Self-assembling peptoids and their applications

Abstract

The present invention discloses a class of cationic symmetrical bacteria-induced antibacterial self-assembling peptoids, whose structure has the following general formula: #imgabs0# The primary structure of this type of molecule is different from that of natural polypeptides. It not only has linear symmetry, but also has antibacterial effects and can also serve as an antibacterial adjuvant for sensitizing antibiotics.

Description

Technical Field

[0001] The present invention relates to the technical field of peptidomimetic self-assembly and its application, in particular to a self-assembling peptidomimetic derivative and its application as an antibacterial agent / antibacterial adjuvant. Background Art

[0002] Peptide self-assembly technology has been a research hotspot in recent years. Through molecular design and solid-phase synthesis, thousands of structurally diverse peptides can be generated. The nanomaterials formed by peptide self-assembly have a wide range of potential applications. However, the peptide chains that can self-assemble are typically long and complex to synthesize, thus limiting their biocompatibility in specific applications. The self-assembly of amino acid-derived peptoids and their modifications has received less attention and research, making their subsequent modification and application development necessary and meaningful.

[0003] At present, self-assembling peptides used for antibacterial purposes can generally be divided into two categories: spontaneous assembly and induced assembly. Spontaneous assembly mainly forms antibacterial materials such as hydrogels, which utilize the properties of the hydrogels themselves (hydrophobic functions and positive charges on the surface of the material) for antibacterial effects. Induced self-assembly is to change the properties of the peptides themselves under specific stimuli (such as enzymes, environmental factors, etc.), thereby promoting the formation of self-assemblies (the most widely studied is alkaline phosphatase-induced self-assembly). This induced self-assembly behavior is specific because it is mediated by specific substances. Therefore, self-assembly antibacterial peptoids mediated by the specificity of bacteria themselves will be more selective (targeting bacteria), which will greatly reduce the non-specific toxicity to mammalian cells.

[0004] Cationic peptides, as antimicrobial peptides, hold promise for overcoming the growing clinical threat of bacterial drug resistance and are widely used in the construction of antimicrobial agents. However, inherent peptide instability and nonspecific cytotoxicity have hindered their development as pharmaceuticals. Peptidomimetics, on the other hand, possess antimicrobial properties similar to those of natural peptides and are generally resistant to enzymatic hydrolysis. Current antimicrobial peptides lack stability, nonspecific cytotoxicity, and poor bacterial selectivity. Furthermore, few reports exist on peptides that possess both antimicrobial activity and antimicrobial adjuvant properties, particularly short peptides that self-assemble under bacterial induction. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a self-assembling peptidomimetic molecule and its application as an antibacterial agent / antibacterial adjuvant.

[0006] A self-assembling peptidomimetic molecule having the following general formula:

[0007]

[0008] wherein the R1 group is any one of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), formula (XI), formula (XII), formula (XIII), formula (XIV), formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), and formula (XX);

[0009]

[0010] The R group is any one of formula (a), formula (b), formula (c), formula (d), formula (e), formula (f), formula (g), formula (h), formula (i), formula (j), formula (k), formula (l), formula (m), formula (n), formula (o), formula (p), formula (q), and formula (r);

[0011]

[0012] The present invention addresses the problems of poor stability, nonspecific cytotoxicity, and poor bacterial selectivity in current antimicrobial peptides, as well as the lack of reports on peptides that simultaneously possess both antimicrobial activity and antimicrobial adjuvant properties, particularly short bacterial-induced self-assembling peptoids. The present invention designs a class of cationic, symmetrical, bacterial-induced antimicrobial self-assembling peptoids. These peptoids differ from natural peptides in their primary structure, possessing linear symmetry, exhibit antimicrobial activity, and can also serve as antimicrobial adjuvants for sensitizing antibiotics. Furthermore, while there have been numerous reports on natural or synthetic peptides with antimicrobial activity, peptoids that simultaneously possess both antimicrobial and antibiotic adjuvant properties are rare, and reports on self-assembling peptoids that induce bacterial membrane perturbations or bacterial-induced aggregation are even rarer. The present invention discloses a self-assembling peptoid molecule with both bacterial-promoting self-assembly properties and antimicrobial performance, and its applications. Based on the rich non-covalent bonding capabilities and good lipid membrane interaction capabilities of modified arginine molecules, the present invention designs and synthesizes self-assembling peptide molecules that can perturb the bacterial membranes of both Gram-positive and Gram-negative bacteria while exhibiting antimicrobial activity. The series of molecules of the present invention can self-assemble and flocculate to form self-assemblies in an aqueous solution at pH 7.4 through strong intermolecular hydrophobic interactions. The interactions between bacteria and the peptidomimetic molecules promote the self-assembly of the peptidomimetic molecules. The self-assembling peptidomimetic molecules of the present invention have good bacterial specificity and antibacterial activity, a single component, and a minimal amino acid sequence (containing only two arginines). Due to their short sequence, good biocompatibility, and ease of synthesis and production, these molecules have the potential to be used as a highly efficient, environmentally friendly, novel self-assembling antibacterial agent / antibacterial adjuvant.

[0013] The series of molecules of the present invention can self-assemble to form liquid-phase separated particles after intermolecular interaction in a phosphate buffered saline solution at pH = 7.5. The self-assembling peptidomimetic molecules of the present invention have good antibacterial activity and sensitizing antibiotic potential, and are excellent in bacterial selectivity compared to the same type of antibacterial peptidomimetic molecules. Specifically, the same type of molecules (pentamidine and chlorhexidine) exhibit nonspecific hemolytic toxicity and mammalian cell toxicity, while this series of self-assembling antibacterial peptidomimetic molecules exhibit extremely low hemolytic effect and cytotoxicity, and even no obvious in vivo toxicity was observed in the mouse model. Due to their biological origin and short sequence, this series of molecules has the advantages of good biocompatibility and easy synthesis and production, and has the potential for application as a new type of efficient and environmentally friendly bacterial antibacterial agent and antibacterial adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The molecular structure diagram of the peptidomimetic;

[0015] Figure 2 H of peptidylcholine molecule g-15 1 NMR spectra;

[0016] Figure 3 Figure 2 shows the toxicity test of peptidomimetics d-15, g-15, j-15, n-15 and molecules with similar structures (pentamidine and chlorhexidine) on mammalian cells;

[0017] Figure 4 To test the self-assembly ability of g-15, the most active peptidomimetic molecule in the series;

[0018] Figure 5 It is the disturbance of the intracellular spatial distribution of the peptidomimetic molecule g-15;

[0019] Figure 6 Animal experiments on the peptidomimetic molecule g-15;

[0020] Figure 7 H of the peptidosome j-14 1 NMR spectra;

[0021] Figure 8 To test the self-assembly ability of the peptidomimetic molecule j-14 with LPS;

[0022] Figure 9 H of the peptidylcholine molecule d-15 1 NMR spectra;

[0023] Figure 10 Screening of IA32 antibiotics for the peptidomimetic molecule d-15;

[0024] Figure 11 Screening for FDA-approved non-antibiotic drugs for the peptidomimetic molecule d-15;

[0025] Figure 12 To test the self-assembly ability of the peptidomimetic molecule d-15 with LPS and preferred drugs. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings. Unless otherwise specified, the components or equipment in the following embodiments are all universal standard parts or components known to those skilled in the art, and their structures and principles are all known to those skilled in the art through technical manuals or conventional experimental methods.

[0027] Based on the rich non-covalent bonding ability and good interaction ability with lipid membranes of modified arginine molecules, the present invention designed and synthesized self-assembling peptidomimetic molecules that can disturb the bacterial membranes of Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, etc.) and Gram-negative bacteria (such as Escherichia coli, Acinetobacter baumannii, etc.) and have bacteria-induced antibacterial activity.

[0028] The first object of the present invention is to provide a bacterial-induced self-assembly peptidomimetic molecule with antibacterial properties and antibacterial adjuvant, the specific structure of which is as follows: Figure 1 As shown, these peptidomimetic molecules have the following general formula:

[0029]

[0030] wherein the R1 group is any one of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), formula (XI), formula (XII), formula (XIII), formula (XIV), formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), and formula (XX);

[0031]

[0032] The R group is any one of formula (a), formula (b), formula (c), formula (d), formula (e), formula (f), formula (g), formula (h), formula (i), formula (j), formula (k), formula (l), formula (m), formula (n), formula (o), formula (p), formula (q), and formula (r);

[0033]

[0034] The second object of the present invention is to provide a method for preparing the self-assembling peptidomimetic molecule, wherein the method adopts a liquid phase peptide synthesis method to couple a linker (diamine linker) with Fmoc or Boc protected arginine (with a modified group on the guanidine group) through acid amine condensation to synthesize the self-assembling peptidomimetic molecule, wherein the guanidine group in the arginine amino acid needs to be modified, and the amino group protected by Fmoc or Boc needs to be deprotected to expose the free amino group. Figure 1 Taking g-15 and j-14 in the above as examples, the synthetic routes are as follows:

[0035]

[0036] The third object of the present invention is to provide the use of the self-assembling peptidomimetic molecule as an antibacterial agent. The self-assembling peptidomimetic molecule directly utilizes its self-assembly characteristics to interact with pathogenic bacteria, thereby significantly inhibiting bacterial growth.

[0037] The fourth object of the present invention is to provide the self-assembling peptidomimetic molecules as adjuvants for current antibiotic sensitization, such as d-15 peptidomimetic molecules used in combination with vancomycin to fight against Gram-negative bacteria (such as Acinetobacter baumannii).

[0038] Example 1: Liquid Phase Synthesis of Peptidomimetic Molecules

[0039] Below is Figure 1 The synthesis of the peptidomimetic molecule g-15 is used as an example to illustrate the liquid phase synthesis method of the peptidomimetic molecule of the present invention. The synthesis of the remaining molecular structures is similar to that of g-15. During the molecular synthesis process, the amino acids are protected with Fmoc or Boc depending on the modification group of the target molecule and the linker.

[0040] In the synthesis of amino acid protecting groups using Fmoc, arginine (Fmoc-Arg(Pbf)-OH) and diamine linker molecules are mixed, and the amino acid and diamine are coupled using an acid-amine condensing agent. The specific preparation process is as follows (taking the preparation of g-15 as an example):

[0041] (1) Weigh 1296 mg (2 mmol) of Fmoc-Arg(Pbf)-OH amino acid into a round-bottom flask and dissolve it in anhydrous DMF.

[0042] (2) Weigh 608 mg (2.2 mM, 1 eq) of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride hydrate, dissolve it in water, and add it to a flask and stir at room temperature for 30 minutes to activate the amino acid.

[0043] (3) Weigh 1 eq of the diamine compound into a flask and continue stirring for 8-10 h. Monitor the reaction by TLC. After the reaction is complete, slowly add the reaction solution directly to the stirring beaker while stirring in ice water. A large amount of white precipitate will be seen. Filter with a Buchner funnel to remove the solvent to obtain a white solid crude product. The crude product is purified by column chromatography (dichloromethane:methanol 20:1) to obtain the target compound.

[0044] (4) The target compound of the previous step was dissolved in tetrahydrofuran solvent, piperidine (final concentration of 20%) was added, and the mixture was stirred at room temperature for 2 h. The solvent was dried to obtain a crude product, which was purified by column chromatography (dichloromethane: methanol 5:1) to obtain the target compound g-15. 1 NMR Figure 2 shown.

[0045] For Boc-protected peptoids, arginine (Boc-Arg(Tip)-OH) and diamine linker molecules were mixed using a liquid phase synthesis method, and the amino acid and diamine were coupled using an acid amine condensing agent. The specific preparation process is as follows (taking the preparation of j-14 as an example):

[0046] (1) Weigh 1080.58 mg (2 mmol) of Boc-Arg(Tip)-OH amino acid into a round-bottom flask and dissolve it in anhydrous DMF.

[0047] (2) Weigh 608 mg (2.2 mM, 1 eq) of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride hydrate, dissolve it in water, and add it to a flask and stir at room temperature for 30 minutes to activate the amino acid.

[0048] (3) Weigh 1 eq of the diamine compound into a flask and continue stirring for 8-10 h. Monitor the reaction by TLC. After the reaction is complete, slowly add the reaction solution directly to the stirring beaker while stirring in ice water. A large amount of white precipitate will be seen. Filter with a Buchner funnel to remove the solvent to obtain a white solid crude product. The crude product is purified by column chromatography (dichloromethane:methanol 20:1) to obtain the target compound.

[0049] (4) The target compound of the previous step was dissolved in dichloromethane solvent, trifluoroacetic acid (final concentration of 20%) was added, and the mixture was stirred at room temperature for 2 h. The solvent was dried by spin drying to obtain a crude product, which was slurried with ether to form a salt, and the organic matter was washed with saturated sodium bicarbonate, and then extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The crude product obtained by vacuum spin drying was purified by column chromatography (dichloromethane: methanol 5:1) to obtain the target compound j-14.

[0050] Example 2: Antibacterial activity and cytotoxicity testing of peptidomimetic molecules

[0051] The purified peptoid molecules were dissolved in DMSO and the antibacterial activity of this series of peptoids against various pathogens was tested using the standard broth dilution method. In testing this series of antibiotics, it was found that the intermediate linker and the modification group on the guanidine group had a significant impact on the antibacterial activity. For the intermediate linker, when the intermediate linker in this series was a single benzene ring, it showed strong antibacterial activity; and for the modification group on the guanidine group, it was found that there was a positive correlation between the hydrophobicity of the modification group and the antibacterial activity, that is, the more hydrophobic the modification group, the stronger its antibacterial ability. The antibacterial results are shown in Table 1.

[0052] The cytotoxicity test of the peptidomimetic molecule is as follows: the peptidomimetic compound is gradiently diluted with sterile PBS in a sterile 96-well plate, and the compound solution of different concentrations is gently added to the cell culture medium. 20 μL of compound solution is added to each well to make the final concentrations (256, 128, 64, 32, 8, 4, 2 μg / mL), respectively, and placed in an incubator for 24 hours. After 24 hours, weigh and prepare the MTT solution. Carefully aspirate the culture medium in the wells of the drug-treated cells and discard it. Add 100 μL of sterile PBS to each well to wash it once, then aspirate the PBS, add 0.5 mg / mL of MTT solution under light-proof environment conditions, and place it in an incubator for 1.5 hours. After the MTT incubation time is sufficient, carefully aspirate the MTT, then add 100 μL of dimethyl sulfoxide to each well and let it stand in a 37-degree incubator. Immediately use an enzyme reader to read the absorbance at 595 nm, and then use GraphPad to process the data. The results are as follows Figure 3 As shown, among the six compounds tested, the peptidomimetic series had no significant toxicity, and thus the cytotoxicity of all peptidomimetic compounds was less than that of pentamidine and chlorhexidine compounds with similar structures (linear symmetry). The toxicity results of the compounds are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] Note: Ef: Enterococcus faecium; Sa: Staphylococcus aureus; MASA: Methicillin-resistant Staphylococcus aureus; Bs: Bacillus subtilis; Pa: Pseudomonas aeruginosa; Kp: Klebsiella pneumonia; Ab: Acinetobacter baumannii; Ec: Escherichia coli; and Ms: Mycobacterium marinum. Strains marked with "-1" are multidrug-resistant strains isolated from clinical settings. HC 50 :The blood cells used are sheep blood cells; IC 50 : The cells used are HEK293T.

[0057] Example 3: Self-assembly test of antimicrobial peptidomimetic g-15 molecules with high antibacterial effect

[0058] First, pure g-15 peptoid was dissolved in DMSO with a stock concentration of 10.24 mg / mL. 25 μL of g-15 stock solution was added to an EP tube filled with 1 mL of PBS (pH = 7) buffer to a final concentration of 256 μg / mL. After mixing and standing, granular assemblies (such as Figure 4 Then, after adjusting the pH of the system to 5 using hydrochloric acid solution, the assembly disappeared, indicating that the assembly of the peptoid molecule is related to the pH value (as shown in a). Figure 4 (a in Figure 1). Thioflavin T (ThT) is a fluorescent molecule with a natural excitation wavelength of 385 nm. When the molecule is inserted into the β-sheet structure, the excitation wavelength shifts to 450 nm and the emission wavelength becomes 490 nm. Therefore, ThT can be used as a fluorescent probe to detect the assembly of g-15 peptoid molecules. The critical assembly concentration (CMC) of g-15 peptoid is 50 μg / mL (e.g. Figure 4 To further observe the formation of particles, laser confocal microscopy was also used to observe the formed particles. Among the different concentrations tested, it was clearly observed that the lowest concentration for forming assemblies was greater than 64 μg / mL, which is consistent with the lowest CMC value observed using various methods. Figure 4The particles formed at a concentration of 128 μg / mL and a ThT concentration of 5 μM are shown in FIG. 3. The particles formed were observed using a scanning electron microscope (SEM). The assembly was a fibrous aggregate (e.g., Figure 4 In order to further determine the size of the assembled particles, dynamic light scattering (DLS) was performed on the particles in the solution state, as shown in FIG. Figure 4 As shown in Figure e, the size of the formed particles is approximately between 100-1000 nm. Since this type of peptoid contains biguanide groups, which are DNA binding groups, the electrophoretic migration ability of g-15 after binding to DNA was tested. It was found that when the concentration of g-15 reached 32μg / mL, it could bind DNA well (such as Figure 4 Therefore, we speculated whether the binding of DNA could promote the self-assembly behavior of the peptoid itself, and tested the CAC value of the peptoid in the presence of DNA. It was found that the CMC value of the peptoid became smaller in the presence of DNA. Therefore, 32 μg / mL of the peptoid was used to test its fluorescence intensity. It was found that its fluorescence intensity in the presence of DNA was 4.7 times that in the absence of DNA, which confirmed that the peptoid binding to DNA could indeed induce its self-assembly (as shown in Figure 2). Figure 4 As shown in g). And because the assembly formed uniform particles, we tried to detect the formed particles using flow cytometry, and quantified them by reading the number of particles in a fixed volume (40 μL). We found that when reading the same volume, the number of particles in the presence of DNA was much greater than that in the absence of DNA (as shown in g). Figure 4 h in the figure).

[0059] Example 4: g-15 perturbs the intracellular spatial distribution

[0060] In order to verify the DNA-induced self-assembly behavior in bacteria, Bacillus subtilis was used as a model strain to conduct experimental observations on the intracellular DNA-induced peptidomimetic self-assembly behavior. The results showed that after the peptidomimetic g-15 was treated with Bacillus subtilis, aggregates were formed in the bacteria ( Figure 5 a in the figure). However, no such aggregation phenomenon was observed in the control group ( Figure 5 (a in the figure). The self-assembly formed within the cell is filled in the bacterial cytoplasm ( Figure 5 a) in the figure, this self-assembly may affect the function of intracellular protein aggregates. To verify this hypothesis, an in vitro experiment was established to use ParB aggregates to perform precise chromatin separation. The experiment confirmed that the presence of DNA will lead to the inhibition of related aggregate functions. For example, the CTP hydrolysis activity of ParB protein, the amount of RNA synthesis and the protein translation output decreased ( Figure 5b). We further hypothesize that after the peptoid molecules are taken up by bacteria, they are induced by bacterial DNA to form toxic assemblies, ultimately leading to bacterial death. First, Staphylococcus aureus treated with peptoid g-15 or PBS were observed by TEM. The results showed that the inner surface of the normal Staphylococcus aureus membrane would have an appropriate wrinkled morphology ( Figure 5 c). This inner membrane wrinkle is the correct form of the relevant protein. However, when treated with peptoids, the wrinkles gradually disappeared, and then the entire bacterial membrane ruptured and the contents leaked, and finally the bacteria died ( Figure 5 c). This bacterial inner membrane disruption phenomenon suggests that the peptidomimetic molecule is most likely induced by DNA in the bacteria to self-assemble, leading to the distribution and destruction of intracellular substances. To verify that this assembly can further cause bacterial cell inner membrane lysis, we designed the following experiment. An E. coli capable of expressing mCherry in the bacterial periplasm was constructed. When the peptidomimetic self-assembled in E. coli by DNA induction, the assembly contacted and interacted with the inner membrane, disrupting the bacterial inner membrane and causing the mCherry protein expressed in the bacterial periplasm to leak ( Figure 5 d in the figure). The experimental results showed that when mCherry was expressed in the bacterial periplasm, the mCherry protein was observed to be concentrated in the bacterial periplasm under confocal conditions ( Figure 5 d), after g-15 treatment, the bacteria were found to be full of mCherry protein, showing that the entire bacteria showed red fluorescence ( Figure 5 d) in the above.

[0061] Example 5: Animal Experiments on g-15

[0062] In vivo antibacterial effect: Given that Staphylococcus aureus is a major pathogen in community and clinical infections, we established three in vivo infection models of Staphylococcus aureus to evaluate the therapeutic potential of antimicrobial peptides. First, we established a mouse epidermal wound Staphylococcus aureus infection model for infection treatment testing and evaluated the bacterial load of the epidermal wound after drug administration ( Figure 6 a), the results showed that the bacterial load of mouse wounds could be significantly reduced after the second day of administration ( Figure 6 The bacterial load in the peptoid-treated group was reduced by 1.4 Lg CFU compared to the PBS-treated group ( Figure 6 b). After 7 days of continuous administration, the healing rate of epidermal wounds in the peptoid treatment group was 73%, which was significantly better than that of the PBS treatment group (58%). Figure 6c) and the wound healing rate during the entire treatment period was significantly better than that of the PBS control group. The weight of the mice increased steadily throughout the treatment process, indicating that the peptidomimetic compound did not show obvious side effects. We then further constructed a subcutaneous abscess model of mice infected with Staphylococcus aureus to evaluate the in vivo efficacy of the peptidomimetic compound. The results showed that the bacterial load in the main organs of the mice was significantly lower than that in the PBS control group. Even in the spleen and kidney, the bacterial load in the peptidomimetic group was lower than that of the control drug vancomycin ( Figure 6 e in). These experimental results further demonstrate that peptidomimetics can be applied to the study of more complex bacterial infection models. We know that Staphylococcus aureus is closely related to blood infections, and sepsis caused by blood infections has a very high mortality rate in clinical practice. Therefore, we extended the in vivo efficacy evaluation to the Staphylococcus aureus blood infection sepsis model. After the peptidomimetics were intravenously injected (10 mg / kg) twice, the mortality rate of mice was observed and counted. The results showed that both the peptidomimetics group and vancomycin group had a survival rate of 50%, with no significant difference, while the PBS group had a mortality rate of 100%, indicating that the drug-treated group can significantly improve the survival rate of mice after infection compared with the PBS group ( Figure 6 f). In summary, the peptoids showed excellent antibacterial activity in vivo and have great potential for drug development.

[0063] Example 6: Synthesis of j-14

[0064] For Boc-protected peptoids, arginine (Boc-Arg(Tip)-OH) and diamine linker molecules were mixed using a liquid phase synthesis method, and the amino acid and diamine were coupled using an acid amine condensation agent. The specific preparation process is as follows (taking the preparation of j-14 as an example):

[0065] (1) Weigh 1080.58 mg (2 mmol) of Boc-Arg(Tip)-OH amino acid into a round-bottom flask and dissolve it in anhydrous DMF.

[0066] (2) Weigh 608 mg (2.2 mM, 1 eq) of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride hydrate, dissolve it in water, and add it to a flask and stir at room temperature for 30 minutes to activate the amino acid.

[0067] (3) Weigh 1 eq of the diamine compound into a flask and continue stirring for 8-10 h. Monitor the reaction by TLC. After the reaction is complete, slowly add the reaction solution directly to the stirring beaker while stirring in ice water. A large amount of white precipitate will be seen. Filter with a Buchner funnel to remove the solvent to obtain a white solid crude product. The crude product is purified by column chromatography (dichloromethane:methanol 20:1) to obtain the target compound.

[0068] (4) The target compound of the previous step was dissolved in dichloromethane solvent, trifluoroacetic acid (final concentration of 20%) was added, and the mixture was stirred at room temperature for 2 h. The solvent was dried by rotary evaporation to obtain a crude product, which was then slurried with ether to form a salt, and the organic matter was washed with saturated sodium bicarbonate, and then extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The crude product after vacuum drying was purified by column chromatography (dichloromethane: methanol 5:1) to obtain the target compound j-14. Figure 7 H for J-14 1 NMR spectrum.

[0069] Example 7: Study on LPS-induced self-assembly of j-14 peptoids

[0070] In order to detect the self-assembly of peptoids, we conducted a Nile red dye test. Nile red is a hydrophobic dye that is insoluble in water. When the compound self-assembles, the emission wavelength of the dye can be detected by fluorescence spectroscopy to determine the minimum concentration of peptoid self-assembly. Figure 8 As shown in a in Figure 1, the assembly concentration (CMC) of the peptoid is 42 μg / mL. Interestingly, in the presence of additional LPS (16 μg / mL), the assembly concentration of the peptoid becomes smaller to 15.6 μg / mL, which indicates that the binding of LPS to the peptoid affects the self-assembly ability of the peptoid, that is, LPS induces the peptoid to self-assemble at a lower concentration. In order to further determine the size of the assembled particles, dynamic light scattering (DLS) characterization of the particles in the solution state is performed, as shown in Figure 1. Figure 8 As shown in b, the size of the particles formed at 128 μg / mL of the peptoid is about 100 nm, while in the presence of LPS, the particle size of the peptoid increases significantly, to about 600 nm. This indicates that LPS affects the particle size of the peptoid and aggregates the peptoid into larger particles. And because the assembly forms uniform particles, an attempt was made to detect the formed particles using a flow cytometer, and the number of particles in a fixed volume (40 μL) was read for quantification. It was found that when reading the same volume, the number of particles in the same volume in the presence of LPS was much greater than that in the absence of LPS (as shown in Figure 4). Figure 8 In order to observe the morphology of the peptoid more intuitively, a scanning electron microscope (SEM) was used to observe the formed particles, as shown in FIG. Figure 8 As shown in Figure d, the peptoids alone are small spheres of approximately 100 nm in size. However, after the addition of additional LPS, the peptoids aggregate to form larger spherical particles ranging in size from 450 to 750 nm. This confirms that peptoids bound to LPS can indeed induce their self-assembly.

[0071] Example 8: Synthesis steps of d-15

[0072] The preparation of d-15 is as follows:

[0073] (1) Weigh 1296 mg (2 mmol) of Fmoc-Arg(Pbf)-OH amino acid into a round-bottom flask and dissolve it in anhydrous DMF.

[0074] (2) Weigh 608 mg (2.2 mM, 1 eq) of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride hydrate, dissolve it in water, and add it to a flask and stir at room temperature for 30 minutes to activate the amino acid.

[0075] (3) Weigh 1 eq of the diamine compound into a flask and continue stirring for 8-10 h. Monitor the reaction by TLC. After the reaction is complete, slowly add the reaction solution directly to the stirring beaker while stirring in ice water. A large amount of white precipitate will be seen. Filter with a Buchner funnel to remove the solvent to obtain a white solid crude product. The crude product is purified by column chromatography (dichloromethane:methanol 20:1) to obtain the target compound.

[0076] The target compound in the previous step was dissolved in tetrahydrofuran solvent, piperidine (final concentration 20%) was added, and the mixture was stirred at room temperature for 2 h. The solvent was evaporated to obtain a crude product, which was purified by column chromatography (dichloromethane: methanol 5:1) to obtain the target compound d-15. Figure 9 H for d-15 1 NMR spectrum.

[0077] Example 9: Antibiotic Sensitization Test of Peptidomimetic Molecule d-15 against Gram-Negative Bacteria

[0078] Using d-15 as a sensitizer and clinical Acinetobacter baumannii (A. baumannii-77-1) as the screening target, 32 important antibiotics and FDA-approved non-antibiotic drugs were screened. The specific antimicrobial activity test experimental process is as follows (the operation process is sterile and all consumables are autoclaved):

[0079] The overnight cultured bacteria were diluted with fresh sterile MH medium to a final concentration of 5 × 10 5 CFU / mL was used as the working solution for a fixed bacterial concentration, where the final solution volume in a 96-well plate was 100 μL, the culture was incubated at 37°C and 220 RPM for 16-18 hours, and its absorbance at a wavelength of 595 nm was measured.

[0080] First, we performed sensitization screening of antibiotics. The antibiotics used were Figure 10The 32 representative important antibiotics with different antibacterial mechanisms shown in a in Figure 1 were tested for their ability to enhance the sensitivity of d-15 to IA32 (Important Antibiotics 32) after determining the minimum inhibitory concentration (MIC) of d-15 under the above culture conditions. The overnight cultured bacteria were diluted with fresh sterile MH medium to a final concentration of 5×10 5 CFU / mL was used as the fixed bacterial concentration of working solution A. After covering a 96-well plate (8×12) with 100 μL of working solution A, two parallel experimental groups were set up for each antibiotic. The drugs were diluted into eight doubling dilutions, with each 96-well plate testing five antibiotics. Two columns were reserved: one containing working solution A as a PC control, and the other containing uninoculated MH medium as an NC control. The cultures were incubated at 37°C and 220 rpm for 16-18 hours, and the absorbance at 595 nm was measured. This determined the antibacterial activity of each antibiotic against the clinical strain A. baumanii-77-1 under these conditions. Next, an equal volume of working solution A was added with d-15 at a concentration of 1 / 4 the MIC to create working solution B. Similarly, the antibacterial activity of IA32 was tested after the addition of d-15 at a concentration of 1 / 4 the MIC. This revealed which antibiotics d-15 could enhance sensitization. The test was repeated three times.

[0081] Through the above tests, we can obtain antibiotics that can be sensitized by d-15 against clinical strain A. baumannii-77-1, as shown in Figure 10c. d-15 can sensitize nine antibiotics, including Rifampicin, Bacitracin, and Vancomycin. To explore the differences in their sensitization abilities, we conducted a checkerboard experiment on these nine antibiotics and d-15, and the resulting checkerboard data is shown in the figure below. Figure 10 As shown in b, the specific experimental operations are as follows:

[0082] The overnight culture of A. baumannii-77-1 was diluted with fresh sterile MH medium to a final concentration of 5 × 10 5CFU / mL was used as the working solution with a fixed bacterial concentration. 100 μL of the working solution with a fixed bacterial concentration was used to cover the remaining wells of the 96-well plate except the last column, and 100 μL of the working solution was used to dilute d-15 10 times vertically and 7 times horizontally in the 96-well plate. Finally, the two drugs formed an 8×11 matrix with different concentration gradient combinations. In addition, 4 parallel fresh sterile MH culture medium wells were added to the last column of the 96-well plate as NC controls, and 4 parallel bacterial working solution wells without any drug treatment were used as PC controls. The 96-well plate was incubated at 220 rpm at 37°C for 16-24 hours, and its absorbance at a wavelength of 595 nm was measured. Each drug combination included at least two independent experiments. Each drug combination included at least two independent experiments. The important indicator for evaluating whether there is a synergistic effect when the drugs are combined is the graded inhibitory concentration index (FICi):

[0083]

[0084] MIC a and MIC b Represent the MIC of drug a and drug b alone; MIC ac and MIC bc Represent the MICs of drug a and drug b, respectively, when used in combination. An FICi ≤ 0.5 indicates synergy between the two drugs (i.e., the sensitizer has a sensitizing effect on the other drug). Each drug combination includes at least two independent checkerboard experiments, and the final FICi value for each drug combination is the average of these multiple measured FICi values.

[0085] Example 10: Sensitization of the peptidomimetic molecule d-15 to an FDA-approved non-antibiotic anti-Gram-negative bacteria test

[0086] We used 1235 non-antibiotic compounds from the FDA compound library (Selleck Chemicals, Houston, Texas) for sensitization screening. The screening process is shown below: Figure 11 As shown in a.

[0087] First, a preliminary screening of 1,375 non-antibiotic compounds in the FDA library was conducted, and 61 preliminary compounds with potential antibacterial sensitization were obtained. The specific experimental steps are as follows: Preparation of working solution: overnight culture of bacteria A. baumannii-77-1 was diluted with fresh sterile MH medium to a final concentration of 5×10 5CFU / mL was used as working solution A, which had a fixed bacterial concentration. An equal volume of working solution A was supplemented with 1 / 2 the MIC of d-15 to create working solution B. Due to the large number of drugs, a 384-well plate was used for preliminary screening. 30 μL of working solution A was added to each well of the 384-well plate, along with 50 μM of drug. Working solution A served as a PC control. A methyl urea base solution served as an NC control. The 96-well plate was incubated at 37°C and 220 rpm for 16-24 hours, and the absorbance at 595 nm was measured. This assay determined whether these FDA-approved compounds possessed antibacterial activity against the clinical strain A. baumanii-77-1. The working solution was then replaced with working solution B containing 1 / 4 the MIC of d-15, serving as a PC control. A methyl urea base solution served as an NC control. The 96-well plate was incubated at 37°C and 220 rpm for 16-24 hours, and the absorbance at 595 nm was measured. This assay determined whether these FDA-approved compounds possessed antibacterial activity against the clinical strain A. baumanii-77-1. The working solution was then replaced with working solution B containing 1 / 4 the MIC of d-15, serving as a PC control. The methyl urea base solution served as an NC control. By comparing the antibacterial differences in different solutions through the above experiments, we obtained 61 drugs that can be sensitized by d-15, that is, they have no antibacterial effect in working solution A, but have antibacterial effects in working solution B. The screening data is as follows Figure 11 As shown in b in FIG. , the evaluation result of the quality of the sensitization screening was good.

[0088] Next, to investigate the ability of these initially screened drugs to be sensitized by d-15, we further investigated their sensitizing antibacterial activity. By comparing the sensitization factor of these drugs by d-15 at 1 / 4 the MIC, we identified 16 top drugs that were at least 4-fold sensitized by d-15. The specific experimental steps were as follows: Working solution A and working solution B containing d-15 at 1 / 4 the MIC were prepared as described for the FDA non-antibiotic preliminary screening. A 96-well plate (8×12) was filled with 100 μL of working solution A. Two parallel groups were set up for each antibiotic. The drugs were diluted in eight doubling dilutions, with each 96-well plate testing five antibiotics. Two columns were reserved: one containing working solution A as a PC control and the other containing uninoculated MH medium as an NC control. The cultures were incubated at 37°C and 220 rpm for 16–18 hours, and the absorbance at 595 nm was measured. In this way, we can know the sensitization multiple of the candidate drug, and thus obtain 16 non-antibiotic drugs that can be sensitized at least four times. And compare the sensitization ability with the common sensitizer pentamidine. Using the same dose of sensitizer, we compare the MIC values ​​of these 16 sensitized non-antibiotics in the presence of sensitizer. The smaller the value, the stronger the sensitization ability of the sensitizer. The sensitization ability comparison data is as follows Figure 11 As shown in d.

[0089] We then conducted a chessboard synergistic antibacterial experiment on these 16 drugs and d-15, and obtained 6 non-antibiotic drugs with FICi values ​​less than 0.2, namely Penfluridol, Cinacalcet HCl, Aripiprazole, Toremifene Citrate, Tamoxifen, and Citrate Ponatinib. Among them, Penfluridol and #15 had the best synergistic antibacterial effect. The chessboard data table is as follows Figure 11 As shown in e.

[0090] Finally, the screening quality of all screening sensitization experiments was evaluated, and the evaluation results were as follows: Figure 11 As shown in c in Figure 2, it can be seen that the quality of the entire screening experiment is high.

[0091] Example 11: Penfluridol promotes the self-assembly of the peptidomimetic molecule d-15 and LPS

[0092] Nile red is an environmentally sensitive, lipophilic dye with an excitation wavelength of 550 nm and an emission wavelength of 590 nm. Nile red exhibits strong fluorescence in lipid-rich environments, but its fluorescence is quenched in aqueous solutions. Since d-15 exhibits a certain degree of hydrophilicity and hydrophobicity, it may possess the ability to self-assemble. Therefore, Nile red can be used as a fluorescent probe to monitor the assembly of d-15 peptidomimetics.

[0093] Step 1: Prepare a fluorescent dye solution containing 5μM Nile Red using ultrapure water. Step 2: Add 150μL of the fluorescent dye solution to 3 x 12 columns of a 96-well plate wrapped in tinfoil. Add #15 to the first well of each column, so that the #15 concentration in the well is 256μg / mL. Then, take another 150μL of the fluorescent dye solution and dilute it row by row, leaving the last row undiluted. This creates a series of 2-fold dilution gradient solutions with an initial #15 concentration of 128μg / mL, with the last row serving as the dye background value. Step 3: After preparing the 96-well plate containing the gradient dilutions of #15 in the fluorescent dye solution, wrap the 96-well plate in tinfoil and incubate in the dark for 30 minutes. Step 4: Use a fluorimeter to test the fluorescence of the solution prepared in step 3 by scanning a fixed range of emission wavelengths at a fixed excitation wavelength. First, test the fluorescent dye solution without d-15 as a background test, then test the solution from low to high concentrations of #15. The test solution volume was 100 μL, the excitation wavelength was set to 550 nm, and the emission wavelength range was scanned from 570 nm to 750 nm. The experiment was repeated twice or more.

[0094] Then, the fluorescence intensity at the maximum emission wavelength was selected to change with the concentration of d-15, such as Figure 12As shown in a in . d-15 has the behavior of a sudden increase in fluorescence intensity, that is, the aggregation behavior of d-15 in an aqueous environment changes, and from the properties of Nile red, it is known that d-15 forms hydrophobic aggregates in an aqueous solution environment. It can be judged that d-15 can self-assemble in this environment, and the concentration of the fluorescence sudden increase is called the critical micelle concentration (CMC) of d-15. Due to the sensitization property of d-15 against Gram-negative bacteria, we further used the lipid LPS unique to Gram-negative bacteria to explore the effect of LPS on the self-assembly behavior of d-15. First, after adding 16 μg / mL of LPS to the fluorescent dye environment, the effect of LPS at this concentration on the luminescence of the fluorescent dye solution can be ignored. The solution intensity is analyzed with the concentration change of d-15, as shown in the figure. Figure 12 As shown in Figure (a), the CMC value of d-15 is smaller after the addition of LPS, indicating that the self-assembly behavior of d-15 is enhanced. Furthermore, at the same d-15 concentration, the fluorescence intensity is greater after the addition of LPS. This indicates that LPS can promote the self-assembly behavior of d-15.

[0095] Based on the previous screening, we know that Penfluridol and d-15 have a better synergistic effect against Gram-negative bacteria. We want to explore whether Penfluridol will affect the self-assembly behavior of d-15 and LPS. We added 52μg / mL of Penfluridol to an environment containing 16μg / mL of LPS fluorescent dye, and analyzed the changes in the fluorescence intensity of the solution with the concentration of d-15 after deducting the background effect. Figure 12 As shown in a, it was found that after the addition of Penfluridol, the CMC value of d-15 was smaller than the CMC value of d-15 with only LPS added, and the solution of d-15 at the same concentration had a higher fluorescence intensity. It can be inferred that Penfluridol can promote the self-assembly of d-15 and LPS.

[0096] In order to further determine the assembly behavior of this multi-species assembly, dynamic light scattering (DLS) characterization of the particles in the solution state was performed, as shown in Figure 12 As shown in b, the size of the particles formed changes with the change of the components in the solution, and D d-15+LPS <D d-15 <D d-15+LPS+Penfluridol. Based on the above fluorescent probe related experiments, it can be inferred that after adding 16μg / mL of LPS, LPS and d-15 formed more small particles compared with d-15 itself, and after adding Penfluridol, Penfluridol promoted the agglomeration behavior between the small particles, forming larger assembled particles. We also tried to detect the formed particles using a flow cytometer, and quantified them by reading the number of particles in a fixed volume (50μL). It was found that when reading the same volume, the number of particles in the same volume was greater when LPS was present than when there was no LPS, which is consistent with our above-mentioned speculation on the assembly behavior of different components (such as Figure 12 (as shown in c in the figure).

[0097] In the above description, many specific details are set forth in order to fully understand the present invention. However, the above description is only a preferred embodiment of the present invention. The present invention can be implemented in many other ways different from those described herein, so the present invention is not limited to the specific implementation disclosed above. At the same time, any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A self-assembling peptidomimetic molecule, characterized in that It has the following structural formula:

2. A method for preparing the self-assembling peptidomimetic molecule according to claim 1, characterized in that: A liquid-phase peptide synthesis method is used to couple a diamine compound with Fmoc- or Boc-protected arginine through acid-amine condensation to synthesize the self-assembling peptidomimetic molecule, wherein the guanidine group in the Fmoc- or Boc-protected arginine carries a modified group, and the amino group in the Fmoc- or Boc-protected arginine is a free amino group that can be deprotected.

3. The preparation method according to claim 2, characterized in that The steps include: 1) dissolving the Fmoc- or Boc-protected arginine in anhydrous DMF; 2) adding an activating agent to the solution of step 1) to activate the Fmoc- or Boc-protected arginine; 3) adding the diamine compound to the solution obtained in step 2) and stirring to obtain a white precipitate, which is then separated.

4. The preparation method according to claim 3, characterized in that The following steps are also included: 4) dissolving the precipitate separated in step 3) in tetrahydrofuran solvent, adding piperidine, stirring at room temperature, and spinning off the solvent to obtain a crude product, which is then purified to obtain the peptidomimetic molecule.

5. An antibacterial agent comprising the self-assembling peptidomimetic molecule according to claim 1. An antibiotic sensitizing adjuvant comprising the self-assembling peptidomimetic molecule according to claim 1 .

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