Use of forfenib in preparation of drug resistance plasmid conjugation transfer inhibitor

Abstract

The application belongs to the technical field of biological medicine, and discloses application of forseritin in preparation of a drug-resistant plasmid conjugation transfer inhibitor. bla NDM The conjugation transfer test proves that forseritin can significantly reduce the conjugation transfer efficiency of IncX3 type plasmid carrying bla NDM The carbapenem-resistant gene in the horizontal transmission risk in the Escherichia coli population. Therefore, forseritin or its active ingredients are used as a drug-resistant plasmid conjugation transfer inhibitor, applied to the survival environment of Escherichia coli, to block bla NDM The spread of the gene. The application provides a new candidate compound and technical idea for controlling the spread of the carbapenem-resistant gene, and has potential application value in the fields of medicine, food safety, veterinary public health and environmental drug resistance prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to the application of fripitinib in the preparation of drug-resistant plasmid conjugation transfer inhibitors. Background Technology

[0002] The global spread of antibiotic resistance has become one of the most serious challenges in modern public health. Resistance genes can spread not only through vertical inheritance but also rapidly across hosts via horizontal gene transfer between different strains and even different species. Among the many horizontal transfer mechanisms, conjugation transfer is one of the main ways in which resistance genes in Gram-negative bacteria spread. It enables the transfer of resistance plasmids between donor and recipient bacteria, thereby accelerating the spread of resistance phenotypes in clinical, aquaculture, and environmentally relevant bacterial populations.

[0003] blue NDM The gene encodes a New Delhi metallo-β-lactamase, which mediates bacterial resistance to carbapenem antibiotics. The IncX3 plasmid is... blue NDM As one of the most representative and prevalent vectors in the global dissemination of genes, it can spread across different host backgrounds via conjugation transfer, thus becoming an important target in research on the horizontal transmission and control of drug-resistant genes. Current antibacterial drugs primarily aim to inhibit or kill bacteria; however, in controlling the transmission of drug-resistant genes, relying solely on bactericidal or bacteriostatic strategies is insufficient to fundamentally block the spread of drug-resistant genes among bacterial populations. Therefore, developing conjugation transfer inhibitors that can reduce the efficiency of conjugation transfer of drug-resistant plasmids is of great significance for intervening in strategies against drug resistance transmission.

[0004] Faritinib is a small-molecule, multi-target receptor tyrosine kinase inhibitor, primarily used as an anti-tumor drug candidate in current technologies. Faritinib regulates tumor cell proliferation, migration, invasion, and angiogenesis by acting on targets such as hepatocyte growth factor receptor (HGFR, also known as c-MET) and vascular endothelial growth factor receptor 2 (VEGFR2) encoded by the MET proto-oncogene. It also affects the expression of cell cycle-related proteins, inducing G2 / M phase arrest in tumor cells, thereby exerting anti-tumor activity. However, to date, there are no reports, either domestically or internationally, on the direct interference or blocking of bacterial resistance plasmid conjugation and metastasis by ferritinib. Therefore, developing new applications for ferritinib in inhibiting resistance plasmid conjugation and metastasis has potential application value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discloses a new use for faritinib, specifically its ability to inhibit genes carrying carbapenem resistance to antimicrobial drugs. blue NDM Application of Escherichia coli IncX3 plasmid conjugation transfer.

[0006] Therefore, the first objective of this invention is to provide a novel drug-resistant plasmid conjugation transfer inhibitor – furitinib.

[0007] A second objective of this invention is to provide the application of faritinib in inhibiting the transfer of carbapenem resistance genes. More specifically, its application in the transfer of resistance genes to carbapenem antimicrobial drugs such as meropenem, imipenem, or ertapenem.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention first provides the application of foretinib in the preparation of drug-resistant plasmid conjugation transfer inhibitors. Foretinib, also known as GSK1363089, CAS number: 849217-64-7, is a small molecule multi-target receptor tyrosine kinase inhibitor with the molecular formula C2. 34 H 34 F2N4O6 is a white to off-white powder that is readily soluble in dimethyl sulfoxide (DMSO), slightly soluble in ethanol, and almost insoluble in water. It is mainly used as an anti-tumor drug candidate and can regulate tumor cell proliferation, migration, and tumor angiogenesis by acting on targets such as MET receptor tyrosine kinase and vascular endothelial growth factor receptor 2 (VEGFR2).

[0010] Preferably, the drug resistance plasmid carries a carbapenem antimicrobial drug resistance gene.

[0011] Preferably, the carbapenem antibacterial drugs include, but are not limited to, meropenem, imipenem, or ertapenem resistance genes.

[0012] Preferably, the drug resistance gene is blue NDM Gene.

[0013] Preferably, the drug resistance plasmid is an IncX3 type plasmid.

[0014] Preferably, the drug resistance plasmid is a drug resistance plasmid of Escherichia coli; the faritinib inhibits conjugation and transfer between Escherichia coli.

[0015] Preferably, the concentration of foripitinib used is 5 μg / mL or 10 μg / mL. However, the present invention is not limited to this, and other concentrations of foripitinib that can effectively inhibit the conjugation and transfer of drug-resistant plasmids (including but not limited to any concentration in the range of 1 to 20 μg / mL) are also within the scope of protection of this application. Those skilled in the art can determine the optimal concentration through conventional experiments based on the actual application scenario and strain background.

[0016] Preferably, foripinib, or a substance containing the active ingredient of foripinib, is used as an inhibitor of drug-resistant plasmid conjugation transfer, acting on the survival environment of *E. coli* to inhibit bladder infection. NDM Drug resistance genes spread horizontally among bacterial populations. Furipinib can be used alone or, preferably, in combination with other synergistic antimicrobial agents or inhibitors.

[0017] Preferably, the formulation of the drug-resistant plasmid conjugation transfer inhibitor is a powder, a spray, or a sustained-release formulation. The inhibitor can be in liquid or solid form; in addition to the active ingredient furitinib, it also includes adjuvants or carriers required to prepare the corresponding dosage form. These adjuvants or carriers are common pharmaceutical materials (such as dispersants, stabilizers, excipients, solvents, etc.), which will not be elaborated further in this application.

[0018] This invention also provides a method for inhibiting the spread of carbapenem resistance genes in bacteria, comprising applying a resistance plasmid conjugation transfer inhibitor directly to the growth environment or clinical environment of poultry and livestock to control... blue NDM The spread of genes among Enterobacteriaceae. As a method to inhibit the spread of carbapenem resistance genes in bacteria, the aforementioned resistance plasmid conjugation transfer inhibitors can be directly applied to the growth environment of poultry and livestock (including but not limited to feed, drinking water, bedding, and livestock housing environment) or clinical environment (including but not limited to medical device surfaces, ward environments, and hand disinfection of medical personnel) to control [the spread of these genes]. blue NDM The transmission of genes among Enterobacteriaceae. The applications of this invention are not limited to this; they can also be extended to the fields of food safety (such as food processing environments), veterinary public health (such as pet hospital environments), and environmental resistance control (such as wastewater treatment systems).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention demonstrates through conjugation transfer assays that faritinib can significantly reduce the risk of carrying carbapenem resistance genes. blue NDM The efficiency of conjugation and transfer of IncX3 type plasmids between E. coli was observed. Therefore, furipinib can be used as a conjugation plasmid for IncX3 type plasmids. blue NDMPositive plasmid conjugation transfer inhibitors are used to suppress carbapenem resistance genes. blue NDM This study aims to mitigate the risk of horizontal transmission among E. coli populations, providing new candidate compounds and technical approaches to block the spread of carbapenem resistance genes. These compounds have potential applications in medicine, food safety, veterinary public health, and environmental drug resistance control. Attached Figure Description

[0021] Figure 1 To test the effects of furipitinib at different final concentrations (0, 5, and 10 μg / mL) on four strains of Escherichia coli carrying the IncX3 type from different sources. blue NDM The influence of drug resistance plasmid conjugation and transfer frequency. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. It should be noted that these embodiments are only for the purpose of helping to understand the present invention and do not constitute a limitation on the scope of protection of the present invention. Furthermore, the technical features involved in the following embodiments can be combined with each other as long as they do not conflict with each other.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are conventional methods in the art; the materials and reagents used are commercially available unless otherwise specified; and the equipment used are conventional experimental equipment unless otherwise specified.

[0024] Foretinib was purchased from Shanghai Taoshu Biochemical Technology Co., Ltd.

[0025] Dimethyl sulfoxide (DMSO) was purchased from Shaanxi Baisha Biotechnology Co., Ltd. and used as a solvent for preparing fripitinib stock solution.

[0026] Streptomycin (STR) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0027] Meropenem (MEM) was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0028] LB agar (Luria-Bertani Agar, batch number HB0129) and LB broth (batch number HB0128) were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0029] The culture medium preparation methods involved in the following examples are as follows:

[0030] LB agar medium containing streptomycin: Cool the LB agar medium, which has been autoclaved at 121 °C for 15 min, to about 55 °C, add the pre-prepared and filtered sterilized streptomycin stock solution (concentration of 30 mg / mL) to make the final concentration of streptomycin 3000 μg / mL, mix thoroughly, and then pour the mixture onto a plate for later use.

[0031] LB agar medium containing meropenem: Cool the LB agar medium, which has been autoclaved at 121 °C for 15 min, to about 55 °C, add the pre-prepared and filtered sterilized meropenem stock solution (concentration of 5 mg / mL) to make the final concentration of meropenem 0.5 μg / mL, mix thoroughly, and then pour the mixture into plates for later use.

[0032] LB agar medium containing streptomycin and meropenem: After autoclaving at 121 °C for 15 min, the LB agar medium was cooled to approximately 55 °C. Pre-prepared and filtered sterilized streptomycin stock solution (concentration 30 mg / mL) and meropenem stock solution (concentration 5 mg / mL) were added to bring the final concentrations of streptomycin and meropenem to 3000 μg / mL and 0.5 μg / mL, respectively. The mixture was thoroughly mixed and then poured onto a plate for later use.

[0033] The following examples use four IncX3-carrying plants. blue NDM The drug-resistant *E. coli* strains were used as donor bacteria, and the streptomycin-resistant *E. coli* C600 strain was used as the recipient bacteria; both were provided by the Department of Pharmacology, College of Veterinary Medicine, South China Agricultural University. The four donor strains were isolated from fish meat samples, environmental samples, chicken feces samples, and chicken meat samples, respectively. The IncX3 strain from the fish meat samples was the most abundant. blue NDM The drug resistance plasmid has been sequenced in its entirety and submitted to the NCBI database, accession number: NZ_MK033578.1; the IncX3 type plasmid was carried by strains from environmental samples, chicken feces samples, and chicken meat samples. blue NDM The drug-resistant plasmid has been confirmed by PCR amplification and sequencing methods in our laboratory. The public may obtain the aforementioned biological materials from the Department of Pharmacology, College of Veterinary Medicine, South China Agricultural University (the applicant) with their consent. These biological materials are solely for the purpose of repeating experiments related to this invention and will not be used for any other purpose.

[0034] Example 1: Determination of the minimum inhibitory concentration (MIC) of furipinib against Escherichia coli donor and recipient bacteria, specifically including the following steps:

[0035] (1) Preparation of drugs

[0036] Weigh 5 mg of foritinib powder, add 1 mL of dimethyl sulfoxide (DMSO) and dissolve it completely to prepare a stock solution with a concentration of 5 mg / mL. After sterilization by filtration through a 0.22 μm sterile filter membrane, store at 4°C in the dark for later use.

[0037] (2) Preparation of bacterial culture

[0038] Using an inoculation loop, purified donor and recipient bacteria and bacterial suspensions were streaked onto LB agar plates containing the corresponding antibiotics. Four donor strains were streaked onto LB agar plates containing meropenem (0.5 μg / mL), and the recipient strain, *E. coli* C600, was streaked onto LB agar plates containing streptomycin (3000 μg / mL). The plates were incubated overnight at 37°C with the plates inverted. Uniform single colonies were picked from the culture medium and inoculated into 10 mL glass tubes containing 2 mL of autoclaved MH broth. The tubes were incubated at 37°C with shaking for 4 h. The OD values ​​of the bacterial suspensions were measured using a microplate reader. 600 Value, and the bacterial culture OD 600 Adjust to approximately 0.5; then dilute with MH broth at a ratio of 1:1000 to bring the bacterial count of the working culture to approximately 1×10⁻⁶. 6 CFU / mL, for later use.

[0039] (3) Broth dilution method

[0040] In a sterile 96-well plate, 190 μL of MH broth was added to each well in the first column, and 100 μL of MH broth was added to each well in the second to last columns. 10 μL of prepared faritinib solution was added to the corresponding well in the first column. Three parallel wells were set up for each strain, along with a positive control (without drug) and a negative control (with sterile broth). After thorough mixing, 100 μL was transferred from the first column to the second column and mixed again. This process was then serially diluted to the last column, and the excess 100 μL in the last column was discarded after mixing. Subsequently, 100 μL of the diluted working bacterial culture was added to each well, gently mixed, and the 96-well plate was incubated at 37°C for 16–18 h. The results were then recorded.

[0041] The minimum inhibitory concentrations (MICs) of furipinib against recipient bacterium C600 and four donor strains are shown in Table 1.

[0042] Table 1

[0043] As shown in Table 1, the minimum inhibitory concentration (MIC) of furipinib against recipient strain C600 and the four donor strains was greater than 125 μg / mL, indicating that under the conditions of 5 μg / mL and 10 μg / mL used in the subsequent conjugation transfer experiments, furipinib did not produce significant antibacterial effects against the donor and recipient strains.

[0044] Example 2: Furetinib for patients with IncX3 type blue NDM The impact of drug-resistant plasmids on the conjugation and transfer ability of E. coli

[0045] This embodiment is used to evaluate the effects of faritinib on patients with IncX3 type 3 antibodies from different sources. blue NDM The influence of drug-resistant plasmids on the conjugation and transfer ability of E. coli donor strains was investigated, and a DMSO solvent control was set up to eliminate potential interference from the drug solvent on the experimental results. The specific experimental procedure is as follows:

[0046] (1) Experimental preparation: Prepare a stock solution of 1 mg / mL furitinib, and prepare LB agar medium containing streptomycin (3000 μg / mL), LB agar medium containing meropenem (0.5 μg / mL), LB agar medium containing meropenem (0.5 μg / mL) and streptomycin (3000 μg / mL), LB broth, 0.9% sodium chloride solution, 1.5 mL centrifuge tubes, etc.

[0047] (2) Strain resuscitation: Four strains carrying IncX3 type were recovered from the strains stored in the laboratory at -80 ℃. blue NDM Adequate bacterial cultures were taken from different donor and recipient strains of *E. coli* C600 containing the drug-resistant plasmid for resuscitation. Four donor strains were streaked onto LB agar containing meropenem (0.5 μg / mL), while the recipient strain *E. coli* C600 was streaked onto LB agar containing streptomycin (3000 μg / mL), and incubated at 37°C for 18 h. Subsequently, single colonies of both donor and recipient strains were picked and inoculated into 2 mL of autoclaved LB broth, and cultured at 37°C and 180 rpm with shaking for 16 h to obtain activated donor and recipient bacterial cultures for subsequent conjugation transfer experiments. Three biological replicates were established for each strain.

[0048] (3) Preparation of bacterial culture: The activated overnight cultures of donor and recipient bacteria were transferred to antibiotic-free fresh LB liquid medium at a ratio of 1:100 and cultured at 37 ℃ and 180 rpm for 4 h to allow the bacterial culture to reach OD. 600 The concentration was adjusted to 0.4-0.6. Subsequently, LB broth was used to adjust the concentration of donor and recipient bacteria, and the ratio of donor to recipient bacteria was kept at 1:1 to ensure that the initial bacterial counts of donor and recipient bacteria in the conjugation system were similar.

[0049] (4) Conjugation transfer: Take 1 mL of the adjusted donor bacterial culture and 1 mL of recipient bacterial C600 culture and mix thoroughly. Then, add the corresponding volume of fripitinib stock solution to each conjugation system to make the final concentrations of fripitinib 0 μg / mL (blank control group, no drug added), 5 μg / mL and 10 μg / mL, respectively.

[0050] To eliminate the potential influence of the drug solvent dimethyl sulfoxide (DMSO) on the conjugation transfer process, this example uses a fish meat sample carrying IncX3 type... blue NDM The donor strain of the drug-resistant plasmid was used as a representative strain, and a DMSO solvent control group was set up. In this group, the same volume of DMSO as the corresponding faritinib treatment group was added to the donor-recipient mixed bacterial solution. Subsequently, the mixed bacterial solutions of each group were incubated at 37 °C for 12 h to construct the conjugation transfer system between Escherichia coli.

[0051] (5) Plate counting: After inoculation, 2 mL of the inoculated bacterial culture was transferred to a 2 mL centrifuge tube and centrifuged at 5000 rpm for 5 min. The supernatant was discarded. 1 mL of autoclaved 0.9% sodium chloride solution was added, and the bacterial cells were vortexed to mix. This was taken as the original concentration bacterial culture. Subsequently, serial dilutions were performed using 0.9% sodium chloride solution at a dilution gradient of 10. -1 10 -2 10 -3 10 -4 and 10 -5 After each dilution, the medium was thoroughly vortexed to ensure uniform distribution of the bacterial culture. 60 µL of the diluted bacterial culture was then dropped onto LB agar containing streptomycin (3000 µg / mL) to count the number of recipient bacteria; simultaneously, 60 µL of the diluted bacterial culture was dropped onto LB agar containing meropenem (0.5 µg / mL) and streptomycin (3000 µg / mL) to count the number of transzygotes. After complete absorption, the culture medium was incubated at 37 °C for 12–16 h, and the number of colonies on the respective media was counted.

[0052] The conjugation frequency was calculated as follows: Conjugation frequency = (number of colonies on the dual-antibody medium × corresponding dilution factor) / (number of colonies on the streptomycin monoclonal antibody medium × corresponding dilution factor). Three replicates were set up for each experiment, and the average value was taken to ensure the reliability of the experimental results. With a DMSO solvent control set up to eliminate solvent interference, the conjugation frequency of the blank control group and the groups treated with different concentrations of fripitinib was compared to evaluate the efficacy of fripitinib against different sources of IncX3-type bacteria. blue NDMThe effect of DMSO solvent on the conjugation and transfer ability of drug-resistant plasmids in E. coli. The results showed that the plasmid conjugation and transfer frequency in the DMSO solvent control group was similar to that in the blank control group, with no significant inhibitory effect observed. This indicates that under the experimental conditions used in this example, the DMSO solvent itself does not significantly inhibit the conjugation and transfer of the IncX3 type plasmid. blue NDM The conjugation and transfer of drug-resistant plasmids were not significantly affected.

[0053] Results Analysis

[0054] Statistical results of conjugation transfer inhibition experiments using donor bacteria from different sources are shown below. Figure 1 . Figure 1 a, Figure 1 b, Figure 1 c and Figure 1 The 'd's correspond to the IncX3 type carried by E. coli from fish, environmental sources, chicken feces, and chicken meat sources, respectively. blue NDM Results of conjugation and transfer of positive drug-resistant plasmids.

[0055] When the final concentration of furipinib was 5 μg / mL, the IncX3 type carried by the four Escherichia coli strains from different sources was reduced. blue NDM The conjugation and transfer frequencies of positive drug-resistant plasmids decreased by approximately 49.9%, 44.4%, 61.8%, and 44.4% respectively compared to the blank control group, and the differences were statistically significant (P < 0.05).

[0056] When the final concentration of foripitinib was 10 μg / mL, the IncX3 type carried by the four Escherichia coli strains from different sources was reduced. blue NDM The conjugation and transfer frequencies of positive drug-resistant plasmids decreased by approximately 46.3%, 75.2%, 67.0%, and 56.9% compared to the blank control group, respectively. Within the tested concentration range, the plasmid conjugation and transfer frequencies of all strains treated with 5 μg / mL and 10 μg / mL faritinib were significantly lower than those of the blank control group (P < 0.01).

[0057] The above results indicate that furipinib can reduce the concentration of IncX3 in Escherichia coli from different sources at both 5 μg / mL and 10 μg / mL. blue NDM The frequency of conjugation and transfer of positive drug-resistant plasmids indicates that foripitinib can inhibit the horizontal spread of such drug-resistant plasmids among Escherichia coli.

[0058] In summary, furipinib can significantly reduce the presence of bacteria carried by different E. coli donor strains. blue NDMThe frequency of IncX3-type resistance plasmid conjugation transfer in the gene was analyzed. These results suggest that furipinib could serve as a carrier of carbapenem resistance genes. blue NDM IncX3 type plasmid conjugation transfer inhibitors are used to inhibit blue NDM The horizontal spread of genes among E. coli provides new candidate compounds and technical approaches for blocking the spread of carbapenem resistance genes.

[0059] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of faritinib in the preparation of drug-resistant plasmid conjugation transfer inhibitors, characterized in that, The resistance plasmid carries a carbapenem resistance gene; the resistance plasmid is carried by *E. coli*. bla NDM Positive IncX3 type drug-resistant plasmid, wherein the faritinib is used to inhibit the conjugation and transfer of the drug-resistant plasmid between Escherichia coli.

2. The application according to claim 1, characterized in that, The carbapenem antibacterial drug is one or more of meropenem, imipenem, or ertapenem.

3. The application according to claim 1, characterized in that, The concentration of furatinib used is 1~20 μg / mL.

4. The application according to claim 1, characterized in that, Furetinib, as a resistance plasmid conjugation transfer inhibitor, acts on the survival environment of *E. coli* to inhibit the aforementioned... bla NDM The positive IncX3 type drug-resistant plasmid spreads horizontally among Escherichia coli populations.

5. The application according to claim 1, characterized in that, The formulation of the drug-resistant plasmid conjugation transfer inhibitor is in the form of powder, spray, or sustained-release agent.

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