Application of MAPK Pathway Inhibitors in Anti-Pathogen Infections

Abstract

The present invention discloses the use of MAPK pathway inhibitors in anti-pathogen infection. Specifically, the present invention discloses the use of a MAPK pathway inhibitor for preparing a pharmaceutical preparation or composition for anti-pathogen infection. The MAPK pathway inhibitor of the present invention controls the reproduction of pathogens in host cells and / or reduces the number of pathogens by promoting the diffusion of vimentin in cells, thereby effectively controlling pathogen infection.

Description

Technical Field

[0001] The present invention relates to the field of anti-infection, and particularly to the application of MAPK pathway inhibitors in anti-pathogen infection. Background Art

[0002] Pathogenic microorganisms are a group of microorganisms that can cause human infections and even severe infectious diseases. They exist in every corner of nature, including air, soil, and water. Pathogenic microorganisms include bacteria, viruses, chlamydia, rickettsia, mycoplasma, spirochetes, and fungi, etc.

[0003] Salmonella is a common zoonotic pathogen and one of the four major causes of diarrheal diseases. Diarrheal diseases are the most common diseases caused by unsafe food, with 550 million people suffering from them every year, including 220 million children under five years old. According to the report of the World Health Organization, the global food contamination by Salmonella is becoming increasingly serious, causing huge economic losses and seriously threatening human health and life safety. Therefore, it has been listed as an important object and index for the detection of pathogenic bacteria in food. In China, food poisoning caused by Salmonella ranks first repeatedly. According to data statistics, 70%-80% of bacterial food poisoning in China is caused by Salmonella, and more than 90% of the foods that cause Salmonella poisoning are animal products such as meat. The symptoms of food poisoning caused by Salmonella mainly include nausea, vomiting, abdominal pain, headache, chills, and diarrhea, etc., and are also accompanied by fatigue, muscle soreness, blurred vision, moderate fever, restlessness, and lethargy, lasting for 2-3 days, with an average fatality rate of 4.1%. Although the overall global health conditions have been greatly improved at present, Salmonella incidents still occur from time to time.

[0004] Salmonella is a Gram-negative, intracellular parasitic intestinal bacterium, widely distributed in nature and with a wide variety. More than 2,500 Salmonella serotypes have been detected so far, and the vast majority of Salmonella are pathogenic to humans and animals. However, due to the abuse of antibiotics, the drug resistance of Salmonella is becoming increasingly serious. At present, it has developed drug resistance to β-lactams, quinolones, tetracyclines, aminoglycosides, sulfonamides, etc., and its drug resistance mechanism is extremely complex. As time goes by, its drug resistance rate will increase significantly, and its drug resistance spectrum will also continue to widen, posing a serious threat to the aquaculture industry and human health in China.

[0005] However, whether the body gets sick after being invaded by pathogens depends on the body's own immunity on the one hand, and on the pathogenicity and the number of invading pathogens on the other hand. Generally, the larger the number, the greater the possibility of getting sick.

[0006] Therefore, in order to effectively control Salmonella infection and the diseases caused by it, there is an urgent need in this field to develop and find compounds that can effectively control its reproduction in host cells and / or reduce the number of pathogens. Summary of the Invention

[0007] The object of the present invention is to provide compounds capable of effectively controlling the reproduction of pathogens in host cells and / or reducing the number of pathogens, and their use in anti-pathogen infections.

[0008] In a first aspect of the present invention, there is provided the use of a MAPK pathway inhibitor for preparing a composition or preparation, which is used for:

[0009] (a) promoting the diffusion of vimentin in cells; and

[0010] (b) inhibiting pathogen infection.

[0011] In another preferred embodiment, the MAPK pathway inhibitor includes teni drugs or pharmaceutically acceptable salts thereof.

[0012] In another preferred embodiment, the MAPK pathway inhibitor is selected from the group consisting of: Trametinib, Refametinib, Selumetinib, Cobimetinib, Pimasertib, TAK-733, AZD8330, BI-847325, GDC-0623, PD318088, or a combination thereof; preferably Trametinib, Refametinib, Selumetinib, more preferably Trametinib.

[0013] In another preferred embodiment, the pharmaceutically acceptable salts include hydrochloride, sulfate, sulfonate, carbonate, acetate, tartrate, or hydroxyethyl sulfonate.

[0014] In another preferred embodiment, the pathogens include: bacteria, chlamydia, rickettsia, mycoplasma, spirochetes, fungi, or a combination thereof.

[0015] In another preferred embodiment, the bacteria include Gram-negative bacteria and Gram-positive bacteria.

[0016] In another preferred embodiment, the bacteria include: Salmonella, Staphylococcus aureus, Escherichia coli, Shigella, Listeria, Streptococcus, Mycobacterium tuberculosis, Klebsiella, Acinetobacter baumannii, or a combination thereof.

[0017] In another preferred embodiment, the bacteria are Salmonella.

[0018] In another preferred embodiment, the composition is a pharmaceutical composition.

[0019] In another preferred embodiment, the pharmaceutical composition contains (i) a MAPK pathway inhibitor or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

[0020] In another preferred embodiment, the MAPK pathway inhibitor is a tinib compound.

[0021] In another preferred embodiment, the MAPK pathway inhibitor is selected from the group consisting of Trametinib, Refametinib, Selumetinib, Cobimetinib, Pimasertib, TAK-733, AZD8330, BI-847325, GDC-0623, PD318088, or a combination thereof; preferably Trametinib, Refametinib, Selumetinib, more preferably Trametinib.

[0022] In another preferred embodiment, the MAPK pathway inhibitor is Trametinib.

[0023] In another preferred embodiment, the pharmaceutically acceptable salts include hydrochloride, sulfate, sulfonate, carbonate, acetate, tartrate, or hydroxyethyl sulfonate.

[0024] In another preferred embodiment, the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0025] In another preferred embodiment, the pharmaceutical composition includes tablets, pills, powders, capsules, syrups, injections, patches, drops, ointments, or sprays.

[0026] In another preferred embodiment, the administration methods of the pharmaceutical composition include oral administration, intramuscular injection, intravenous injection, intravenous drip, intratumoral injection, enema, spraying, external application, or intraperitoneal injection.

[0027] In another preferred embodiment, the "inhibiting pathogen infection" includes inhibiting pathogen infection at the cellular level and / or inhibiting pathogen infection at the animal level.

[0028] In another preferred embodiment, the "inhibiting pathogen infection" is selected from the group consisting of inhibiting pathogen replication in host cells, reducing infected cell death caused by pathogens, reducing the number of pathogen-infected cells, reducing the number of pathogens released by assembled pathogen-infected cells, inhibiting the expression of pathogen proteins in infected cells, or a combination thereof.

[0029] In another preferred embodiment, the "inhibiting pathogen infection" further includes inhibiting the bacterial load of pathogens in animal tissues and / or inhibiting tissue lesions caused by pathogens.

[0030] In a second aspect of the present invention, there is provided a method for non-therapeutically inhibiting the infection of host cells by a pathogen in vitro, comprising the step of adding a MAPK pathway inhibitor or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, to a host cell culture system containing the pathogen, thereby inhibiting the infection of host cells by the pathogen.

[0031] In another preferred embodiment, the pathogen includes: bacteria, chlamydia, rickettsia, mycoplasma, spirochete, fungus, or a combination thereof.

[0032] In another preferred embodiment, the pathogen is Salmonella.

[0033] In another preferred embodiment, the host cell is a human or non-human mammalian cell.

[0034] In another preferred embodiment, the MAPK pathway inhibitor is a teni-class drug.

[0035] In another preferred embodiment, the MAPK pathway inhibitor is selected from the group consisting of: Trametinib, Selumetinib, Refametinib, Cobimetinib, Pimasertib, TAK-733, AZD8330, BI-847325, GDC-0623, PD318088, or a combination thereof; preferably Trametinib, Refametinib, Selumetinib, and more preferably Trametinib.

[0036] In another preferred embodiment, the MAPK pathway inhibitor is Trametinib.

[0037] In a third aspect of the present invention, there is provided a method for combating pathogen infection, the method comprising the step of administering a safe and effective amount of a MAPK pathway inhibitor or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, to a subject in need thereof, thereby inhibiting pathogen infection.

[0038] In another preferred embodiment, the subject in need thereof is a human or non-human mammal.

[0039] In another preferred embodiment, the subject in need thereof is infected with a pathogen or at risk of being infected with a pathogen.

[0040] In another preferred embodiment, the pathogen includes: bacteria, chlamydia, rickettsia, mycoplasma, spirochete, fungus, or a combination thereof.

[0041] In another preferred embodiment, the pathogen is Salmonella.

[0042] In another preferred embodiment, the MAPK pathway inhibitor is a teni-class compound.

[0043] In another preferred embodiment, the MAPK pathway inhibitor is selected from the group consisting of: Trametinib, Selumetinib, Refametinib, Cobimetinib, Pimasertib, TAK-733, AZD8330, BI-847325, GDC-0623, PD318088, or a combination thereof; preferably Trametinib, Refametinib, Selumetinib, more preferably Trametinib.

[0044] In another preferred embodiment, the MAPK pathway inhibitor is Trametinib.

[0045] In another preferred embodiment, the safe and effective amount refers to: 0.001 - 50 mg / kg (body weight), preferably 0.04 - 5 mg / kg (body weight), more preferably 0.04 - 1 mg / kg (body weight), most preferably 0.04 mg / kg (body weight).

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

[0047] Figure 1 shows that small molecule inhibitors of the MAPK pathway can significantly promote the diffusion of cytoplasmic vimentin. Among them Figure 1A shows the significant difference in the effect of small molecule compounds on the intracellular vimentin area; the small molecule compounds with log2FC > 0.3 (FC > 1.2) and -log 10 Pvalue > 1 (P value < 0.1) are boxed in green, and the small molecule compounds with log2FC > 0.5 (FC > 1.4) and -log 10 Pvalue > 2 (P value < 0.01) are boxed in orange. Figure 1B and Figure 1C shows the result of signal pathway enrichment for the Figure 1A small molecule compounds therein.

[0048] Figure 2 shows that MEK1 / 2 small molecule inhibitors in the MAPK pathway can significantly promote the diffusion of vimentin. Figure 2A It is a pie chart showing the proportion of small molecule inhibitors of the MAPK pathway belonging to different inhibition targets. Figure 2B shows the chemical structures of 9 MEK1 / 2 small molecule inhibitors therein. Figure 2CShows the effects of the top 6 compounds that promote vimentin diffusion on vimentin morphology.

[0049] Figure 3 shows that the MEK1 / 2 small molecule inhibitor can inhibit Salmonella infection. Figure 3A Shows the effects of 6 compounds on cell viability after 24 h of treatment at different concentrations (0.046 μM - 100 μM). Figure 3B Shows that 5 compounds with no obvious toxicity to cells at a concentration of 1 μM for 24 h can all inhibit Salmonella replication, expressed as colony-forming unit (CFU), and trametinib has the most obvious effect. Among them: **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0050] Figure 4 shows that the MEK1 / 2 inhibitor trametinib can promote the diffusion of cytoplasmic vimentin. Figure 4A Shows the inhibitory effect of trametinib on the MEK1 / 2 downstream kinase ERK1 / 2. Figure 4B Shows the immunofluorescence staining results of cytoplasmic vimentin after treatment with gradient concentrations of trametinib. The label length of the top legend is 65 μm, and the label length of the ROI magnified legend is 10 μm. Figure 4C Is Figure 4B The quantitative statistical results of, with the number of cells in each group n = 100 - 114. Among them: ***p < 0.001.

[0051] Figure 5 shows that trametinib can inhibit Salmonella infection. Figure 5A Shows that after treatment with 1 μM trametinib, Salmonella forms dispersed small Salmonella-containing follicles (microSCV) in host cells, with a label length of 10 μm. Figure 5B Is Figure 5A The quantitative statistical results of. Figure 5C Shows that the half-maximal inhibitory concentration of trametinib on Salmonella replication is 4.579 μM. Among them: ****p < 0.0001.

[0052] Figure 6 Shows that trametinib can inhibit the number of bacteria replicated in mice. Figure 6 A shows that trametinib significantly inhibits the number of Salmonella in mouse feces (CFU / g). Figure 6 B shows that trametinib significantly inhibits the number of Salmonella in the small intestine of mice (CFU / g). Figure 6 C shows that trametinib significantly inhibits the number of Salmonella in the spleen of mice (CFU / organ). There are 5 mice in each group. Among them: **p < 0.01, ***p < 0.001.

[0053] Figure 7 It shows that trametinib can improve splenomegaly in mice caused by Salmonella. Figure 7 A shows an example diagram of the changes in the spleens of mice. After intragastric administration of Salmonella (1×10 5 CFU) for 28 h, the spleens of the mice were enlarged; after intragastric administration of trametinib, the morphology of the spleens of the mice recovered. Figure 7 B shows a statistical chart of the weights of the spleens of mice, with 5 mice in each group. Among them: ns, p>0.05, *p<0.05, **p<0.01.

[0054] Figure 8 It shows that trametinib can improve the colonic lesions caused by Salmonella. Figure 8 A shows that after intragastric administration of Salmonella (1×10 5 CFU) for 28 h, obvious pathological changes occurred in the colons of the mice, including a significant increase in the mucosal length and edema in the submucosa. After intragastric administration of trametinib for 24 h, the pathological changes in the colons of the mice were significantly improved. The upper legend label length is 100 μm, and the lower legend label length is 25 μm. Figure 8 B shows a quantitative statistical chart of the significant improvement of trametinib on the thickening of the colonic mucosa in mice. Figure 8 C shows a quantitative statistical chart of the significant improvement of trametinib on the submucosal edema in mice. Among them: ns, p>0.05, *p<0.05, **p<0.01, ****p<0.0001. Detailed implementation manners

[0055] Through extensive and in-depth research, the present inventors unexpectedly discovered for the first time that MAPK pathway inhibitors can significantly promote the intracellular distribution of vimentin in mammalian cells, and by promoting the significant diffusion of vimentin in the cytoplasm, achieve resistance to pathogen infection, reduce the number of pathogens invading and replicating, thereby significantly reducing the pathogenicity of pathogens. On this basis, the present invention was completed.

[0056] Specifically, the experiments of the present invention show that multiple small molecule inhibitors related to the MAPK signaling pathway can cause significant diffusion of cytoplasmic vimentin, and show a dose-dependence. For example, the inhibitor of the present invention can effectively inhibit Salmonella infection by promoting the significant diffusion of vimentin in the cytoplasm. The inhibitor of the present invention can disrupt the integrity of the follicular SCV containing Salmonella to effectively inhibit Salmonella replication. In addition, the inhibitor of the present invention can inhibit the bacterial load of Salmonella in mouse tissues, and can effectively improve splenomegaly and colonic lesions caused by Salmonella.

[0057] Terms

[0058] To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0059] The term "about" refers to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined. As used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0060] As used herein, the terms "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the said terms also include "consisting essentially of", or "consisting of".

[0061] MAPK pathway and its inhibitors

[0062] The MAPK signaling pathway is an important signal transduction system in eukaryotic cells that mediates extracellular signals into intracellular responses. The signals related to the MAPK pathway are a highly conserved family of protein kinases. A total of 19 MAPKKKs (also known as MAP3Ks or MEKKs), 7 MAPKKs (MAP2Ks), and 14 MAPKs have been discovered. They are three-tiered cascade signaling molecules, where MAPKKK activates MAPKK, and the latter then activates MAPK, that is, extracellular signal → MAPK kinase kinase (MKKK) → MAPK kinase (MKK) → MAPK, regulating various physiological processes such as cell growth, differentiation, apoptosis, and death. The MAPK signaling pathway mainly includes the classical MEK / ERK signaling pathway, the JNK / p38MAPK signaling pathway, and the ERK5 signaling pathway. Among them, the earliest discovered classical MEK / ERK signal transduction pathway is involved in the signal transduction after the activation of various growth factors, cytokines, mitogens, and hormone receptors, and has an important regulatory role in cell proliferation, growth, and differentiation.

[0063] The present invention provides the use of MAPK pathway inhibitors, which are used for anti-pathogen infection.

[0064] As used herein, the terms "compounds of the present invention", "MAPK pathway inhibitors of the present invention", and "inhibitors of the present invention" are used interchangeably and refer to MAPK pathway inhibitors that can be used for anti-pathogen infection.

[0065] As used herein, the term "MAPK pathway inhibitor" refers to an inhibitor that can inhibit the MAPK pathway. Representative ones include (but are not limited to): TKI drugs (including TKI drugs, or their analogs, or their pharmaceutically acceptable salts).

[0066] As used herein, the terms "tinib drugs" and "tinib compounds" are used interchangeably.

[0067] In a preferred embodiment of the present invention, the MAPK pathway inhibitor is selected from the group consisting of: Trametinib, Refametinib, Selumetinib, Pimasertib, Cobimetinib, TAK-733, AZD8330, BI-847325, GDC-0623, PD318088, or a combination thereof.

[0068] Tinib drugs are a class of approved "tinib" drugs, and the indications of this class of drugs mainly focus on tumors, and secondly also involve skin diseases, blood system diseases, immune system diseases, and musculoskeletal and connective tissue diseases. The important targets and pathways of tinib drugs are systematic and complex, and the main targets include tyrosine-protein kinase, tyrosine-protein kinase receptor, various growth factor receptors, and mitogen-activated proteinkinase kinase.

[0069] Trametinib used in the examples of the present invention is an inhibitor of mitogen-activated proteinkinase kinase MEK1 / 2, and is approved by the US FDA for the treatment of unresectable or metastatic melanoma with BRAF600E or V600K gene mutations and in combination with dabrafenib for the treatment of metastatic non-small cell lung cancer (NSCLC) with BRAFV600E mutations.

[0070] Vimentin

[0071] Vimentin is one of the proteins of intermediate filaments. Intermediate filaments are an important structural feature of eukaryotic cells. They, together with microtubules and actin microfilaments, form the cytoskeleton.

[0072] In the examples of the present invention, using the MAPK pathway inhibitor of the present invention can promote the diffusion of vimentin in cells and play a role in anti-pathogen infection.

[0073] Anti-pathogen infection / Inhibition of pathogen infection

[0074] The present invention provides the use of MAPK pathway inhibitors in the prevention of pathogen infections, where the pathogens include bacteria, chlamydia, rickettsia, mycoplasma, spirochetes, fungi, or combinations thereof.

[0075] In another preferred embodiment, the bacteria include Gram-negative bacteria and Gram-positive bacteria.

[0076] Specifically, the bacteria are selected from the group consisting of Salmonella, Staphylococcus aureus, Escherichia coli, Shigella, Listeria, Streptococcus, Mycobacterium tuberculosis, Klebsiella, Acinetobacter baumannii, or combinations thereof.

[0077] In the present invention, the pathogens include wild-type, or its subtypes, or its mutants.

[0078] As used herein, the terms "prevention of pathogen infection" and "inhibition of pathogen infection" are used interchangeably.

[0079] In another preferred embodiment, the "inhibition of pathogen infection" includes inhibiting pathogen infection at the cellular level, and / or inhibiting pathogen infection at the animal level.

[0080] In another preferred embodiment, the "inhibition of pathogen infection" is selected from the group consisting of inhibiting pathogen replication in host cells, reducing infected cell death caused by the pathogen, reducing the number of cells infected by the pathogen, reducing the number of pathogens released by the assembly of infected cells, inhibiting the expression of pathogen proteins in infected cells, or combinations thereof.

[0081] In another preferred embodiment, the "inhibition of pathogen infection" further includes inhibiting the bacterial load of the pathogen in animal tissues, and / or inhibiting tissue lesions caused by the pathogen.

[0082] In one embodiment of the present invention, the MAPK pathway inhibitor inhibits Salmonella infection. Specifically, the MAPK pathway inhibitor (such as trametinib) effectively inhibits Salmonella intracellular replication by promoting the intracellular diffusion of vimentin and disrupting the integrity of the Salmonella-containing vacuole SCV. Further, it inhibits the bacterial load of Salmonella in mouse tissues and improves tissue lesions in mice (including the spleen and colon).

[0083] Pharmaceutical composition

[0084] As used herein, the term "pharmaceutical composition" refers to a composition that is administered for a specific purpose.

[0085] For the purposes of the present invention, the pharmaceutical composition contains a MAPK pathway inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier and is used for the prevention of pathogen infection. The MAPK pathway inhibitors include teni drugs and their analogs.

[0086] The term "pharmaceutically acceptable" refers to substances that are listed and recognized in the pharmacopoeia or by government drug regulatory agencies and can be used in vertebrates, especially in humans. Generally, a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatible" herein means that the components in the composition can be admixed with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0087] There is no particular limitation on the mode of administration of the active ingredient or pharmaceutical composition of the present invention. Representative modes of administration include (but are not limited to): oral administration, intramuscular injection, intravenous injection, intravenous drip, intratumoral injection, enema, spraying, topical application, or intraperitoneal injection.

[0088] Solid dosage forms for oral administration include tablets, pills, powders, granules, or capsules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or calcium phosphate, or is mixed with the following components: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxypropylmethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0089] Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared with coatings and shell materials, such as enteric coatings and other materials well known in the art. They can contain opacifying agents, and the release of the active ingredient or compound in such a composition can be delayed and released in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric materials and wax materials. If necessary, the active ingredient can also be in the form of microcapsules with one or more of the above excipients.

[0090] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms can contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifying agents, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances, etc.

[0091] In addition to these inert diluents, the composition can also contain adjuvants, such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and fragrances.

[0092] In addition to the active ingredient, the suspension can contain suspending agents, for example, ethoxylated isooctadecanol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum monostearate, and agar, or mixtures of these substances, etc.

[0093] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or non - aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for re - dissolving into sterile injectable solutions or dispersions. Suitable aqueous and non - aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and their suitable mixtures.

[0094] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required under sterile conditions.

[0095] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds. For example, the compounds of the present invention can be used in combination with other antibacterial drugs, or drugs that enhance immunity.

[0096] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal in need of treatment (such as a human), and the dosage during administration is an effective dosage considered pharmaceutically. For a person weighing 60 kg, the daily dosage is usually 1 - 2000 mg, preferably 6 - 600 mg. Of course, the specific dosage should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician. Usually, the "safe and effective amount" refers to: an amount of the compound sufficient to significantly improve the condition without causing serious side effects. In addition, the pharmaceutical composition or active ingredient of the present invention can also be administered together with other anti-pathogen infection therapeutic agents.

[0097] The main advantages of the present invention are as follows:

[0098] (1) The present invention discovers for the first time that MAPK pathway inhibitors have the efficacy of anti-pathogen (such as Salmonella) infection;

[0099] (2) The present invention discovers for the first time the regulation of vimentin distribution by the MAPK signaling pathway;

[0100] (3) The present invention discovers for the first time that MAPK pathway inhibitors inhibit pathogen-infected cells by promoting the diffusion of vimentin in cells;

[0101] (4) The compounds of the present invention include drugs such as trametinib and selumetinib that have been used clinically, with guaranteed biosafety, and the cycle of putting old drugs into new clinical uses is greatly shortened.

[0102] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the materials and reagents used in the embodiments of the present invention are all commercially available products. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0103] Example 1

[0104] Confirmation of the effect that small molecule inhibitors of the MAPK pathway significantly promote the diffusion of cytoplasmic vimentin

[0105] Experimental method:

[0106] The vimentin-GFP and actin-RFP double-labeled osteosarcoma cell line U2OS was cultured in a 384-well plate, where vimentin was the target protein and actin was used to label the cell contour. When the cells adhered and reached a density of 70-80%, approximately 1700 test compounds from the selleck small molecule inhibitor compound library were added to each well. Based on previous studies, the compound administration concentration was determined to be 1 μM, and the drug application time was 12 h. Three replicate wells were set for each compound, and the control wells were 0.1% DMSO, WFA (known to cause vimentin aggregation in cells), and hypotonic medium (medium diluted with deionized water (medium: deionized water = 1:9), treated for 5-7 minutes, resulting in obvious diffusion of vimentin in cells). After fixation and DAPI nuclear staining, cell imaging and data quantitative analysis were performed using a PerkinElmer high-content imaging system and Harmony imaging and analysis software.

[0107] Experimental results:

[0108] The average area ratio of vimentin / actin in the 0.1% DMSO control group was normalized to 1, and the effects of all compounds on the vimentin area were analyzed, with the fold change (FC) and P value as reference indicators.

[0109] As Figure 1A shown, with log2FC as the abscissa and -log 10 Pvalue as the ordinate, the differential significance of the effects of compounds on the vimentin area was analyzed. Among them, the small molecule compounds (a total of 196) with log2FC > 0.3 (FC > 1.2) and -log 10 Pvalue > 1 (P value < 0.1) were boxed in green; the small molecule compounds (a total of 19) with log2FC > 0.5 (FC > 1.4) and -log 10 Pvalue > 2 (Pvalue < 0.01) were boxed in orange. This indicates that these compounds caused significant diffusion of cytoplasmic vimentin.

[0110] The above 196 small molecule compounds were subjected to signal pathway enrichment analysis. With -log 10 Pvalue as the abscissa and signal pathway as the ordinate, it was found that the MAPK signal pathway had the most significant enrichment, as Figure 1B and Figure 1C shown. The above results indicate that multiple small molecule inhibitors related to the MAPK signal pathway can cause significant diffusion of cytoplasmic vimentin.

[0111] Example 2

[0112] Confirmation of the significant effect of small molecule inhibitors of MEK1 / 2 in the MAPK pathway on promoting vimentin diffusion

[0113] Experimental method:

[0114] Since the MAPK signaling pathway contains a large number of protein kinases, further in-depth analysis was carried out on the MAPK signaling pathway that causes cytoplasmic vimentin diffusion (Foldchange > 1.2, P value < 0.1), and different target protein inhibitors were classified.

[0115] Experimental results:

[0116] As Figure 2A shown, among the 18 small molecule inhibitors of the MAPK pathway that cause cytoplasmic vimentin diffusion with FC > 1.2 and P value < 0.1, there are 9 MEK1 / 2 inhibitors (accounting for 50%), 4 RAF inhibitors (accounting for 22%), 3 p38 MAPK inhibitors (accounting for 17%), and 1 ERK and JNK inhibitor each (accounting for 6% each).

[0117] Further reducing the P value (< 0.05), there are a total of 14 small molecule inhibitors of the MAPK pathway, among which there are 9 MEK1 / 2 inhibitors (accounting for 65%), 2 RAF inhibitors (accounting for 14%), 2 p38 MAPK inhibitors (accounting for 14%), and 1 JNK inhibitor (accounting for 7%).

[0118] Furthermore, among the small molecule inhibitors of the MAPK pathway that cause cytoplasmic vimentin diffusion with FC > 1.4 and P value < 0.01, there are a total of 6, and all are MEK1 / 2 inhibitors (accounting for 100%).

[0119] Figure 2B The chemical structural formulas of 9 MEK1 / 2 inhibitors that significantly promote vimentin diffusion are shown, including Refametinib, Pimasertib, AZD8330, Trametinib, TAK-733, BI-847325, GDC-0623, PD318088, Selumetinib. Among them, 8 (except BI-847325) have similar structures (aniline with halogen at the ortho and para positions). In addition, for Refametinib, Pimasertib, AZD8330, Trametinib, TAK-733, BI-847325, the FC of cytoplasmic vimentin significant diffusion > 1.4. The examples of the promotion of vimentin diffusion by these 6 MEK1 / 2 inhibitors are asFigure 2C as shown

[0120] The results showed that multiple MEK1 / 2 small molecule inhibitors in the MAPK pathway could change the intracellular distribution of vimentin and promote its significant diffusion.

[0121] Example 3

[0122] Confirmation of the effect of MEK1 / 2 inhibitors on Salmonella replication

[0123] Experimental method:

[0124] To exclude the influence of MEK1 / 2 inhibitors on bacterial replication by affecting cell viability, we first used a CCK-8 kit to detect the effects of 6 inhibitors on cell viability at different concentrations. Cells were seeded in 96-well plates at a density of 2×10 3 . When the cells adhered and reached a density of 70 - 80%, compounds with concentration gradients (0.046 μM - 100 μM, 3-fold concentration gradient) were added, 100 μl per well. After the compounds acted for 24 h, 10 μl of CCK-8 reagent was added to each well, and the plates were further incubated at 37 °C for 0.5 - 4 h. Then the absorbance at 450 nm was measured.

[0125] Since BI-847325 had an inhibitory effect on cell viability of about 12% when acting at a concentration of 1 μM for 24 h, we only detected the inhibitory effects of the other 5 MEK1 / 2 inhibitors on Salmonella replication. U2OS cells were seeded in 24-well plates at a density of 5×10 4 . After the cells adhered overnight, they were washed 3 times with PBS and then added to serum-free medium. They were infected with Salmonella Typhimurium at a multiplicity of infection (MOI) of 10. After 1 h of bacterial infection, they were washed 3 times with PBS and then changed to serum-free medium containing gentamicin (50 ng / μl), and 1 μM of MEK1 / 2 inhibitors were added respectively. After 24 h of incubation, the cells were collected, washed 2 times with PBS, then 500 μl of deionized sterile water was added, and after lysing for 10 - 15 minutes, the lysate was transferred to a 1.5 ml EP tube. The cell lysate was diluted by an appropriate multiple and then plated. The plates were incubated in an incubator at 37 °C overnight. When the colony size was appropriate, the number of colonies was counted to analyze the effect of the drug on Salmonella replication in host cells.

[0126] Experimental results:

[0127] As Figure 3AAs shown, all six compounds have cytotoxicity with a concentration gradient. When the concentration of the compounds was 1 μM, except for BI-847325 which had an inhibitory effect of about 12% on cell viability, the other compounds had no obvious cytotoxicity. Therefore, we detected the inhibitory effects of the other five compounds: Refametinib, Pimasertib, AZD8330, Trametinib, and TAK-733 on Salmonella at a concentration of 1 μM.

[0128] As Figure 3B shown, all five compounds showed significant effects in inhibiting Salmonella replication, among which Trametinib had the most significant inhibitory effect.

[0129] The above results indicate that MEK1 / 2 inhibitors have a general inhibitory effect on Salmonella, and among them, Trametinib has the most significant anti-Salmonella effect.

[0130] Example 4

[0131] Confirmation of the effect of Trametinib in inhibiting MEK1 / 2 and promoting the diffusion of cytoplasmic vimentin

[0132] Experimental method:

[0133] Human osteosarcoma cells U2OS were seeded in 6-well plates. When the cell density reached 70 - 80%, Trametinib at a concentration of 1 μM was added, and 0.1% DMSO was added to the control wells (Vehicle). After treatment for 3 h, 6 h, 12 h, and 24 h, the cells were lysed and cell samples were collected. Using Western blotting, the effects of Trametinib on the expression levels of ERK1 / 2, p-ERK1 / 2 (phosphorylated ERK1 / 2), and vimentin were detected, with GAPDH as an internal reference.

[0134] In addition, human osteosarcoma cells U2OS were seeded on glass coverslips. When the cell density reached 70 - 80%, the cells were treated with Trametinib at different concentrations (0.5 μM, 1 μM, 2 μM, 5 μM), and 0.1% DMSO was added to the control wells (Vehicle). After treatment for 12 h, the cells were fixed, and endogenous vimentin (green fluorescence) and actin (red fluorescence) in the cells were stained using immunochemistry. The cell nuclei were stained with DAPI (blue fluorescence). The promoting effect of Trametinib on the diffusion of cytoplasmic vimentin at different concentrations was confirmed. Finally, photographs were taken using a laser confocal fluorescence microscope (Olympus FV1200), and the relative area change of cytoplasmic vimentin was analyzed using cell profilier software.

[0135] Experimental results:

[0136] As Figure 4A shown, after treating cells with trametinib (1 μM) for 3 h, it produced a significant effect of inhibiting downstream ERK1 / 2 activity, inhibited the phosphorylation of EKR1 / 2, and had no obvious effect on the expression of total ERK1 / 2 and vimentin proteins.

[0137] As Figure 4B and 4C shown, under the action of different concentration gradients of trametinib (0.5 - 5 μM), the cytoplasmic vimentin area increased in a concentration-dependent manner, and when treated with 5 μM trametinib, the cytoplasmic vimentin area expanded to about 1.6 times the original area. Among them Figure 4C is the quantitative statistical result of Figure 4B , and the number of cells in each group is n = 100 - 114.

[0138] This result verified the effect of trametinib in inhibiting MEK1 / 2 activity, and at the same time verified the reliability of the small molecule compound screening results in Example 1. In addition, it was again confirmed that one of the MEK1 / 2 inhibitors, trametinib, could significantly promote the diffusion of cytoplasmic vimentin and showed a dose dependence.

[0139] Example 5

[0140] Confirmation of the in vitro effect of trametinib in inhibiting Salmonella infection

[0141] After Salmonella invades host cells, it replicates in the Salmonella-containing vacuole (SCV). The integrity of the SCV is damaged, which will affect the replication and survival of Salmonella in the cell. Therefore, through immunofluorescence imaging, using lysosome-associated membrane glycoprotein 1 (LAMP1) as a surface marker of SCV, we observed the effect of trametinib on the integrity of SCV.

[0142] Experimental method:

[0143] U2OS cells were seeded on glass coverslips. After the cells adhered overnight, they were washed three times with PBS and then cultured in serum-free medium. The cells were infected with Salmonella typhimurium labeled with the red fluorescent protein mCherry (S.Tm-mCherry) at a multiplicity of infection (MOI) of 10. After 1 hour of bacterial infection, the cells were washed three times with PBS and then changed to serum-free medium containing gentamicin (50 ng / μl). At the same time, 1 μM trametinib was added, and 0.1% DMSO was added to the control wells (Vehicle). After 24 hours of treatment, the cells were fixed, and endogenous LAMP1 and vimentin in the cells were stained by immunochemistry. The cell nuclei were stained with DAPI. Finally, the cells were photographed using a laser confocal fluorescence microscope (Olympus FV1200) to observe the effect of trametinib on the integrity of SCV.

[0144] In addition, the half-maximal inhibitory concentration (IC50) of trametinib against Salmonella was detected by CFU. U2OS cells were seeded in 24-well plates at a density of 5×10 4 . After the cells adhered overnight, they were washed three times with PBS and then cultured in serum-free medium. The cells were infected with Salmonella typhimurium at a multiplicity of infection (MOI) of 10. After 1 hour of bacterial infection, the cells were washed three times with PBS and then changed to serum-free medium containing gentamicin (50 ng / μl). At the same time, trametinib with a concentration gradient (0.04 μM - 30 μM, 3-fold concentration gradient) was added. After 24 hours of treatment, the cells were collected, washed twice with PBS, and then 500 μl of deionized sterile water was added. After lysis for 10 - 15 minutes, the cell lysate was transferred to a 1.5 ml EP tube. After appropriate dilution, the cell lysate was plated. The plates were incubated overnight in a 37°C incubator. When the colony size was appropriate, the number of colonies was counted to analyze the IC50 of trametinib against Salmonella.

[0145] Experimental results:

[0146] As Figure 5A and 5B shown, in the control group, approximately 76% of the infected cells formed intact SCVs. After treatment with trametinib for 24 hours, the proportion of cells forming intact SCVs decreased to approximately 47%, and the integrity of SCVs in approximately 53% of the infected cells was disrupted, forming multiple small SCVs (microSCVs).

[0147] In addition, when the concentration of trametinib was 4.579 μM and the cells were treated for 24 hours, the inhibition rate of Salmonella replication reached 50%, that is, the IC50 of trametinib against Salmonella replication was 4.579 μM.

[0148] This result preliminarily explored the mechanism of action of trametinib in inhibiting Salmonella infection: it disrupted the integrity of the SCV and determined its half-maximal inhibitory concentration to be 4.579 μM.

[0149] Example 6

[0150] Confirmation of the effect of trametinib in inhibiting Salmonella infection in mice

[0151] Experimental method:

[0152] Eight-week-old male C57 / B6j mice weighing 20 - 25 g were randomly divided into a control group and a drug administration group, with 5 mice in each group. After the mice were fasted and water-deprived for 4 h, they were gavaged with streptomycin (20 mg / mouse) to eliminate other dominant bacteria in the gastrointestinal tract, and then returned to normal diet and water. After 24 h, they were fasted and water-deprived for 4 h again. Then they were gavaged with Salmonella (1×10 5 CFU). Four hours later, they were gavaged with trametinib (2 mg / kg) or an equal volume of the solvent DMSO. The solvent or drug was dissolved in corn oil for gavage administration. After administration, they were returned to normal diet and water.

[0153] After 24 h, the mice were sacrificed, and feces, small intestine, and spleen tissues were collected, weighed, ground, diluted, and plated for Salmonella CFU analysis.

[0154] Experimental results:

[0155] As Figure 6 shown in A, the Salmonella load per gram of feces in the control group was 1.4×10 9 CFU, while after administration of trametinib, the Salmonella load per gram of feces decreased to 6.6×10 7 CFU. As Figure 6 shown in B, the Salmonella load per gram of small intestine in the control group was 1.0×10 5 CFU, while after administration of trametinib, the Salmonella load per gram of small intestine decreased to 1.0×10 4 CFU. As Figure 6 shown in C, the Salmonella load per spleen in the control group of mice was 713 CFU, while after administration of trametinib, the Salmonella load per spleen decreased to 72 CFU.

[0156] The above results indicate that trametinib can significantly reduce the Salmonella load in the feces, small intestine, and spleen of mice.

[0157] Example 7

[0158] Confirmation of the effect of trametinib in improving splenomegaly caused by Salmonella in mice

[0159] Experimental method:

[0160] Male C57 / B6j mice at 8 weeks old with a body weight of 20 - 25 g were randomly divided into an uninfected group (Mock), an infected control group, and an infected drug - administered group, with 5 mice in each group. After the mice were fasted and water - deprived for 4 h, streptomycin (20 mg / mouse) was administered by gavage to eliminate other dominant bacteria in the gastrointestinal tract, and then normal diet and water were restored. After 24 h, the mice were fasted and water - deprived for 4 h again. The infected group was inoculated with Salmonella (1×10 5 CFU) by gavage, and the uninfected group was given an equal volume of PBS by gavage. After 4 h, the infected group was administered trametinib (2 mg / kg) or an equal volume of the solvent DMSO by gavage. The solvent or drug was dissolved in corn oil for gavage administration, and the uninfected group was given an equal volume of the solvent. After administration, normal diet and water were restored.

[0161] The spleen was taken, weighed, photographed, and the effect of trametinib on improving Salmonella - induced splenomegaly was observed.

[0162] Experimental results:

[0163] As Figure 7 shown in Figures A and 7B, the average weight of the spleen of normal mice was 0.072 g. After infection with Salmonella, the spleen of the mice became enlarged, and the average weight increased significantly to 0.11 g. After administration of trametinib, this phenomenon was improved, and the spleen weight decreased to a weight close to that of normal mice, with an average of 0.079 g.

[0164] The above results indicate that trametinib can significantly improve Salmonella - induced splenomegaly in mice.

[0165] Example 8

[0166] Confirmation of the effect of trametinib on improving Salmonella - induced colonic lesions

[0167] Experimental method:

[0168] Male C57 / B6j mice at 8 weeks old with a body weight of 20 - 25 g were randomly divided into an uninfected group (Mock), an infected control group, and an infected drug - administered group, with 5 mice in each group. After the mice were fasted and water - deprived for 4 h, streptomycin (20 mg / mouse) was administered by gavage to eliminate other dominant bacteria in the gastrointestinal tract, and then normal diet and water were restored. After 24 h, the mice were fasted and water - deprived for 4 h again. The infected group was inoculated with Salmonella (1×10 5 CFU) by gavage, and the uninfected group was given an equal volume of PBS by gavage. After 4 h, the infected group was administered trametinib (2 mg / kg) or an equal volume of the solvent DMSO by gavage. The solvent or drug was dissolved in corn oil for gavage administration, and the uninfected group was given an equal volume of the solvent. After administration, normal diet and water were restored.

[0169] The colon was taken, fixed with 4% PFA, embedded in paraffin, sectioned, and stained with HE to observe the pathological changes of the colon.

[0170] Experimental results:

[0171] As Figure 8 shown in FIGS. A and 8B, after infection with Salmonella, the mucosal length of the mouse colon increased significantly, while after administration of trametinib, the mucosa recovered to a length similar to that of normal mice. As Figure 8 shown in FIGS. A and 8C, after infection with Salmonella, obvious edema appeared in the submucosa of the mouse colon, while after administration of trametinib, the edema in the submucosa was significantly improved.

[0172] The above results indicate that trametinib can significantly improve the pathological changes of the mouse colon caused by Salmonella.

[0173] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a MEK1 / 2 inhibitor in the preparation of a preparation for inhibiting Salmonella infection, Among them, wherein the MEK1 / 2 inhibitor is selected from the group consisting of trametinib, refametinib, Pimasertib, TAK-733, AZD8330, or a combination thereof, or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that, The MEK1 / 2 inhibitor is selected from trametinib or refametinib, or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, characterized in that, The MEK1 / 2 inhibitor is selected from trametinib or refametinib.

4. The use according to claim 1, characterized in that, The MEK1 / 2 inhibitor is trametinib.

5. The use according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts include hydrochloride, sulfate, sulfonate, carbonate, acetate, or tartrate.

6. The use according to claim 1, characterized in that, The preparation contains (i) a MEK1 / 2 inhibitor or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

7. The use according to claim 1, characterized in that, The "inhibition of Salmonella infection" includes inhibiting Salmonella infection at the cellular level and / or inhibiting Salmonella infection at the animal level.

8. The use according to claim 7, characterized in that, The "inhibition of Salmonella infection" is selected from the group consisting of inhibiting the replication of Salmonella in host cells, reducing the death of infected cells caused by Salmonella, reducing the number of cells infected with Salmonella, reducing the number of Salmonella released from the assembly of infected cells, inhibiting the expression of Salmonella proteins in infected cells, or a combination thereof.

9. The use according to claim 7, characterized in that, The "inhibition of Salmonella infection" also includes inhibiting the bacterial load of Salmonella in animal tissues and / or inhibiting tissue lesions caused by Salmonella.

10. An in vitro non-therapeutic method for inhibiting Salmonella infection of host cells, comprising the step of adding a MEK1 / 2 inhibitor or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, to a host cell culture system containing Salmonella, thereby inhibiting Salmonella infection of host cells; Among them, wherein the MEK1 / 2 inhibitor is selected from the group consisting of trametinib, refametinib, Pimasertib, TAK-733, AZD8330, or a combination thereof.

11. The method according to claim 10, characterized in that, The host cells are human or non-human mammalian cells.

12. The method according to claim 10 or 11, characterized in that, The MEK1 / 2 inhibitor is trametinib or refametinib, or a pharmaceutically acceptable salt thereof.

13. The method according to claim 12, wherein The MEK1 / 2 is selected from trametinib or refametinib.

14. The method according to claim 12, wherein The MEK1 / 2 inhibitor is trametinib.

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