Cyclopeptide for inhibiting drug-resistant bacterium AcrB protein as well as delivery vector and application of cyclopeptide
The problem of antibiotic resistance is solved by designing a cyclic peptide that can bind AcrB protein with high affinity and bringing it into the bacterial membrane through a delivery vehicle, and improving antibiotic sensitivity is achieved.
Patent Information
- Application Number
- CN202510282150.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-03-14
- Filing Date
- 2025-03-11
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to effectively inhibit AcrB protein in gram-negative bacteria, resulting in antibiotic resistance problems.
A cyclic peptide was designed to inhibit its efflux of antibiotics by binding to AcrB protein and to bring the cyclic peptide into the bacterial membrane through a delivery vehicle, enhancing the sensitivity of the antibiotics.
By inhibiting the efflux function of AcrB protein, the minimum inhibitory concentration (MIC) of antibiotics is reduced, the sensitivity of bacteria to antibiotics is improved, and antibiotic resistance is reversed.
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Figure CN120098087A_ABST
Abstract
Description
[0001] This application claims the priority of patent application No. 202410290640.4 (the filing date of the prior application is March 14, 2024, and the name of the invention is A cyclic peptide for inhibiting AcrB protein of drug-resistant bacteria and its delivery vector and application). Technical Field
[0002] The present invention belongs to the technical field of biomedicine and relates to a cyclic peptide for inhibiting AcrB protein of drug-resistant bacteria and a delivery vector and application thereof. Background Art
[0003] According to statistics, about 4.95 million people died from diseases caused by bacterial antibiotic resistance. Among them, the multidrug resistance of Gram-negative bacteria is increasing. Its isolation rate in ICU wards is as high as 34%, and its multidrug resistance rate is as high as 44%. It often causes serious infections in various parts of the human body such as the digestive tract, respiratory tract, blood, urinary system, postoperative and wounds. Among them, AcrB is a typical protein in the multidrug resistance efflux mechanism of Gram-negative bacteria. It is located in the bacterial inner membrane, and AcrA is located in the periplasmic space, connecting AcrB and Tolc, crossing the bacterial inner and outer membranes in the form of trimers, and actively pumping substances in the bacteria out of the cell. This tripartite complex is the most clinically relevant efflux pump. Inhibiting the efflux of antibiotics by AcrB protein is an important strategy to reverse antibiotic resistance. Summary of the invention
[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a cyclic peptide for inhibiting the AcrB protein of drug-resistant bacteria and its delivery vector and application.
[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:
[0006] In a first aspect, the present invention provides a cyclic peptide for inhibiting the AcrB protein of drug-resistant bacteria, wherein the amino acid sequence of the cyclic peptide comprises any one of the sequences shown in SEQ ID No.1-SEQ ID No.15.
[0007] SEQ ID No. 1: MVIP.
[0008] SEQ ID No. 2: TTNYTVYVLVQIIQL.
[0009] SEQ ID No. 3: VYWLLSIVAYPSISA.
[0010] SEQ ID No. 4: VYWLLSIVAYPSLSS.
[0011] SEQ ID No. 5: VYWLLSIVAYPSIST.
[0012] SEQ ID No. 6: VYWLLSIVAYPSLSP.
[0013] SEQ ID No. 7: VYWLLSIVAYPSISP.
[0014] SEQ ID No. 8: VYWLLSIVAYPSISS.
[0015] SEQ ID No.9: KLVCSLYTRF.
[0016] SEQ ID No. 10: AIACSIIWTL.
[0017] SEQ ID No. 11: KLTCLILKTF.
[0018] SEQ ID No. 12: AIACTLIKGWAEL.
[0019] SEQ ID No. 13: SIACKLLTLWKQ.
[0020] SEQ ID No. 14: KLACTIGRLLW.
[0021] SEQ ID No. 15: KLICQLTCYVYGW.
[0022] The polypeptides corresponding to the amino acid sequences shown in SEQ ID No.1 to SEQ ID No.15 are named L1 to L15 respectively.
[0023] Preferably, the amino acid sequence of the cyclic peptide includes any one of the sequences shown in SEQ ID No.1-SEQ ID No.10.
[0024] The above cyclic peptide can bind to AcrB protein with a binding rate of more than 20% and a binding constant of less than 100 nM.
[0025] In a second aspect, the present invention provides a method for synthesizing a cyclic peptide for inhibiting the AcrB protein of drug-resistant bacteria according to the first aspect, the method comprising: synthesizing the polypeptide by a polypeptide solid phase synthesis method, adding an amino acid to the N-terminus and the C-terminus of the polypeptide respectively, and forming a ring from head to tail.
[0026] Preferably, the cyclization method includes disulfide bond cyclization, molecular fragment connection cyclization or thioether bond connection cyclization.
[0027] Preferably, the amino acid added to the N-terminus of the polypeptide includes cysteine or tyrosine.
[0028] Preferably, the amino acid added to the C-terminus of the polypeptide includes cysteine or glycine.
[0029] The peptide chain is cyclized in the following manner:
[0030] Ringing method 1:
[0031] A cysteine (C) is added to the N-terminus and the C-terminus of the peptide chain sequence, and a disulfide bond is formed through the two cysteines at the head and tail to form a ring. Taking polypeptide L1 as an example, the chemical formula of the cyclic peptide formed by polypeptide L1 in this way is shown in Formula I.
[0032]
[0033] Ring forming method 2:
[0034] A cysteine (C) is added to each of the N-terminus and the C-terminus of the peptide chain sequence, and the two cysteines at the head and tail are connected by a molecular fragment. Taking polypeptide L1 as an example, polypeptide L1 is ring-formed in this way, and the chemical formula of the cyclic peptide formed is shown in Formula II.
[0035]
[0036] Ringing method three:
[0037] A tyrosine (Y) is added to the N-terminus of the peptide chain sequence, a glycine (G) is added to the C-terminus, and the tyrosine and cysteine at the head and tail are connected by a thioether bond. Taking polypeptide L1 as an example, polypeptide L1 is ring-formed in this way, and the chemical formula of the cyclic peptide formed is shown in Formula III.
[0038]
[0039] The polypeptides L1-L10 are cyclized by the third method to obtain cyclic peptides H1-H10; the polypeptides L11-L15 are cyclized by the first method to obtain cyclic peptides H11-H15.
[0040] In a third aspect, the present invention provides a delivery vector-encapsulated cyclic peptide, wherein the delivery vector-encapsulated cyclic peptide comprises the cyclic peptide described in the first aspect and a delivery vector, wherein the cyclic peptide is encapsulated inside the delivery vector.
[0041] The delivery carrier loaded with cyclic peptide of the present invention can be stably dispersed in the aqueous solution in the form of nanoparticles, and the size of the nanoparticles is 100-180nm. The delivery carrier loaded with cyclic peptide can enhance the sensitivity of bacteria to antibiotics and reduce the MIC of antibiotics.
[0042] Preferably, the molar ratio of the cyclic peptide to the delivery vector is (1-8):8.
[0043] The specific point values in (1-8) can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, etc. Other specific point values within the above numerical range can be selected and will not be listed here one by one.
[0044] Preferably, the delivery vehicle comprises LIPO-20 and LIPO-S.
[0045] The LIPO-20 comprises palmitic acid, a polypeptide sequence 1 and a polypeptide sequence 2 which are connected in sequence.
[0046] The LIPO-S comprises palmitic acid, polypeptide sequence 1 and polypeptide sequence 3 which are connected in sequence.
[0047] The amino acid sequence of polypeptide sequence 1 includes the sequence shown in SEQ ID No.16, the amino acid sequence of polypeptide sequence 2 includes the sequence shown in SEQ ID No.17, and the amino acid sequence of polypeptide sequence 3 includes the sequence shown in SEQ ID No.18,
[0048] SEQ ID No. 16: LVFFA.
[0049] SEQ ID No. 17: KKRAKKFFKKPRVIGVSIPF.
[0050] SEQ ID No. 18: SGSGSGSGSG.
[0051] Preferably, the molar ratio of LIPO-20 to LIPO-S is 1:(0.2-1).
[0052] The specific point values in (0.2-1) can be selected as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc. Other specific point values within the above numerical range can be selected, and they will not be described one by one here.
[0053] In a fourth aspect, the present invention provides a method for preparing the delivery vector-loaded cyclic peptide according to the third aspect, the preparation method comprising: mixing the delivery vector with a solvent, rotary evaporating, and then mixing with the cyclic peptide solution to obtain the product.
[0054] Preferably, the solvent comprises methanol and water.
[0055] Preferably, the cyclic peptide solution comprises a cyclic peptide and a cosolvent.
[0056] Preferably, the co-solvent comprises dimethyl sulfoxide and hexafluoroisopropanol.
[0057] Preferably, the volume of the cosolvent is 1%-2% of the volume of the cyclic peptide solution, for example 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc. Other specific values within the above numerical range can be selected and will not be described one by one here.
[0058] Preferably, the mixing with the cyclic peptide solution is performed under ultrasonic conditions.
[0059] In a fifth aspect, the present invention provides a use of the cyclic peptide for inhibiting the AcrB protein of drug-resistant bacteria according to the first aspect or the cyclic peptide encapsulated by the delivery vector according to the third aspect in the preparation of a drug for enhancing the sensitivity of bacteria to antibiotics.
[0060] Preferably, the antibiotics include quinolone antibiotics, cephalosporin antibiotics, aminoglycoside antibiotics or penicillin antibiotics.
[0061] Preferably, the bacteria include Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa or Klebsiella pneumoniae.
[0062] Compared with the prior art, the present invention has the following beneficial effects:
[0063] The present invention discloses a cyclic peptide inhibitor for reversing antibiotic resistance. First, a cyclic peptide that can form a high affinity with the AcrB protein is designed and screened; second, the delivery carrier encapsulates the cyclic peptide to the bacterial membrane, causing membrane disturbance, and the cyclic peptide enters the bacterial membrane and binds to the AcrB on the inner membrane to inhibit the efflux of antibiotics, thereby enhancing the sensitivity of bacteria to antibiotics and reducing the MIC of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS
[0064] Figure 1 It is the DLS of cyclic peptide H7 and delivery vector before and after encapsulation.
[0065] Figure 2 It is the DLS of cyclic peptide H2 and delivery vector before and after encapsulation.
[0066] Figure 3 It is the DLS of cyclic peptide H5 and delivery vector before and after encapsulation.
[0067] Figure 4 Figure 1 is the TEM of cyclic peptide H8 and the delivery vector before and after encapsulation, where Figure A is the TEM before encapsulation and Figure B is the TEM after encapsulation.
[0068] Figure 5 Figure 1 is the TEM of cyclic peptide H4 and the delivery vector before and after encapsulation, where Figure A is the TEM before encapsulation and Figure B is the TEM after encapsulation.
[0069] Figure 6Figure 1 is the TEM of cyclic peptide H1 and the delivery vector before and after encapsulation, where Figure A is the TEM before encapsulation and Figure B is the TEM after encapsulation.
[0070] Figure 7 It is the MIC of the delivery vector-encapsulated cyclic peptide H7 and quinolones (ciprofloxacin) against Escherichia coli.
[0071] Figure 8 It is the MIC of the delivery vector-encapsulated cyclic peptide H6 and cephalosporin (cefazolin) against Pseudomonas aeruginosa.
[0072] Fig. 9 It is the MIC of the delivery vector-encapsulated cyclic peptide H9 and aminoglycoside (kanamycin) against Klebsiella pneumoniae.
[0073] Fig.10 It is the MIC of the delivery vector-encapsulated cyclic peptide H15 and penicillins (amoxicillin) against Acinetobacter baumannii.
[0074] Fig.11 It is the binding rate of the cyclic peptide H1-H15 designed by the present invention and the AcrB protein.
[0075] Fig.12 It is the binding constant between the cyclic peptide H1-H15 designed by the present invention and the AcrB protein. DETAILED DESCRIPTION
[0076] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.
[0077] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.
[0078] The LIPO-20 and LIPO-S involved in the following embodiments are artificially synthesized. For example, the following preparation method can be used:
[0079] (1) A peptide solid phase synthesis method using the FMOC strategy, using amino acids with FMOC protection at the amino terminus as raw materials, sequentially connecting the required amino acids in the peptide sequence from right to left to obtain a peptide, and then adding palmitic acid to connect the palmitic acid to the peptide (connection is performed by a solid phase synthesis method);
[0080] The synthetic reagents are as follows:
[0081] (a) Carrier resin: wang resin;
[0082] (b) Deprotection reagent: 5% (mass fraction) anhydrous piperazine solid + 2% (volume fraction) 1,8-diazacycloundecene-7 (DBU) + 98% (volume fraction) DMF;
[0083] (c) Coupling reagent used in the condensation reaction: 5% N-methylmorpholine + 95% DMF;
[0084] (2) Prepare cleavage solution: 0.125 mL of deionized water + 0.125 mL of triisopropylsilane + 4.75 mL of trifluoroacetic acid. Add the prepared cleavage solution to a cleavage bottle containing polypeptide monomer molecules. Add a magnetic rotor to the cleavage bottle for stirring. Place the cleavage bottle in a 0°C ice water bath at a speed of 300-400 r / min and stir for 3 hours to cleave the polypeptide monomer molecules from the carrier resin. Then wash (mainly to remove the cleavage solution) and dry to obtain the product.
[0085] Example 1
[0086] This embodiment provides a delivery vector and a cyclic peptide encapsulated therein:
[0087] The preparation method of the delivery vector is as follows:
[0088] LIPO-20 and LIPO-S were dissolved in DMSO respectively to obtain mother solutions. The DMSO mother solutions of LIPO-20 and LIPO-S were respectively taken, mixed, and ultrapure water was added while vortexing. The mixture was assembled at room temperature (25°C) for 1 hour. The concentration of LIPO-20 in the assembly mixture was 25 μM, and the concentration of LIPO-S was 25 μM. The volume of DMSO accounted for 1% of the total volume of the assembly mixture.
[0089] The preparation method of the delivery carrier-loaded cyclic peptide is as follows:
[0090] LIPO-20 and LIPO-S were dissolved in hexafluoroisopropanol, and the cyclic peptide was dissolved in DMSO to obtain mother liquors. The hexafluoroisopropanol mother liquors of LIPO-20 and LIPO-S were respectively taken, mixed and added with methanol while vortexing, and then evaporated to form a film attached to flask 1. The DMSO mother liquor of H7 was taken and added with ultrapure water while ultrasonicating. The cyclic peptide solution was added to flask 1 while ultrasonicating, and H7 was loaded into the carrier. The concentration of LIPO-20 in the assembly mixture was 25 μM, the concentration of LIPO-S was 25 μM, the concentration of cyclic peptide was 3.125 μM, and the volume of hexafluoroisopropanol and DMSO accounted for 1% of the total volume of the assembly mixture.
[0091] The size of the delivery carrier before and after encapsulation of the cyclic peptide was characterized. Figure 1 (DLS results), as shown in the figure, the hydrate particle size measured by DLS is about 68.1±23.2nm before encapsulation and about 106±16nm after encapsulation.
[0092] Example 2
[0093] This example provides a delivery vector and a cyclic peptide encapsulated therein. The only difference between this example and Example 1 is that the cyclic peptide encapsulated therein is H2, and its concentration is 6.25 μM. The size of the delivery vector before and after encapsulation of the cyclic peptide is characterized. Figure 2 (DLS results), as shown in the figure, the hydrate particle size measured by DLS was about 78.8±23.6nm before encapsulation and about 145.7±37.8nm after encapsulation.
[0094] Example 3
[0095] This embodiment provides a delivery vector and a cyclic peptide encapsulated therein. The only difference between this embodiment and embodiment 1 is that the cyclic peptide encapsulated therein is H5. The size of the delivery vector before and after the cyclic peptide encapsulation is characterized. Figure 3 (DLS results), as shown in the figure, the hydrate particle size measured by DLS was about 58.8±19.1nm before encapsulation and about 120.1±30.68nm after encapsulation.
[0096] Example 4
[0097] This embodiment provides a delivery vector and a cyclic peptide encapsulated therein. The difference between this embodiment and embodiment 1 is that the cyclic peptide encapsulated therein is H8, and its concentration is 6.25 μM. The morphology of the delivery vector before and after the cyclic peptide encapsulation is characterized. Figure 4 (TEM results) show that the loading was successful.
[0098] Example 5
[0099] This embodiment provides a delivery vector and a cyclic peptide encapsulated therein. The difference between this embodiment and embodiment 1 is that the cyclic peptide encapsulated therein is H4, and its concentration is 6.25 μM. The morphology of the delivery vector before and after the cyclic peptide encapsulation is characterized. Figure 5 (TEM results) show that the loading was successful.
[0100] Example 6
[0101] This embodiment provides a delivery vector and a cyclic peptide encapsulated therein. The difference between this embodiment and embodiment 1 is that the cyclic peptide encapsulated therein is H1. The morphology of the delivery vector before and after the cyclic peptide encapsulation is characterized. Figure 6 (TEM results) show that the loading was successful.
[0102] Test Example 1
[0103] Combination of delivery vector-loaded cyclic peptides and antibiotics to combat the MIC of drug-resistant bacteria
[0104] The final concentration of the cyclic peptide encapsulated by the delivery vector was determined to be 25 μM. The concentration of the antibiotic was changed, and the cyclic peptides and antibiotics encapsulated by different delivery vectors were added to a 96-well plate in turn. The same volume of bacterial solution and culture medium were added and cultured in a 37-degree incubator for 18 hours. The bacterial survival rate was scanned with an ELISA instrument and plotted.
[0105] The results are as follows Figure 7-10 As shown, compared with the use of antibiotics alone, the combination of the cyclic peptide encapsulated by the delivery vector and the antibiotics significantly enhanced the sensitivity of bacteria to the antibiotics and reduced the MIC of the antibiotics.
[0106] Test Example 2
[0107] Fifteen cyclic peptides were screened by mRNA display technology. The results are as follows Fig.11 shown.
[0108] Test Example 3
[0109] The two classic molecular docking technologies, AutoDockVina and SMINA, were combined, and AcrB protein (PDB: 6ZO7) was selected as the analysis object. The docking process used Monte Carlo sampling to search the conformational space of the analysis object. The KD values of the binding of 15 cyclic peptides to AcrB protein were calculated by computer simulation. The results are as follows Fig.12 shown.
[0110] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the cyclic peptide for inhibiting the AcrB protein of drug-resistant bacteria and its delivery vector and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
[0111] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.
[0112] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.
Claims
1. A cyclic peptide for inhibiting AcrB protein of drug-resistant bacteria, characterized in that: The amino acid sequence of the cyclic peptide includes any one of the sequences shown in SEQ ID No.1-SEQ ID No.
15.
2. The cyclic peptide for inhibiting AcrB protein of drug-resistant bacteria according to claim 1, characterized in that: The amino acid sequence of the cyclic peptide includes any one of the sequences shown in SEQ ID No.1-SEQ ID No.
10.
3. A method for synthesizing a cyclic peptide for inhibiting the AcrB protein of drug-resistant bacteria according to claim 1 or 2, characterized in that: The method comprises: synthesizing the polypeptide by using a polypeptide solid phase synthesis method, adding an amino acid to the N-terminus and the C-terminus of the polypeptide respectively, and forming a ring from head to tail to obtain the polypeptide.
4. The method for synthesizing a cyclic peptide for inhibiting the AcrB protein of drug-resistant bacteria according to claim 3, characterized in that: The ring-forming method includes disulfide bond ring-forming, molecular fragment connection ring-forming or thioether bond connection ring-forming; Preferably, the amino acid added to the N-terminus of the polypeptide includes cysteine or tyrosine; Preferably, the amino acid added to the C-terminus of the polypeptide includes cysteine or glycine.
5. A delivery vector containing a cyclic peptide, characterized in that: The delivery vector-loaded cyclic peptide comprises the cyclic peptide according to claim 1 or 2 and a delivery vector, and the cyclic peptide is loaded inside the delivery vector.
6. The delivery carrier carrying cyclic peptide according to claim 5, characterized in that: The molar ratio of the cyclic peptide to the delivery vector is (1-8):8; Preferably, the delivery vector comprises LIPO-20 and LIPO-S; Preferably, the molar ratio of LIPO-20 to LIPO-S is 1:(0.2-1) 。 7. A method for preparing a delivery carrier-loaded cyclic peptide according to claim 5 or 6, characterized in that: The preparation method comprises: mixing the delivery carrier with a solvent, rotary evaporating the mixture into a film, and then mixing the mixture with a cyclic peptide solution to obtain the product.
8. The method for preparing a delivery carrier-loaded cyclic peptide according to claim 7, characterized in that: The solvent includes methanol and water; Preferably, the cyclic peptide solution comprises a cyclic peptide and a cosolvent; Preferably, the cosolvent comprises dimethyl sulfoxide and hexafluoroisopropanol; Preferably, the volume of the co-solvent is 1%-2% of the volume of the cyclic peptide solution.
9. Use of the cyclic peptide for inhibiting AcrB protein of drug-resistant bacteria according to claim 1 or 2 or the cyclic peptide encapsulated by the delivery vector according to claim 5 or 6 in the preparation of a drug for enhancing the sensitivity of bacteria to antibiotics.
10. The use according to claim 9, characterized in that: The antibiotics include quinolone antibiotics, cephalosporin antibiotics, aminoglycoside antibiotics or penicillin antibiotics; Preferably, the bacteria include Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa or Klebsiella pneumoniae.