300 bacterial solution at a ratio of MOI = 10 and co-infect at 37°C for 6 hours.

[0046] (3) Intracellular bacterial count: After the infection is completed, cells are lysed and plated for counting according to the method in step (5) of the "Therapeutic drug administration experiment".

[0047] 3. Experimental Results 3.1 Results of therapeutic drug administration experiments like Figure 1 As shown, compared with the solvent control group (DMSO treatment), the number of USA300 colonies in infected RAW264.7 cells was significantly reduced after 2 μM SAL therapeutic administration. p The value <0.001 indicates that SAL administration after infection can effectively clear MRSA that has entered the host cells.

[0048] 3.2 Results of prophylactic drug administration experiment like Figure 2 As shown, after SAL pretreatment for 6 h, challenge with bacteria resulted in a significantly lower intracellular bacterial load compared to the control group. p <0.05); After SAL pretreatment for 12 h, the intracellular bacterial load was significantly reduced ( p <0.01).

[0049] The above results indicate that salinomycin not only combats intracellular MRSA, but this effect is not derived from direct bactericidal action, but rather through long-term regulation of host cells.

[0050] Example 2: Interaction between salinomycin and RAB7A 1. Molecular docking and molecular dynamics simulation The docking simulation between SAL and RAB7A was performed using the molecular docking software AutoDock Vina. First, the 3D crystal structure of the RAB7A protein was downloaded from the RCSB Protein Data Bank. Water molecules and existing ligands were removed using PyMOL, followed by the addition of polar hydrogen atoms and saving as a receptor file. The small molecule 2D structure of SAL was obtained from PubChem, and the receptor and ligand were converted to PDBQT format files using AutoDock Tools. The docking grid parameters, including center coordinates and box size, were set according to the location of the RAB7A active pocket. Molecular docking calculations were then performed in AutoDock Vina using default parameters. Finally, the conformation with the lowest binding energy was selected as the optimal binding mode, and the protein-ligand interaction mode, including hydrogen bonding and hydrophobic interactions, was visualized and analyzed using PyMOL.

[0051] like Figure 3 As shown, SAL forms a stable direct interaction with the host target protein RAB7A. The SAL ligand primarily binds to the hydrophobic pocket of the RAB7A protein's GTPase domain, forming a 2.2 Å hydrogen bond with glycine at position 66 (GLY-66) and a strong 1.9 Å hydrogen bond with glutamine at position 71 (GLN-71). These two key residues are located in the Switch I / II functional region of RAB7A, which is the core interface regulating RAB7A conformational transitions and effector binding. Furthermore, the polyether backbone of SAL also undergoes extensive van der Waals contacts with surrounding hydrophobic residues, further stabilizing the complex structure.

[0052] 2. Isothermal titration calorimetry The pET-30a(+) plasmid encoding RAB7A was transformed into... E. coli Prokaryotic expression and purification were performed in BL21(DE3). A 100 μM RAB7A protein solution and a 1 mM SAL solution were prepared. 350 μL of the degassed protein solution was slowly aspirated using a syringe and injected into the reaction chamber. 52 μL of the degassed small molecule solution was aspirated using a titration needle, and the titration volume was 2.5 μL, repeated 20 times. The results were then processed using ITC instrument software.

[0053] like Figure 4 As shown, the binding process of SAL to RAB7A protein is a spontaneous and enthalpy-driven process. The binding enthalpy change (ΔH) obtained through fitting analysis is approximately -80 kcal / mol, and the Gibbs free energy change (ΔG) is approximately -6.6 kcal / mol. The binding entropy change (-TΔS) corresponds to the difference between ΔH and ΔG. The relatively small absolute value of ΔH and the proximity of ΔG to -1.00 kcal / mol shown in the figure suggest a binding interaction between SAL and RAB7A. This ITC result provides thermodynamic evidence for the direct binding of SAL to RAB7A.

[0054] Example 3: The effect of bidirectional regulation of RAB7A protein on intracellular MRSA infection To verify the key role of RAB7A protein in host resistance to intracellular MRSA infection, this embodiment employs two strategies—overexpression and RNA interference—to bidirectionally regulate the expression level of RAB7A in RAW264.7 macrophages and observe changes in intracellular bacterial load.

[0055] 1. Experimental Methods Overexpression experiment: RAB7A overexpression plasmid (pcDNA3.1(+)-RAB7A) and empty vector control were transfected into RAW264.7 cells. Cells were collected 48 h after transfection and the overexpression efficiency was verified by Western blot.

[0056] Interference experiment: RAW264.7 cells were transfected with RAB7A-specific siRNA, namely si-RAB7A (designed by a commissioned company, GGAAGAAAGUGUUGCUGAA / dT / / dT / and UUCAGCAACACUUUCUUCC / dT / / dT / ), and negative control siRNA (si-NC). Cells were collected 48 h after transfection, and the knockdown efficiency was verified by Western blot.

[0057] Intracellular bacterial infection detection: 48 h after transfection, MRSA USA300 bacterial suspension was added at MOI=10, and the infection was carried out for a total of 6 h. Subsequently, cells were lysed and serially diluted and plated on TSA plates, and intracellular colony-forming units (CFU / mL) were counted.

[0058] 2. Experimental Results Overexpression of RAB7A significantly inhibits intracellular MRSA: such as Figure 5 As shown, compared with the empty vector control group, the intracellular bacterial load in the RAB7A overexpression group was significantly reduced ( p The value <0.01 indicates that increasing RAB7A expression levels can enhance the host's ability to clear intracellular MRSA.

[0059] Interference with RAB7A eliminates protective effects: such as Figure 6 As shown, compared with the si-NC control group, knocking down RAB7A expression significantly increased intracellular bacterial load ( p <0.01), indicating that loss of RAB7A function weakens the host's defense against intracellular MRSA.

[0060] Bidirectional genetic validation through overexpression and interference confirmed that RAB7A is a key positive regulator of host resistance to intracellular MRSA infection. This result further supports the mechanism by which SAL exerts its host-directed anti-infection effect by targeting and activating RAB7A.

Claims

1. Use of salinomycin in the preparation of drugs for the treatment or prevention of intracellular methicillin-resistant Staphylococcus aureus (MRSA) infections.

2. The application according to claim 1, characterized in that, The drug exerts its anti-intracellular MRSA infection effect by targeting the RAB7A protein in host cells.

3. The application according to claim 1, characterized in that, The drug is an agonist or enhancer of the RAB7A protein.

4. The application according to claim 1, characterized in that, The intracellular MRSA infection includes chronic or recurrent infections mediated by intracellular MRSA retention.

5. The application according to claim 4, characterized in that, The chronic or recurrent infection is selected from one or more of the following diseases: mastitis, osteomyelitis, endocarditis, necrotizing pneumonia, and sepsis.

6. The application according to claim 1, characterized in that, The drug is a formulation made with salinomycin as the active ingredient, in combination with a pharmaceutically acceptable carrier or excipient.

7. The application according to claim 6, characterized in that, The preparation is selected from injections, powder injections, tablets, capsules, granules, oral liquids, external preparations, or inhalants.

8. The application according to claim 1, characterized in that, The drug is used for preventative or therapeutic administration.

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