Application of 5-fluorodeoxycytidine and medicine

Abstract

The invention discloses application of 5-fluorodeoxycytidine and a medicine, relates to the technical field of biological medicines, and particularly provides application of 5-fluorodeoxycytidine in preparation of a medicine for resisting staphylococcus aureus. The 5-fluorodeoxycytidine can act on staphylococcus aureus to replicate an initiator protein DnaA, inhibit the initiator protein DnaA and cause DNA replication disorder of the staphylococcus aureus, so that the activity of the staphylococcus aureus is effectively inhibited, and a bactericidal effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application and drug of 5-fluorodeoxycytidine. Background Art

[0002] Bacterial infection is a common clinical disease, which 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, which can cause various infectious diseases, such as endocarditis, osteomyelitis, bacteremia, septicemia and fatal pneumonia. Penicillin and methicillin were initially used as the main antibiotics for the treatment of Staphylococcus aureus infections. However, bacteria rapidly acquire drug resistance by producing penicillinase to hydrolyze the β-lactam ring of penicillin and encoding a low-affinity penicillin-binding protein PBP2a. More seriously, Staphylococcus aureus is resistant to all β-lactam antibiotics and has spread rapidly and widely globally, which has become an important public health problem. Staphylococcus aureus has been listed by the World Health Organization as a pathogen that requires priority research and development of new antibiotics. Therefore, it is crucial to develop new antibiotics against Staphylococcus aureus.

[0003] In previous pharmacological studies, 5-fluorodeoxycytidine, as a tumor-selective prodrug of thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (5-fluoro-2'-dUMP), has the function of inhibiting DNA methyltransferase (DNMT) and belongs to the class of fluoropyrimidine nucleoside analogs in structure. However, so far, there has been no report on the antibacterial effect of 5-fluorodeoxycytidine, and there is no antibacterial drug with 5-fluorodeoxycytidine as the main active ingredient. Summary of the Invention

[0004] Based on the above deficiencies of the prior art, the purpose of the present invention is to provide the application and drug of 5-fluorodeoxycytidine, aiming to develop new anti-Staphylococcus aureus drugs and at the same time broaden the application of 5-fluorodeoxycytidine.

[0005] The technical solution of the present invention is as follows:

[0006] In the first aspect of the present invention, there is provided the application of 5-fluorodeoxycytidine in the preparation of drugs against Staphylococcus aureus.

[0007] In the second aspect of the present invention, there is provided the application of 5-fluorodeoxycytidine in the preparation of drugs for preventing and / or treating Staphylococcus aureus infection.

[0008] Optionally, the Staphylococcus aureus includes at least one of methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0009] The third aspect of the present invention provides an anti-Staphylococcus aureus drug, which comprises a first active ingredient and a pharmaceutically acceptable first carrier, wherein the first active ingredient comprises 5-fluorodeoxycytidine.

[0010] Optionally, the first carrier includes at least one of a glidant, a diluent, a wetting agent, a suspending agent, a solvent and an emulsifier.

[0011] Optionally, the drug further comprises a first additive, wherein the first additive comprises at least one of a preservative, a colorant, a flavoring agent, a stabilizer and an isotonic agent.

[0012] In a fourth aspect, the present invention provides a drug for preventing and / or treating Staphylococcus aureus infection, which comprises a second active ingredient and a pharmaceutically acceptable second carrier, wherein the second active ingredient comprises 5-fluorodeoxycytidine.

[0013] Optionally, the pharmaceutically acceptable second carrier includes at least one of a glidant, a diluent, a wetting agent, a suspending agent, a solvent and an emulsifier.

[0014] Optionally, the medicine further comprises a second additive, wherein the second additive comprises at least one of a preservative, a colorant, a flavoring agent, a stabilizer and an isotonic agent.

[0015] Optionally, the dosage form of the drug includes an oral dosage form, an injection dosage form or a skin administration dosage form.

[0016] Beneficial effects: 5-fluorodeoxycytidine in the present invention can act on the replication initiation protein DnaA of Staphylococcus aureus, inhibit the initiation protein DnaA, cause DNA replication disorder of Staphylococcus aureus, thereby effectively inhibiting the activity of Staphylococcus aureus and playing a bactericidal role. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figures are the analysis graphs of the genomic gene copy numbers of the strains in different groups in Example 3, wherein (a) is the logarithmic phase WT control group and the logarithmic phase WT+5FDC group, (b) is the logarithmic phase tolerant strain control group and the logarithmic phase tolerant strain+5FDC group, (c) is the logarithmic phase WT control group and the logarithmic phase tolerant strain control group, (d) is the stable phase WT control group and the stable phase WT+5FDC group, (e) is the stable phase tolerant strain control group and the stable phase tolerant strain+5FDC group, and (f) is the stable phase WT control group and the stable phase tolerant strain control group.

[0018] Figure 2 TEM images of bacteria in different treatment groups in Example 5.

[0019] Figure 3It is a graph showing the results of cell parameters of bacteria in different groups in Example 5. Among them, (a) is the graph of the percentage of cell septum, (b) is the graph of cell area, and (c) is the graph of cell wall thickness.

[0020] Figure 4 It is a surface plasmon resonance (SPR) graph showing the interaction of 5-fluorodeoxycytidine with the DnaA protein of the WT strain and the resistant strain in Example 6. Among them, (a) is the WT strain and (b) is the resistant strain.

[0021] Figure 5 It is a graph of the bacterial load in the wound of a mouse skin infection model after treatment with different drugs in Example 7. Detailed implementation manners

[0022] The present invention provides the application and drug of 5-fluorodeoxycytidine. To make the purpose, technical solution and effects of the present invention clearer and more definite, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific implementation manners and are not intended to limit the present invention.

[0024] If there are descriptions involving "first", "second", etc. in the embodiments of the present invention, such descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the quantity of the indicated technical features.

[0025] The embodiments of the present invention provide the application of 5-fluorodeoxycytidine in the preparation of drugs against Staphylococcus aureus.

[0026] The chemical name of 5-fluorodeoxycytidine is 5-Fluoro-2'-deoxycytidine, and the CAS number is 10356-76-0. The specific structural formula is as follows:

[0027]

[0028] DnaA is an essential component for bacterial survival and a major initiator protein for bacterial replication. DnaA is highly conserved, and its monomer can recognize the replication origin by binding to double-stranded DNA sequences (DnaA-box). 5-Fluorodeoxycytidine can act on the replication initiator protein DnaA of Staphylococcus aureus, affect the function of DnaA, and then cause bacterial DNA replication disorders, thereby affecting the survival of bacteria. 5-Fluorodeoxycytidine has good antibacterial effects, can effectively inhibit the activity of Staphylococcus aureus, and play a bactericidal role. Therefore, 5-fluorodeoxycytidine can be used to prepare drugs against Staphylococcus aureus.

[0029] The embodiment of the present invention also provides the use of 5-fluorodeoxycytidine in the preparation of drugs for preventing and / or treating Staphylococcus aureus infection. 5-Fluorodeoxycytidine has good antibacterial effects, can effectively inhibit the activity of Staphylococcus aureus, and play a bactericidal role. Therefore, 5-fluorodeoxycytidine can be used to prepare drugs for preventing and / or treating Staphylococcus aureus infection.

[0030] In some embodiments, the Staphylococcus aureus includes at least one of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA).

[0031] 5-Fluorodeoxycytidine has good antibacterial effects, can effectively inhibit the activity of Staphylococcus aureus, especially has significant positive effects on MRSA infection, and has great clinical significance.

[0032] The embodiment of the present invention also provides a drug against Staphylococcus aureus, which includes a first active ingredient and a pharmaceutically acceptable first carrier, and the first active ingredient includes 5-fluorodeoxycytidine.

[0033] In some embodiments, the first carrier includes at least one of a glidant, a diluent, a wetting agent, a suspending agent, a solvent, and an emulsifier.

[0034] In some embodiments, the drug further includes a first additive, and the first additive includes at least one of a preservative, a coloring agent, a flavoring agent, a stabilizer, and an isotonic agent.

[0035] The embodiment of the present invention also provides a drug for preventing and / or treating Staphylococcus aureus infection, which includes a second active ingredient and a pharmaceutically acceptable second carrier, and the second active ingredient includes 5-fluorodeoxycytidine.

[0036] In some embodiments, the pharmaceutically acceptable second carrier includes at least one of a glidant, a diluent, a wetting agent, a suspending agent, a solvent, and an emulsifier.

[0037] In some embodiments, the drug further comprises a second additive, and the second additive comprises at least one of a preservative, a colorant, a flavoring agent, a stabilizer, and an isotonic agent.

[0038] In some embodiments, the dosage form of the drug against Staphylococcus aureus includes an oral dosage form, an injection dosage form, or a topical dosage form, and the dosage form of the drug for preventing and / or treating Staphylococcus aureus infection includes an oral dosage form, an injection dosage form, or a topical dosage form.

[0039] In some embodiments, the oral dosage form includes one of a solution, a pill, a tablet, a capsule, a powder, a lozenge, and a paste. The topical dosage form includes one of an ointment, a cream, and a patch.

[0040] The present invention will be further described below through specific examples.

[0041] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the quantitative tests in the following examples are all set with three repeated experiments, and the results are averaged.

[0042] Unless otherwise specified, the materials, reagents, equipment, etc. used in the following examples can all be obtained through commercial channels.

[0043] Among them, Staphylococcus aureus ATCC 29213 is commercially available, and the source is the American Type Culture Collection; Methicillin-resistant Staphylococcus aureus MRSA 252, MRSA 49008, MRSA 48973, and MRSA 48966 are described in the literature (Pyrimirhodomyrtone inhibits Staphylococcus aureus by affecting the activity of NagA, Wei Huang, Biochemical Pharmacology 210(2023)115455).

[0044] The 5-fluorodeoxycytidine (5FDC) used in the following examples was purchased from Shanghai TaoSu Biochemical Technology Co., Ltd. The vancomycin used in the following examples was purchased from MedChemexpress Biotechnology Co., Ltd., USA. The bacterial genomic DNA extraction kit and bacterial total RNA isolation kit used in the following examples were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0045] The components and preparation of the LB broth medium used in the following examples are as follows:

[0046] Weigh 8 g of tryptone powder, 8 g of sodium chloride, and 4 g of yeast extract powder, dissolve them in 800 mL of distilled water, and autoclave at 121 °C for 15 minutes. Store at 4 °C for later use.

[0047] The components and preparation of the LB agar plates used in the following examples are as follows:

[0048] Weigh 8 g of tryptone powder, 8 g of sodium chloride, 4 g of yeast extract powder, and 12 g of agar powder, dissolve them in 800 mL of distilled water, autoclave at 121 °C for 15 minutes, pour into sterile petri dishes for bacteria, and cool and solidify. Store at 4 °C for later use.

[0049] The components and preparation of the MH broth medium used in the following examples are as follows:

[0050] Weigh 21.0 g of MH broth medium, dissolve it in 1000 mL of distilled water, and autoclave at 121 °C for 15 minutes. Store at 4 °C for later use.

[0051] The meanings of some symbols in the following implementation are as follows:

[0052] OD 600 : refers to the absorbance value at a wavelength of 600 nm;

[0053] DMSO: dimethyl sulfoxide;

[0054] PBS: phosphate buffer solution.

[0055] Example 1 Minimum inhibitory concentration test of 5-fluorodeoxycytidine (abbreviated as 5FDC)

[0056] Inoculate Staphylococcus aureus ATCC 29213 and methicillin-resistant Staphylococcus aureus (MRSA252, MRSA49008, MRSA48973, and MRSA48966 respectively) into LB broth medium, and resuscitate overnight at 37 °C and 220 rpm. Then inoculate the overnight culture into 5 mL of fresh LB broth medium at a volume ratio of 1:100 and shake culture until the bacteria grow to the logarithmic growth phase (OD 600 = 0.6 - 0.8) to obtain a bacterial culture.

[0057] The specific steps for determining the minimum inhibitory concentration (MIC) are as follows:

[0058] (1) Adjust the bacterial cultures with OD 600 = 0.6 - 0.8 to OD 600 = 0.001 with MH broth medium;

[0059] (2) Add 2 μL of the 5FDC stock solution with a concentration of 1 mg / mL (solvent DMSO) and 198 μL of the bacterial culture obtained in step (1) to a 96-well plate. Then, serially dilute the bacterial culture with an OD 600 = 0.001 two-fold. The volume in each well is 100 μL, and the final concentrations of 5FDC are 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.313 μg / mL, 0.156 μg / mL, 0.078 μg / mL, and 0 μg / mL, respectively. Repeat each concentration 3 times as the experimental group.

[0060] (3) Set up a blank group (add 100 μL of MH broth medium) and a control group (add 100 μL of the bacterial culture with an OD 600 = 0.001) in the 96-well plate;

[0061] (4) After incubating the 96-well plate in a 37 °C incubator for 18 hours, add 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) with a concentration of 5 mg / mL to each well. Incubate in the 37 °C incubator in the dark for 30 minutes. Finally, measure the OD 600 .

[0062] Calculate the bacterial inhibition rate according to the formula: Bacterial inhibition rate (%) = 1 - (OD 实验组 - OD 空白 ) / (OD 对照组 - OD 空白 ) × 100%. The MIC of 5FDC against Staphylococcus aureus is the lowest compound concentration that inhibits 90% of bacterial growth. Use the same method as above to test the MIC of vancomycin against Staphylococcus aureus.

[0063] The results are shown in Table 1. The MICs of 5FDC against Staphylococcus aureus ATCC 29213, MRSA 252, MRSA 49008, MRSA48973, and MRSA 48966 are 0.02 μg / mL, 0.02 μg / mL, 0.04 μg / mL, 0.02 μg / mL, and 0.02 μg / mL, respectively, which are significantly lower than those of the existing antibacterial drug vancomycin. Thus, it can be shown that 5FDC can effectively inhibit the activity of Staphylococcus aureus.

[0064] Table 1. MIC of 5FDC against various bacteria

[0065]

[0066] Example 2

[0067] The mechanism of 5-FDC inhibiting Staphylococcus aureus activity was studied through a resistant strain screening experiment, including the following steps:

[0068] (1) Resistant strain screening: Inoculate 50 μL of Staphylococcus aureus ATCC 29213 (OD 600 = 0.5) into 5 mL of LB broth medium and resuscitate overnight at 37 °C and 220 rpm; inoculate the overnight culture into 2 mL of fresh LB broth medium at a volume ratio of 1:100, add 5-fluorodeoxycytidine at a concentration of 0.1625 μg / mL, and culture at 37 °C and 220 rpm until OD 600 > 1; inoculate the above culture into 2 mL of fresh LB broth medium at a volume ratio of 1:100, add 5-fluorodeoxycytidine at a concentration of 0.3125 μg / mL (the solvent is DMSO), and culture at 37 °C and 220 rpm until OD 600 > 1; repeat the step of doubling the concentration of 5-FDC in a ladder until the concentration of 5-FDC is 5 μg / mL. Dip the inoculation loop into the bacterial solution and streak it on an LB agar plate (containing 5 μg / mL of 5-FDC);

[0069] (2) MIC determination of resistant strains: Pick a single colony from step (1) and inoculate it into LB broth medium, and determine the MIC according to the method in Example 1.

[0070] (3) Extract genomic DNA: Select strains with MIC determination values > 5 μg / mL, culture them overnight in LB broth medium, and extract genomic DNA using a bacterial genomic DNA extraction kit.

[0071] (4) Sequencing: After the DNA sample is qualified, randomly fragment it using a Covaris ultrasonic disruptor, and then complete the entire library preparation work through steps such as end repair, adding A tails, adding sequencing adapters, purification, and PCR amplification. After the library construction is completed, first perform a preliminary quantification using Qubit 2.0, dilute the library to 1.5 ng / μL, and then use Agilent 2100 to detect the insert size of the library. After the insert size meets the expectations, accurately quantify the effective concentration of the library using the Q-PCR method (the effective concentration of the library > 2 nM). After the library is qualified, pool different libraries into the flowcell according to the requirements of the effective concentration and the target output data volume. After clustering by cBOT (cluster generation workstation), use the Illumina high-throughput sequencing platform (HiSeq / MiSeq) for sequencing to generate raw sequencing data (.raw).

[0072] (5) Sequence analysis: Use the Spades software to assemble the raw data, use Prokka to annotate the assembled data, and use the Snippy software to perform site analysis on the annotated sequences.

[0073] After bioinformatics analysis, the results are shown in Table 2. A mutation from T to G occurred at position 399 of the DnaA gene or a mutation from G to A occurred at position 826. It is speculated that 5-fluorodeoxycytidine exerts its antibacterial activity by acting on the DnaA protein.

[0074] Table 2. Mutation site analysis of 5FDC-resistant strains

[0075]

[0076] Example 3

[0077] Study the mechanism of 5FDC inhibiting the activity of Staphylococcus aureus through genomic analysis experiments, including the following steps:

[0078] (1) Genomic DNA extraction

[0079] Divide the wild-type Staphylococcus aureus ATCC 29213 and the resistant strain (the 276th amino acid mutated from Val to Ile, obtained from Example 2) grown to OD 600 = 0.4 into 6 parts, each part being 10 mL. After the following treatments respectively, centrifuge to remove the supernatant, and extract the total DNA using a bacterial genomic DNA extraction kit.

[0080] Logarithmic-phase wild-type strain (WT) control group: Add 3 μL of DMSO and culture with shaking at 37°C until OD 600 = 0.6 - 0.8.

[0081] Logarithmic-phase WT + 5FDC group: Add 2 μL of 5FDC solution with a concentration of 1 mg / mL (solvent DMSO) and culture with shaking at 37°C until OD 600 = 0.6 - 0.8.

[0082] Logarithmic-phase resistant strain control group: Add 3 μL of DMSO and culture with shaking at 37°C until OD 600 = 0.6 - 0.8.

[0083] Logarithmic-phase resistant strain + 5FDC group: Add 2 μL of 5FDC solution with a concentration of 1 mg / mL (solvent DMSO) and culture with shaking at 37°C until OD 600 = 0.6 - 0.8.

[0084] Stationary-phase WT control group: Add 3 μL of DMSO and culture with shaking at 37°C for 24 h.

[0085] Stationary-phase WT + 5FDC group: Add 2 μL of 5FDC solution with a concentration of 1 mg / mL (solvent DMSO), and culture with shaking at 37 °C for 24 h.

[0086] Stationary-phase resistant strain control group: Add 3 μL of DMSO, and culture with shaking at 37 °C for 24 h.

[0087] Stationary-phase resistant strain + 5FDC group: Add 2 μL of 5FDC solution with a concentration of 1 mg / mL (solvent DMSO), and culture with shaking at 37 °C for 24 h.

[0088] (2) DNA fragmentation and library preparation

[0089] A total of 200 ng of DNA from each sample was used for DNA sample preparation. The QuarPrep EZ DNA Library Kit was used to generate the sequencing library. The genomic DNA was randomly fragmented to a size of 350 bp, and then the DNA fragments were end-processed, A-tailed, and ligated to adapters for Illumina sequencing. The PCR products were purified using magnetic beads and then amplified for 5 - 6 cycles. The size distribution of the library was analyzed by an Agilent 2200 Bioanalyzer and quantified using Qubit. Finally, the library was sequenced using an Illumina NovaSeq 6000 with 150 bp paired ends.

[0090] (3) WGS data analysis

[0091] The raw reads were filtered to obtain clean reads by removing sequencing adapters, short reads (length < 30 bp), and low-quality reads using Fastp (v0.12.4). Then, quality control was performed using Fastp. The clean reads were aligned to the reference genome using BWA-MEM2 (v2.2.0), samtools (v1.5), and FastQC (q 30 calculation, v0.11.9). The Genome Analysis Toolkit (GATK, v4.2.2.0) was used for variant discovery. Control-FREEC (v11.6) was used to call CNVs. Lumpy (v0.2.13) and Svtyper (v0.7.1) were used to analyze SVs. The detected sequence variants were functionally annotated by ANNOVAR (2018 / 4 / 16). All software was used with default parameters.

[0092] (4) SV detection and annotation

[0093] Structural variants (SVs) are genomic variations with relatively large (>50 bp) mutations, including deletions, duplications, insertions, inversions, and translocations. Based on the reference genome mapping results and the detected insert fragment sizes, Lumpy and svtyper software were used to detect insert (INS), deletion (DEL), inversion (INV), intrachromosomal translocation (ITX), and interchromosomal translocation (CTX) mutations. The detected SVs were filtered by removing those with fewer than 2 supporting paired-end (PE) reads, and INS, DEL, and INV were further annotated by ANNOVAR.

[0094] (5) CNV detection and annotation

[0095] Copy number variation (CNV) is a structural variation that shows deletions or duplications in the genome. Based on the read depth of the reference genome, Control-FREEC was used to detect potential deletion and duplication CNVs with default parameters. The detected CNVs were further annotated by ANNOVAR.

[0096] After genomic analysis, the results are as Figure 1 shown. After treatment with 5FDC, the wild-type strain grew to the logarithmic phase, and the relative copy number of genes near the replication origin oriC increased significantly, while this phenomenon was not observed in the tolerant strain bacteria. There were no obvious changes in the genomic copy numbers of the wild-type and tolerant strain bacteria during the stationary phase.

[0097] Example 4

[0098] The mechanism of 5FDC inhibiting the activity of Staphylococcus aureus was studied through transcriptome analysis experiments, including the following steps:

[0099] (1) Bacterial total RNA isolation

[0100] The wild-type Staphylococcus aureus ATCC 29213 and the tolerant strain (the 276th amino acid mutated from Val to Ile, obtained from Example 2) grown to OD 600 = 0.4 were each divided into 3 portions, 10 mL each. After the following treatments, the supernatant was removed by centrifugation, and total RNA was extracted using a bacterial RNA extraction kit.

[0101] Wild-type strain (WT) control group: 3 μL of DMSO was added, and the cells were cultured with shaking at 37 °C until OD 600 = 0.6 - 0.8.

[0102] WT + 5FDC group: 2 μL of a 5FDC solution with a concentration of 1 mg / mL (solvent DMSO) was added, and the cells were cultured with shaking at 37 °C until OD 600 = 0.6 - 0.8.

[0103] Tolerant strain control group: Add 3 μL of DMSO and culture with shaking at 37 °C until OD 600 = 0.6 - 0.8.

[0104] Tolerant strain + 5FDC group: Add 2 μL of 5FDC solution with a concentration of 1 mg / mL (solvent DMSO) and culture with shaking at 37 °C until OD 600 = 0.6 - 0.8.

[0105] (2) RNA quality detection and library construction

[0106] Detect the RNA quality by 0.8% agarose gel electrophoresis and spectrophotometry. High-quality RNA with an absorbance ratio of 260 / 280 nm of 1.8 - 2.2 is used for library construction and sequencing. Construct the Illumina library according to the instructions (Illumina, USA). Oligo-dT primers are used to cross-cut mRNA to obtain cDNA (APExBIO, catalog number K1159). Amplify the cDNA to synthesize the second strand of cDNA. Purify the cDNA product by the AMPure XP system (Beckman Coulter, Beverly, USA). After library construction, enrich the library fragments by PCR amplification and select according to the fragment size of 350 - 550 bp. Use the Agilent 2100 Bioanalyzer (Agilent, USA) to evaluate the quality of the library. Sequence the library using the Illumina NovaSeq 6000 sequencing platform (paired-end 150) to generate raw reads.

[0107] (3) RNA-seq data analysis

[0108] By calling the Cutadapt tool, TrimGalore filters the original paired-end fastq reads to discard adapters and low-quality bases. Then use HISAT2 to align the obtained clean reads with the mm10 / hg19 mouse / human genome, and then use StringTie for reference genome-guided transcriptome assembly and gene expression quantification. Identify differentially expressed genes (DEGs) by DESeq2 (for samples with replicates) or edgeR (for samples without replicates), with a threshold of log2 fold change > 1 and p-adjusted < 0.05. clusterProfiler is used for functional enrichment analysis of the annotated important DEGs and potential genes in the identification module based on gene ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway categories. Terms with p value < 0.05 are considered significant. Perform gene set enrichment analysis (GSEA) through the functions in the clusterProfiler package, and sort the gene list by log2 fold change.

[0109] After RNA-seq data analysis, the results are shown in Table 3. After 5FDC treatment, compared with the tolerant strain, the cell components in the periplasmic space surrounded by the cell wall and outer membrane of the wild strain were significantly enriched.

[0110] Table 3. Results of GO gene set enrichment analysis (Cellular component - GSEA)

[0111]

[0112] Example 5

[0113] The mechanism of 5FDC inhibiting the activity of Staphylococcus aureus was studied by transmission electron microscopy observation experiment, including the following steps:

[0114] (1) Bacterial sample preparation

[0115] The wild-type Staphylococcus aureus ATCC 29213 and the tolerant strain (the 276th amino acid mutated from Val to Ile, obtained from Example 2) grown to OD 600 = 0.4 were each divided into 3 portions, 10 mL each. After the following treatments respectively, the supernatant was removed by centrifugation, and the bacterial cells were stored at 4 °C with 2.5% glutaraldehyde.

[0116] Wild-type (WT) control group: Add 3 μL of DMSO and incubate at 37 °C with shaking for 1 h.

[0117] WT + 5FDC group: Add 2 μL of 5FDC solution with a concentration of 1 mg / mL (solvent DMSO) and incubate at 37 °C with shaking for 1 h.

[0118] Tolerant strain control group: Add 3 μL of DMSO and incubate at 37 °C with shaking for 1 h.

[0119] Tolerant strain + 5FDC group: Add 2 μL of 5FDC solution with a concentration of 1 mg / mL (solvent DMSO) and incubate at 37 °C with shaking for 1 h.

[0120] (2) Bacterial sample treatment

[0121] The bacterial samples were fixed with 2.5% glutaraldehyde at 4 °C for 2 hours and further washed with PBS (washed four times in total, 30 minutes each time). The cells were fixed with 1% osmium tetroxide at 4 °C for 2 hours and then further washed with ddH2O (washed three times in total, 10 minutes each time). The cells were dehydrated at 4 °C through the following steps: 50% acetone (15 minutes); 70% acetone (15 minutes); 90% acetone (15 minutes); 100% acetone (2 × 15 minutes). Then the cells were infiltrated through the following steps: infiltrated in a mixture of 100% acetone and Epon812 (volume ratio of the two is 1:1, Epon812 is a glycidyl aliphatic epoxy resin) for 2 h; infiltrated in a mixture of 100% acetone and Epon812 (volume ratio of the two is 1:2) for 2 h; overnight at 37 °C in 100% Epon812. The Epon812 resin-embedded polymerized cells were maintained at 37 °C for 24 hours, 45 °C for 24 hours, and 60 °C for 48 hours, and then 60 nm sections were cut using a Leica UC7 (an ultramicrotome). The samples were further mixed with uranyl acetate and lead citrate and imaged under a transmission electron microscope HITACHI HT7700 (Hitachi, Japan).

[0122] The results of the transmission electron microscopy test are as Figure 2 and Figure 3 shown, Figure 3 in which, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001. After the wild type was treated with 5FDC, the cell area increased significantly, the cell wall thickness decreased significantly, and the cell septum ratio decreased significantly. No such changes were observed after the mutant was treated with the drug.

[0123] Example 6

[0124] The mechanism of 5FDC inhibiting the activity of Staphylococcus aureus was studied through SPR experiments, including the following steps:

[0125] Using a Biacore TM 8K system and an S series CM5 chip to detect the kinetic and affinity data of the binding of small molecules to proteins. A Cytiva amino coupling kit was used for the coupling of DnaA protein. The Cytiva coupling buffer was 10 mM sodium acetate with a pH of 4.5.

[0126] (1) Prepare the running buffer and the solvent calibration curve: The running buffer for the small molecule sample was 1×PBS containing 5% DMSO. Take 105 mL of 10×PBS and dilute it to 1 L with deionized water to prepare 1.05×PBS. And according to Table 4, 1.05×PBS and DMSO were used to prepare 5% DMSO running buffer and 4.5% DMSO and 5.8% DMSO calibration mother liquors.

[0127] Table 4. Preparation of running buffer and calibration target solution

[0128]

[0129] According to Table 5, mix 4.5% DMSO and 5.8% DMSO calibration stock solution to prepare a calibration curve with a 5% DMSO concentration.

[0130] Table 5. Preparation of calibration curve with 5% DMSO concentration

[0131] Buffer / Sequence 1 2 3 4 5 6 7 8 4.5% DMSO 0 200 400 600 800 1000 1200 1400 5.8% DMSO 1400 1200 1000 800 600 400 200 0

[0132] (2) Small molecule sample preparation: Dilute the 10 mM small molecule stock solution (i.e., DMSO solution of 5FDC) 20-fold with 1.05×PBS buffer without DMSO to obtain small molecules in 1×PBS containing 5% DMSO at 500 μM. Then, dilute 5FDC to 150 μM with the prepared running buffer containing 5% DMSO as the highest injection concentration, and serially dilute it by half seven concentration gradients downward, such as 150 μM, 75 μM, 37.5 μM, 18.75 μM, 9.375 μM, 4.6875 μM, 2.34 μM. Set a repeated concentration at intervals and add a 0 concentration.

[0133] (3) Automatically run the coupling of DnaA protein and the injection program of 5FDC according to the instrument operation procedures.

[0134] (4) Use Biacore TM 8K Control Software for experimental result fitting and analysis calculation.

[0135] The SPR results are as Figure 4 shown. It can be seen that the binding affinity of the mutant DnaA protein of the resistant strain to 5FDC becomes weaker compared with that of the wild-type (WT) DnaA protein.

[0136] Example 7

[0137] Use 5FDC to test in a mouse skin infection model, including the following steps:

[0138] (1) Establish a skin infection model: Remove the back hair of 20 female BALB / c mice (about 20 g) aged 6 - 8 weeks with an electric shaver. Then, excise the back skin of each mouse to create a 0.6 cm × 0.6 cm wound. Inject 50 μL of MRSA 252 cells (10 7 CFUs) intradermally into the wound of the mouse. After 12 hours, inject 50 μL of MRSA 252 cells (10 7 CFUs) intradermally into the wound of the mouse again.

[0139] (2) Group treatment: Female mice were randomly divided into three groups (n = 5 mice / group). After reinfection in step (1) for 24 hours, drug treatment was administered. The drug was applied to the wound area once a day for 3 days.

[0140] Among them, in the three groups, there were a solvent control group (applied with DMSO), treatment group I (applied with 0.1 mg of mupirocin per kg of mice, with the solvent being physiological saline), and treatment group II (applied with 0.1 mg of 5FDC per kg of mice, with the solvent being DMSO). On the 5th day of infection, each mouse's wound was scraped ten times with a sterile cotton swab respectively. Then the cotton swab was placed in 1 mL of sterile physiological saline and stirred to release the bacteria. After continuous dilution 20-fold with sterile physiological saline, 5 μL of each sample was taken and spread on an LB plate. After incubation at 37 °C for 24 hours, the number of bacteria on each plate was counted. The results are as Figure 5 shown (where, ** indicates p < 0.01). Compared with the control group, in treatment group II, after treatment with 5-fluorodeoxycytidine, the bacterial load at the skin infection site of the mice was significantly reduced.

[0141] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description. All such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. Use of 5-fluorodeoxycytidine in the preparation of a medicament against Staphylococcus aureus.

2. Use of 5-fluorodeoxycytidine in the preparation of a medicament for preventing and / or treating Staphylococcus aureus infection.

3. The application according to claim 1 or 2, characterized in that, The Staphylococcus aureus includes at least one of methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

4. A drug against Staphylococcus aureus, characterized in that, It includes a first active ingredient and a pharmaceutically acceptable first carrier, and the first active ingredient includes 5-fluorodeoxycytidine.

5. The medicament according to claim 4, characterized in that, The first carrier includes at least one of a glidant, a diluent, a wetting agent, a suspending agent, a solvent and an emulsifier.

6. The drug according to claim 4, characterized in that, The medicament further includes a first additive, and the first additive includes at least one of a preservative, a coloring agent, a flavoring agent, a stabilizer and an isotonic agent.

7. A drug for preventing and / or treating Staphylococcus aureus infection, characterized in that, It includes a second active ingredient and a pharmaceutically acceptable second carrier, and the second active ingredient includes 5-fluorodeoxycytidine.

8. The drug according to claim 7, wherein The pharmaceutically acceptable second carrier includes at least one of a glidant, a diluent, a wetting agent, a suspending agent, a solvent and an emulsifier.

9. The drug according to claim 7, characterized in that, The medicament further includes a second additive, and the second additive includes at least one of a preservative, a coloring agent, a flavoring agent, a stabilizer and an isotonic agent.

10. The drug according to any one of claims 4-9, characterized in that, The dosage form of the medicament includes an oral dosage form, an injection dosage form or a topical dosage form.

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