Low-toxic broad-spectrum antimicrobial peptide with palindromic structure and application thereof

Abstract

This invention discloses a low-toxicity, broad-spectrum antimicrobial peptide with a palindromic structure and its applications. The invention uses two Dabs (D-Dab) as the center of symmetry, introducing one Leu (D-Leu) and two Trp (D-Trp) on each side to obtain an L-type or D-type antimicrobial peptide with a palindromic structure. This structure is novel, simple to design, low in manufacturing cost, and easy to use for clinical antimicrobial drug screening. In vitro antimicrobial and hemolysis experiments show that the palindromic antimicrobial peptide of this invention has strong inhibitory effects on both Gram-positive and Gram-negative bacteria, exhibiting broad-spectrum antimicrobial activity and low hemolytic toxicity. Therefore, it has good application prospects in the preparation of clinical antimicrobial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and relates to two low-toxicity, broad-spectrum antimicrobial peptides with palindromic structures. This invention also relates to the application of the said antimicrobial peptides in the preparation of clinical antimicrobial drugs. Background Technology

[0002] The discovery of antibiotics is considered one of the greatest revolutions in medical history, but due to the improper use of antibiotics, bacteria have developed resistance. To date, all antibiotics used clinically have shown bacterial resistance, and antibiotic resistance has become a major global public health problem (Lancet Infect Dis. 2021, 21(1):10-11), posing a great threat to global economic development and human health and safety. In 2019 alone, bacterial resistance caused approximately 5 million deaths worldwide (Lancet. 2022, 399(10325):629-655), and this threat is increasing year by year. It is estimated that by 2050, antibiotic resistance will cause approximately 300 million premature deaths (NPJ Antimicrob Resist. 2023, 1(1):17-38), increase medical costs by $1 trillion, and cause global economic losses of $100 trillion (NPJ Antimicrob Resist. 2023, 1(1):17-38; The World Bank, 2017). Therefore, there is an urgent need to develop new antibacterial agents to address the global life safety crisis caused by drug-resistant bacteria.

[0003] Antimicrobial peptides (AMPs), as small-molecule bioactive peptides, are widely found in organic organisms such as insects, animals, and microorganisms. They possess strong broad-spectrum antimicrobial activity and exhibit good antimicrobial activity against drug-resistant strains (Curr Med Chem. 2014, 21(20):2299-321). Antimicrobial peptides can exert their antimicrobial activity through non-receptor-mediated membrane action mechanisms, or by binding to bacterial DNA, RNA, proteins, or nucleic acids, or by achieving bactericidal effects through immune regulation. Due to their multiple mechanisms of action, antimicrobial peptides are less likely to induce bacterial resistance and are considered one of the ideal alternatives to traditional antibiotics (Nat Rev Microbiol. 2023, 4627-4740; Nat Biotechnol. 2006, 24(12):1551-1557).

[0004] In recent years, the design strategy of palindromic peptides has achieved good results. Many studies have shown that AMPs with palindromic structures usually exhibit high antibacterial activity and selectivity. Shan's group has conducted a lot of research on palindromic AMPs. The group obtained the antimicrobial peptide KL4A6 (LLKAAAKAAAKLL-NH2) with high bacterial selectivity by designing the palindromic peptide template XXYXXXYXXXYXX (X is Leu or Ala, Y is Lys) (Amino acids. 2016, 48(2): 403-417). Furthermore, the group designed the palindromic peptide template WXMXW-NH2 (M is KAAAKAAAK, X is Val, Phe, Trp or Ile) centered on "KAAAKAAAK". The antimicrobial peptide synthesized based on this template improved the antimicrobial activity of KL4A6 and broadened the antimicrobial spectrum (Int J MolSci. 2019, 20(6): 1417-1430). The group also designed the palindromic peptide template R with imperfect amphiphilicity. n (XRXXXRX)R n (n = 1 or 2; X is Ile, Phe, Trp or Leu), resulting in antifungal peptides with high activity, low toxicity, and good salt ion and serum tolerance (J Med Chem. 2018, 61(9): 3889-3907). Kim et al. designed palindromic antimicrobial peptides PST11-RK(KKFPWWWPFKK-NH2) and PST13-RK(KKKFPWWWPFKKK-NH2) with broad-spectrum antimicrobial activity centered on "WWW" (Int J Antimicrob Agents. 2006, 27(4): 325-330). The Ni group designed palindromic antimicrobial peptide templates such as XRRWWWRRX, XWRRRWX, XWWRRRWWX, and XRWRRRWRX (X = Ala, Val, Phe, Ile, Leu, or Trp), obtaining palindromic peptides FRRW (FRRWWWRRF-NH2) and LWWR (LWWRRRWWL-NH2) with high antimicrobial activity and low toxicity (Biochem Pharmacol. 2021, 186: 114470-114494; Acta Biomater. 2022, 154: 145-167). Based on these studies, this invention designs palindromic antimicrobial peptides with two "Dab" or "D-Dab" residues as the center of symmetry. Summary of the Invention

[0005] One of the objectives of this invention is to provide a low-toxicity, broad-spectrum antimicrobial peptide with a palindromic structure that is simple to design, easy to synthesize, has low preparation cost, and strong antibacterial activity.

[0006] Another object of the present invention is to provide the application of the above-mentioned low-toxicity broad-spectrum antimicrobial peptide with palindromic structure in the preparation of clinical antimicrobial drugs.

[0007] To achieve its purpose, the present invention adopts the following technical solution:

[0008] I. Structural Design of Low-Toxicity Broad-Spectrum Antimicrobial Peptides with Palindromic Structures

[0009] The low-toxicity, broad-spectrum antimicrobial peptide involved in this invention has a palindromic structure, with two Dabs or D-Dabs as symmetry centers, exhibiting a palindromic structure. Its general structural formula is as follows:

[0010] YYZXXXXZYY-NH2, denoted as P-YYZXX;

[0011] Where X is Dab or D-Dab; and when X is Dab, Y is Trp, Z is Leu, denoted as P-YYZXX-L, the antimicrobial peptide structure is D-Trp-D-Trp-D-Leu-D-Dab-D-Dab-D-Dab-D-Leu-D-Trp-D-Trp; when X is D-Dab, Y is D-Trp, Z is D-Leu, denoted as P-YYZXX-D, the antimicrobial peptide structure is Trp-Trp-Leu-Dab-Dab-Dab-Dab-Leu-Trp-Trp.

[0012] II. Application of Low-Toxicity Broad-Spectrum Antimicrobial Peptides with Palindromic Structures in the Preparation of Clinical Antimicrobial Drugs

[0013] 1. In vitro antibacterial test

[0014] The classic micro-dilution method was used to determine the minimum inhibitory concentration (MIC) of the above-mentioned antimicrobial peptides against Gram-positive strains (S. aureus ATCC25923, B. subtilis ATCC 23857, S. epidermidis ATCC 12228, E. faecalis ATCC 29212) and Gram-negative strains (E. coli ATCC 25922, K. pneumoniae ATCC 700603, P. aeruginos ATCC27853, A. baumannii ATCC 19606), thus evaluating their antimicrobial activity. Specifically, the experimental bacteria grown to mid-log phase were diluted to 1×10⁻⁶ using MH broth. 6CFU / mL bacterial suspensions were prepared. Antimicrobial peptides were dissolved in sterile water and diluted twice with MH broth to prepare a series of antimicrobial peptide analog solutions of different concentrations. These solutions were then mixed with an equal volume of bacterial suspension and added to 96-well culture plates. After incubation at 37°C for 18 hours, the lowest concentration at which no significant bacterial growth was observed was defined as the minimum inhibitory concentration (MIC) of the antimicrobial peptide analog. Polymyxin B was used as a positive control. The experiment was repeated three times in parallel, and the results are shown in Table 1.

[0015] Table 1. Minimum inhibitory concentrations of the low-toxicity, broad-spectrum palindromic antimicrobial peptides of this invention against standard strains.

[0016]

[0017] The results in Table 1 show that the palindromic antimicrobial peptides P-YYZXX-D and P-YYZXX-L designed in this invention have strong antimicrobial activity against common experimental strains and have a strong inhibitory effect on both Gram-positive and Gram-negative bacteria, exhibiting broad-spectrum antimicrobial activity.

[0018] 2. Hemolysis test

[0019] Fresh blood (1000g) from healthy mice was centrifuged at 4℃ for 10 min. The supernatant plasma was discarded, and the lower blood cell layer was washed with physiological saline to prepare a suspension containing 8% blood cells. 100 μL / well was added to a 96-well plate. Antimicrobial peptide analogs were dissolved in physiological saline and diluted twice to prepare peptide solutions of different concentrations. Equal volumes of these solutions were added to the 96-well plates containing the blood cell suspension and incubated at 37℃ for 1 h. After centrifugation at 1200g for 15 min, 100 μL / well of the supernatant was transferred to a new 96-well plate, and the absorbance at 490 nm was measured using a microplate reader. The physiological saline group served as a negative control, and 1% Triton X-100 served as a positive control. The formula was: Hemolysis rate (%) = [(OD...] 490nm peptides -OD 490nm negative control ) / (OD 490nm positive control -OD 490nm negative control )]×100%, calculate the hemolysis rate, and the result is as follows Figure 1 .

[0020] Figure 1 The results showed that the palindromic antimicrobial peptides P-YYZXX-D and P-YYZXX-L of the present invention both had low hemolytic toxicity, with a hemolysis rate of less than 10% at the highest detection concentration (256 μM), indicating high safety.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention uses two Dabs (D-Dab) as the center of symmetry, introducing one Leu (D-Leu) and two Trp (D-Trp) on each side to obtain L-type or D-type antimicrobial peptides with palindromic structures. The structures are novel, simple to design, low in manufacturing cost, and easy to use for screening clinical antimicrobial drugs. In vitro antimicrobial and hemolysis experiments show that the palindromic antimicrobial peptides of this invention have strong inhibitory effects on both Gram-positive and Gram-negative bacteria, exhibiting broad-spectrum antimicrobial activity and low hemolytic toxicity. Therefore, they have promising applications in the preparation of clinical antimicrobial drugs. Attached Figure Description

[0023] Figure 1 The figure shows the hemolytic toxicity test results of the palindromic antimicrobial peptide of this invention;

[0024] Figure 2 This is the mass spectrum of the palindromic antimicrobial peptide P-YYZXX-D of the present invention;

[0025] Figure 3 This is the mass spectrum of the palindromic antimicrobial peptide P-YYZXX-L of this invention. Detailed Implementation

[0026] The synthesis methods of the low-toxicity broad-spectrum antimicrobial peptides P-YYZXX-D and P-YYZXX-L with palindromic structures of the present invention will be described in detail below through specific embodiments.

[0027] Example 1: Synthesis of antimicrobial peptide P-YYZXX-D

[0028] (1) Resin activation and pretreatment

[0029] Accurately weigh 0.444 g (0.2 mmol) of MBHA resin (substitution value 0.45 mmol / g) and place it in the synthesizer. After swelling with dichloromethane solution and washing with DMF, the resin was tested using the ninhydrin colorimetric method. The resin was colorless and transparent, indicating that the resin was normal.

[0030] (2) Synthesis of P-YYZXX-D-resin

[0031] The MBHA resin that passed the above test was treated with a DMF solution (v / v) containing 20% ​​piperidine to remove the Fmoc protecting group. The resin was tested using the ninhydrin colorimetric method; a blue-purple color indicated that the Fmoc protecting group had been removed. After washing with DMF, three times the excess (0.6 mmol) of Fmoc-D-Trp(Boc)-OH, three times the excess (0.6 mmol) of HBTU and HOBT, and six times the excess (1.2 mmol) of DIEA were dissolved in redistilled DMF and added to the synthesizer. The condensation reaction was carried out for 1 hour. The resin was tested using the ninhydrin colorimetric method; a colorless and transparent color indicated that the condensation reaction was successful, yielding Fmoc-D-Trp(Boc)-resin.

[0032] Following the above method, the following amino acids were sequentially condensed: Fmoc-D-Trp(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Dab(Boc)-OH, Fmoc-D-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-D-Dab(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, and Fmoc-D-Trp(Boc)-OH, with the same amounts of amino acids, HOBT, HBTU, and DIEA as above, to obtain Fmoc-D-Trp(Boc)-D-Trp(Boc)-D-Leu-D-Dab(Boc)-D-Dab(Boc)-D-Dab(Boc)-D-Dab(Boc)-D-Leu-D-Trp(Boc)-D-Trp(Boc)-resin.

[0033] The terminal Fmoc protecting group was removed by a DMF solution containing 20% ​​piperidine to obtain D-Trp(Boc)-D-Trp(Boc)-D-Leu-D-Dab(Boc)-D-Dab(Boc)-D-Dab(Boc)-D-Leu-D-Trp(Boc)-D-Trp(Boc)-resin.

[0034] (3) Peptide cleavage

[0035] The obtained D-Trp(Boc)-D-Trp(Boc)-D-Leu-D-Dab(Boc)-D-Dab(Boc)-D-Dab(Boc)-D-Dab(Boc)-D-Leu-D-Trp(Boc)-D-Trp(Boc)-resin was washed sequentially with dichloromethane and methanol, thoroughly compressed and dried, and then the polypeptide chain was cleaved by adding 60%-90% trifluoroacetic acid solution to finally obtain D-Trp-D-Trp-D-Leu-D-Dab-D-Dab-D-Dab-D-Leu-D-Trp-D-Trp-NH2. After extraction with ice-cold ether and water, it was freeze-dried to obtain crude peptide lyophilized powder.

[0036] (4) Peptide purification

[0037] The crude peptide lyophilized powder obtained above was separated and purified by RP-HPLC. The eluent was collected, then freeze-dried, and identified by mass spectrometry as P-YYZXX-D with a molecular weight of 1387.77 Da. The mass spectrum is shown below. Figure 2 The RP-HPLC purification conditions were as follows: mobile phase A: 0.1% TFA / water; mobile phase B: 0.1% TFA / acetonitrile; linear gradient elution, and collection of the eluent with the main absorption peak.

[0038] Example 2: Synthesis of P-YYZXX-L

[0039] (1) Resin activation and pretreatment

[0040] Same as Example 1.

[0041] (2) Synthesis of P-YYZXX-L-resin

[0042] The following amino acids, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH and Fmoc-Trp(Boc)-OH, were sequentially condensed using the same method as in Example 1. The terminal Fmoc protecting group was removed with a DMF solution containing 20% ​​piperidine to obtain Trp(Boc)-Trp(Boc)-Leu-Dab(Boc)-Dab(Boc)-Dab(Boc)-Leu--Trp(Boc)--Trp(Boc)-resin.

[0043] (3) Peptide cleavage

[0044] Same as Example 1.

[0045] (4) Peptide purification

[0046] Same as in Example 1, P-YYZXX-L was identified by mass spectrometry with a molecular weight of 1387.77 Da. The mass spectrum is shown below. Figure 3 .

Claims

1. A low-toxicity broad-spectrum antibacterial peptide having a palindromic structure, characterized in that, The antimicrobial peptide has a palindromic structure with two Dabs or D-Dabs as its symmetry center, and its general structural formula is as follows: YYZXXXXZYY-NH2, denoted as P-YYZXX; Where X is Dab or D-Dab; and when X is Dab, Y is Trp and Z is Leu, denoted as P-YYZXX-L; when X is D-Dab, Y is D-Trp and Z is D-Leu, denoted as P-YYZXX-D.

2. The application of the low-toxicity, broad-spectrum antimicrobial peptide with a palindromic structure as described in claim 1 in the preparation of clinical antimicrobial drugs, characterized in that, The bacteria inhibited by the antibacterial drug are Gram-positive or Gram-negative bacteria; the Gram-positive bacteria are Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis and / or Enterococcus faecalis, and the Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and / or Acinetobacter baumannii.

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