Effect of N-methylated and fatty acid conjugation on analogs of antimicrobial peptide Anoplin

https://doi.org/10.1016/j.ejps.2020.105453Get rights and content

Abstract

With the increment of drug-resistant bacteria and the slow development of novel antibiotics, antimicrobial peptides have gained increasing attention as a potential antibiotic alternative. They not only displayed a broad-spectrum antimicrobial activity but also were difficult to induce resistance development because of their unique membrane-lytic activity. Herein, to improve the limitations of Anoplin, the N-methyl amino acids were first used to replace the amino acids of Anoplin at sensitive enzymatic cleave sites (Leu, Ile, Lys and Arg). Afterward, the N-methylated analogs M3.6/M4.7/M5.7 with high stability were screened out and further modified by N-terminal fatty acid conjugation to develop new antimicrobial peptide analogs with both potent antimicrobial activity and high proteolytic stability, and 12 new Anoplin analogs Cn-M3.6/M4.7/M5.7 (n = 8,10,12,14) were designed and synthesized. Our results showed that compared with native Anoplin, the stability of these N-methylated lipopeptides against trypsin and chymotrypsin degradation were increased by 104–106 times. Besides, they still possessed potent antimicrobial activity under physiological salts and serum environment. Among them, the new designed analogs C12-M3.6/M4.7/M5.7 showed the optimal antimicrobial activity, synergy and additive effects were also observed when they were combined with traditional antibiotics polymyxin B, rifampin, and kanamycin. Moreover, they could effectively inhibit the formation of biofilms by P. aeruginosa and S. aureus. The antimicrobial mechanism studied revealed that these N-methylated lipopeptides could display a rapid bactericidal effect by destroying the bacterial cell membrane. Notably, no detectable resistance of these new designed peptides was developed after continuous cultured with E. coli for 20 passages. In summary, we have designed a new class of antimicrobial peptide analogs with potent antimicrobial activity and high proteolytic stability through N-methyl amino acids substitution and N-terminal fatty acid conjugation. This study also provides new ideas and methods for the modification of antimicrobial peptides in the future.

Introduction

In recent years, the high incidence and mortality of bacterial infectious diseases have posed a serious threat to global health, and antibiotics have been widely used to treat infections caused by various pathogenic bacteria since 1928. However, with the emerging of drug-resistance bacteria caused by the abuse of antibiotics, it is an urgent need to develop novel antimicrobial agents to solve this problem. Antimicrobial peptides (AMPs) have gained considerable attention as a promising antibiotic candidate to combat against drug-resistance bacteria (Hancock, 1997; Ghosh et al., 2019; Ageitos et al., 2017). They exhibited antimicrobial activity through unique membrane-lytic action, other than conventional antibiotics which targeted specific sites (Mishra et al., 2017). Therefore, it is difficult for bacteria to modify their membrane components to develop resistance. Moreover, AMPs exhibit various bioactivities against not only Gram-negative and Gram-positive bacteria, but also fungus and parasite.

Several AMPs have been applied in clinical application to treat against bacterial infection. For example, polymyxin B and colistin (polymyxin E) were used to treat urinary system infection caused by Gram-negative bacteria (Sierra et al., 2017). Besides, Daptomycin, a cyclic lipopeptide, was approved to treat complicated skin and skin structure infections caused by Gram-positive bacteria (Carpenter and Chambers, 2004). However, there are still many limitations that restrict further development of natural AMPs. The first is their poor stability towards proteolytic degradation, which resulted in low bioavailability (Kim et al., 2014). Another is their sensibility to physiological salt and serum conditions (Yu et al., 2011). Besides, low selectivity between the bacterial membrane and mammalian cells membrane, high manufacture cost, and weak pharmacokinetics property also limit their clinical applications (Mourtada et al., 2019; Molhoek et al., 2011). To develop novel AMPs with potent antimicrobial activity, low cytotoxicity, and high proteolytic stability, a considerable number of strategies have been applied. For example, the use of unnatural amino acids such as D-form amino acids and N-methyl amino acids, cyclization, PEG modification, fatty acids conjugation, the application of prodrug strategy and peptidomimetic (Molhoek et al., 2011; Li et al., 2017; Malmsten, 2016; Skovbakke et al., 2015; Cao et al., 2018).

N-methyl amino acids were widely present in natural peptides. For example, Cyclosporin A, a cyclic peptide with seven N-methylation amino acids, possessed potent bioactivity and good oral bioavailability (Biron et al., 2008). It was demonstrated that N-methylation could significantly improve various bioactivities of peptides, including therapeutic efficacy, proteolytic stability, membrane affinity, and receptor selectivity (Biron et al., 2008; Haviv et al., 1993; Harris et al., 2009).

Additionally, it was well known that lipidation could significantly enhance the antimicrobial activity of AMPs (Lee et al., 2019). Siriwardena et al. reported that conjugation fatty acid with peptide dendrimer to develop TNS18, which displayed not only potent antimicrobial activity against Gram-negative multidrug-resistant bacteria and MRSA but also possessed high serum stability (Siriwardena et al., 2017). Previous research demonstrated that the addition of fatty acid increased hydrophobicity, thus improved hydrophobic interaction between peptides and membrane (Datta et al., 2016). However, peptides with high hydrophobicity were also along with high toxicity towards mammalian cells, possibly due to their non-selective to the bacterial and eukaryotic cell membranes (Chu-Kung et al., 2010).

Anoplin (GLLKRIKTLL-NH2) is one of the shortest natural AMPs isolated from the venom of the solitary wasp Anoplius samariensis (Konno et al., 2001). It adopted an amphipathic α-helix structure and displayed a broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, moreover, it displayed low hemolytic activity towards human erythrocytes. Structure-activity relationship studies demonstrated that amino acid substitution at specific positions could regulate its antimicrobial activity and hemolysis activity (Uggerhøj et al., 2015; Ifrah et al., 2005; Munk et al., 2013). However, truncation analogs of Anoplin lost bioactivity because of the decrease of hydrophobicity (Salas et al., 2018). Chionis et al. showed that conjugated with a fatty acid at the N-terminal of Anoplin enhanced antimicrobial activity against both Gram-negative bacteria and fungi (Chionis et al., 2016). Besides, N-lipidated analogs also improved stability towards trypsin degradation. General, D-amino acid substitution was a useful approach to enhance enzymatic stability. Previous research has reported that all D-amino acids analog of Anoplin showed increased resistance towards trypsin and serum (Wang et al., 2014).

A large number of studies have focused on enhancing the antimicrobial activity of Anoplin (Chionis et al., 2016; Sahariah et al., 2015; Libardo et al., 2015; Liu et al., 2017; Slootweg et al., 2013). However, detail studies on its enzymatic cleave sites are few. In this study, we performed an N-methylated scan of Anoplin to determine the critical cleave sites. Afterward, a combination approach of N-methyl amino acid replacement and N-terminal fatty acid conjugation was used to develop both the antimicrobial activity and their resistance towards proteolytic degradation of the novel analogs of Anoplin. Furthermore, the mechanism of action of these new designed peptides were investigated using membrane permeabilization assay, cytoplasmic membrane depolarization, PI uptake, leakage of nucleic acid, and SEM. Overall, our results indicated that the approach of combining N-methylated with N-lipidated was a promising method to improve both antimicrobial activity and proteolytic stability of nature AMPs.

Section snippets

Peptide design and synthesis

All peptides used in this study were synthesized using standard solid-phase peptide synthesis on rink amide MBHA resin using Fmoc chemistry (Fields and Noble, 1990). The purity of the peptides was assessed using RP-HPLC (Waters Massachusetts, USA) on a C18 column. And the molecular mass was determined by electrospray ionization-mass spectrometry (ESI-MS, MaXis 4G, Bruker, USA).

Bacterial strains and animals

E. coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 23857), Pseudomonas aeruginosa (ATCC

Peptide design and synthesis

In this study, we aimed to develop novel analogs of Anoplin with both potent antimicrobial activity and high resistance towards enzymatic degradation. Firstly, in order to improve the protease stability of Anoplin, single and multiple N-methyl amino acids substitution were performed to screen potential enzymatic activity sites. Subsequently, analogs M3.6, M4.7, and M5.7 which had high proteolytic stability but lost their antimicrobial activity were chosen for further modification. Fatty acids

Discussion

Antimicrobial peptides are promising candidates to combat against antibiotics resistant bacterial due to their unique mechanism of disrupting the bacterial membrane (Hancock, 1997). However, the clinical application of AMPs was limited by many factors, such as high manufacture cost, low selective towards bacterial and host cells, and poor stability against proteolytic degradation (Mourtada et al., 2019). The unnatural amino acid substation was a practical and straightforward approach to improve

Conclusion

In summary, we initially introduced N-methyl amino acids replacement in nature AMP Anoplin and screened out the possible enzymatic activity sites, (Fig. 14). Moreover, the results of this study indicated that the reduction of hydrophobicity and α-helix content of these N-methylated analogs were related with the decreased of their antimicrobial effect. Afterward, an approach of combination N-methyl amino acids substitution with N-terminal fatty acid conjugation was employed to develop the new

Author statement

Tianqi Liu - design, synthesis and characterization of peptides, tests of antimicrobial activity, stability assays, mechanism assays and paper writing.

Ningyi Zhu - synthesis, tests of antimicrobial activity assays and hemolytic activity assays.

Chao Zhong - peptides design, synthesis and characterization of peptides.

Yuewen Zhu - tests of antimicrobial activity assays and data analysis.

Sanhu Gou - peptides design, synthesis and characterization of peptides.

Linlin Chang - cytotoxic activity and

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This study was supported by the grants from the National Natural Science Foundation of China (nos. 81773564) and the Key Science and Technology Foundation of Gansu Province (nos. 17YF1FA125).

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