CN111773216A - Use of C-JUN N-terminal kinase inhibitor SU3327 - Google Patents

Abstract

The present invention relates to the use of a C-JUN N-terminal kinase (JNK) inhibitor SU3327, wherein the C-JUN N-terminal kinase inhibitor SU3327 is used for the bacteriostasis of different animal and human species. The C-JUN N-terminal kinase inhibitor SU3327 acts on bacterial strains isolated from humans or/and animals or clinically isolated from infected individuals to perform sterilization and bacteriostasis.

Description

Use of C-JUN N-terminal kinase inhibitor SU3327

technical field

The present invention relates to the use of a C-JUN N-terminal kinase (JNK) inhibitor SU3327.

Background technique

Antibiotics are the most effective drugs that humans have discovered from soil and the metabolites of various microorganisms to treat bacterial infections. With the abuse of antibiotics, some bacteria have begun to appear resistant to antibiotics and become drug-resistant bacteria, which increases the difficulty of treating human and animal diseases. In 2019, my country issued the feed "anti-antibiotic prohibition" order (Announcement No. 194), which clearly prohibits the use of feed additives containing growth-promoting drugs in the breeding industry, and actively promotes the breeding industry to achieve "anti-antibiotic prohibition" in feed and "anti-antibiotic reduction" in breeding. , The development direction of "antibiotic-free" products, encourage the research and development of safe and efficient new research and development of antibacterial drugs to replace traditional antibiotics. As long as new antibiotics can be developed, drug-resistant bacteria can be killed. However, the development of antibiotics needs to be screened in the soil and various products of microorganisms in it. This process requires high cost and a very long time. The rate of growth is much faster than the rate of research and development of new antibiotics.

SU3327, a c-Jun N-terminal kinase (JNK) inhibitor, can be used for diabetes treatment. Its molecular formula is C5H3N5O2S3, and its molecular weight is 261.313 as shown in Figure 1.

SU3327 is disclosed, and the references as a preclinical research drug for diabetes treatment are as follows:

J Med Chem (2009 Apr 9), Titled Design, Synthesis, and Structure-Activity Relationship of Substrate Competitive, Selective, and in Vivo ActiveTriazole and Thiadiazole inhibitors of the c-Jun N-Terminal Kinase, Main Content: In the article SU3327 is compound 9, which is a synthetic JNK inhibitor. Protein kinases account for 2% of the mammalian genome and catalyze the transfer of the γ-phosphoryl group of ATP to specific protein substrates. c-Jun N-terminal kinase (JNK) is a series of serine/threonine protein kinases of the mitogen-activated protein kinase (MAPK) family. Upregulation of JNK activity is associated with many diseases, such as type-2 diabetes, obesity, cancer, and stroke. JNK inhibitors can inhibit the activity of JNK, therefore, JNK inhibitors have anti-disease effects.

Journal of Acta Pharmacol Sin (2014 Mar), titled TNF-α induces CXCL1 chemokineexpression and release in human vascular endothelial cells in vitro via twodistinct signaling pathways, main content: The article found that TNF-α (2, 5 ng/mL) in the concentration and Induction of CXCL1 release and mRNA expression in cells in a time-dependent manner. TNF-α causes JNK, p38 MAPK, PI3K and Akt activation, whereas pretreatment with JNK inhibitors (SP600125 and SU3327), p38 MAPK inhibitor (SB202190) or PI-3K inhibitor (LY294002) significantly inhibited TNF- Alpha-induced CXCL1 release from cells, SU3327 was used as a JNK inhibitor in this paper.

Microvasc Res journal (2012 Sep), titled Post-transcriptional regulation of placenta growth factor mRNA by hydrogen peroxide, main content: Oxidative stressors such as hydrogen peroxide activate multiple kinase pathways that affect transcription, RNA stability and translation process. The kinase pathway contributes to the superoxide-induced increase of PLGF mRNA level, and SU3327, as a JNK inhibitor, can significantly inhibit the superoxide-induced increase of PLGF mRNA level.

Redox Biol journal (Dec 2015), titled Critical role of c-jun N-terminal protein kinase in promoting mitochondrial dysfunction and acute liver injury, main content: The article mainly studies the role of c-jun N-terminal protein kinase (JNK) in promoting mitochondrial dysfunction and acute liver injury and critical role in acute liver injury. The article used SU3327-treated mice as a control, that is, JNK activity was inhibited, and found that SU3327 pretreatment significantly reduced the levels of p-JNK, mitochondrial phosphoprotein and liver damage in CCl4-exposed mice.

SUMMARY OF THE INVENTION

The present invention provides the use of a C-JUN N-terminal kinase (JNK) inhibitor SU3327, for example, the C-JUN N-terminal kinase inhibitor SU3327 is used for the bactericidal and bacteriostatic effects of different animal and human species.

Further: the C-JUN N-terminal kinase inhibitor SU3327 acts on the strains isolated from humans or/and animals or clinically isolated from infected persons and then cultured to perform sterilization and bacteriostasis.

In some embodiments, the SU3327 is bactericidal against a variety of bacteria including Escherichia coli ATCC25922, Staphylococcus aureus CMCC(B) 26003, Staphylococcus aureus MRSA ATCC43300, Acinetobacter baumannii ATCC19606, Clostridium sporogenes CMCC (B) 64941, Bacillus subtilis CMCC (B) 63501, Candida albicans ATCC10231. Moreover, compared with gentamicin sulfate, an aminoglycoside broad-spectrum antibiotic, the SU3327 has better bacteriostatic effect.

The above Escherichia coli ATCC25922, Staphylococcus aureus CMCC (B) 26003, Staphylococcus aureus MRSA ATCC43300, Acinetobacter baumannii ATCC19606, Clostridium sporogenes CMCC (B) 64941, Bacillus subtilis CMCC (B) 63501, Candida albicans ATCC10231 are all derived from strains isolated from humans or/and animals or clinically isolated from infected individuals and then cultured.

Description of drawings

Figure 1 is the structural formula of the N-terminal kinase inhibitor SU3327 of the present invention.

Detailed ways

Based on the deep learning technology, the present invention independently finds out potential new antibiotics from the existing safe drug database or in the clinical trial stage. The bacterial susceptibility test confirmed that SU3327 has broad-spectrum antibiotic activity. The present invention provides a novel antibiotic, which further brings important influence to the field of medicine.

Bacterial susceptibility test to determine SU3327 against Escherichia coli ATCC25922, Staphylococcus aureus CMCC (B) 26003, Staphylococcus aureus MRSA ATCC43300, Acinetobacter baumannii ATCC19606, Clostridium sporogenes CMCC (B) 64941, Bacillus subtilis CMCC (B) Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 63501, Candida albicans ATCC10231. It can be seen from the experimental results that SU3327 has good bacteriostatic ability and can be used as a broad-spectrum antibiotic to effectively reduce the mortality rate of patients after pathogenic bacteria infection.

The following mainly introduces the drug susceptibility test of SU3327 to various bacteria, namely the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), to explain the present invention. It should be noted that the following specific experimental protocols are for illustrative purposes only and do not specifically limit the scope of the present invention.

In the following examples, the used test protocols are conventional methods unless otherwise specified.

In the following examples, the materials, reagents, etc. used are obtained from commercial sources unless otherwise specified.

1. Strain culture and concentration adjustment

Pick out the pure bacteria solution cultured for 18-24h and dissolve it in 2-5ml sterile saline, and adjust its turbidity to match the turbidity of a 0.5 McFarland turbidity tube.

2. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

(1) Experimental equipment

96-well plate, test tube, 5uL-50uL micropipette, 100uL-1000uL micropipette, and matching pipette tips, 10mL centrifuge tube, McFarland turbidimetric tube, ultra-clean workbench, autoclave, blast Drying box, biochemical incubator, MH broth medium, PBS buffer, sterile water.

(2) Experimental method

Step 1: Put the prepared MH medium, PBS buffer, sterile water, matching pipette tips, and centrifuge tubes into an autoclave for sterilization, and sterilize at 121°C for 25 minutes. Put the test tubes together in a blast drying oven for sterilization. Sterilize at 170°C for 2h. The 96-well plate was UV sterilized on an ultra-clean workbench for more than 30 minutes.

Step 2: Take out the strain to be tested from the refrigerator and activate it for 0.5h, add 5mL of PBS buffer, gently shake it on the slant for 80 times on the palm to wash the strain completely, pour it into a test tube and shake gently ; Draw 1 mL of bacterial solution and add it to 4 mL of PBS buffer, then draw 1 mL of the diluted bacterial suspension to carry out 5-fold gradient dilution in turn, and select a bacterial suspension of about 10 ⁸ CFU/mL or a spore of about 10 ⁶ CFU/mL Suspension (compared to 0.5 McFarland turbidity tube), dilute the selected bacterial suspension ten-fold serially and select a second test tube (that is, bacterial suspension with a concentration of about 10 ⁶ CFU/mL or 10 ⁴ CFU/mL about the spore suspension), then pipette 0.5ml into 4.5ml MH medium, and blow it well with a pipette tip for later use.

Step 3: Use dimethyl sulfoxide to prepare the SU3327 liquid with a concentration of 10.5 mg/ml; use the test tube double dilution method to add the SU3327 liquid, bacterial liquid and MH broth medium to a 96-well culture plate for overnight culture. The concentration of the drug solution groups are all in parallel to ensure the reliability of the experimental data. Incubate the 96-well culture plate in a 37°C incubator for 16 hours, observe the turbidity of the liquid in the well, and take the lowest concentration that can completely inhibit bacterial growth as the MIC value. Take 100 μl of the culture from each well and apply the solid medium by double dilution. After overnight incubation in a 37°C incubator, observe the colony growth, and take the lowest concentration at which the bacteria are completely killed as the MBC value.

The experimental results of the above experiments are shown in Table 1. As shown in Table 1, the bacteriostatic effect and bactericidal effect of SU3327 on the bacterial species used in the experiment are better than those of gentamicin sulfate.

have to be aware of is:

1. The double-dilution method for determining the bacterial MIC value can be replaced by the disc diffusion method, that is, the paper disc contaminated with a certain concentration of medicinal liquid is placed on the bacterial culture medium, and the formation between the bacterial growth area on the medium and the paper disc is measured. The inhibition zone of bacteria is used to determine the MIC value of bacterial susceptibility;

2. The above-mentioned MH broth medium can be replaced with other medium, such as agar medium;

3. The above-mentioned double dilution method for MBC value determination can be replaced by the intrabasal inoculation method.

Table 1. MIC and MBC values of SU3327 and gentamicin sulfate against different strains

As shown in Table 1 above, the bacteriostatic and bactericidal effects of SU3327 on Gram-negative pathogenic bacteria and Gram-positive pathogenic bacteria are better than gentamicin sulfate.

Although the present invention is disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection shall be subject to the scope defined by the claims of the present invention.

Claims (10)

1. The C-JUN N-terminal kinase inhibitor SU3327 has antibiotic use. 2 . The use of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 , wherein the C-JUN N-terminal kinase inhibitor SU3327 is used for the bactericidal and bacteriostatic effects of different animal and human species. 3 . 3. The purposes of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 2, characterized in that: the C-JUN N-terminal kinase inhibitor SU3327 acts on human or/and animal in vivo isolation or clinically obtained from SU3327. The strains obtained after isolation from the infected person are cultured for sterilization and bacteriostasis. 4. The purposes of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 or 2, wherein the C-JUN N-terminal kinase inhibitor SU3327 is used for the inhibition and killing of Pseudomonas aeruginosa . The use of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 or 2, characterized in that: the C-JUN N-terminal kinase inhibitor SU3327 is used for the inhibition and killing of Staphylococcus aureus. The use of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 or 2, characterized in that: the C-JUN N-terminal kinase inhibitor SU3327 is used for the inhibition and killing of Bacillus subtilis. The use of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 or 2, characterized in that: the C-JUN N-terminal kinase inhibitor SU3327 is used for the inhibition and killing of Escherichia coli. The use of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 or 2, characterized in that: the C-JUN N-terminal kinase inhibitor SU3327 is used for the inhibition and killing of Candida albicans. 9. The purposes of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 or 2, wherein the C-JUN N-terminal kinase inhibitor SU3327 is used for the inhibition and killing of Acinetobacter baumannii . The use of the C-JUN N-terminal kinase inhibitor SU3327 according to claim 1 or 2, characterized in that: the C-JUN N-terminal kinase inhibitor SU3327 is used for the inhibition and killing of Clostridium sporogenes.

Images