Small-molecule inhibitor of targeted bacterial DsbA enzyme and application of small-molecule inhibitor
By designing small molecule inhibitors targeting bacterial DsbA enzyme, the problem of weak transmembrane targeting ability in existing technologies was solved, and efficient inhibition of DsbA enzyme and antibiotic sensitization effects were achieved, with good biocompatibility.
Patent Information
- Application Number
- CN202510902586.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-01
- Publication Date
- 2025-09-23
AI Technical Summary
Existing DsbA enzyme inhibitors have weak transmembrane targeting capabilities, resulting in low bioavailability and difficulty in effectively inhibiting bacterial pathogenicity and drug resistance.
A small molecule inhibitor targeting bacterial DsbA enzyme was designed, which includes membrane targeting and transmembrane functional modules, a connection module and a DsbA enzyme targeting functional module. Through artificial intelligence technology screening and experimental verification, a polypeptide fragment with nM level affinity was formed and prepared by solid-phase peptide synthesis.
It achieves efficient targeted inhibition of the DsbA enzyme, significantly reduces bacterial toxicity and drug resistance, enhances the sensitization effect of antibiotics, and has good biocompatibility and cell safety.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule inhibitor targeting bacterial DsbA enzyme and applications thereof. Background Art
[0002] With the widespread use of antibiotics, the development of bacterial resistance is becoming increasingly serious. Resistance is particularly prominent in the Gram-negative bacterium Escherichia coli, with some bacteria even showing resistance to nearly all antibiotics. New antimicrobial targets or new antimicrobial agents are urgently needed to address this resistance. Current antimicrobial strategies include β-lactamase inhibitors, antimicrobial peptides, and outer membrane permeability enhancers. However, these approaches still face challenges such as the development of resistance and toxic side effects.
[0003] DsbA (Disulfidebond A) is an oxidoreductase located in the bacterial periplasm, primarily responsible for disulfide bond formation in bacterial proteins. Disulfide bonds, formed by covalent bonds between two cysteine residues through a sulfur atom, are a stabilizing structural feature of many proteins, playing a particularly important role in outer membrane proteins, secretory proteins, and certain cytokines. By catalyzing disulfide bond formation within the cell, DsbA participates in the conformational activation of hundreds of periplasmic proteins, including those involved in bacterial pathogenicity, such as the secretory system, flagella, and pili; extracellular virulence factors such as Shiga toxins and adhesins; and antibiotic-modifying enzymes such as β-lactamases. These functional proteins are closely linked to bacterial pathogenicity and drug resistance. Furthermore, DsbA is highly conserved among various Gram-negative bacteria (e.g., Escherichia coli, Klebsiella, Pseudomonas aeruginosa, and Acinetobacter baumannii). Compared to traditional antibiotics, DsbA inhibitors pose no selective pressure for survival. Therefore, the development of targeted inhibitors for DsbA enzyme has important research value in bacterial pathogenicity, drug resistance and new drug development.
[0004] Existing reports on inhibitors of the Escherichia coli DsbA enzyme can be roughly divided into two categories. One is peptide analogs of the DsbA molecular chaperone DSBB, and the other is small molecule analogs of benzofuran. However, the affinity of these two types of DsbA enzyme inhibitors at the protein level is at the μM level, and in verification experiments at the bacterial level, hundreds of μM or mM are required to see significant inhibition, resulting in low bioavailability. Combined with existing reported literature, it is speculated that the reason for this phenomenon is the weak ability of the molecules to target across the bacterial outer membrane and the periplasmic DsbA.
[0005] Therefore, this application combines the good biocompatibility characteristics of the polypeptide itself, and uses artificial intelligence technology combined with experimental experience to design a dual-targeting mechanism polypeptide with targeting across the bacterial outer membrane and targeting DsbA from scratch. Summary of the Invention
[0006] The purpose of the present invention is to provide a small molecule inhibitor targeting bacterial DsbA enzyme and its application to solve the problem of carbapenem resistance in bacteria and reduce the infectivity and toxicity of bacteria in infection.
[0007] To achieve the above objectives, the present invention provides a small molecule inhibitor targeting bacterial DsbA enzyme, which consists of the following three functional modules:
[0008] (1) Membrane targeting and transmembrane functional module: comprising an amphipathic cationic α-helical transmembrane peptide, the amino acid sequence of which is shown in SEQ ID NO. 1;
[0009] (2) Linker module: including one of lysine K, alanine A, serine S, glycine G, or no linker amino acid;
[0010] (3) Targeting functional module for DsbA enzyme: A 10-amino acid peptide fragment was designed for the active site of Escherichia coli K-12 DsbA enzyme through virtual screening technology. The peptide fragment formed a disulfide bond with the DsbA enzyme and the affinity was verified to be at the nM level through Gnina docking.
[0011] Preferably, the active sites of the Escherichia coli K-12 DsbA enzyme are: 36, 64, 148, 63, 147, 150, 149, 30, 151, 178, 174, 32.
[0012] Preferably, the sequence of the DsbA enzyme targeting functional module is shown as SEQ ID NO.2.
[0013] Preferably, the structure of the small molecule inhibitor is as shown below:
[0014]
[0015] The sequence of the small molecule inhibitor is shown in SEQ ID NO.3.
[0016] In another aspect, the present invention provides a method for designing the above-mentioned small molecule inhibitor targeting bacterial DsbA enzyme, comprising the following steps:
[0017] S1. Screening membrane targeting and transmembrane functional modules, connection modules and DsbA enzyme targeting functional modules respectively;
[0018] S2. The membrane targeting and transmembrane functional modules were spliced with the DsbA enzyme targeting functional module through the linker module. AlphaFold2 modeling was used to verify that an α-helical structure was formed, a disulfide bond was formed with the DsbA enzyme, and the docking affinity was at the nM level.
[0019] S3. Functional verification: Through reverse verification at the protein level and bacterial level experiments, a full-length peptide with dual-targeting function is obtained.
[0020] On the other hand, the present invention provides a method for preparing the above-mentioned small molecule inhibitor targeting bacterial DsbA enzyme, which adopts a solid phase peptide synthesis method, and the specific steps include:
[0021] S1. Weigh 300-600 mg of resin and calculate the amount of amino acid to be used according to the formula: resin loading × resin mass × 10 times excess;
[0022] S2. Mixing amino acids with O-benzotriazole-tetramethyluronium hexafluorophosphate in a molar ratio of 1:1, and sequentially linking the amino acid sequences of the membrane targeting and transmembrane functional modules, the linker module, and the DsbA enzyme targeting functional module to the resin for peptide synthesis;
[0023] S3. After the synthesis is completed, the protecting groups on the amino acid side chains are removed and the peptide is cleaved from the resin using hydrogen bromide cleavage solution to obtain a powdered peptide;
[0024] S4. Wash the powdered polypeptide with ether, dry, and purify to obtain the target polypeptide.
[0025] On the other hand, the present invention provides a use of the above-mentioned small molecule inhibitor targeting bacterial DsbA enzyme in the preparation of antibacterial drugs.
[0026] Therefore, the present invention provides a small molecule inhibitor targeting bacterial DsbA enzyme and its application, which has the following beneficial effects:
[0027] (1) The present invention combines experimental experience with artificial intelligence technology to design peptide inhibitors targeting bacterial DsbA enzymes from scratch, and ultimately obtains inhibitors that show good attenuation and sensitization effects through screening;
[0028] (2) The small molecule inhibitor targeting bacterial DsbA enzyme prepared by the present invention has bacterial targeting properties and can stably cross the bacterial outer membrane and be captured by the DsbA enzyme in the periplasmic space, thereby reducing bacterial toxicity and solving the problem of bacterial resistance. The KD of protein affinity detection is 288nM. Under the condition of using 40μM of the small molecule inhibitor targeting bacterial DsbA enzyme, a significant inhibitory effect can be observed;
[0029] (3) The small molecule inhibitor targeting bacterial DsbA enzyme prepared by the present invention inhibits the activity of β-lactamase from the upstream, and the dosing interval is longer. In addition, since DsbA modifies a large number of substrate proteins, the small molecule inhibitor targeting bacterial DsbA enzyme prepared by the present invention can also inhibit the formation of biofilms and has different degrees of sensitization effects on different types of antibiotics. The highest synergistic multiple can sensitize carbapenem-resistant Escherichia coli by 16 times.
[0030] (4) The small molecule inhibitor targeting bacterial DsbA enzyme prepared by the present invention has good biocompatibility. No hemolysis occurred under the condition of 160 μM in the hemolysis experiment. In the cytotoxicity experiment, in the cytotoxicity detection using mouse fibroblasts (L929) and human umbilical vein endothelial cells (HUVEC), the cell survival rate was greater than 90% under the condition of 200 μM.
[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.
[0033] Figure 1 Schematic diagram of the molecular design of the small molecule inhibitor targeting bacterial DsbA enzyme of the present invention;
[0034] Figure 2 This is the ESI result diagram of the small molecule inhibitor targeting bacterial DsbA enzyme of the present invention;
[0035] Figure 3 The results of the experimental study on the inhibitory effect of the combination of the small molecule inhibitor targeting bacterial DsbA enzyme and antibiotics on bacterial proliferation are shown in the figure.
[0036] Figure 4 The results are experimental results of the bacterial killing effect of the combination of the small molecule inhibitor targeting bacterial DsbA enzyme and antibiotics of the present invention;
[0037] Figure 5 The results of the synergistic sensitization test on Escherichia coli by combining the small molecule inhibitor targeting bacterial DsbA enzyme and antibiotics of the present invention are as follows;
[0038] Figure 6 These are the cytotoxicity test results of the small molecule inhibitor targeting bacterial DsbA enzyme of the present invention, where A is the survival rate of HUVEC cells and B is the survival rate of L929 cells;
[0039] Figure 7 Figure 3 is the biofilm inhibition rate of the small molecule inhibitor targeting bacterial DsbA enzyme of the present invention on carbapenem-resistant multidrug-resistant Escherichia coli, wherein A is a crystal violet staining diagram of the untreated group; B is the biofilm content of the groups treated with different drug concentrations;
[0040] Figure 8The results of the molecular binding force determination between the small molecule inhibitor targeting bacterial DsbA enzyme of the present invention and DsbA enzyme;
[0041] Figure 9 These are the hemolytic assay results of the small molecule inhibitors targeting bacterial DsbA enzymes of the present invention. DETAILED DESCRIPTION
[0042] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.
[0043] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.
[0044] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.
[0045] Example 1
[0046] A small molecule inhibitor targeting bacterial DsbA enzyme is a polypeptide molecule. The molecular design diagram is shown in the figure Figure 1 As shown, the polypeptide molecule includes a membrane targeting and transmembrane functional module, a DsbA enzyme targeting functional module and a connection module between the two functional modules.
[0047] The membrane targeting and transmembrane functional module is an amphipathic cationic α-helical transmembrane peptide, and its amino acid sequence is shown in SEQ ID NO.1.
[0048] SEQ ID NO. 1: N-KKRAKKFFKKPRVIGVSIPF-C.
[0049] The linker module includes lysine (K), alanine (A), serine (S), glycine (G), or no linker amino acid is added as the intermediate linker.
[0050] The DsbA enzyme targeting functional module is a DsbA enzyme inhibitory peptide. DsbA of Escherichia coli K-12 (PDB Code: 1FVK), which has high similarity to DsbA enzymes of different subtypes of Gram-negative bacteria, is selected as the receptor protein.
[0051] The DsbA enzyme from Escherichia coli K-12 has a known crystal-resolved active site. The Colab version of AfDesign-binder+hotspot test was used to design a targeting functional fragment targeting the DsbA enzyme.
[0052] The active sites were set to "36, 64, 148, 63, 147, 150, 149, 30, 151, 178, 174, 32" and the length was set to 10 amino acids. The formation of disulfide bonds and the affinity of Gnina docking simulation at the nM level were used as screening conditions to obtain a functional module, whose sequence is shown in SEQ ID NO. 2.
[0053] SEQ ID NO. 2: PFNPNDCQPF.
[0054] The amphipathic cationic α-helical transmembrane peptide and DsbA enzyme inhibitory peptide were spliced through the connection module and modeled by the Colab version of AlphaFold2 using MMseqs2 to form a disulfide bond with DsbA, the affinity after docking using the docking software was at the nM level, and the single molecule had an α-helical structure as screening conditions to obtain the full-length molecule.
[0055] The splicing method adopts solid phase peptide synthesis method, which includes the following steps:
[0056] (1) Weigh 300-600 mg of resin, which is the support for solid-phase peptide synthesis.
[0057] (2) Calculation of amino acid dosage: The required amount of amino acid for each connection is calculated according to "resin loading amount × resin mass × 10 times excess". Excess amino acid is used to ensure that the reaction can proceed fully.
[0058] (3) The molar ratio of amino acid to HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate) was determined to be 1:1.
[0059] (4) Linking synthesis: According to the preferred amino acid sequences of the membrane targeting and transmembrane functional modules and the DsbA enzyme targeting functional module, amino acids are linked to the resin in sequence for synthesis.
[0060] (5) Side chain removal and cleavage: After the synthesis is completed, the protecting groups on the amino acid side chains are first removed, and then the synthesized polypeptide is cleaved from the resin using hydrogen bromide cleavage solution.
[0061] (6) Post-treatment: The polypeptide is washed with ether to obtain a powdered polypeptide, which is then dried and purified to obtain a high-purity target polypeptide (P1), the sequence of which is shown in SEQ ID NO. 3.
[0062] SEQ ID NO. 3: KKRAKKFFKKKPFNPNDCQPF.
[0063] Its structure is:
[0064]
[0065] The structure of peptide P1 was verified by ESI. Figure 2 shown.
[0066] Test Example 1
[0067] The steps for the bacterial proliferation inhibition experiment are as follows:
[0068] The bacterial proliferation was determined by broth culture. A single clone of clinically isolated multidrug-resistant Escherichia coli (E. coli MDR ESBL-1 (Clinical), published in ACS Nano. 2022, 16, (12): 20545-20558) was incubated in LB broth at 37°C with a shaker at 180 rpm overnight. The next day, 1% of the bacterial suspension was added to fresh LB broth and cultured to a standard absorbance of OD 600nm =0.1(1.0×10 8 CFU / mL) and diluted to 1.5×10 6 CFU / mL.
[0069] In a 96-well plate, add 100 μL of the above bacterial suspension and 100 μL of different drugs to each well. Set up the following drug groups: control (PBS buffer), meropenem (MER) group, peptide inhibitor group (P1), and P1 + MER combination group. Incubate at 37°C and measure absorbance at 600 nm using a microplate reader at 1, 2, 4, 6, 8, 10, 12, and 15 hours. Repeat the experiment at least three times.
[0070] The results are as follows Figure 3 As shown, compared with the control group, the use of MER or P1 alone could not inhibit bacterial proliferation; while the combination of P1 and MER could significantly inhibit bacterial proliferation.
[0071] Test Example 2
[0072] The bactericidal kinetics experiment steps are as follows:
[0073] The bacterial proliferation was determined using the broth culture method. A single clone of clinically isolated multidrug-resistant E. coli (E. coli MDR ESBL-1 (Clinical)) was incubated in LB broth at 37°C with a shaker at 180 rpm overnight. The next day, 1% of the bacterial suspension was added to fresh LB broth and incubated until the absorbance reached OD 0. 600nm =0.1(1.0×10 8 CFU / mL) and diluted to 1.5×10 6 CFU / mL.
[0074] In a 96-well plate, add 100 μL of the above bacterial suspension and 100 μL of different drugs to each well. The drug groups were set as follows: control group (PBS buffer), meropenem (MER) group, peptide inhibitor group (P1), and P1 + MER combination group. Incubate at 37°C, set time points, and use the plate count method to record bacterial survival at 0.5, 1, 2, 4, and 6 hours: add 100 μL to a LB solid agar plate, spread evenly with a spreader, and incubate inverted at 37°C for 18 hours. The colony-forming units (CFU) were counted.
[0075] The results are as follows Figure 4 As shown, it can be seen that the combination of P1 and MER can quickly kill bacterial pathogens within 1 hour; while the use of MER or P1 alone cannot effectively kill bacteria within 6 hours.
[0076] Test Example 3
[0077] Antibiotic synergy test, the steps are as follows:
[0078] The broth dilution method was used to determine the synergistic effect of P1 and the antibiotics tetracycline (TET), meropenem (MER), ampicillin (AMP), kanamycin sulfate (KAN), tetracycline (TET), and levofloxacin (LVFX). Monoclonal clinically isolated multidrug-resistant Escherichia coli (E. coli MDR), standard competent strain E. coli B2, and urinary tract infection E. coli UPEC were incubated in M63 broth medium at 37°C and shaken at 180 rpm overnight. The next day, 1% of the above bacterial suspension was added to fresh M63 broth medium and standardized to an absorbance of OD 600nm =0.1(1.0×10 8 CFU / mL) and diluted to 1.5×10 6 CFU / mL.
[0079] Take a 96-well plate and add 100 μL of the above bacterial suspension and different antibiotics to each well. Set 4 replicates for each concentration. Set blank culture medium as blank group and no drug group as control group. After incubation at 37°C for 18 hours, measure OD 600nm The absorbance was measured and the FIC index was calculated.
[0080] The results are as follows Figure 5 As shown, it can be seen that P1, when used in combination with antibiotics TEX, LVFX, KAN or MER, has a synergistic sensitization effect on a variety of Escherichia coli, with a synergistic index (FIC Index) <0.5; there is no significant synergistic sensitization effect on AMP.
[0081] Test Example 4
[0082] Cytotoxicity assay, steps are as follows:
[0083] (1) Cell culture: L929 cells (mouse epithelial fibroblasts) and HUVECs (human umbilical vein endothelial cells) were first selected and, after trypsinization, resuspended in 1640 complete medium containing 10% fetal bovine serum and 1% triple antibodies (penicillin, streptomycin, and amphotericin B). After counting using a hemocytometer, the cells were seeded into 96-well plates at 8,000 cells / 100 μL per well. 200 μL PBS was added to the outermost wells of the 96-well plate to prevent marginalization. The 96-well plate was placed in a cell culture incubator and cultured for 24 h before use. The next step of the experiment was performed after the cells adhered to the wall.
[0084] (2) Drug incubation: Add P1 molecules with a concentration gradient of 200, 160, 80, 40, 20, and 10 μM prepared using complete culture medium. Set up 6 replicate wells for each concentration, add 100 μL of drug solution, and place in a CO2 incubator. After incubation with cells for 24 hours, proceed to the next experiment.
[0085] (3) Add CCK-8 reagent: After 24 hours of drug treatment, remove the medium containing the drug and rinse with PBS to remove the drug. Add 100 μL of CCK-8 diluent (diluted to V CCK-8 ∶V 培养基 =1∶9).
[0086] (4) Detection: After adding CCK-8 diluent for 2 hours, the absorbance of each well at 450nm was measured using a microplate reader. s In addition, a blank control well A was also set up. b (no cells, no drug treatment), control well A c (with cells, without drug treatment).
[0087] The formula for calculating cell viability is:
[0088]
[0089] Among them, A s is the absorbance of the experimental well at 450 nm, A b A is the absorbance of the blank control well at 450 nm. c The absorbance of the control wells was at 450 nm.
[0090] The results are as follows Figure 6 As shown in the figure, A is the survival rate of HUVEC cells and B is the survival rate of L929 cells. It can be seen that the polypeptide monomer molecule P1 has no toxic side effects on cells (L929 and HUVEC). The cell survival rate of P1 at a dose of 200 μM is above 80%, indicating good biosafety.
[0091] Test Example 5
[0092] The biofilm removal experiment steps are as follows:
[0093] (1) Preparation of bacterial suspension: Carbapenem-resistant multidrug-resistant Escherichia coli (E. coli MDR ESBL) was used as the test bacteria. Bacteria in the logarithmic growth phase were taken and washed with PBS. The bacterial count was measured in PBS and the OD was controlled. 600 =0.1(1×10 8 CFU / mL), and then diluted to 3×10 6 CUF / mL for later use.
[0094] (2) Preparation of high-glucose TSB: Prepare a high-concentration sterile β-glucose solution and a TSB broth containing (w / v) 1% glucose.
[0095] (3) Drug preparation: Prepare P1 according to the concentration gradient of 320, 160, 80 and 40 μM for use.
[0096] (4) Drug incubation: In a 96-well plate, 50 μL of the bacterial solution in (1), 50 μL of PBS, and 100 μL of high-glucose TSB broth were taken as the positive control group. 100 μL of PSB and 100 μL of high-glucose TSB broth were taken as the negative control group. 50 μL of the bacterial solution in (1), 50 μL of different concentrations of P1 solution, and 100 μL of high-glucose TSB broth were taken as the experimental group, with three replicates set for each concentration.
[0097] (5) Static culture: Incubate in a 37°C constant temperature incubator for 24 h.
[0098] (6) Crystal violet staining: First, after 24 h of culture, the culture medium was discarded from the 96-well plate and the plate was washed twice with PBS. The plate was inverted and dried at 37°C for 30 min. Then, the biofilm was fixed with 100 μL of anhydrous methanol for 15 min, and the plate was inverted and dried at 37°C for 20 min. Then, 50 μL of 0.1% (w / v) crystal violet solution was added to each well for staining for 15 min, and the excess crystal violet was washed with PBS, and the plate was dried. Finally, 100 μL of 95% ethanol was added to each well to dissolve the crystal violet bound to the biofilm.
[0099] (7) Biofilm detection: The absorbance of the experimental group, positive control group, and negative control group at 600 nm was measured using an enzyme-labeled instrument, and the biofilm content was calculated according to the following formula:
[0100]
[0101] Among them, A sis the absorbance of the experimental group at 600 nm, A 阴 is the absorbance of the negative control group at 600 nm, A 阳 is the absorbance of the positive control group at 600 nm.
[0102] The results are as follows Figure 7 As shown, the peptide inhibitor P1 was concentration-dependent and effectively removed bacterial biofilms. At 80 μM, the biofilm content was less than 20%.
[0103] Test Example 6
[0104] The biofilm interferometry (BLI) binding assay was performed as follows:
[0105] (1) Probe activation: Select a nickel ion sensor probe. The probe surface is functionalized with nickel ions and has a certain affinity to bind to the histidine tag (His) on the target protein.
[0106] (2) Sample preparation: Prepare solutions of the peptide to be tested at different concentrations. The concentration needs to be adjusted according to the experimental needs, usually by diluting the sample in a gradient dilution.
[0107] (3) Baseline determination: Before starting the experiment, a baseline determination was performed using PBST buffer. The purpose of this step is to ensure that the optical signal of the instrument is stable and provide a reference baseline for subsequent data measurements.
[0108] (4) Molecular binding: The molecule to be tested is brought into contact with the target molecule immobilized on the surface. The intermolecular binding reaction is observed, and the BLI instrument obtains binding kinetic information by monitoring the changes in the spectral signal in real time. The binding process usually lasts for several minutes. During this time, an increase in the thickness of the probe surface and an increase in the signal value can be observed.
[0109] (5) Molecular dissociation: The sample is eluted from the sensor surface and the bound molecules are removed from the surface using PBS wash buffer. By monitoring the signal drop, the kinetics of dissociation can be obtained. The duration and rate of the dissociation process are also recorded and analyzed in real time.
[0110] (6) Data analysis: Based on the obtained binding and dissociation curves, the following key parameters can be calculated:
[0111] Association rate constant (ka): the rate of association between molecules.
[0112] Dissociation rate constant (kd): The rate at which a molecule dissociates after binding.
[0113] Affinity constant (KD): Binding affinity, usually the ratio of ka and kd (KD = kd / ka), reflects the strength of the intermolecular interaction.
[0114] like Figure 8 As shown, the binding force is 288.8 nM.
[0115] Test Example 7
[0116] The hemolytic assay is performed as follows:
[0117] Fresh blood was collected from ICR mice to evaluate the hemolytic activity of P1 molecule.
[0118] (1) Blood treatment: Freshly drawn ICR mouse blood was slowly added with normal saline at a volume ratio of 1:2. The blood containing normal saline was centrifuged at 1500 rpm and 4°C for 15 min. The precipitate was retained and resuspended in an equal volume of normal saline. This step was repeated three times until the supernatant was clear. 200 μL of the precipitate, i.e., red blood cells, was taken and added to 10 mL of normal saline to prepare a 2% blood cell count.
[0119] (2) Drug preparation: 1 mL of P1 molecules with a final concentration gradient of 160, 80, 40, 20, 10, and 5 μM were prepared using normal saline;
[0120] (3) Hemolytic assay: 1 mL of material solution with different concentrations was mixed with 1 mL of red blood cell suspension, incubated at 37°C for 3 h, and centrifuged at 4000 rpm and 4°C for 2 min. 200 μL of supernatant was taken from each centrifuge tube and added to a 96-well plate. The absorbance As of each drug concentration at 540 nm was read using a microplate reader. A negative control was set up using physiological saline (A b ), deionized water was used as positive control (A c ), the hemolysis rate is calculated as follows:
[0121]
[0122] like Figure 9 As shown in the figure, P1 did not show hemolysis at a concentration of 160 μM, indicating that P1 has good biocompatibility.
[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A small molecule inhibitor targeting bacterial DsbA enzyme, characterized in that It consists of the following three functional modules composition: (1) Membrane targeting and transmembrane functional module: comprising an amphipathic cationic α-helical transmembrane peptide, the amino acid sequence of which is shown in SEQ ID NO. 1; (2) Linker module: including one of lysine K, alanine A, serine S, glycine G, or no linker amino acid; (3) Targeting functional module for DsbA enzyme: A 10-amino acid peptide fragment was designed for the active site of Escherichia coli K-12 DsbA enzyme through virtual screening technology. The peptide fragment formed a disulfide bond with the DsbA enzyme and the affinity was verified to be at the nM level through Gnina docking.
2. A small molecule inhibitor targeting bacterial DsbA enzyme according to claim 1, characterized in that The active sites of Escherichia coli K-12 DsbA enzyme are: 36, 64, 148, 63, 147, 150, 149, 30, 151, 178, 174, 32.
3. A small molecule inhibitor targeting bacterial DsbA enzyme according to claim 1, characterized in that: The sequence of the DsbA enzyme targeting functional module is shown in SEQ ID NO.
2.
4. A small molecule inhibitor targeting bacterial DsbA enzyme according to claim 1, characterized in that: The structure of small molecule inhibitors is shown below: The sequence of the small molecule inhibitor is shown in SEQ ID NO.
3.
5. A method for designing a small molecule inhibitor targeting bacterial DsbA enzyme according to any one of claims 1 to 4, characterized in that: The steps include: S1. Screening membrane targeting and transmembrane functional modules, connection modules and DsbA enzyme targeting functional modules respectively; S2. The membrane targeting and transmembrane functional modules were spliced with the DsbA enzyme targeting functional module through the linker module. AlphaFold2 modeling was used to verify that an α-helical structure was formed, a disulfide bond was formed with the DsbA enzyme, and the docking affinity was at the nM level. S3. Functional verification: Through reverse verification at the protein level and bacterial level experiments, a full-length peptide with dual-targeting function is obtained.
6. A method for preparing a small molecule inhibitor targeting bacterial DsbA enzyme according to any one of claims 1 to 4, characterized in that: The solid phase peptide synthesis method is used, and the specific steps include: S1. Weigh 300-600 mg of resin and calculate the amount of amino acid to be used according to the formula: resin loading × resin mass × 10 times excess; S2. Mixing amino acids with O-benzotriazole-tetramethyluronium hexafluorophosphate in a molar ratio of 1:1, and sequentially linking the amino acid sequences of the membrane targeting and transmembrane functional modules, the linker module, and the DsbA enzyme targeting functional module to the resin for peptide synthesis; S3. After the synthesis is completed, the protecting groups on the amino acid side chains are removed and the peptide is cleaved from the resin using hydrogen bromide cleavage solution to obtain a powdered peptide; S4. Wash the powdered polypeptide with ether, dry, and purify to obtain the target polypeptide.
7. Use of the small molecule inhibitor targeting bacterial DsbA enzyme according to any one of claims 1 to 4 in the preparation of antibacterial drugs.