Use of midostaurin in the preparation of a medicament for inhibiting or combating listeria monocytogenes

Abstract

This invention belongs to the field of medical pharmaceutical technology, specifically the application of midostaurin in the preparation of drugs that inhibit or combat Listeria monocytogenes. This invention provides a novel medical use of midostaurin, a drug for treating FLT3 mutation-positive acute myeloid leukemia (AML) and invasive systemic mastocytosis, in the prevention or treatment of Listeria monocytogenes infection. By targeting the key virulence factor PlcA in Listeria monocytogenes, it reduces phagocytic escape and proliferation within bacterial cells, thereby decreasing bacterial pathogenicity. In in vitro and in vivo therapeutic trials, midostaurin demonstrated significant therapeutic effects against Listeria monocytogenes infection, indicating broad medical applications.

Description

Technical Field

[0001] This invention relates to the field of medical pharmaceutical technology, specifically the use of midostaurin in the preparation of drugs that inhibit or combat Listeria monocytogenes. Background Technology

[0002] Listeria monocytogenes is a common and important foodborne pathogen worldwide. It is a Gram-positive opportunistic pathogen that can infect humans and various animals, causing fatal diseases such as abortion, stillbirth, sepsis, and meningitis. This intracellular pathogen escapes phagosomes by secreting virulence factors (such as LLO, PlcA, and PlcB) within the cell, thereby replicating and multiplying within the host cell. Currently, treatment for this bacterial infection mainly relies on antibiotics, but existing antibiotic therapies carry a serious risk of bacterial resistance, and direct-acting bactericidal drugs may also cause side effects. Therefore, developing drugs that treat this bacteria through novel mechanisms is of great value. Phagosome escape is a key pathway for the intracellular replication and proliferation of Listeria monocytogenes, and PlcA plays a crucial promoting role in this process. Currently, there are no clinical drugs that target PlcA to inhibit Listeria monocytogenes infection.

[0003] Midotulin, a multi-target tyrosine kinase inhibitor, is FDA-approved for the treatment of FLT3-mutant positive acute myeloid leukemia (AML) and aggressive systemic mastocytosis. This invention explores for the first time its novel application in clearing bacterial infections by targeting the key bacterial virulence factor PlcA to inhibit intracellular escape from Listeria monocytogenes. This strategy, through "drug repurposing," not only effectively avoids the drawbacks of high costs and long development cycles in new drug development but also reduces the pressure of bacterial resistance by blocking the bacterial infection process rather than through traditional direct bactericidal mechanisms, providing a new strategy and approach for the development of anti-infective drugs. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0006] The use of midostaurin in the preparation of drugs that inhibit or combat Listeria monocytogenes.

[0007] As a preferred embodiment of the use of midostaurin as described in this invention in the preparation of a drug for inhibiting or combating Listeria monocytogenes, the drug can effectively inhibit the intracellular escape process of Listeria monocytogenes in infected cells and enhance the host cell's ability to clear pathogens.

[0008] As a preferred embodiment of the use of midostaurin as described in this invention in the preparation of a medicament for inhibiting or combating Listeria monocytogenes, the medicament comprises midostaurin and a pharmaceutically acceptable formulation.

[0009] As a preferred embodiment of the use of midostaurin as described in this invention in the preparation of a drug for inhibiting or combating Listeria monocytogenes, the drug is administered via a route including gastrointestinal administration and non-gastrointestinal administration.

[0010] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a novel medical application of midostaurin, a drug used to treat FLT3 mutation-positive acute myeloid leukemia (AML) and aggressive systemic mastocytosis, in the prevention or treatment of Listeria monocytogenes infection. By targeting the key virulence factor PlcA in Listeria monocytogenes, it reduces phagocytic escape and proliferation within bacterial cells, thereby reducing bacterial pathogenicity. In in vitro and in vivo therapeutic trials, midostaurin has shown significant therapeutic effects against Listeria monocytogenes infection, indicating its broad medical applications. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0012] Figure 1 The following are the experimental results of midostaurin binding to and inhibiting PlcA activity in this invention: (A) protein thermal drift; (B) PlcA hydrolysis phosphatidylinositol activity inhibition test; (C) Listeria monocytogenes in vitro growth curve.

[0013] Figure 2 The following are the results of the protective clearance of bacterial infection by midostaurin at the cellular level in this invention: (A) LAMP2-labeled bacterial statistics; (B) intracellular bacterial growth curve; (C) bacterial infection-induced cell damage assay.

[0014] Figure 3 Figure 1 shows the results of the in vivo protection test of midostaurin in this invention ((A) Survival test of mice infected with Listeria monocytogenes; (B) Histopathological evaluation). Detailed Implementation

[0015] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0017] This invention discloses a new use of midotulin, a multi-target tyrosine kinase inhibitor and a drug already approved by the FDA for the treatment of FLT3 mutation-positive acute myeloid leukemia (AML) and invasive systemic mastocytosis. Specifically, it reduces the escape and proliferation of phagosomes within bacterial cells by targeting the key virulence factor PlcA of Listeria monocytogenes, thereby reducing the pathogenicity of the bacteria and can be used to prevent or treat Listeria monocytogenes infection.

[0018] The present invention provides midotoxin that can bind to Listeria monocytogenes and exert a key virulence factor, PlcA, during the pathogenic process, thereby inhibiting the escape of intracellular phagosomes and enhancing the host's clearance of the infecting bacteria.

[0019] In this embodiment, protein drift technology was used to screen potential candidate compounds that bind to the PlcA protein by utilizing changes in protein thermostability, and midostaurin was identified as a potential inhibitor of PlcA through activity inhibition. Phosphatidylinositol (PI) hydrolysis experiments demonstrated that midostaurin effectively inhibited PlcA activity and had no inhibitory effect on the in vitro growth of Listeria monocytogenes. Late endosome fluorescent probe (LAMP2) was used to quantitatively analyze bacterial markers in Listeria monocytogenes-infected cells after midostaurin treatment to evaluate bacterial intracellular escape. Intracellular growth curves and cytotoxicity assays confirmed that midostaurin effectively inhibited bacterial intracellular escape, reduced intracellular proliferation, and effectively alleviated damage to infected cells. A mouse model of Listeria monocytogenes infection was established, demonstrating that midostaurin effectively reduced the mortality rate of infected mice and alleviated pathological damage to tissues.

[0020] Example 1

[0021] Midottolin is a novel inhibitor that targets the key force factor PlcA of Listeria monocytogenes, reduces intracellular escape of infecting bacteria, and enhances host activity. It is available in any formulation form suitable for clinical use.

[0022] Example 2

[0023] Midottolin, as an inhibitor of the key virulence factor PlcA of Listeria monocytogenes, is used in the preparation of drugs for the treatment of infectious diseases.

[0024] Example 3

[0025] Midottolin is used as an inhibitor of Listeria monocytogenes virulence factor PlcA to treat infectious diseases caused by bacteria, especially infections associated with the key virulence factor PlcA of Listeria monocytogenes.

[0026] Experiment 1

[0027] Protein thermal drift binding, activity inhibition assay and in vitro antibacterial activity analysis:

[0028] Recombinant PlcA protein with a histidine tag was co-incubated with different concentrations of midostaurin. After incubation, an orange fluorescent probe dye (which binds to the hydrophobic groups of the protein) was added to the co-incubation system. The thermostability curves of the PlcA protein in different co-incubation systems were detected using real-time quantitative PCR, and the melting temperature (Tm) was calculated. Figure 1 A). Engineered *E. coli* carrying the PlcA protein expression plasmid were plated on phosphatidylinositol lecithin plates containing midostaurin and DMSO (solvent control) under induction conditions, and incubated at 37°C. Changes in the phospholipid hydrolysis rings around the colonies were observed. Figure 1 B). Listeria monocytogenes strain was co-cultured with different concentrations of midostaurin, and bacterial growth was monitored by ultraviolet absorbance values. Figure 1 C).

[0029] Conclusion: Midottolin can directly bind to PlcA protein to enhance its thermal stability, effectively inhibit the activity of PlcA in hydrolyzing phosphatidylinositol, and does not affect the in vitro growth of Listeria monocytogenes.

[0030] Experiment 2

[0031] Analysis of the effect of midostaurin at the cellular level on Listeria monocytogenes infection:

[0032] HeLa cells were seeded onto cell crawling slides and infected with bacteria at an MOI of 100. The infection systems were incubated with midostaurin and the control solvent DMSO for 1 h. Immunofluorescence staining was performed using Listeria monocytogenes antibody and the late endosome marker LAMP2 antibody, and the percentage of bacteria co-localized with LAMP2 in different groups was calculated. Figure 2 A). HeLa cells were incubated with midostaurin and DMSO (the solvent control) to infect Listeria monocytogenes (MOI=100). Colonies were counted on lysed cells at different time points. Figure 2 B). HeLa cells were co-incubated with midostaurin and the solvent control DMSO for 6 h to infect Listeria monocytogenes, and the release of lactate dehydrogenase (LDH) in the culture supernatant was measured. Figure 2 C).

[0033] Conclusion: Midottolin can inhibit intracellular escape of Listeria monocytogenes, enhance the labeling of phagocytic markers, inhibit intracellular bacterial proliferation, and reduce damage to infected cells.

[0034] Experiment 3

[0035] Evaluation of the in vivo effects of midostaurin:

[0036] 6-8 week old BALB / c mice were intraperitoneally injected with 1×10⁻⁶ Listeria monocytogenes. 7 CFU (Case-Free Factor Analysis) or 5×10 6 An infection model was established using CFU (cartopathological analysis), followed by subcutaneous injection of midostaurin (100 mg / kg). The drug was administered every 8 hours post-infection, with mortality monitored for 96 hours and tissue damage monitored for 36 hours.

[0037] Conclusion: Compared with the infection group, midostaurin effectively reduced the mortality rate of Listeria monocytogenes-infected mice. Figure 3 Midotuximab (A) significantly alleviates histopathological damage caused by bacterial infection. This suggests that midotuximab holds promise as a novel candidate drug for treating Listeria monocytogenes infection.

[0038] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Application of midostaurin in the preparation of drugs that inhibit or combat Listeria monocytogenes.

2. The use of midostaurin according to claim 1 in the preparation of drugs that inhibit or combat Listeria monocytogenes, characterized in that, The drug can effectively inhibit the intracellular escape process of Listeria monocytogenes in infected cells and enhance the host cell's ability to clear pathogens.

3. The use of midostaurin according to claim 1 in the preparation of drugs for inhibiting or combating Listeria monocytogenes, characterized in that, The drug comprises midostaurin and pharmaceutically acceptable formulations.

4. The use of midostaurin according to claim 1 in the preparation of drugs for inhibiting or combating Listeria monocytogenes, characterized in that, The drug can be administered via gastrointestinal or non-gastrointestinal routes.

Images