Use of indole compounds with electron-withdrawing groups for inhibiting plasmid conjugative transfer

Abstract

This invention discloses the application of indole compounds with electron-withdrawing groups in inhibiting plasmid conjugation transfer. The invention initially screened 12 indole compounds and found that indole compounds carrying electron-withdrawing side chain groups are an effective class of plasmid conjugation transfer inhibitors. Subsequently, indole compounds carrying electron-withdrawing side chain groups were further investigated. blue NDM‑5 , tight (X4) and mcr‑ 1 Using clinical strains of the plasmid as donors, the effects of indoleacetic acid (IAA), 7-bromoindole, and 5-nitroindole on inhibiting the conjugation and transfer of clinical plasmids were further verified. It was found that the three compounds significantly inhibited the conjugation and transfer of the three clinically relevant plasmids. Furthermore, in vivo conjugation and transfer experiments using IAA as an example showed that the frequency of RP4-7 plasmid conjugation and transfer in the liver and spleen of mice treated with IAA was significantly reduced. This invention provides an effective method for preventing the spread of drug resistance and aims to offer reference and guidance for research on drug resistance control strategies.

Description

Technical Field

[0001] This invention relates to the application of indole compounds with electron-withdrawing groups in inhibiting plasmid conjugation and transfer, belonging to the field of pharmaceutical technology. This invention is a divisional application of patent application number 2025102611525 entitled "Application of Indole Compounds with Electron-Withdrawing Groups in Inhibiting Plasmid Conjugation and Transfer," specifically relating to the application of indole compounds with electron-withdrawing groups in the preparation of drugs that inhibit plasmid conjugation and transfer in vivo or block the intestinal spread of drug-resistant bacteria. Background Technology

[0002] Antibiotics are highly effective at killing pathogenic microorganisms and play a crucial role in modern medicine, saving countless lives. However, in recent years, the overuse of antibiotics has led to a surge in bacterial resistance, posing a significant challenge to human health and production safety. Bacterial resistance can develop in two ways: one is through repeated exposure to antibiotics, resulting in resistance gene mutations and the acquisition and spread of resistance via mobile genetic components such as resistance plasmids and transposons. Plasmid-mediated horizontal transfer of resistance genes is the most prevalent pathway for the spread of antibiotic resistance genes in humans, animals, and the environment. Plasmid conjugation transfer inhibitors are one of the effective strategies for controlling the spread of resistance genes. Common plasmid conjugation transfer inhibitors include fatty acids, polysaccharide antibiotics, and nanomaterials. Most plasmid conjugation transfer inhibitors primarily act on the bacterial type IV secretion system, while a few inhibitors block plasmid conjugation transfer by reprogramming bacterial metabolism.

[0003] Indole and its derivatives are compounds formed by the symmetric combination of pyrrole and benzene, widely distributed in nature, amino acids (such as tryptophan), animal hormones (such as melatonin), and plant hormones. Indole compounds possess a variety of biological activities, including antiviral, anti-inflammatory, anticancer, antioxidant, antimicrobial, antituberculosis, antidiabetic, and antimalarial effects, demonstrating significant potential for clinical application. Furthermore, as an intercellular signaling molecule, indole participates in multiple bacterial physiological processes, including sporulation, plasmid stability, drug resistance, and biofilm formation. The molecular formula of indole is C8H7N. The chemical structural formula of indole is shown in Formula 1.

[0004]

[0005] Indole compounds, with their advantages of being naturally sourced, readily available, and low in toxicity, are undoubtedly ideal candidates for plasmid conjugation transfer inhibitors. However, currently, no studies have been conducted to screen effective conjugation transfer inhibitors based on the structure-activity relationship of indole compounds. Summary of the Invention

[0006] Objective of the invention: The technical problem to be solved by the present invention is to provide the application of indole compounds with electron-withdrawing groups in inhibiting plasmid binding and transfer.

[0007] Technical solution: To solve the above-mentioned technical problems, this invention provides the application of indole compounds with electron-withdrawing groups in inhibiting plasmid binding and transfer.

[0008] The general structural formula of the indole compound having an electron-withdrawing group is as follows:

[0009]

[0010] Wherein, R1, R2 or R3 are electron-withdrawing groups, selected from one of halogen, carboxyl or nitro groups.

[0011] The indole compounds having electron-withdrawing groups include one or more of 5-iodoindole, 3-indoleacetic acid, 7-bromoindole, 7-chloroindole, 7-fluoroindole, 5-nitroindole, 3-indolecarboxylic acid, 3-indolecarboxaldehyde, 3-indolepropionic acid, and 3-indolebutyric acid.

[0012] Wherein, the plasmid conjugation transfer is a plasmid transfer between bacteria, and the bacteria are Gram-negative bacteria.

[0013] The plasmid conjugation transfer includes engineered plasmids (RP4-7) and clinical plasmids (bla). NDM-5 , tet(X4) and mcr-1).

[0014] The donor bacteria include bacteria of the same genus (Escherichia coli) and bacteria of different genus (Klebsiella pneumoniae).

[0015] The concentration of the indole compound used is 0.1 μg / mL to 10 μg / mL.

[0016] The present invention also includes a method for inhibiting in vitro plasmid conjugation and transfer, comprising the following steps: contacting bacteria with an effective concentration of an indole compound having an electron-withdrawing group to inhibit plasmid conjugation and transfer between the bacteria.

[0017] The concentration of the indole compound with electron-withdrawing groups is 0.1 μg / mL to 10 μg / mL.

[0018] In this invention, the inhibition of plasmid conjugation transfer refers to the inhibition of plasmid conjugation transfer in vitro. In vitro, as described in this invention, refers to an environment or space outside of a living organism, such as outside of a human or animal body.

[0019] The present invention also includes the use of indole compounds with electron-withdrawing groups in the preparation of drugs that inhibit plasmid conjugation transfer in vivo or block the intestinal spread of drug-resistant bacteria.

[0020] The term "in vivo" in this invention refers to the body of a living organism, specifically the body of a human or animal.

[0021] The in vivo plasmid conjugation transfer refers to plasmid transfer between bacteria in vivo, and preferably, the bacteria are Gram-negative bacteria.

[0022] Among them, the drug-resistant bacteria include drug-resistant E. coli DH5α carrying the RP4-7 plasmid, and drug-resistant bacteria carrying the bla... NDM-5 Drug-resistant bacteria carrying plasmids include K. pneumoniae C12 or E. coli L65, drug-resistant bacteria carrying the tet(X4) plasmid include E. coli RS3-1 or E. coli RF2-1, and drug-resistant bacteria carrying the mcr-1 clinical plasmid include E. coli LD67-1.

[0023] The drug that inhibits in vivo binding and transfer includes drugs that reduce the frequency of plasmid binding and transfer in the liver and / or spleen of mice.

[0024] The concentration of the indole compound with electron-withdrawing groups used is 0.5 mg / kg mouse.

[0025] The present invention describes blocking the intestinal spread of drug-resistant bacteria by inhibiting the conjugate transfer frequency of plasmids while maintaining a constant bacterial count, thereby blocking their spread.

[0026] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The present invention discloses for the first time the effect of indole compounds with electron-withdrawing groups in inhibiting plasmid conjugation and transfer. Specifically, indole compounds with electron-withdrawing groups can effectively inhibit engineered plasmids (RP4-7) and three clinical plasmids (bla). NDM-5 The study investigated the conjugation transfer of plasmids tet(X4) and mcr-1. In vivo conjugation transfer experiments using IAA as an example showed a significant decrease in the frequency of plasmid conjugation transfer in the liver and spleen of mice treated with IAA. This invention provides a new perspective for the development of novel drug resistance control strategies and offers a new method for blocking the spread of increasingly serious drug resistance genes. Attached Figure Description

[0027] Figure 1 Screening for indole compounds to inhibit plasmid conjugation transfer.

[0028] Figure 2 The effects of 12 different concentrations of indole compounds on the conjugation transfer frequency of RP4-7 plasmid were investigated.

[0029] Figure 3 To evaluate the efficacy of indoleacetic acid, 7-bromoindole, and 5-nitroindole in inhibiting clinical plasmid conjugation and transfer.

[0030] Figure 4 To evaluate the in vivo efficacy of indoleacetic acid in inhibiting plasmid conjugation and transfer. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings. It should be noted that those skilled in the art can make several modifications and improvements without departing from the principle of the present invention, and these should also be considered within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. The quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged.

[0032] The LB broth medium used in the examples was an aqueous solution containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the MHB broth medium was an aqueous solution containing 2 g / L beef meal, 1.5 g / L soluble starch, and 17.5 g / L acid-hydrolyzed casein; and the DMEM basal medium was a solution containing 2 mM L-glutamic acid, 1 mM sodium pyruvate, glucose, amino acids, inorganic salts, and other basic nutrients.

[0033] CD-1 female mice were purchased from the Comparative Medicine Center of Yangzhou University.

[0034] All 12 indole compounds were purchased from Shanghai Yuanye Bio-Technology, with catalog numbers S43256 (5-iodoindole), V34639 (3-indoleacetic acid), S49050 (5-methylindole), S47797 (7-bromoindole), S48076 (7-chloroindole), S46713 (4-fluoroindole), S49039 (5-nitroindole), S31552 (3-indolecarboxylic acid), S30763 (3-indolecarboxaldehyde), S18033 (3-indolepropionic acid), T90319 (3-indolebutyric acid), and S30160 (5-hydroxyindoleacetic acid).

[0035] All strains used in the examples were strains preserved in our laboratory.

[0036] Example 1: Screening of indole compounds that inhibit plasmid conjugation transfer

[0037] Conjugation transfer frequency determination: E. coli DH5α carrying the RP4-7 plasmid was used as the donor, and E. coli C600 as the recipient to determine the conjugation transfer frequency. (Donor and recipient bacteria are from the article Zhang M, Yang B, Shi J, Wang Z, Liu Y. Host defense peptides mitigate the spread of antibiotic resistance in physiologically relevant condition. Antimicrob Agents Chemother. 2024 Apr).

[0038] 3; 68(4):e0126123). A single colony was inoculated into 1 mL of LB medium and incubated overnight. A 1:100 volume ratio was inoculated into 10 mL of LB medium and incubated at 37°C and 200 rpm in a shaker until the logarithmic growth phase. The bacterial OD was adjusted using a microplate reader. 600nm Up to 0.25. Pipe 1 mL of donor E. coli DH5α and 1 mL of recipient E. coli C600 into a new 5 mL EP tube. Add conjugates, i.e., different concentrations of indole compounds (5-iodo-1H-indole (5 mg / mL): Purify, S43256 - 100 mg; Indole-3-lactic acid (5 mg / mL): Purify, V34639 - 100 mg; 5-Methylindole (5 mg / mL): Purify, S49050 - 100 mg; 7-bromo-1H-indole (5 mg / mL): Purify, S47797 - 100 mg; 7-chloro-1H-indole (5 mg / mL): Purify, S48076 - 100 mg). mg; 7-fluoro-1H-indole (7-fluoroindole, 5 mg / mL): Purify, S46713-100 mg; 5-Nitroindole (5-nitroindole, 5 mg / mL): Purify, S49039-100 mg; Indole-3-carboxylic acid (3-indolecarboxylic acid, 5 mg / mL): Purify, S31552-100 mg; Indole-3-carboxaldehyde (3-indolecarboxaldehyde, 5 mg / mL): Purify, S30763-100 mg; Indole-3-propionicacid (3-indolepropionic acid, 5 mg / mL): Purify, S30763-100 mg;

[0039] (mg / mL): Purify, S18033-100 mg; Indole-3-butyric acid (3-indolebutyric acid, 5 mg / mL): Purify, T90319-100 mg; 5-Hydroxyindole-3-acetic acid (5-hydroxyindoleacetic acid, 5 mg / mL): Purify, T90319-100 mg;

[0040] (mg / mL): Purify, S30160-100 mg; to final concentrations of 0.1, 1, and 10 μg / mL, and incubate at 37°C and 200 rpm for 15 h. Afterward, dilute the conjugated bacteria with appropriate concentration gradients and plate them onto rifampicin single-drug agar plates and ampicillin / rifampicin dual-drug agar plates to screen for recipient bacteria and conjugates. Incubate the agar plates overnight at 37°C. Then, count the bacteria on the agar plates to calculate the conjugation transfer frequency. Conjugation transfer frequency = number of conjugates / number of recipient bacteria.

[0041] Minimum inhibitory concentration (MIC) determination: Single clones of zygotes, donor bacteria, and recipient bacteria were inoculated into 1 mL of LB medium and cultured overnight. 100 μL of MHB medium was transferred to a 96-well plate. Rifampin or ampicillin was added to the first well to a final concentration of 2048 μg / mL, and the mixture was thoroughly mixed. 100 μL was then transferred to the second well, and this process was repeated serially until the last well, discarding the first 100 μL. The overnight cultured bacteria were diluted 1:1000 in MHB medium, and 100 μL of the diluted bacterial suspension was transferred to a 96-well plate. The lowest concentration at which no significant bacterial growth was observed after 18 h of incubation at 37°C was defined as the MIC concentration.

[0042] Polymerase chain reaction (PCR) analysis: The reaction system consisted of 2×Ex Tag Premix (25 μL), 1 μL each of upstream primer 5'-TCGGACGTTTGACCGGAA-3' and downstream primer 5'-GCTTCGTGTGTTTCAGCAA-3', 1 μL of DNA template, and ddH2O added to a final volume of 50 μL. The reaction program was 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, for 30 cycles; 72℃ for 5 min. The PCR amplification products were detected by agarose gel electrophoresis at a constant voltage of 120V for 15 min, and the presence of the desired target fragment was determined based on the marker bands.

[0043] The analysis results are shown below. Figure 1 and Figure 2 The results showed that, except for 5-methylindole and 5-hydroxyindoleacetic acid, the other 10 indole compounds could effectively inhibit plasmid conjugation transfer. Indole compounds carrying electron-withdrawing groups exhibited a stronger inhibitory effect on plasmid conjugation transfer.

[0044] Example 2: Evaluation of the efficacy of indoleacetic acid, 7-bromoindole, and 5-nitroindole in inhibiting clinical plasmid conjugation and transfer.

[0045] Clinical plasmid conjugation transfer frequency determination: using plasmids carrying b... NDM-5 Clinical strains of K. pneumoniae C12, E. coli L65, E. coli RS3-1, E. coli RF2-1, and E. coli LD67-1 were used as donors, and E. coli C600 was used as the recipient to determine the conjugation transfer frequency (all of the above clinical strains were derived from the article Zhang M, Yang B, Shi J, Wang Z, Liu Y. Host defense peptides mitigate the spread of antibiotic resistance in physiologically relevant condition. Antimicrob Agents Chemother. 2024 Apr 3; 68(4):e0126123). A single colony was inoculated into 1 mL of LB medium and cultured overnight. Inoculated at a ratio of 1:100 into 10 mL of LB medium and cultured in a shaker at 37°C and 200 rpm until the logarithmic phase. The OD of the bacteria was adjusted using an enzyme-linked immunosorbent assay (ELISA) reader. 600nm To a final concentration of 0.25, transfer 1 mL of donor bacteria and 1 mL of recipient bacteria into new 5 mL EP tubes. Add different concentrations of the drug (Indole-3-lactic acid, 5 mg / mL: Purify, V34639-100 mg; 7-bromo-1H-indole, 5 mg / mL: Purify, S47797-100 mg; 5-Nitroindole, 5 mg / mL: Purify, S49039-100 mg) to a final concentration of 0.1, 1, or 10 μg / mL. Incubate at 37°C and 200 rpm for 15 h. Afterward, dilute the conjugated bacteria to appropriate concentration gradients and plate them onto agar plates containing antibiotics to screen for recipient bacteria and conjugates. Incubate the agar plates overnight at 37°C. Count the bacteria on the agar plates to calculate the conjugation transfer frequency. Conjugation transfer frequency = number of conjugates / number of recipient bacteria.

[0046] The analysis results are shown below. Figure 3 The results showed that indoleacetic acid, 7-bromoindole, and 5-nitroindole significantly inhibited bla NDM-5 Conjugation and transfer of three clinical plasmids: tet(X4) and mcr-1.

[0047] Example 3: Evaluation of the in vivo effect of indoleacetic acid in inhibiting plasmid conjugation and transfer.

[0048] In vivo conjugation transfer model in mice: Sixteen female CD-1 mice (Comparative Medicine Center, Yangzhou University, 6-8 weeks old, weighing 20±2g) were randomly divided into a Control group and an IAA treatment group (IAA, 0.5mg / kg), with 8 mice in each group. They were acclimatized for 3 days with free access to food and water. Donor bacteria E. coli DH5α and recipient bacteria E. coli C600 were cultured overnight, and bacterial OD was then adjusted. 600 nm To a concentration of 0.5. First, 100 μL of donor and recipient bacteria were injected intraperitoneally. 15 min after infection, 100 μL of PBS and IAA (0.5 mg / kg) were injected intraperitoneally again. 24 h later, the mice were euthanized, and their livers and spleens were collected and ground. The ground organs were diluted with appropriate concentration gradients. The diluted homogenate was plated on corresponding antibiotic-containing agar plates, and colony counting was performed after 12 h to calculate the conjugation and transfer frequency of bacteria in the organs.

[0049] The analysis results are shown below. Figure 4 The results showed that, compared with the Control group, the frequency of bacterial conjugation and transfer in the liver and spleen of mice treated with IAA was significantly reduced. Meanwhile, the number of recipient bacteria in the liver and spleen did not change significantly in the IAA-treated group, indicating that IAA suppressed the frequency of plasmid conjugation and transfer without affecting the bacterial count.

[0050] In summary, indole compounds with electron-withdrawing groups can significantly inhibit the conjugation and transfer of engineered and clinical plasmids, exhibiting good inhibitory effects both in vivo and in vitro. This invention provides a new perspective for the development of novel drug resistance control strategies and offers a new method for blocking the spread of increasingly serious drug resistance genes.

Claims

1. The application of indole compounds with electron-withdrawing groups in the preparation of drugs that block the intestinal spread of drug-resistant bacteria, characterized in that, The indole compound with an electron-withdrawing group is selected from one or more of 5-iodoindole, 7-bromoindole, 7-chloroindole, and 7-fluoroindole; the drug-resistant bacteria are selected from drug-resistant bacteria carrying the RP4-7 plasmid. E. coli DH5α, carrying bla NDM-5 Plasmid-resistant bacteria K. pneumoniae C12 or E. coli L65, Carrying tet (X4) Plasmid-resistant bacteria E. coli RS3-1 or E. coli RF2-1, Carry mcr-1 Drug-resistant bacteria with clinical plasmids E. coli LD67-1.

2. The application according to claim 1, characterized in that, The concentration of the indole compound with electron-withdrawing groups used is 0.5 mg / kg.

3. The application according to claim 1, characterized in that, The method of blocking the intestinal spread of drug-resistant bacteria includes inhibiting the conjugate transfer frequency of plasmids while maintaining a constant bacterial count, thereby blocking their spread.

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