Application of NTP42 in preparation of antibacterial drugs

Abstract

The invention discloses application of NTP42 in preparation of antibacterial drugs, and belongs to the technical field of drugs. Research finds that NTP42 has an antibacterial effect on staphylococcus aureus, can effectively inhibit the activity of methicillin-sensitive or drug-resistant staphylococcus aureus, and is an antibacterial drug with a very good development prospect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to application of NTP42 in the preparation of antibacterial drugs. Background Art

[0002] Bacterial infection is a common clinical disease that has seriously threatened people's lives and health and has become a medical and health problem that needs to be solved urgently. Staphylococcus aureus is one of the main pathogens of hospital and community infections and has become a worrying health threat worldwide. Staphylococcus aureus can cause a variety of infectious diseases, such as endocarditis, osteomyelitis, bacteremia, sepsis and fatal pneumonia. Penicillin and methicillin were originally used as the main antibiotics for the treatment of Staphylococcus aureus infections.

[0003] However, bacteria rapidly acquire drug resistance by producing penicillinase to hydrolyze the β-lactam ring of penicillin and encode the low-affinity penicillin-binding protein PBP2a. More seriously, methicillin-resistant Staphylococcus aureus is resistant to all β-lactam antibiotics, and methicillin-resistant Staphylococcus aureus (MRSA) has spread rapidly and widely around the world and has become a major public health problem. It has strong drug resistance and can usually cause a variety of infections, bringing great difficulties and challenges to the removal and prevention of pathogens. Therefore, it is crucial to develop new antibiotics against Staphylococcus aureus.

[0004] In previous pharmacological studies, NTP42 is a thromboxane (TXA2) receptor antagonist that can be used to treat pulmonary arterial hypertension (PAH) and is a highly potential drug lead compound. However, to date, there have been no reports of NTP42's antibacterial effects, let alone antibacterial drugs with NTP42 as the main active ingredient. Summary of the invention

[0005] The purpose of the present invention is to provide an application of NTP42 in the preparation of antibacterial drugs. Experiments have found that NTP42 has good antibacterial effects and can effectively inhibit the activity of methicillin-sensitive and -resistant Staphylococcus aureus.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides the use of NTP42 or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial drug or a drug for preventing and / or treating a bacterial infection disease;

[0008] The chemical name of NTP42 is:

[0009] N-(tert-Butylcarbamoyl)-5-cyano-2-((4'-(difluoromethoxy)-[1,1'-biphenyl]-3-yl)oxy)benzenesulfonamide.

[0010] The PubChem CID number of the NTP42 is: 124147085.

[0011] The structural formula of the NTP42 is as follows:

[0012]

[0013] In the application, the antibacterial drug can inhibit the activity of Staphylococcus aureus.

[0014] In the application, the bacteria is Staphylococcus aureus.

[0015] In the application, the Staphylococcus aureus is at least one of methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, such as MRSA 252, MRSA48973, MRSA49008, MRSA48706, and MRSA48966.

[0016] In the application, the minimum inhibitory concentration of NTP42 against methicillin-sensitive Staphylococcus aureus is 0.3125 μg / mL;

[0017] In the application, the minimum inhibitory concentration of NTP42 against methicillin-resistant Staphylococcus aureus is 0.156 μg / mL.

[0018] In the application, the minimum effective concentration of NTP42 against methicillin-resistant Staphylococcus aureus at the animal level is 2.5 mg / kg.

[0019] In the application, the dosage form of the antibacterial drug is an oral preparation and / or an injection.

[0020] In the application, the dosage form of the drug for preventing and / or treating bacterial infection diseases is an oral preparation and / or an injection.

[0021] In the application, the NTP42 inhibits the activity of the bacteria by acting on MurJ, a protein essential for the survival of the bacteria.

[0022] The present invention further provides an antibacterial drug, the active ingredient of which is the NTP42.

[0023] The antibacterial drug is a drug that inhibits the activity of Staphylococcus aureus. The Staphylococcus aureus is at least one of methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0024] The present invention further provides a drug for preventing and / or treating bacterial infection diseases, wherein the active ingredient of the drug is the NTP42.

[0025] The bacteria is Staphylococcus aureus, and the Staphylococcus aureus is at least one of methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0026] The inventors of the present invention have found in long-term research that compound NTP42 can act on lipid flippase MurJ. MurJ is an essential component for bacterial survival and is mainly responsible for transporting lipid II, an important component of cell wall peptidoglycan, from the cytoplasm to the periplasmic space. Therefore, inhibiting the function of MurJ can cause a synthetic disorder of bacterial peptidoglycan, thereby affecting bacterial survival. The present invention finds that NTP42 has a good antibacterial effect and can effectively inhibit the growth of methicillin-sensitive and -resistant Staphylococcus aureus.

[0027] The present invention has the following beneficial effects:

[0028] (1) In the present invention, NTP42 is used as an antibacterial agent for treating Staphylococcus aureus infection, which can effectively inhibit the activity of Staphylococcus aureus; in particular, it has a significant positive effect on methicillin-resistant Staphylococcus aureus (MRSA) infection, and has great clinical significance.

[0029] (2) In the present invention, NTP42 can act on MurJ, an essential protein for bacterial survival, inhibit the synthesis of bacterial peptidoglycan, and has significant antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a comparative graph of the survival rate of NTP42 in treating the methicillin-resistant Staphylococcus aureus (MASR 252) infection model in BALB / c female mice in Example 3. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0032] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Staphylococcus aureus ATCC 29213 in the following examples is commonly available on the market from the American Type Culture Collection.

[0034] The methicillin-resistant Staphylococcus aureus MRSA 252, MRSA 48973, MRSA 49008, MRSA 48706 and MRSA 48966 in the following embodiments are recorded in the literature (Pyrimirhodomyrtone inhibits Staphylococcus aureus by affecting the activity of NagA, Wei Huang, Biochemical Pharmacology 210 (2023) 115455), which can be obtained by the public from the applicant. The above-mentioned biological materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0035] The LB broth medium used in the following examples was composed and prepared as follows: 8 g of tryptone powder, 8 g of sodium chloride, and 4 g of yeast extract powder were weighed and dissolved in 800 mL of distilled water, and sterilized by autoclaving at 121° C. for 15 minutes and stored at 4° C. for later use.

[0036] LB agar plate: weigh 8g of tryptone powder, 8g of sodium chloride, 4g of yeast extract powder, and 12g of agar powder and dissolve them in 800mL of distilled water. Autoclave at 121℃ for 15 minutes, pour into a sterile bacterial culture dish, cool and solidify. Store at 4℃ for later use.

[0037] MH broth medium: Weigh 21.0 g of MH broth medium, dissolve in 1000 mL of distilled water, and sterilize by autoclaving at 121°C for 15 minutes. Store at 4°C for later use.

[0038] The NTP42 used in the following examples was purchased from Shanghai Taosu Biochemical Technology Co., Ltd.

[0039] Vancomycin used in the following examples was purchased from MedChemexpress Biotechnology Company, USA.

[0040] The bacterial genomic DNA extraction kit used in the following examples was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0041] Example 1

[0042] In this example, Staphylococcus aureus ATCC 29213 and methicillin-resistant Staphylococcus aureus (MRSA 252) were inoculated into LB broth medium, revived overnight at 37°C and 220 rpm, and then inoculated into 5 mL of fresh LB broth medium at a volume ratio of 1:100 and cultured with shaking until the bacteria grew to the logarithmic growth phase (OD 600 =0.6~0.8), and obtain bacterial culture.

[0043] The specific steps for determining the minimum inhibitory concentration (MIC) are as follows:

[0044] 1) OD 600 = 0.6 to 0.8 of bacterial culture medium was adjusted to OD 600 =0.001;

[0045] 2) Add 2 μL of 1 mg / mL NTP42 stock solution (solvent DMSO) and 198 μL of the bacterial culture obtained in step 1) to a 96-well plate, and then use OD 600 =0.001 was serially diluted 2-fold, the volume of each well was 100 μL, the final concentrations of NTP42 were 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0 μg / mL, and each concentration was repeated 3 times.

[0046] 3) Set up a blank group (add 100 μL MH broth medium), a control group (add 100 μL OD 600 0.001 for bacterial cultures);

[0047] 4) Place the 96-well plate in a 37°C incubator for 18 hours, then add 10 μL of 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to each well. Incubate in a 37°C incubator in the dark for 30 minutes. Finally, measure the OD value on a microplate reader. 600 .

[0048] According to the bacterial inhibition rate (%) = 1-(OD 实验组 -OD 空白 ) / (OD 对照组 -OD 空白 )×100% to calculate the bacterial inhibition rate, and MIC was recorded as the lowest compound concentration that inhibited 90% bacterial growth.

[0049] The results are shown in Table 1. The MICs of NTP42 against Staphylococcus aureus ATCC 29213, MRSA 252, MRSA 48973, MRSA 49008, MRSA 48706 and MRSA 48966 were 0.3125, 0.3125, 0.156, 0.3125, 0.156 and 0.156 μg / mL, respectively.

[0050] Table 1 The results of NTP42's inhibition on various bacteria

[0051]

[0052] Example 2

[0053] The mechanism of NTP42 inhibiting the activity of Staphylococcus aureus was explored through tolerant strain screening experiments to further illustrate the present invention.

[0054] 1) Tolerant strain screening: 50 μL of Staphylococcus aureus ATCC 29213 (OD 600 =2.5) was inoculated into 5 mL LB broth medium and revived overnight at 37°C and 220 rpm; the overnight culture was inoculated into 2 mL LB broth medium at a volume ratio of 1:100, NTP42 at a concentration of 0.1625 μg / mL was added, and cultured at 37°C and 220 rpm until OD 600 >1; the above culture was inoculated into 2 mL LB broth at a volume ratio of 1:100, and NTP42 at a concentration of 0.3125 μg / mL was added, and cultured at 37°C and 220 rpm until OD 600 >1; Repeat the process of increasing the NTP42 concentration by 2 times until the concentration of NTP42 reaches 5μg / mL. Use an inoculation loop to streak the bacterial solution on an LB agar plate (containing 5μg / mL NTP42);

[0055] 2) MIC determination of tolerant strains: A single colony in step 1) was picked and inoculated into LB broth medium, and the MIC was determined according to the method of Example 1.

[0056] 3) Extraction of genomic DNA: strains with MIC values ​​> 5 μg / mL were selected, cultured overnight in LB broth medium, and genomic DNA was extracted using a bacterial genomic DNA extraction kit.

[0057] 4) Sequencing: After the DNA sample is qualified, it is randomly interrupted using a Covaris ultrasonic crusher, and then the entire library preparation is completed through end repair, A-tailing, sequencing adapter addition, purification, PCR amplification and other steps. After the library is constructed, Qubit2.0 is used for preliminary quantification, and the library is diluted to 1.5ng / uL. Then, the insert size of the library is detected using Agilent2100. After the insert size meets expectations, the effective concentration of the library is accurately quantified using the Q-PCR method (library effective concentration>2nM). After the library is qualified, different libraries are pooled to flowcell according to the effective concentration and target data volume. After cBOT clustering, Illumina high-throughput sequencing platform (HiSeq / MiSeq) is used for sequencing to generate sequencing raw data (.raw)

[0058] 5) Sequence analysis: Spades software was used to assemble the raw data, Prokka was used to annotate the assembled data, and Snippy software was used to perform site analysis on the annotated sequences.

[0059] After bioinformatics analysis, the results are shown in Table 2. The 142nd position of the murJ gene mutated from G to T. It is speculated that NTP42 exerts its antibacterial activity by acting on the MurJ protein.

[0060] Table 2: Analysis of mutation sites of NTP42 resistant strains

[0061]

[0062] Example 3

[0063] In this example, NTP42 was used in a mouse MRSA infection model to further illustrate the present invention.

[0064] 1) Establishment of infection model: 40 6-8 week old BALB / c female mice (weighing about 20 g) were injected via tail vein with 1.4×10 8 CFU of MRSA 252 bacteria.

[0065] 2) Group treatment: Female mice were randomly divided into five groups (n = 8 mice / group) and the drug was administered once by intraperitoneal injection 2 hours before infection and again by intraperitoneal injection 24 hours and 48 hours after infection.

[0066] Among them, the four groups included a blank control group (injected with an equal amount of DMSO), a positive control group (injected with 5 mg / kg vancomycin, the solvent was water), treatment group I (injected with 2.5 mg / kg NTP42, the solvent was DMSO), treatment group II (injected with 5 mg / kg NTP42, the solvent was DMSO), and treatment group III (injected with 10 mg / kg NTP42, the solvent was DMSO).

[0067] The death of female mice was observed every 12 hours for a total of 156 hours.

[0068] The results are as follows Figure 1 As shown, both the positive control group and the treatment group significantly increased the survival rate of infected mice compared with the solvent control.

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of NTP42 or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial drug or a drug for preventing and / or treating a bacterial infection; The structural formula of the NTP42 is as follows:

2. The use according to claim 1, characterized in that: The antibacterial drug is a drug that inhibits the activity of Staphylococcus aureus; The bacteria is Staphylococcus aureus.

3. The use according to claim 2, characterized in that: The Staphylococcus aureus is at least one of methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

4. The use according to any one of claims 1 to 3, characterized in that: The minimum inhibitory concentration of NTP42 against methicillin-sensitive Staphylococcus aureus is 0.3125 μg / mL; The minimum inhibitory concentration of the NTP42 against methicillin-resistant Staphylococcus aureus is 0.156 μg / mL.

5. The use according to any one of claims 1 to 4, characterized in that: The minimum effective concentration of NTP42 against methicillin-resistant Staphylococcus aureus at the animal level is 2.5 mg / kg.

6. The use according to any one of claims 1 to 5, characterized in that: The dosage form of the antibacterial drug is an oral preparation and / or an injection; The dosage form of the drug for preventing and / or treating bacterial infection diseases is an oral preparation and / or an injection.

7. The use according to any one of claims 1 to 6, characterized in that: The NTP42 inhibits bacterial activity by acting on the protein MurJ.

8. An antibacterial drug, the active ingredient of which is the NTP42.

9. A drug for preventing and / or treating bacterial infection diseases, wherein the active ingredient of the drug is the NTP42.

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