Application of caffeic acid phenethyl ester in preparation of ribosome large subunit protein L1 targeting inhibitor

Abstract

The invention discloses the technical field of biological medicine, and particularly relates to application of caffeic acid phenethyl ester in preparation of a ribosome large subunit protein L1 targeted inhibitor. According to the present invention, the drug affinity induced target stability discovers that the ribosome large subunit protein L1 target inhibitor can directly target the ribosome large subunit protein L1 in bacteria, and can be adopted as the competitive inhibitor to competitively combine with the RRF L1 so as to inhibit the ribosome recovery process, such that the bacterial translation process is inhibited, and the bacterial protein synthesis blocking and death are caused. The mechanism enables the ribosome large subunit protein L1 to become a ribosome target with therapeutic potential, provides a new target and theoretical basis for clinical treatment of bacterial infection, and provides new support for screening of new broad-spectrum antibiotics.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of caffeic acid phenethyl ester in the preparation of L1-targeting inhibitors of ribosomal large subunit protein. Background Technology

[0002] Currently used antibiotics primarily exert their effects by inhibiting bacterial protein synthesis, mainly targeting the 30S or 50S subunits of the ribosome. For example, antibiotics such as chloramphenicol and macrolides act on the 50S subunit, interfering with peptidyl transferase centers or nascent peptide chain channels; tetracyclines and tigecycline bind to the 30S subunit, affecting translation initiation or tRNA localization. However, bacteria can develop cross-resistance to these drugs through various mechanisms, including rRNA modification and the production of drug-modifying enzymes, leading to treatment failure based on these drugs. Therefore, exploring protein synthesis processes not yet targeted by existing antibacterial drugs has become an important direction for developing antibiotics with novel mechanisms. Among these, the ribosome recycling process after translation termination, as a key step in maintaining translation cycle efficiency, has not yet been fully explored as an antibacterial target.

[0003] Ribosome recycling refers to the dissociation of the 70S ribosome into 30S and 50S subunits after translation termination. This process consumes GTP and relies on the synergistic action of elongation factor G (EF-G) and ribosome recycling factor (RRF). The L1 protein in the 50S subunit forms the core of the L1 stalk, and its conformational changes participate in the release of deacylated tRNA from the E site. Although the roles of RRF and EF-G in ribosome dissociation are well understood, the interaction mechanism between RRF and 50S subunit proteins (especially L1) remains unclear, and whether the dynamic movement of the L1 stalk synergistically regulates the recycling process with RRF function also needs to be elucidated. Revealing the molecular details of this process is expected to provide new targets for antimicrobial drug development.

[0004] Helicobacter pylori ( Helicobacter pylori Helicobacter pylori (H. pylori) is a pathogenic bacterium that colonizes the gastric mucosa and is closely related to the development of chronic gastritis, peptic ulcers, and gastric cancer. With the global spread of multidrug-resistant strains of H. pylori, the efficacy of traditional quadruple therapy for H. pylori is increasingly declining. At the same time, due to pharmacokinetic and safety limitations, the types of antibiotics available to special populations such as the elderly, children, and pregnant women are even more limited, making the treatment of H. pylori infection a serious challenge.

[0005] Therefore, there is an urgent need to develop novel broad-spectrum antibacterial drugs with entirely new mechanisms of action, that can avoid cross-resistance, achieve sustained clearance in refractory infections, and have good safety profiles, for the broad-spectrum killing of pathogenic bacteria.

[0006] Based on the above situation, this invention proposes to use the large ribosomal subunit protein L1 as a new target for the development of broad-spectrum antibacterial drugs. Furthermore, it reveals a new mechanism by which L1-targeting inhibitors specifically bind to key residues of the L1 protein, blocking the binding of RRF to the 50S subunit, thereby inhibiting ribosome recycling, leading to translational arrest and bacterial death. This provides a new direction and theoretical basis for the design and screening of novel antibacterial drugs. Summary of the Invention

[0007] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the objective of this invention is to provide a ribosomal large subunit protein L1 targeting inhibitor and its application. This invention discovers the application of ribosomal large subunit protein L1 as a target for treating bacterial infections, reveals the therapeutic potential of ribosomal large subunit protein L1 target inhibitors in treating bacterial infections, and, taking one such ribosomal large subunit protein L1 target inhibitor—caffeic acid phenethyl ester—as an example, explores its actual therapeutic effect and broad-spectrum antibacterial activity. In this invention, through drug affinity-induced target stability experiments, it was found that the ribosomal large subunit protein L1 target inhibitor can directly target ribosomal large subunit protein L1 in bacteria. As a competitive inhibitor, it competitively binds to L1 with RRF, inhibiting the ribosome reuptake process, thereby inhibiting bacterial translation, leading to bacterial protein synthesis inhibition and death. This mechanism makes ribosomal large subunit protein L1 a ribosomal target with therapeutic potential, providing a new target and theoretical basis for the clinical treatment of bacterial infections, and providing new support for the screening of new broad-spectrum antibiotics.

[0008] In a first aspect, the invention provides the use of a ribosomal large subunit protein L1 targeting inhibitor in the preparation of a broad-spectrum antibacterial product.

[0009] In some embodiments of the present invention, the ribosomal large subunit protein L1 targeting inhibitor includes caffeic acid phenethyl ester.

[0010] In some embodiments of the present invention, the ribosomal large subunit protein L1 targeting inhibitor is caffeic acid phenethyl ester.

[0011] In some embodiments of the present invention, the L1-targeting inhibitor of the large ribosomal subunit protein inhibits the bacterial ribosomal recycling process, inhibits the bacterial translation process, and kills bacteria by competitively binding to the L1 protein with the ribosomal recycling factor.

[0012] This invention provides a novel antibacterial strategy targeting the interaction between bacterial ribosomal large subunit protein L1 and ribosome recycling factor RRF. Using an inhibitor of ribosomal large subunit protein L1 as the active molecule (e.g., caffeic acid phenethyl ester), the strategy inhibits ribosome recycling by suppressing the interaction between L1 and RRF, thereby inhibiting bacterial translation and leading to bacterial growth suppression and / or death. This addresses the problems of rising resistance to existing antibacterial drugs and the lack of effective inhibitors targeting key steps in ribosome recycling.

[0013] In this invention, "ribosomal large subunit protein L1", "L1" and "L1 protein" can be used interchangeably. "ribosome recovery factor RRF", "RRF" and "RRF protein" can also be used interchangeably.

[0014] In some embodiments of the present invention, the ribosomal large subunit protein L1 targeting inhibitor binds to the L1-RRF interaction interface region.

[0015] In some embodiments of the invention, the binding is a competitive binding, thereby blocking or weakening the interaction between L1 and RRF.

[0016] In some embodiments of the present invention, the L1-RRF interaction interface region refers to the bonding region formed by the contact surface between L1 and RRF.

[0017] In some embodiments of the present invention, the binding region on L1 includes at least one or more interfacial key residues selected from A56, D57, Q58, M59, R130, P134, and M138.

[0018] In some embodiments of the present invention, the binding region on L1 contains at least two or more key interfacial residues selected from A56, D57, Q58, M59, R130, P134, and M138.

[0019] In some embodiments of the present invention, the binding site of the ribosomal large subunit protein L1 targeting inhibitor on L1 includes at least one of D167, K168 and / or R165.

[0020] In some embodiments of the present invention, D167, K168 and / or R165 together constitute a binding pocket for accommodating a ribosomal large subunit protein L1 targeting inhibitor, which binds to the binding pocket to inhibit the interaction between L1 and RRF.

[0021] In this invention, the aforementioned site of action was verified by mutating key sites of L1. Specifically, after replacing the aforementioned key interface residues and / or key residues of the binding pocket of L1, the binding effect of the ribosomal large subunit protein L1 targeting inhibitor on L1 was significantly reduced, indicating that the site of action of the ribosomal large subunit protein L1 targeting inhibitor is located in the binding pocket region.

[0022] In some embodiments of the present invention, the bacterial ribosome recovery system can characterize the accumulation of post-termination complexes and the decrease in ribosome recycling rate caused by ribosome recovery obstruction.

[0023] In some embodiments of the present invention, the bacterial ribosome recovery system includes any one or more of the following: a purified ribosome system, a post-termination complex system, a cell-free translation system, or a bacterial cell system.

[0024] Unless otherwise stated, the "ribosome recovery system" described in this invention includes, but is not limited to: purified 50S subunit system, purified 70S ribosome system, post-termination complex (PTC) system, cell-free translation system, and bacterial cell system.

[0025] In some embodiments of the present invention, inhibiting bacterial ribosome recycling can lead to inhibition of bacterial translation. In this invention, translation inhibition can manifest as a decrease in protein synthesis rate, a reduction in translation reporter signals, and / or impaired ribosome recycling after translation termination.

[0026] In some embodiments of the present invention, the translation inhibition results in inhibited bacterial growth and / or bacterial death.

[0027] In some embodiments of the present invention, the broad-spectrum antibacterial product includes: antibacterial drugs or surface bactericides.

[0028] In this invention, the term "surface disinfectant" refers to a broad-spectrum, highly effective disinfectant, primarily used for the control of microbial contamination on various food or non-food contact surfaces. Depending on the specific field and location of its application, its components may be adjusted to some extent, but it must contain at least one substance with broad-spectrum bactericidal activity. In this invention, the substance with broad-spectrum bactericidal activity is a ribosomal large subunit protein L1 targeting inhibitor. In some embodiments of this invention, the substance with broad-spectrum bactericidal activity is phenethyl caffeate.

[0029] In some embodiments of the present invention, the broad-spectrum antibacterial product can inhibit or kill bacteria.

[0030] In some embodiments of the present invention, the bacteria include bacteria that host animals or plants.

[0031] In some embodiments of the present invention, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria.

[0032] In some embodiments of the present invention, the bacteria also include drug-resistant strains.

[0033] In some embodiments of the present invention, the bacteria include, but are not limited to: Escherichia coli, Bacillus cereus, Listeria, Salmonella, Streptococcus agalactiae, Streptococcus faecium, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, and Helicobacter pylori.

[0034] In some embodiments of the present invention, the dosage form of the antibacterial drug includes: oral dosage form, injection dosage form, or topical dosage form.

[0035] In some embodiments of the present invention, the oral preparations include, but are not limited to: tablets (such as coated tablets, dispersible tablets, effervescent tablets, chewable tablets, lozenges, sublingual tablets, etc.), capsules (such as hard capsules, soft capsules, enteric-coated capsules, etc.), granules, powders, pills, drop pills, ointments, gels, solutions, suspensions, emulsions, syrups, elixirs, etc.

[0036] In some embodiments of the present invention, the injectable agent includes, but is not limited to, injection solution, injection suspension, injection emulsion, powder for injection, etc.

[0037] In some embodiments of the present invention, the external agents include, but are not limited to: ointments, creams, gels, pastes, tinctures / lactoses, lotions, films, patches (oral patches, skin patches), ophthalmic preparations (such as eye ointments or eye drops), drops, sprays / aerosols, mouthwashes, suppositories, etc.

[0038] In some embodiments of the present invention, the surface bactericide includes, but is not limited to, powders, granules, tablets, capsules, emulsions, aqueous solutions, and aerosols.

[0039] In some embodiments of the present invention, the broad-spectrum antibacterial product further includes pharmaceutically acceptable excipients.

[0040] In some embodiments of the present invention, the pharmaceutically acceptable excipients are rationally selected based on factors such as the product form of the pharmaceutical composition, the intended use, and the route of administration, and include, but are not limited to: buffers, coenzymes, enzyme protectants, metal ions, catalysts, defoamers, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), chelating agents (such as disodium EDTA), etc.

[0041] In some embodiments of the present invention, the broad-spectrum antibacterial product further includes a second active substance.

[0042] In some embodiments of the present invention, the second active substance comprises one or more antibacterial drugs, which can produce synergistic or additive antibacterial effects when used in combination with a large ribosomal subunit protein L1 targeting inhibitor, thereby reducing the dosage and / or slowing down the development of drug resistance.

[0043] A second aspect of the invention provides the use of a ribosomal large subunit protein L1 targeting inhibitor in the preparation of a medicament for the prevention of bacterial infections.

[0044] In some embodiments of the present invention, the ribosomal large subunit protein L1 targeting inhibitor includes caffeic acid phenethyl ester.

[0045] In some embodiments of the present invention, the bacteria include: Escherichia coli, Bacillus cereus, Listeria, Salmonella, Streptococcus agalactiae, Streptococcus faecium, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, and Helicobacter pylori.

[0046] A third aspect of the invention provides the use of a ribosomal large subunit protein L1 targeting inhibitor in the preparation of a medicament for the treatment or adjuvant treatment of bacterial infections.

[0047] In some embodiments of the present invention, the ribosomal large subunit protein L1 targeting inhibitor includes caffeic acid phenethyl ester.

[0048] In some embodiments of the present invention, the bacteria include: Escherichia coli, Bacillus cereus, Listeria, Salmonella, Streptococcus agalactiae, Streptococcus faecium, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, and Helicobacter pylori.

[0049] A fourth aspect of the present invention provides a method for killing bacteria in vitro, the method comprising: contacting a ribosomal large subunit protein L1 targeting inhibitor with an object to be sterilized, thereby killing bacteria thereon.

[0050] In some embodiments of the present invention, the ribosomal large subunit protein L1 targeting inhibitor includes caffeic acid phenethyl ester.

[0051] In some embodiments of the present invention, the bacteria include: Escherichia coli, Bacillus cereus, Listeria, Salmonella, Streptococcus agalactiae, Streptococcus faecium, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, and Helicobacter pylori.

[0052] In some embodiments of the present invention, an effective amount of the ribosomal large subunit protein L1 targeting inhibitor is used to contact the object to be sterilized.

[0053] In this invention, the “effective amount” refers to the amount of ribosomal large subunit protein L1 that is capable of producing detectable inhibition of L1-RRF interaction, detectable inhibition of ribosome recovery, and / or detectable inhibition of translation in any of the above systems.

[0054] In some embodiments of the present invention, the amino acid sequences of L1 and RRF may be as shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, and the relevant residue numbers shall be based on the sequence shown in the corresponding SEQ ID. In the following embodiments, residues such as D167, K168, R130, A56, D57, Q58, M59, P134, and M138 are all numbered according to SEQ ID NO:1.

[0055] In this invention, in order to verify the action site and mechanism of action of the L1 targeting inhibitor of ribosomal large subunit protein, the inventors also constructed an L1 mutant. The mutation site includes, but is not limited to, one or more of the key interface residues A56, D57, Q58, M59, R130, P134, and M138, and one or more of the key binding pocket residues D167, K168, and / or R130.

[0056] In some embodiments of the present invention, the mutation methods include point mutations (such as Ala scans), deletions, or insertions.

[0057] In some embodiments of the present invention, methods including but not limited to the following can be used to detect whether the ribosome recovery process is hindered: sucrose density gradient centrifugation analysis of changes in ribosome components after CAPE treatment of Helicobacter pylori, polysome accumulation detection, or determination of ribosome recirculation kinetic parameters.

[0058] A fifth aspect of the present invention provides a method for screening ribosome recovery inhibitors, comprising the following steps: A system containing the large ribosomal subunit protein L1 and the ribosomal regeneration factor RRF was constructed. The test compound was added, and the inhibitory effect of the test compound on the interaction between L1 and RRF was detected, thereby screening out compounds that can inhibit the interaction as candidate ribosomal regeneration inhibitors.

[0059] In some embodiments of the present invention, L1 and RRF are mixed and incubated to form an L1–RRF complex, thereby obtaining a system containing the ribosomal large subunit protein L1 and the ribosomal recycling factor RRF.

[0060] In some embodiments of the present invention, the inhibition effect includes: binding occurring in the L1-RRF interaction interface region and / or the binding pocket / binding groove region defined by D167, K168 and / or R165.

[0061] In some embodiments of the present invention, the system may further contain a ribosomal large subunit protein L1 targeting inhibitor, thereby characterizing the inhibitory effect based on competitive binding and / or occupancy binding.

[0062] In some embodiments of the present invention, protein-small molecule binding detection methods can be used to verify the binding of the ribosomal large subunit protein L1 targeting inhibitor to L1. The detection methods include, but are not limited to, one or more of the following: microthermophoresis (MST), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), differential scanning fluorescence (DSF), or fluorescence quenching.

[0063] In some embodiments of the present invention, the binding site of the ribosomal large subunit protein L1 targeting inhibitor on L1 can be located based on one or more of structural modeling, molecular docking, molecular dynamics modeling, cryo-electron microscopy density matching and / or crystal structure analysis.

[0064] In some embodiments of the present invention, protein interaction detection methods are used to evaluate the interaction between L1 and RRF and the inhibitory effect of the large ribosomal subunit protein L1. These methods include, but are not limited to, one or more of the following: pull-down assay, co-immunoprecipitation (Co-IP), biolayer interference (BLI), SPR, FRET / fluorescence polarization, or gel migration assay.

[0065] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention, through drug affinity-induced target stability experiments, identified that the ribosomal large subunit protein L1 targeting inhibitor can directly target the bacterial ribosomal large subunit protein L1. Then, proteomics analysis revealed a significant upregulation of ribosome recycling factor RRF expression, indicating that it affects the functional state of RRF by targeting L1. Furthermore, based on the interaction and binding activity between L1 and RRF, this invention reveals that their binding ensures the efficient recycling of ribosomes from a post-termination state to a reusable state.

[0066] 2. In this invention, CAPE is used as an example to verify the effects of the ribosomal large subunit protein L1 targeting inhibitor on inhibiting bacterial ribosome recycling and inhibiting bacterial translation, demonstrating its potential in the treatment or prevention of bacterial infections.

[0067] 3. This invention reveals the mechanism of action of ribosomal large subunit protein L1 and ribosomal regeneration factor RRF in bacterial infection, with Helicobacter pylori (Helicobacter pylori) as an example. H. pylori Taking this as an example, it was found that L1-targeting inhibitors of the large ribosomal subunit can act as competitive inhibitors, competitively binding to RRF and inhibiting the ribosome reuptake process. Figure 7 As shown, the elucidation of this molecular mechanism establishes L1 as a new ribosome site of action, with the potential for therapeutic or adjuvant therapy and alleviating bacterial resistance, providing a novel target and theoretical basis for breaking through the bottleneck of new ribosome-based clinical diagnosis and treatment. Attached Figure Description

[0068] Figure 1 The results show the direct interaction between caffeic acid phenethyl ester (CAPE) and the bacterial ribosomal large subunit protein L1. A represents the Coomassie brilliant blue stained gel image and statistical analysis of differentially expressed protein bands from the Drug Affinity-Induced Target Stability (DARTS) experiment; B represents the silver stained gel image and statistical analysis of differentially expressed protein bands from the DARTS experiment; C represents the characterization results of L1 protein prepared using the E. coli prokaryotic system; and D represents the in vitro direct binding constant of CAPE to L1 protein determined by surface plasmon resonance.

[0069] Figure 2 This is a 3D model of the molecular dynamics simulation of CAPE and L1 protein stably binding at 1000 ns.

[0070] Figure 3 This is the confirmation result of CAPE's direct action on L1 and key amino acid sites. Among them, A is a 2D model of the stable binding of CAPE and L1 protein at 1000 ns in molecular dynamics simulation; B is the expression and purification results of mutant proteins at key amino acid sites (R165, D167, K168) of L1; C is the determination of the binding activity of CAPE and L1 mutant protein through biomembrane interference experiments; D is the fitting result of the binding constant Kd value of CAPE and L1 mutant protein in biomembrane interference experiments.

[0071] Figure 4 This is the result of DARTS combined with proteomics analysis of the mechanism by which CAPE exerts its effects by targeting L1. Among them, A is the volcano plot of differentially expressed proteins after CAPE treatment of Helicobacter pylori ATCC43504 and the statistics of the top 10 proteins upregulated and downregulated; B is the molecular docking diagram of the interaction between L1 and RRF; C is the characterization results of the expression and purification of ribosome recovery factor RRF in the E. coli prokaryotic expression system; D is the determination of the direct binding of L1 and RRF at the in vitro protein level through biomembrane interference experiments; E is the determination of the in vivo interaction between L1 and RRF in Helicobacter pylori ATCC43504 in the form of immunoprecipitation experiments.

[0072] Figure 5 The results show that CAPE inhibits the interaction between L1 and RRF by targeting L1. Specifically, A is the inhibitory effect of CAPE on the binding activity of RRF and L1 at the protein level by biomembrane interference assay; B is the inhibition of RRF-L1 interaction by CAPE at the cellular level by immunoprecipitation assay; and C is the inhibitory effect of CAPE on L1-RRF interaction by polyribosome analysis.

[0073] Figure 6 These are the in vitro anti-Helicobacter pylori activity results of CAPE. Among them, A is the inhibition curve of CAPE against Helicobacter pylori ATCC43504; B is the inhibition curve of CAPE against Helicobacter pylori SS1; C is the MIC value of CAPE against Helicobacter pylori ATCC43504; and D is the MIC value of CAPE against Helicobacter pylori SS1.

[0074] Figure 7 This is a molecular mechanism diagram of the present invention. Detailed Implementation

[0075] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0076] In this invention, caffeic acid phenethyl ester is used as an exemplary L1-targeting inhibitor of the large ribosomal subunit protein to demonstrate the broad-spectrum antibacterial effect of L1-targeting inhibitors of the large ribosomal subunit protein. It should be understood that this invention is not intended to limit caffeic acid phenethyl ester itself; other L1-targeting inhibitors of the large ribosomal subunit protein are also within the scope of protection of this invention.

[0077] The chemical structural formula of caffeic acid phenethyl ester (CAPE) is shown in formula (I), and its molecular formula is C1. 15 H 14 O4, molecular weight 284.306, CAS Registry Number: 100981-80-4.

[0078]

[0079] Formula (I).

[0080] Example 1 In this embodiment, using Helicobacter pylori as an example, the direct-acting protein of CAPE targeting bacteria was identified, and the binding activity of CAPE to L1 was measured. In this embodiment, caffeic acid phenethyl ester was used as an exemplary inhibitor targeting the large ribosomal subunit protein L1.

[0081] The specific experimental method is as follows: Using a bacterial protein extraction kit (Kanglang), Helicobacter pylori ATCC43504 (ATCC) was treated according to the instructions to obtain total protein lysis buffer (final concentration approximately 1.5 mg / mL). CAPE (final concentrations of 250 μg / mL or 500 μg / mL) was added to the total protein lysis buffer and incubated at room temperature for 90 min. Pronase E was then added at a mass ratio of 1:450 (Pronase E:total protein lysis buffer) and incubated at 37 °C for 45 min. 5 μL of a protease inhibitor (Cocktail, Yisheng Biotechnology) was added, and the reaction was terminated by placing the incubated mixture on ice. Coomassie brilliant blue staining and silver staining were used for colorimetric observation. After observation and recording, the target band was excised and subjected to destaining, DTT reduction, iodoacetamide alkylation, and then trypsin digestion overnight to obtain the target protein.

[0082] After further purification and desalting, the target protein was analyzed using nanoLC-MS / MS. Specifically, an Eksigent Ultra 2D liquid chromatography system coupled with a TripleTOF 5600 mass spectrometer was used. Separation was performed on a 75 μm × 20 cm C18 column at a flow rate of 300 nL / min, with a gradient elution of 5–80% acetonitrile for 60 min. The full scan range of MS1 was set to m / z 350–1500. The top 30 precursor ions with the highest intensities were selected and fragmented under dynamic exclusion conditions using rolling collision energy to obtain MS / MS spectra. The data were compared with the reference proteome data of Helicobacter pylori ATCC 43504 in the database for protein identification.

[0083] A commercially available pET32a plasmid containing the coding sequence of the large ribosomal subunit protein L1 (amino acid sequence MAKKVFKRLEKLFSKIQNDKAYGVEQGVEVVKSLASAKFDETVEVALRLGVDPRHADQMVRGAVVLPHGTGKKVRVAVFAKDIKQDEAKNAGADVVGGDDLAEEIKNGRIDFDMVIATPDMMAVVGKVGRILGPKGLMPNPKTGTVTMDIAKAVSNAKSGQVNFRVDKKGNVHAPIGKASFPEEKIKENMLELVKTINRLKPSSAKGKYIRNAALSLTMSPSVSLDAQELMDI (SEQ ID NO: 1)) (with a His tag) was transformed into Escherichia coli BL21 Star (DE3) strain. L1 protein expression was induced by 0.5 mM isopropyl BD-thiomannoside (IPTG) and incubated for 18 hours at 18°C ​​under a shaker at 200 rpm. Bacterial cells were lysed by sonication, and the supernatant was collected and loaded onto a Ni-NTA superfluid affinity column. Finally, the His-tagged recombinant L1 protein was eluted with 300 mM imidazole. The purity of the eluted recombinant L1 protein was analyzed by SDS-PAGE.

[0084] Further quantitative detection of the binding of ribosomal large subunit protein L1 to CAPE was performed using surface plasmon resonance (SPR) (based on the Biacore 1K sensor chip and Biacore T200 Evaluation Software). The experimental temperature was set at 25 °C. 1×PBST + 5% DMSO was used as the mobile phase buffer. Specifically, ribosomal large subunit protein L1 (10 μg / mL) was immobilized on the CM5 chip surface using a standard amine coupling method, with an immobilization amount of 1718 response units (RU). Subsequently, CAPE at different concentrations (0, 0.25, 0.5, 1, and 2 μM) was injected, and the binding response signals were recorded. The data were fitted using the Biacore T200 Evaluation Software based on the 1:1 Langmuir binding model and the steady-state affinity model, respectively, and the equilibrium dissociation constant was calculated. K d ).

[0085] It should be understood that, in this embodiment, the source of the ribosomal large subunit protein L1 is not limited; it can be the ribosomal large subunit protein L1 with the above-described amino acid sequence, or any commercially available recombinant L1 protein or L1 protein prepared based on bacterial ribosomes. Its selection does not affect the dissociation constant ( ) in this embodiment. Kd ) calculation.

[0086] The results are shown in the table below. Figure 1 As shown.

[0087] Table 1. Mass spectrometry identification results of the differential bands

[0088] Based on the Coomassie brilliant blue staining, silver staining, and mass spectrometry identification results, it can be determined that CAPE can directly target the large ribosomal subunit protein L1 to exert its function. Furthermore, through surface plasmon resonance (SPR) experiments, the binding constant (KD) of L1 and CAPE at the protein level was obtained as 1 μM, which further proves that CAPE can specifically bind to L1.

[0089] Example 2 In this embodiment, the key binding site of CAPE on the large ribosomal subunit protein L1 was further verified.

[0090] The specific experimental method is as follows: Amber24 molecular dynamics simulation software and the ff19SB force field were used to perform all-atom molecular dynamics simulations of the protein system. The initial structural model was constructed using AlphaFold3, and the conformation with the highest confidence score was selected for subsequent molecular dynamics (MD) simulations. All simulations were performed in Amber24, and the protein parameters were obtained using the ff19SB force field. The simulation workflow consisted of three stages: ligand parameterization, system construction, and production-stage kinetic simulation. The ligand parameters were generated using an Amber-compatible workflow [JQ1.1] to ensure consistency with the force field. All-atom resolution MD simulations were conducted in explicit solvents under periodic boundary conditions. Trajectory processing and post-simulation analysis were performed using the CPPTRAJ module in the AmberTools suite.

[0091] To characterize non-covalent interactions in depth, the MDAnalysis-based analysis pipeline incorporated the ProLIF plugin to analyze the trajectories, extracting 1000 frames at 1 ns intervals and statistically analyzing the frequency of various interaction types (including hydrophobic contacts, hydrogen bonds, π-π stacking, cation-π, anion-π interactions, and salt bridges). All preprocessing and analysis steps were implemented using custom Python scripts, plotting was done using Matplotlib, and representative protein conformations were visualized using ChimeraX.

[0092] Further based on the methods described in the above embodiments, an *E. coli* BL21 Star(DE3) transformant expressing the L1 protein mutant was constructed. The plasmid construction and *E. coli* transfection methods were the same as in the above embodiments, except that the L1 protein mutant was obtained by mutating the L1 protein shown in SEQ ID NO: 1 with R165, D167, and K168 mutations (all mutated to alanine A). Expression was induced using 0.5 mM isopropyl BD-thiomannoside (IPTG) and incubated at 18°C ​​on a shaker at 200 rpm for 18 hours. Bacterial cells were lysed by sonication, the supernatant was collected, and loaded onto a Ni-NTA superfluid affinity column. Finally, elution was performed with 300 mM imidazole. The eluted mutant (L1...) MT The purity of the protein was analyzed by SDS-PAGE.

[0093] Kinetic measurements were performed using a Gator Plus instrument (Gator Bio) based on biolayer interferometry (BLI). The L1 mutant protein was immobilized using an APS biosensor (Gator Bio, model 20-5056) and then bound to different concentrations of CAPE (250 μM, 125 μM, 62.5 μM, and 31.25 μM). All data were processed using Gator One software, and kinetic parameters (kon, koff, and KD) were calculated using a global 1:1 fitting model.

[0094] The results are as follows Figure 2 and 3 As shown.

[0095] It was observed that CAPE stably binds to L1 at 1000 ns, with CAPE binding to L1 within a binding pocket region surrounded by D167, K168, and R165. Mutating the key binding sites D167, K168, and R165 to alanine residues revealed that the binding constant (KD) of CAPE to L1 decreased from 1 μM to 357 μM, indicating that D167, K168, and R165 are key binding sites for CAPE targeting L1, thus providing a structural basis for its inhibition of L1-RRF interactions.

[0096] Example 3 In this embodiment, the interaction between the large ribosomal subunit protein L1 and the ribosomal recycling factor RRF was verified.

[0097] The specific experimental method is as follows: Quantitative proteomics (TMT) analysis was performed: Helicobacter pylori ATCC43504 was treated with 100 μg / mL CAPE and then incubated at 37±1℃ under microaerobic conditions (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 6 hours. Parallel treatments were performed simultaneously, with a control group containing 0.25% (v / v) DMSO. The mass spectrometer used in this example was a Q-Exactive HFX. Screening was performed at the spectral level, with a PSM-level FDR ≤ 1%. Further screening was conducted at the protein level, with a protein-level FDR <= 1%. A fold change > 1.2 and a p-value < 0.05 were used as screening criteria for significantly differentially expressed proteins.

[0098] Meanwhile, the three-dimensional structures of L1 and RRF were established using a homology modeling method. The three-dimensional structures of the acceptor were dehydrogenated and dehydrated using the Autodock tool, followed by molecular docking analysis using the AutoDock vina tool. Finally, docking analysis of L1 and RRF was performed using PyMOL software, and the docking data were visualized.

[0099] Then, following the same method as for the L1 protein in the above examples, a pET32a plasmid expressing the RRF protein (amino acid sequence MLQAIYNETKDLMQKSIQALNRDFSTLRSAKVSVNILDHIKVDYYGTPTALNQVGSVMSLDATTLQISPWEKNLLKEIERSIQEANIGVNPNNDGETIKLFFPPMTSEQRKLIAKDAKAMGEKAKVAVRNIRQDANNQVKKLEKDKEISEDESKKAQEQIQKITDEAIKKIDESVKNKEDAILKV (SEQ ID NO: 2)) was constructed and transformed into *E. coli* BL21 Star (DE3) strain. Expression was induced using IPTG and incubated at 18°C ​​on a shaker at 200 rpm for 18 hours. Bacterial cells were lysed by sonication, the supernatant was collected, and loaded onto a Ni-NTA superfluid affinity column. Finally, elution was performed with 300 mM imidazole. The eluted mutant (L1) MT The purity of the protein was analyzed by SDS-PAGE.

[0100] Kinetic analysis was performed using a Gator Plus instrument (purchased from Gator Bio) based on biolayer interferometry (BLI). Specifically, the RRF protein was diluted to 10 μg / mL with PBS. The L1 protein was then diluted in PBS containing 0.2% BSA in a concentration gradient (2-fold serial dilution) to obtain L1 protein dilutions ranging from 10 to 1.25 μM. The RRF protein was immobilized using an APS biosensor (purchased from Gator Bio), and subsequently bound to different concentrations of L1 protein dilutions, followed by dissociation in PBS containing 0.2% BSA. All data were processed using Gator One software, and kinetic parameters (kon, koff, and KD) were calculated using a global 1:1 fitting model.

[0101] Disperse and suspend rProtein A / G MagPoly magnetic beads, then transfer 20 μL of the bead suspension (0.2 mg) to a 1.5 mL tube. Equilibrate and wash the beads, perform magnetic separation, and remove the supernatant with 1× lysis / wash buffer (enhanced). Add 30 μg of L1 antibody (Agrisera, catalog AS111738) and RRF antibody (MyBioSource, catalog MBS7140371), and adjust the volume to 500 μL with antibody storage buffer or 1× lysis / wash buffer. Incubate on ice at 70 rpm for 4 hours. Collect the beads magnetically and wash at least twice with 500 μL of 1× lysis / wash buffer (enhanced), mixing for 1 minute each time. Then add 500 μL of bacterial lysis buffer for the RRF transformants and incubate on ice at 70 rpm for 12 hours. After binding, the magnetic beads were washed twice with 500 μL of 1× lysis / wash buffer (enhanced) and transferred to a new test tube for magnetic separation. The proteins were then washed with 50 μL of elution buffer at room temperature for 10 minutes, and then immediately neutralized with 15 μL of neutralization buffer for Western blotting experiments.

[0102] The results are as follows Figure 4 As shown.

[0103] TMT proteomic analysis revealed that CAPE treatment of Helicobacter pylori significantly upregulated RRF expression, indicating that CAPE can target L1 and thus affect RRF function. Simultaneously, molecular docking revealed a good binding activity between L1 and RRF. Further RRF protein preparation and biomembrane interference assays determined the binding constant (KD) of RRF to L1 at the protein level to be 4.2 μM. Finally, at the cellular level, immunoprecipitation experiments further determined the interaction between L1 and RRF, demonstrating an interaction between L1 and RRF that plays an important role in bacterial ribosome translation.

[0104] Example 4 In this embodiment, the inhibitory effect of the L1-targeting inhibitor of the large ribosomal subunit protein on L1-RRF interaction was verified, and it significantly affected the ribosome recycling process.

[0105] The specific experimental method is as follows: Kinetic analysis was performed using a Gator Plus instrument (purchased from Gator Bio) based on biolayer interferometry (BLI). Specifically, the RRF protein was diluted to 10 μg / mL with PBS. The L1 protein was then diluted in PBS containing 0.2% BSA in a concentration gradient (2-fold serial dilution) to obtain L1 protein dilutions ranging from 10 to 1.25 μM. The RRF protein was immobilized using an APS biosensor (purchased from Gator Bio), and subsequently bound to different concentrations of L1 protein dilutions, followed by dissociation in PBS containing 0.2% BSA. All data were processed using Gator One software, and kinetic parameters (kon, koff, and KD) were calculated using a global 1:1 fitting model.

[0106] Then, using the same method, the RRF protein was first diluted to a working concentration (10 μg / mL) with PBS, and then CAPE was serially diluted (2-fold sequentially) with PBS containing 0.05% Tween 20 to obtain CAPE dilutions ranging from 20 to 2.5 μM. L1 protein was added to each CAPE dilution to a final concentration of 5 μM. The RRF protein was immobilized using an APS biosensor (purchased from Gator Bio), and then bound to the CAPE dilutions containing L1 protein. The raw data were processed and exported using GatorOne software, and then the IC50 was calculated using GraphPad Prism. 50 value.

[0107] Resuspend rProtein A / G MagPoly magnetic beads, then transfer 20 μL of the beads (0.2 mg) to a 1.5 mL tube. Equilibrate and wash the beads, using magnetic separation and removing the supernatant with 1× lysis / wash buffer (enhanced). Add antibody (30 μg) and adjust the volume to 500 μL with antibody storage buffer or 1× lysis / wash buffer, then incubate on ice at 70 rpm for 4 h. Collect the beads magnetically and wash at least twice with 500 μL of 1× lysis / wash buffer (enhanced), mixing for 1 min each time. Then add CAPE (100 μg / mL) and DMSO to treat the bacterial lysis buffer (500 μL), and incubate on ice at 70 rpm for 12 h. After binding is complete, wash the beads twice with 500 μL of 1× lysis / wash buffer (enhanced) and transfer to a new tube for magnetic separation. Wash the protein with 50 μL of elution buffer for 10 minutes at room temperature, then immediately neutralize the elution buffer with 15 μL of neutralization buffer for Western blotting experiments.

[0108] Remove the bacterial samples treated with CAPE and DMSO, add sufficient liquid nitrogen for grinding; then add 300 μL-500 μL of polysome profiling cell lysis buffer and gently vortex. Lyse on ice for 30 min; centrifuge the cell lysis buffer at 13000g for 10 min at 4℃, and collect the supernatant for sample loading. Preparation of sucrose gradient: Take a new SW41 ultracentrifuge tube, add 10% sucrose buffer to each tube, then slowly add 45% sucrose to the bottom of the tube, adjust the parameters, and prepare the density gradient. Sample loading: Put the prepared density gradient sucrose solution into the ultracentrifuge tube sleeve, add 100 μL of cell lysis supernatant, balance the tube, and centrifuge; adjust the ultracentrifuge parameters to 36000 rpm and 3 h. After centrifugation, carefully remove the ultracentrifuge tube, separate and collect the components, and measure the absorbance at 260 nm.

[0109] The results are as follows Figure 5 As shown.

[0110] Biomembrane interference experiments revealed that by adding a fixed amount of RRF (or L1) protein to the system and progressively increasing the CAPE concentration, CAPE could compete with RRF (or L1) for binding sites, indicating that CAPE exhibits a competitive binding characteristic in inhibiting the L1-RRF interaction. Immunoprecipitation experiments also validated the inhibitory effect of CAPE on the L1-RRF interaction at the cellular level. Furthermore, polyribosome analysis showed that CAPE treatment significantly inhibited ribosome regeneration, leading to ribosome accumulation on the mRNA chain and a significant upregulation of the polyribosome ratio, thereby inhibiting bacterial translation.

[0111] Example 5 In this embodiment, the antibacterial effect of the ribosomal large subunit protein L1 targeting inhibitor was verified.

[0112] The experimental methods are shown below.

[0113] (1) Antibacterial effect of L1-targeting inhibitors of ribosomal large subunit protein: Following the CLSI M45 guidelines, the minimum inhibitory concentration (MIC) of CAPE was determined using the agar dilution method. Specifically, the Helicobacter pylori culture grown on Columbia blood agar was adjusted to approximately 2.0 McFarland (approximately 1 × 10⁻⁶). 8 CFU / mL). 1 μL of bacterial culture was inoculated onto a Mueller-Hinton agar plate containing 5% defibrinated sheep blood and CAPE serially diluted twice. The plate was incubated at 37 °C in an ELECTROTEK anaerobic workstation for 72 h under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) to obtain the MIC value. The MIC is defined as the lowest drug concentration that can completely inhibit the growth of visible colonies.

[0114] The results are as follows Figure 6 As shown.

[0115] The results showed that CAPE significantly inhibited the growth of Helicobacter pylori ATCC43504 and Helicobacter pylori SS1, with MIC values ​​of 50 and 100 μg / mL, respectively.

[0116] Example 6 In this embodiment, the broad-spectrum antibacterial effect of the ribosomal large subunit protein L1 targeting inhibitor was further verified.

[0117] The specific experimental method is as follows: Take 40 μL of each test strain preserved in glycerol and inoculate it into the corresponding sterile liquid culture medium. Activate the culture at 37 °C and 180 rpm for 8 h with shaking. If necessary, inoculate again into fresh culture medium to expand the culture to the logarithmic growth phase (OD). 600 =0.6-0.8). Dilute the activated bacterial solution with sterile culture medium at a ratio of 1:1000 and set aside for later use.

[0118] Take a sterile 96-well plate and add 200 μL of sterile water to each of the outermost wells to reduce evaporation. Add 100 μL of sterile culture medium to each of the remaining wells. Then, serially dilute CAPE in the wells (mix by pipetting and transferring 100 μL each time, discarding the last 100 μL) to obtain the desired concentration gradient (e.g., 400, 200, 100, 50, 25, 12.5, 6.25 μg / mL). Use 1% DMSO as a negative control. Then, add 100 μL of the diluted bacterial culture (approximately 10 μL) to each well. 6 -10 7 (CFU / mL), with 3 parallel wells in each group, incubated at 28 ℃ for 16 h, absorbance at 600 nm was measured, and inhibition rate was calculated.

[0119] Among them, the tested strains included Escherichia coli O157:H7 ( Escherichia coli O157:H7 Purchased from ATCC, strain number ATCC 35150); Bacillus cereus ATCC 14579, purchased from ATCC; Listeria monocytogenes (… Listeria monocytogenes ATCC 19115, purchased from ATCC; Salmonella enterica subsp. enterica ( Salmonella enterica Subsp. enterica) ATCC 14028, purchased from ATCC; Lactobacillus-free ( Streptococcus agalactiae ATCC 27956, purchased from ATCC; Streptococcus lactis ( Streptococcus dysgalactiae ATCC27957, purchased from ATCC; PXO99A, the pathogen of rice bacterial blight, purchased from the Chinese Academy of Agricultural Sciences; citrus canker pathogen, purchased from the Chinese Academy of Agricultural Sciences; Ralstonia solanacearum, purchased from the Chinese Academy of Agricultural Sciences.

[0120] The results are shown in the table below.

[0121] Table 2. Inhibitory effects of CAPE on different test strains

[0122] It was found that CAPE had a certain inhibitory effect on all tested strains, with the best effect against strains such as *PXO99A*, *Streptococcus faecium*, and *Bacillus cereus*, with a MIC reaching 25 μg / mL. This demonstrates that the L1-targeting inhibitor of the large ribosomal subunit protein can achieve a good broad-spectrum antibacterial effect through the aforementioned translational inhibition.

[0123] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of caffeic acid phenethyl ester in the preparation of L1-targeting inhibitors of ribosomal large subunit protein, characterized in that, The L1-targeting inhibitor of the large ribosomal subunit protein inhibits bacterial ribosomal recycling and translation processes by competitively binding to the L1 protein with ribosomal recycling factors, thereby inhibiting or killing bacteria. The bacteria include: Bacillus cereus, Listeria, Streptococcus agalactiae, Streptococcus dysgalactiae, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, Salmonella, and Helicobacter pylori.

2. The use according to claim 1, characterized in that, The ribosomal large subunit protein L1 targeting inhibitor is used to prepare antibacterial drugs or surface bactericides.

3. The use according to claim 1, characterized in that, The amino acid sequence of the ribosomal large subunit protein L1 is shown in SEQ ID NO:

1.

4. The use according to claim 2, characterized in that, The dosage forms of the ribosomal large subunit protein L1 targeting inhibitor include: oral, injectable, or topical formulations.

5. The use according to claim 2, characterized in that, The dosage forms of the surface bactericide include: powder, granules, tablets, capsules, emulsions, aqueous solutions, and aerosols.

6. The use of caffeic acid phenethyl ester as a ribosomal large subunit protein L1 targeting inhibitor in the preparation of drugs for the prevention of bacterial infections.

7. The use according to claim 6, characterized in that, The bacteria include: Bacillus cereus, Listeria, Streptococcus agalactiae, Streptococcus dysgalactiae, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, Salmonella, and Helicobacter pylori.

8. Use of caffeic acid phenethyl ester as a ribosomal large subunit protein L1 targeting inhibitor in the preparation of drugs for the treatment or adjuvant treatment of bacterial infections.

9. The use according to claim 8, characterized in that, The bacteria include: Bacillus cereus, Listeria, Salmonella, Streptococcus agalactiae, Streptococcus dysgalactiae, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, and Helicobacter pylori.

10. A method for killing bacteria in vitro, characterized in that, The method includes: contacting a ribosomal large subunit protein L1 targeting inhibitor with an object to be sterilized, thereby inhibiting or killing bacteria on it; The ribosomal large subunit protein L1 targeting inhibitor is caffeic acid phenethyl ester; The bacteria include: Bacillus cereus, Listeria, Salmonella, Streptococcus agalactiae, Streptococcus dysgalactiae, rice bacterial blight pathogen, citrus canker pathogen, Ralstonia solanacearum, and Helicobacter pylori.

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