CN116159140A - Use of MAPK pathway inhibitors against pathogen infection - Google Patents

Abstract

The invention discloses the use of MAPK pathway inhibitors against pathogen infection. In particular, the invention discloses the use of MAPK pathway inhibitors for the preparation of pharmaceutical formulations or compositions against pathogenic infection. The MAPK pathway inhibitors of the present invention are effective in controlling pathogen infection by promoting intracellular wave protein (vimentin) diffusion, thereby controlling pathogen proliferation and/or reducing pathogen numbers in host cells.

Description

Application of MAPK pathway inhibitors in anti-pathogen infection

technical field

The invention relates to the field of anti-infection, in particular to the application of MAPK pathway inhibitors in anti-pathogen infection.

Background technique

Pathogenic microorganisms are groups of microorganisms that can cause human infection or even serious 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.

Salmonella is a common zoonotic pathogen and one of the four major causes of diarrheal diseases. Diarrheal disease is the most common disease caused by unsafe food, affecting 550 million people each year, including 220 million children under the age of five. According to the report of the World Health Organization, Salmonella food contamination is becoming more and more serious around the world, causing huge economic losses and seriously threatening human health and life safety. Therefore, it has been listed as an important object and indicator for the detection of pathogenic bacteria in food. In my country, food poisoning caused by Salmonella has repeatedly ranked first. According to statistics, 70%-80% of bacterial food poisoning in our country is caused by Salmonella, and more than 90% of the food that causes Salmonella poisoning is caused by animal products such as meat. The symptoms of food poisoning caused by Salmonella mainly include nausea, vomiting, abdominal pain, headache, chills and diarrhea, etc., accompanied by fatigue, muscle aches, blurred vision, moderate fever, restlessness and lethargy, lasting for 2 to 3 days. The average fatality rate was 4.1%. Although the overall global health conditions have been greatly improved, Salmonella incidents still occur from time to time.

Salmonella is a Gram-negative, intracellular parasitic enteric bacterium that is widely distributed in nature and has a variety of species. More than 2,500 Salmonella serotypes have been detected so far, most of which are pathogenic to humans and animals. However, due to the abuse of antibiotics, the drug resistance of Salmonella is becoming more and more serious. At present, it has drug resistance to β-lactams, quinolones, tetracyclines, aminoglycosides, sulfonamides and other drugs, and its drug resistance mechanism is also extremely complicated. . With the passage of time, the drug resistance rate will rise sharply, and the drug resistance spectrum will continue to widen, posing a serious threat to my country's breeding industry and human health.

However, whether the body becomes ill after being attacked by pathogens is related to its own immunity on the one hand, and depends on the pathogenicity of the pathogens and the number of invasions on the other hand. Generally, the greater the number, the greater the possibility of disease.

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

Contents of the invention

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 infection.

The first aspect of the present invention provides a use of a MAPK pathway inhibitor for preparing a composition or preparation, and the composition or preparation is used for:

(a) promote the diffusion of vimentin (vimentin) in the cell; and

(b) Inhibition of pathogenic infection.

In another preferred example, the MAPK pathway inhibitor includes tinib or a pharmaceutically acceptable salt thereof.

In another preferred example, 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, Rafatinib, Selumetinib, more preferably Trametinib.

In another preferred embodiment, the pharmaceutically acceptable salt includes hydrochloride, sulfate, sulfonate, carbonate, acetate, tartrate, or isethionate.

In another preferred example, the pathogens include: bacteria, chlamydia, rickettsia, mycoplasma, spirochetes, fungi, or combinations thereof.

In another preferred example, the bacteria include Gram-negative bacteria and Gram-positive bacteria.

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

In another preferred example, the bacteria are Salmonella.

In another preferred example, the composition is a pharmaceutical composition.

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.

In another preferred example, the MAPK pathway inhibitor is a tinib compound.

In another preferred example, 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, Rafatinib, Selumetinib, more preferably Trametinib.

In another preferred example, the MAPK pathway inhibitor is trametinib.

In another preferred embodiment, the pharmaceutically acceptable salt includes hydrochloride, sulfate, sulfonate, carbonate, acetate, tartrate, or isethionate.

In another preferred example, the pharmaceutical composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

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

In another preferred example, the administration of the pharmaceutical composition includes oral administration, intramuscular injection, intravenous injection, intravenous drip, intratumoral injection, enema, spray, external application, or intraperitoneal injection.

In another preferred example, the "inhibiting pathogen infection" includes inhibiting pathogen infection at the cell level, and/or inhibiting pathogen infection at the animal level.

In another preferred example, the "inhibition of pathogen infection" is selected from the group consisting of inhibiting the replication of pathogens in host cells, reducing the death of infected cells caused by pathogens, reducing the number of cells infected by pathogens, and reducing the number of cells infected by pathogens. The amount of pathogen released by cellular assembly, the expression of proteins that inhibit the pathogen in infected cells, or a combination thereof.

In another preferred example, the "inhibiting pathogen infection" also includes inhibiting the bacterial load of pathogens in animal tissues, and/or inhibiting tissue lesions caused by pathogens.

The second aspect of the present invention provides a method for in vitro non-therapeutic inhibition of pathogen infection of host cells, comprising the steps of: adding a MAPK pathway inhibitor or a pharmaceutically acceptable salt thereof to the host cell culture system containing the pathogen, or A pharmaceutical composition containing it, thereby inhibiting pathogens from infecting host cells.

In another preferred example, the pathogens include: bacteria, chlamydia, rickettsia, mycoplasma, spirochetes, fungi, or combinations thereof.

In another preferred example, the pathogen is Salmonella.

In another preferred embodiment, the host cells are human or non-human mammalian cells.

In another preferred example, the MAPK pathway inhibitor is a tinib drug.

In another preferred example, 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, Rafatinib, Selumetinib, more preferably Trametinib.

In another preferred example, the MAPK pathway inhibitor is trametinib.

The third aspect of the present invention provides a method for resisting pathogenic infection, said method comprising the step of: administering a safe and effective amount of a MAPK pathway inhibitor or a pharmaceutically acceptable salt thereof, or a drug containing it to a subject in need composition, thereby inhibiting pathogenic infection.

In another preferred example, the subject in need is a human or a non-human mammal.

In another preferred example, the subject in need is infected by a pathogen or is at risk of being infected by a pathogen.

In another preferred example, the pathogens include: bacteria, chlamydia, rickettsia, mycoplasma, spirochetes, fungi, or combinations thereof.

In another preferred example, the pathogen is Salmonella.

In another preferred example, the MAPK pathway inhibitor is a tinib compound.

In another preferred example, 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, Rafatinib, Selumetinib, more preferably Trametinib.

In another preferred example, the MAPK pathway inhibitor is trametinib.

In another preferred example, the safe and effective dose 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), and most preferably 0.04mg/kg (body weight).

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 in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

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 significance of the difference in the effect of small molecule compounds on the area of vimentin in the cell; the green line box shows the small molecule compounds with log ₂ FC>0.3 (FC>1.2) and -log ₁₀ Pvalue>1 (P value<0.1) , small molecule compounds with log ₂ FC>0.5 (FC>1.4) and -log ₁₀ Pvalue>2 (P value<0.01) are shown in the orange line. Figure 1B and Figure 1C show the results of signaling pathway enrichment for the small molecule compounds in Figure 1A.

Figure 2 shows that small molecule inhibitors of MEK1/2 in the MAPK pathway can significantly promote the diffusion of vimentin. Figure 2A is a pie chart showing the proportion of small molecule inhibitors of MAPK pathway belonging to different inhibitory targets. Figure 2B shows the chemical structure formulas of nine MEK1/2 small molecule inhibitors among them. Figure 2C shows the effect of the top 6 compounds that promote vimentin diffusion on vimentin morphology.

Figure 3 shows that small molecule inhibitors of MEK1/2 can inhibit Salmonella infection. Fig. 3A shows the effects of 6 compounds on cell viability after being treated for 24 hours at different concentrations (0.046 μM-100 μM). Figure 3B shows that when the concentration is 1μM for 24 hours, five compounds without obvious toxicity to cells can inhibit the replication of Salmonella, expressed in colony-forming units (colony-forming units, CFU), among which trametinib is the most effective obvious. Where: **p<0.01, ***p<0.001, ****p<0.0001.

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 MEK1/2 downstream kinase ERK1/2. Figure 4B shows the results of immunofluorescence staining of cytoplasmic vimentin after treatment with gradient concentrations of trametinib, in which the label length of the uppermost legend is 65 μm, and the label length of the ROI enlarged legend is 10 μm. Fig. 4C is the quantitative statistical result of Fig. 4B, and the number of cells in each group is n=100-114. Where: ***p<0.001.

Figure 5 shows that Trametinib can inhibit Salmonella infection. Figure 5A shows that after 1 μM trametinib treatment, Salmonella formed dispersed small Salmonella-containing follicles (microSCVs) in host cells with a tag length of 10 μm. Fig. 5B is the quantitative statistical result of Fig. 5A. Figure 5C shows that trametinib has a median inhibitory concentration of 4.579 μM for Salmonella replication. Where: ****p<0.0001.

Figure 6 shows trametinib can inhibit the bacterial copy number in mice. Figure 6A shows that trametinib significantly inhibited the number of Salmonella in mouse feces (CFU/g). Figure 6B shows that trametinib significantly inhibited the number of Salmonella in the small intestine of mice (CFU/g). Figure 6C shows that trametinib significantly inhibited the number of Salmonella in mouse spleen (CFU/organ). There were 5 mice in each group. Where: **p<0.01, ***p<0.001.

Figure 7 shows that trametinib can improve spleen enlargement in mice caused by Salmonella. Figure 7A shows an example of the changes in the mouse spleen. After intragastric administration of Salmonella (1×10 ⁵ CFU) for 28 hours, the spleens of mice were enlarged; and after intragastric administration of trametinib, the spleen morphology of mice recovered. Figure 7B shows the weight statistics of the spleens of mice, 5 mice in each group. Where: ns, p>0.05, *p<0.05, **p<0.01.

Figure 8 shows that trametinib can improve colonic lesions caused by Salmonella. Fig. 8A shows that after intragastric administration of Salmonella (1×10 ⁵ CFU) for 28 hours, significant pathological changes occurred in the mouse colon, including a significant increase in the length of the mucosa and edema of the submucosa. However, 24 hours after intragastric administration of trametinib, the pathological changes of the mouse colon were significantly improved. The length of the upper legend label is 100 μm, and the length of the lower legend label is 25 μm. Figure 8B shows the quantitative statistical graph of trametinib significantly improving the thickening of the colonic mucosa in mice. Figure 8C shows the quantitative statistical graph of trametinib significantly improving the submucosa edema in mice. Where: ns, p>0.05, *p<0.05, **p<0.01, ****p<0.0001.

Detailed ways

After extensive and in-depth research, the inventors unexpectedly found for the first time that MAPK pathway inhibitors can significantly promote the intracellular distribution of vimentin (vimentin) in mammalian cells, and by promoting the significant diffusion of vimentin in the cytoplasm, achieve Resist pathogen infection, reduce the number of pathogen invasion and replication, thereby significantly reducing the pathogenicity of pathogens. The present invention has been accomplished on this basis.

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 in a dose-dependent manner. 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 destroy the integrity of follicular SCV containing Salmonella to effectively inhibit the replication of Salmonella. 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.

the term

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

The term "about" refers to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. As used herein, the expression "about 100" includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprises" or "includes (comprising)" can be open, semi-closed and closed. In other words, the term also includes "consisting essentially of", or "consisting of".

MAPK pathway and its inhibitors

The MAPK signaling pathway is an important signal transduction system for eukaryotic cells to mediate extracellular signals to intracellular responses. The MAPK pathway-related signal is a highly conserved family of protein kinases. A total of 19 MAPKKKs (also known as MAP3Ks or MEKKs), 7 MAPKKs (MAP2Ks) and 14 MAPKs were found. Between them are the upper, middle and lower cascade signaling molecules, MAPKKK activates MAPKK, and the latter reactivates MAPK, that is, extracellular signal → MAPK kinase kinase (MKKK) → MAPK kinase (MKK) → MAPK, to regulate cell growth , differentiation, apoptosis and death and other physiological processes. The MAPK signaling pathway mainly includes three signaling pathways: the classic MEK/ERK signaling pathway, the JNK/p38MAPK signaling pathway and the ERK5 signaling pathway. As well as the signal transduction after hormone receptor activation, it plays an important regulatory role in cell proliferation, growth and differentiation.

The present invention provides the use of MAPK pathway inhibitors, which are used to fight pathogen infection.

As used herein, the terms "compound of the present invention", "MAPK pathway inhibitor of the present invention", and "inhibitor of the present invention" are used interchangeably and refer to MAPK pathway inhibitors that can be used to fight pathogenic infections.

As used herein, the term "MAPK pathway inhibitor" refers to an inhibitor capable of inhibiting the MAPK pathway. Representative ones include (but not limited to): tinib drugs (including tinib drugs, or analogs thereof, or pharmaceutically acceptable salts thereof).

As used herein, the terms "tinib drug" and "tinib compound" are used interchangeably.

In a preferred example of the present invention, the MAPK pathway inhibitor is selected from the group consisting of Trametinib, Refametinib, Selumetinib, Pimasertib, Cometinib (Cobimetinib), TAK-733, AZD8330, BI-847325, GDC-0623, PD318088, or a combination thereof.

Tini drugs are a class of "tinib" drugs approved for marketing. The indications of this type of drugs are mainly focused on tumors, and secondly involve skin diseases, blood system diseases, immune system diseases, musculoskeletal and connective tissue diseases. The important targets and pathways of tinib drugs are systematic and complicated, and the main targets include tyrosine-protein kinase (tyrosine-protein kinase), tyrosine-protein kinase receptor (tyrosine-protein kinase receptor), and various growth factor receptors. body (growth factor receptor) and mitogen-activated protein kinase kinase (mitogen-activated protein kinase kinase).

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

vimentin

Vimentin is one of the proteins of the intermediate filament. Intermediate filaments are an important structural feature of eukaryotic cells. Together with microtubules and actin filaments, they form the cytoskeleton.

In the embodiment of the present invention, the use of the MAPK pathway inhibitor of the present invention can promote the diffusion of vimentin in cells, and exert the effect of resisting pathogenic infection.

Anti-pathogen infection/inhibition of pathogen infection

The present invention provides the application of MAPK pathway inhibitor in anti-pathogen infection, and said pathogen includes bacteria, chlamydia, rickettsia, mycoplasma, spirochetes, fungi, or combinations thereof.

In another preferred example, the bacteria include Gram-negative bacteria and Gram-positive bacteria.

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

In the present invention, the pathogen includes wild type, or its subtype, or its mutant type.

As used herein, the terms "anti-pathogenic infection" and "inhibiting pathogenic infection" are used interchangeably.

In another preferred example, the "inhibiting pathogen infection" includes inhibiting pathogen infection at the cell level, and/or inhibiting pathogen infection at the animal level.

In another preferred example, the "inhibition of pathogen infection" is selected from the group consisting of inhibiting the replication of pathogens in host cells, reducing the death of infected cells caused by pathogens, reducing the number of cells infected by pathogens, and reducing the number of cells infected by pathogens. The amount of pathogen released by cellular assembly, the expression of proteins that inhibit the pathogen in infected cells, or a combination thereof.

In another preferred example, the "inhibiting pathogen infection" also includes inhibiting the bacterial load of pathogens in animal tissues, and/or inhibiting tissue lesions caused by pathogens.

In one embodiment of the present invention, the MAPK pathway inhibitor inhibits Salmonella infection, specifically, the MAPK pathway inhibitor (such as Trametinib) destroys the integrity of the SCV containing Salmonella follicle by promoting the diffusion of vimentin in the cell, effectively Inhibit the replication of Salmonella in cells; further, inhibit the bacterial load of Salmonella in mouse tissues, and improve the pathological changes of mouse tissues (including spleen and colon).

pharmaceutical composition

As used herein, the term "pharmaceutical composition" refers to a composition to be administered for a specific purpose.

For the purpose 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 fighting pathogen infection. The MAPK pathway inhibitors include tinib drugs and their analogs.

)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。The term "pharmaceutically acceptable" refers to a substance listed and approved by a government drug regulatory agency or in a pharmacopoeia for use in vertebrates, especially humans. Generally, a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and low toxicity. "Compatibility" here means that each component in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, 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.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as

), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

The mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but not limited to): oral administration, intramuscular injection, intravenous injection, intravenous drip, intratumoral injection, enema, spray, external application, or intraperitoneal injection.

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 dicalcium phosphate, or with: (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard 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.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and the release of the active ingredient or compound from such compositions may be in a certain part of the alimentary canal in a delayed manner. Examples of usable embedding components are polymeric substances and waxy substances. The active ingredient can also be in the form of microcapsules with one or more of the above-mentioned excipients, if necessary.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, 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.

Besides such inert diluents, the compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredient, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

Dosage forms for topical administration of a compound of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required, if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds. For example, the compound of the present invention can be used in combination with other antibacterial drugs or drugs for improving immunity.

The use of the pharmaceutical composition is to apply a safe and effective amount of the compound of the present invention to a mammal (such as a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage when administered, and for a person with a body weight of 60kg, the daily dose The dosage is usually 1-2000 mg, preferably 6-600 mg. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians. Usually, the "safe and effective amount" refers to: the amount of the compound is enough 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 therapeutic agents for anti-pathogenic infection.

The main advantages of the present invention are:

(1) The present invention has discovered for the first time that MAPK pathway inhibitors have the effect of resisting pathogenic (such as Salmonella) infection;

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

(3) The present invention finds for the first time that the MAPK pathway inhibitor promotes the diffusion of vimentin in the cell, thereby inhibiting the pathogen from infecting the cell;

(4) The compounds of the present invention include drugs such as trametinib and selumetinib that have been used in clinical practice, and the biological safety is guaranteed, and the cycle of putting old clinical drugs into new use is greatly shortened.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended 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 examples of the present invention are all commercially available products. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer.

Example 1

Confirmation of the effect of small molecule inhibitors of MAPK pathway significantly promoting the diffusion of cytoplasmic vimentin

experimental method:

The vimentin-GFP and actin-RFP double-labeled osteosarcoma cell line U2OS was cultured in a 384-well plate, in which vimentin was the target protein, and actin was used to mark the cell outline. When the cells adhered to the wall and reached 70-80% density, about 1700 compounds to be tested in the selleck small molecule inhibitor compound library were added to each well. Based on existing studies, it was determined that the compound administration concentration was 1 μM, and the drug application time was 12 h. Three replicate wells were set up for each compound, and the control wells were 0.1% DMSO, WFA (known to cause vimentin to accumulate in cells), low Infiltration medium (medium diluted with deionized water (medium: deionized water = 1:9), the treatment time is 5-7 minutes, causing vimentin to diffuse significantly in the cells). The nuclei were fixed and stained with DAPI, and the PerkinElmer high-content imaging system and Harmony imaging and analysis software were used for cell imaging and data quantitative analysis.

Experimental results:

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

As shown in Figure 1A, with log ₂ FC as the abscissa and -log ₁₀ Pvalue as the ordinate, the significance of the difference in the effect of the compounds on the area of vimentin was analyzed. Among them, the small molecule compounds with log ₂ FC>0.3 (FC>1.2) and -log ₁₀ Pvalue>1 (P value<0.1) are shown in the green line frame (total 196); the orange line frame shows log ₂ FC>0.5 (FC >1.4) and -log ₁₀ Pvalue>2 (Pvalue<0.01) small molecule compounds (total 19). It was shown that these compounds caused a significant diffusion of cytoplasmic vimentin.

The signal pathway enrichment analysis of the above 196 small molecule compounds was carried out, with -log ₁₀ Pvalue as the abscissa and the signal pathway as the ordinate, and it was found that the signal pathway of MAPK had the most significant enrichment, as shown in Figure 1B and Figure 1C. These results suggest that multiple small-molecule inhibitors of MAPK signaling lead to significant proliferation of cytoplasmic vimentin.

Example 2

Confirmation of the effect of MEK1/2 small molecule inhibitors in the MAPK pathway to significantly promote the diffusion of vimentin

experimental method:

Since the MAPK signaling pathway contains a large number of protein kinases, the MAPK signaling pathway that causes cytoplasmic vimentin to diffuse (Foldchange>1.2, P value<0.1) was then analyzed in depth to classify different target protein inhibitors.

Experimental results:

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

Further reduce the P value (<0.05), 14 small molecule inhibitors of MAPK pathway, including 9 MEK1/2 inhibitors (65%), 2 RAF inhibitors (14%), p38 MAPK inhibition There are 2 agents (14%) and 1 JNK inhibitor (7%).

Further, the cytoplasmic vimentin was diffused with FC>1.4 and P value<0.01. Among the small molecule inhibitors, there were 6 small molecule inhibitors of MAPK pathway, and all of them were MEK1/2 inhibitors (accounting for 100%).

Figure 2B shows the chemical structural formulas of 9 MEK1/2 inhibitors that significantly promote the diffusion of vimentin, including Refametinib, Pimasertib, AZD8330, Trametinib, TAK-733, BI-847325, GDC -0623, PD318088, Selumetinib. Eight of them (except BI-847325) have a similar structure (aniline with halogen in the ortho-para position). In addition, Refametinib, Pimasertib, AZD8330, Trametinib, TAK-733, BI-847325 significantly diffused cytoplasmic vimentin with FC>1.4, and these six MEK1/2 inhibitors promoted vimentin A plot of the diffusion is shown in Figure 2C.

This result suggests that multiple small molecule inhibitors of MEK1/2 in the MAPK pathway can alter the intracellular distribution of vimentin and promote its significant diffusion.

Example 3

Confirmation of the effect of MEK1/2 inhibitors on inhibiting the replication of Salmonella

experimental method:

In order to rule out that MEK1/2 inhibitors affect bacterial replication by affecting cell viability, we first used the CCK-8 kit to detect the effects of six inhibitors on cell viability at different concentrations. Seed the cells in a 96-well culture plate at a density of 2×10 ³ , and when the cells adhere to the wall and reach a density of 70-80%, add a compound with a concentration gradient (0.046 μM-100 μM, 3-fold concentration gradient), 100 μl per well . After the compound was reacted for 24 hours, 10 μl of CCK-8 reagent was added to each well, and kept at 37°C for 0.5-4 hours of incubation. Absorbance at 450 nm was then measured.

Since BI-847325 has an inhibitory effect on cell viability of about 12% when treated at a concentration of 1 μM for 24 hours, we only tested the inhibitory effect of other 5 MEK1/2 inhibitors on Salmonella replication. The U2OS cells were plated in a 24-well plate at a density of 5×10 ⁴ . After the cells adhered to the wall overnight, they were washed 3 times with PBS, and serum-free medium was added. Infection was carried out with Salmonella Typhimurium, and the multiplicity of infection (MOI) was 10. After 1 hour of bacterial infection, wash 3 times with PBS, replace with serum-free medium containing gentamicin (50 ng/μl), and add 1 μM MEK1/2 inhibitor respectively at the same time. After acting for 24 hours, collect the cells, wash them twice with PBS, add 500 μl of deionized sterile water, lyse for 10-15 minutes, transfer to a 1.5ml EP tube, dilute the cell lysate to an appropriate multiple, and spread on the plate. Place in a 37°C incubator and incubate overnight. When the colony size is appropriate, count the number of colonies to analyze the effect of the drug on the replication of Salmonella in the host cells.

Experimental results:

As shown in Figure 3A, all six compounds were cytotoxic in a concentration gradient. When the concentration of the compound was 1 μM, except for BI-847325 which inhibited the cell activity by about 12%, the other compounds had no obvious cytotoxicity. Therefore, we tested the inhibitory effect of five other compounds: Refametinib, Pimasertib, AZD8330, Trametinib, TAK-733 on Salmonella at a concentration of 1 μM.

As shown in Figure 3B, all five compounds showed significant inhibition of Salmonella replication, among which trametinib had the most significant inhibitory effect.

The above results indicated that MEK1/2 inhibitors have universal inhibitory effect on Salmonella, and trametinib has the most significant anti-Salmonella effect.

Example 4

Confirmation of the effect of trametinib inhibiting MEK1/2 and promoting the proliferation of cytoplasmic vimentin

experimental method:

Human osteosarcoma cells U2OS were plated in a 6-well plate, and when the cell density reached 70-80%, trametinib (1 μM) was added, and 0.1% DMSO was added to the control well (Vehicle). After administration for 3h, 6h, 12h, 24h, cells were lysed and cell samples were collected. Western blotting was used to detect the effect of trametinib on the expression levels of ERK1/2, p-ERK1/2 (phosphorylated ERK1/2) and vimentin, and GAPDH was used as an internal reference.

In addition, human osteosarcoma cells U2OS were spread on glass slides, and 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 the control wells (Vehicle ) was added with 0.1% DMSO. After 12 hours of administration, the cells were fixed, and endogenous vimentin (green fluorescence) and actin (red fluorescence) in the cells were stained by immunochemical method. Nuclei were stained with DAPI (blue fluorescence). To confirm the diffusion-promoting effect of trametinib on cytoplasmic vimentin at different concentrations. Finally, the laser confocal fluorescence microscope (OlympusFV1200) was used to shoot, and the relative area change of cytoplasmic vimentin was analyzed by cell profilier software.

Experimental results:

As shown in Figure 4A, trametinib (1 μM) not only significantly inhibited the downstream ERK1/2 activity, but also inhibited the phosphorylation of EKR1/2 after treating the cells for 3 hours, while affecting the total ERK1/2 and Vimentin protein expression had no obvious effect.

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

This result verified the effect of trametinib on inhibiting MEK1/2 activity, and at the same time verified the reliability of the screening results of small molecule compounds in Example 1. In addition, trametinib, one of the MEK1/2 inhibitors, was once again confirmed to significantly promote the diffusion of cytoplasmic vimentin in a dose-dependent manner.

Example 5

Confirmation of in vitro effect of trametinib against Salmonella infection

After Salmonella invades the host cell, it will replicate in the Salmonella-containing vacuole (SCV), and the integrity of the SCV will be destroyed, which will affect the replication and survival of Salmonella in the cell. Therefore, we used immunofluorescence imaging to observe the effect of trametinib on the integrity of SCV using lysosome-associated membrane protein 1 (LAMP1) as a surface marker of SCV.

experimental method:

Spread U2OS cells on glass slides. After the cells adhere to the wall overnight, they are washed 3 times with PBS, and serum-free medium is added. Salmonella typhimurium (S.Tm-mCherry) labeled with red fluorescent protein mCherry was used for infection, and the multiplicity of infection (MOI) was 10. After 1 hour of bacterial infection, wash 3 times with PBS, replace with serum-free medium containing gentamicin (50 ng/μl), add 1 μM trametinib at the same time, add 0.1% DMSO to the control well (Vehicle). After acting for 24 hours, the cells were fixed, and endogenous LAMP1 and vimentin in the cells were stained by immunochemical method. Nuclei were stained with DAPI. Finally, laser confocal fluorescence microscopy (Olympus FV1200) was used to observe the effect of trametinib on the integrity of SCV.

In addition, the half maximal inhibitory concentration of trametinib against Salmonella was detected by CFU. The U2OS cells were plated in a 24-well plate at a density of 5×10 ⁴ . After the cells adhered to the wall overnight, they were washed 3 times with PBS, and serum-free medium was added. The infection was carried out with Salmonella typhimurium, and the multiplicity of infection (MOI) was 10. After 1 hour of bacterial infection, wash 3 times with PBS, replace with serum-free medium containing gentamicin (50ng/μl), and add trametinib with concentration gradient (0.04μM-30μM, 3-fold concentration gradient) at the same time. After acting for 24 hours, collect the cells, wash them twice with PBS, add 500 μl of deionized sterile water, lyse for 10-15 minutes, transfer to a 1.5ml EP tube, dilute the cell lysate to an appropriate multiple, and plate. Place in a 37°C incubator and incubate overnight. When the colony size is appropriate, count the number of colonies and analyze the half inhibitory concentration of trametinib against Salmonella.

Experimental results:

As shown in Figures 5A and 5B, in the control group, the infected cells that formed complete SCV accounted for about 76% of the total number of infected cells, and after adding trametinib for 24 hours, the proportion of cells that formed complete SCV decreased to about 47%. %, about 53% of the SCV integrity in the infected cells was destroyed, forming multiple small SCV (microSCV).

In addition, when the concentration of trametinib was 4.579 μM, the inhibition rate of Salmonella replication reached 50% after 24 hours of treatment on the cells, that is, the half inhibitory concentration of trametinib to the replication of Salmonella was 4.579 μM.

This result preliminarily explored the mechanism of trametinib's inhibition of Salmonella infection: it destroyed the integrity of SCV, and its half inhibitory concentration was determined to be 4.579 μM.

Example 6

Confirmation of the effect of trametinib on inhibiting Salmonella infection in mice

experimental method:

Eight-week-old male C57/B6j mice with a body weight of 20-25 g were randomly divided into a control group and a treatment group, with 5 mice in each group. After the mice were fasted for 4 hours, streptomycin (20 mg/mouse) was administered orally to remove other dominant flora in the stomach, and then normal diet and drinking water were resumed. After 24 hours, food and water were fasted for 4 hours. Salmonella (1×10 ⁵ CFU) was inoculated by intragastric administration. Trametinib (2 mg/kg) or an equivalent amount of solvent DMSO was administered by intragastric administration after 4 hours, wherein the solvent or drug was dissolved in corn oil for intragastric administration. After administration, resume normal eating and drinking.

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

Experimental results:

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

The above results indicated that trametinib could significantly reduce the load of Salmonella in mouse feces, small intestine and spleen.

Example 7

Confirmation of the effect of trametinib on improving splenomegaly caused by Salmonella in mice

experimental method:

Eight-week-old male C57/B6j mice with a body weight of 20-25 g were randomly divided into an uninfected group (Mock), an infected control group, and an infected administration group, with 5 mice in each group. After the mice were fasted for 4 hours, streptomycin (20 mg/mouse) was administered orally to remove other dominant flora in the stomach, and then normal diet and drinking water were resumed. After 24 hours, food and water were fasted for 4 hours. The infected group was intragastrically inoculated with Salmonella (1×10 ⁵ CFU), and the uninfected group was intragastrically inoculated with the same amount of PBS. After 4 hours, the infected group was intragastrically administered trametinib (2 mg/kg) or an equivalent amount of solvent DMSO, wherein the solvent or drug was dissolved in corn oil for intragastric administration, and the non-infected group was intragastrically administered an equal volume of solvent. After administration, resume normal eating and drinking.

The spleen was taken, weighed, and photographed to observe the effect of trametinib on improving splenomegaly caused by Salmonella.

Experimental results:

As shown in Figures 7A and 7B, the average weight of the spleen of a normal mouse is 0.072 g, but after infection with Salmonella, the spleen of the mouse was enlarged, and the average weight was significantly increased to 0.11 g. After administration of trametinib, this phenomenon was improved, and the weight of the spleen was reduced to a weight close to that of the spleen of normal mice, with an average of 0.079 g.

The above results indicated that trametinib could significantly improve the splenomegaly in mice caused by Salmonella.

Example 8

Confirmation of the effect of trametinib in improving colonic lesions caused by Salmonella

experimental method:

Eight-week-old male C57/B6j mice with a body weight of 20-25 g were randomly divided into an uninfected group (Mock), an infected control group, and an infected administration group, with 5 mice in each group. After the mice were fasted for 4 hours, streptomycin (20 mg/mouse) was administered orally to remove other dominant flora in the stomach, and then normal diet and drinking water were resumed. After 24 hours, food and water were fasted for 4 hours. The infected group was intragastrically inoculated with Salmonella (1×10 ⁵ CFU), and the uninfected group was intragastrically inoculated with the same amount of PBS. After 4 hours, the infected group was intragastrically administered trametinib (2 mg/kg) or an equivalent amount of solvent DMSO, wherein the solvent or drug was dissolved in corn oil for intragastric administration, and the non-infected group was intragastrically administered an equal volume of solvent. After administration, resume normal eating and drinking.

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

Experimental results:

As shown in Figures 8A and 8B, after infection with Salmonella, the length of the mucous membrane of the mouse colon was significantly increased, and after administration of trametinib, the mucous membrane returned to a length similar to that of normal mice. As shown in Figures 8A and 8C, after infection with Salmonella, the submucosa of the colon of mice was significantly edematous, and after administration of trametinib, the edema of the submucosa was significantly improved.

The above results indicated that trametinib could significantly improve the pathological changes of mouse colon caused by Salmonella.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. Use of a MAPK pathway inhibitor for the preparation of a composition or formulation for:

(a) Promoting the diffusion of vimentin (vimentin) in cells; and

(b) Inhibit pathogen infection.

2. The use of claim 1, wherein the MAPK pathway inhibitor comprises a teniposide or a pharmaceutically acceptable salt thereof.

3. The use of claim 1, wherein the MAPK pathway inhibitor is selected from the group consisting of: trametinib (Trametinib), lei Fa tenib (Refametinib), semetinib (selemetinib), coumetinib (Cobimetinib), pimasertib, TAK-733, AZD8330, BI-847325, GDC-0623, PD318088, or combinations thereof; preferably, it is trimetinib, lei Fati ni, semantenib, more preferably trimetinib.

4. The use according to claim 2, wherein the pharmaceutically acceptable salt comprises a hydrochloride, sulfate, sulfonate, carbonate, acetate, tartrate, or isethionate salt.

5. The use of claim 1, wherein the pathogen comprises: bacteria, chlamydia, rickettsia, mycoplasma, spirochete, fungi, or combinations thereof.

6. The use according to claim 5, wherein said bacteria comprise: salmonella, staphylococcus aureus, escherichia coli, shigella, listeria, streptococcus, mycobacterium tuberculosis, klebsiella, acinetobacter baumannii, or a combination thereof.

7. The use according to claim 1, wherein the composition is a pharmaceutical composition comprising (i) a MAPK pathway inhibitor or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

8. The use of claim 1, wherein said inhibiting pathogen infection comprises inhibiting infection by a pathogen at a cellular level and/or inhibiting infection by a pathogen at an animal level.

9. A method of non-therapeutically inhibiting pathogen infection of a host cell in vitro comprising the steps of: the MAPK pathway inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, is added to a host cell culture system containing a pathogen, thereby inhibiting infection of the host cell by the pathogen.

10. The method of claim 9, wherein the pathogen comprises: bacteria, chlamydia, rickettsia, mycoplasma, spirochete, fungi, or combinations thereof.

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