Novel design of simplified ß-hairpin antimicrobial peptide as a potential food preservative based on Trp-pocket backbone.

Food contamination from microbial deterioration requires the development of potent antimicrobial peptides (AMPs). The deployment of approved AMPs as dietary preservatives is limited due to barriers such as instability, toxicity, and high synthetic costs. This exploration utilizes the primary structural elements of the Trp-pocket backbone to engineer a series of ß-hairpin AMPs (XWRWRPGXKXXR-NH2, X representing I, V, F, and/or L). Peptides WpLF, with Phe as X and Leu arranged at the 11th position, demonstrated exceptional selectivity index (SI = 123.08) and sterilization effects both in vitro and in vivo. WpLF consistently exhibited stable bacteriostasis, regardless of physiological salts, serum, and extreme pH. Mechanistic analysis indicated that the peptide penetrates microbial cell membranes, inducing membrane disruption, thereby impeding drug resistance evolution. Conclusively, AMPs engineered by the Trp-pocket skeleton hold substantial potential as innovative biological preservatives in food preservation, providing valuable insights for sustainable and safe peptide-based food preservatives.

Ultrashort All-Hydrocarbon Stapled α-Helix Amphiphile as a Potent and Stable Antimicrobial Compound.

The ravaging effect of drug-resistant bacteria has heightened the need for the development of membrane-soluble antimicrobial peptides (AMPs). However, their potential for clinical use is hindered by issues such as poor biocompatibility, salt sensitivity, and proteolytic lability. In this study, a series of ultrashort stapled cyclization heptapeptides were obtained by inserting all-hydrocarbon staples. StRRL with the highest therapeutic index (TI = 36.3) was selected after evaluating its antibacterial and toxic activity. Furthermore, stRRL demonstrated exceptional performance in high-protease and high-salt environments, making it an effective weapon against bacteria like Escherichia coli in a mouse peritonitis-sepsis model. The membrane lytic mechanism of stRRL, which operates from outside to inside, gives it a low drug-resistant tendency. This suggests that stRRL has the potential to replace antibiotics as a powerful candidate in tackling bacterial infection. In conclusion, the ultrashort all-hydrocarbon stapled antimicrobial amphiphiles inaugurated a novel entrance to the advancements of highly stable peptide compounds.

The design of cell-selective tryptophan and arginine-rich antimicrobial peptides by introducing hydrophilic uncharged residues.

Antimicrobial peptides (AMPs) are considered to be powerful weapons in the fight against traditional antibiotic resistance due to their unique membrane-disruptive mechanism. The combination of traditional and classical hydrophobic tryptophan (W) residues and hydrophilic charged arginine (R) residues is considered as the first choice for the minimalist design of AMPs due to its potent performance in antibacterial activity. However, some W- and R-rich AMPs that are not rationally designed and contain excessive repeats of W and R residues may cause severe cytotoxicity and hemolysis. To address this issue, we designed the (WRX)n (where X = hydrophilic uncharged amino residues; n = number of repeat units) series engineered peptides with high cell selectivity by introducing hydrophilic uncharged threonine (T), serine (S), glutamine (Q) or asparagine (N) residues into the minimalist design of W- and R-rich AMPs. The results showed that the introduction of these hydrophilic uncharged amino residues, especially T residues, significantly improved the cell selectivity of the W- and R-rich engineered peptides. Among (WRX)n series engineered peptides, T6 presents a mixture structure of ß-turn and α-helix. It has broad spectrum and potent antibacterial activity (no activity against probiotics), good biocompatibility, high selectivity index, strong tolerance (physiological salts, serum acid, alkali, and heat conditions), rapid and efficient time-kill kinetics, and no tendency of resistance. Studies on antibacterial mechanism show that T6 exert antibacterial activity mainly by disrupting bacterial cell membrane and inducing the accumulation of reactive oxygen species in bacterial cells. Furthermore, T6 exhibited potent antibacterial and antiinflammatory capabilities in vivo in a mouse peritonitis-sepsis model infected with Escherichia coli. In conclusion, our study confirms an effective strategy for the minimalist design of highly cell selective W- and R-rich AMPs by introducing hydrophilic uncharged T residues, which may trigger widespread attention to hydrophilic uncharged amino acid residues, including T residues, and provide new insights into the design of peptide-based antibacterial biomaterials. STATEMENT OF SIGNIFICANCE: We have introduced hydrophilic uncharged T, S, Q or N residues into the minimalist design of W- and R-rich engineered peptides and found that the introduction of these hydrophilic uncharged amino residues, especially the T residues, can significantly improve the cell selectivity of W- and R-rich engineered peptides. The target compound T6 showed potent antibacterial activity, high cell selectivity, strong tolerance, good in vivo efficacy and killed bacteria through multiple mechanisms mainly membrane-disruptive. These findings may spark widespread interest in hydrophilic uncharged amino acid residues, and provide new insights into the design of peptide-based antimicrobial biomaterials.

Optimization of Antibacterial Activity in Tibetan Swine α-Helix Peptide TP by Site-Directed Mutagenesis.

Antimicrobial peptides (AMPs) have attracted extensive attention because of their broad-spectrum antibacterial activity and low level of induced bacterial resistance. However, the development of some natural AMPs does not consider the perfect balance of structural characteristics, resulting in some empirical and controversial practices still existing. To further explore and complete the relationship between parameters and function of α-helix peptide, in this study, the natural antimicrobial peptide TP secreted from Bacillus strain of Tibetan pigs was selected as a template to investigate the effect of systematic mutations in the hydrogen bond formation site of the α-helical antimicrobial peptide on the activity and cell selectivity of the antimicrobial peptide. The target peptide TP(i+4) 1&2&5 with modification of two pairs of positively charged amino acids and a pair of hydrophobic amino acids showed excellent antibacterial ability and the best selectivity index (SI = 64) in vitro. At the same time, TP(i+4) 1&2&5 remained active in the presence of physiological salts and serum. The results of fluorescence, flow cytometry, and electron microscopy showed that the optimized sequences showed good antibacterial activity by membrane infiltration and membrane destruction. The potential of TP(i+4) 1&2&5 in vivo was tested in a mouse peritonitis model. Organ bacterial loads in the liver, kidney, spleen, and lungs of mice treated with TP(i+4) 1&2&5 were significantly lower compared to the infected group (p < 0.05). Overall, these findings contribute to the design and optimization of antimicrobial peptides with high activity and low toxicity and may accelerate the clinical application of antimicrobial peptides.

Fabrication of Supramolecular Antibacterial Nanofibers with Membrane-Disruptive Mechanism.

By studying the principles of self-assembly and combining the structural parameters required for the asymmetric distribution of antimicrobial peptides (AMPs), we newly designed and screened the high-activity and low-toxicity AMP F2I-LL. This peptide can form a supramolecular hydrogel with a nanofiber microstructure in a simulated physiological environment (phosphate buffered saline), which exhibits broad-spectrum antibacterial activity. Compared with monomeric peptides, the introduction of a self-assembly strategy not only improved the bactericidal titer but also enhanced the serum stability of AMPs. Mechanistic studies showed that the positive charge enriched on the surface of the nanofiber was conducive to its rapid binding to the negatively charged part of the outer membrane of bacteria and further entered the inner membrane, increasing its permeability and ultimately leading to cell membrane rupture and death. This work provides insights into the design of nanopeptides with broad-spectrum antibacterial activity and provides new results for the development of biomedicine.

Self-Assembling Peptide Dendron Nanoparticles with High Stability and a Multimodal Antimicrobial Mechanism of Action.

Self-assembling nanometer-scale structured peptide polymers and peptide dendrimers have shown promise in biomedical applications due to their versatile properties and easy availability. Herein, self-assembling peptide dendron nanoparticles (SPDNs) with potent antimicrobial activity against a range of bacteria were developed based on the nanoscale self-assembly of an arginine-proline repeat branched peptide dendron bearing a hexadecanoic acid chain. The SPDNs are biocompatible, and our most active peptide dendron nanoparticle, C16-3RP, was found to have negligible toxicity after both in vitro and in vivo studies. Furthermore, the C16-3RP nanoparticles showed excellent stability under physiological concentrations of salt ions and against serum and protease degradation, resulting in highly effective treatment in a mouse acute peritonitis model. Comprehensive analyses using a series of biofluorescence, microscopy, and transcriptome sequencing techniques revealed that C16-3RP nanoparticles kill Gram-negative bacteria by increasing bacterial membrane permeability, inducing cytoplasmic membrane depolarization and drastic membrane disruption, inhibiting ribosome biogenesis, and influencing energy generation and other processes. Collectively, C16-3RP nanoparticles show promising biocompatibility and in vivo therapeutic efficacy without apparent resistance development. These advancements may facilitate the development of peptide-based antibiotics in clinical settings.

Binding loop of sunflower trypsin inhibitor 1 serves as a design motif for proteolysis-resistant antimicrobial peptides.

Although antimicrobial peptides (AMPs) have become powerful drug candidates in the post-antibiotic era, but their low protease stability hinders their clinical application. In the present study, the natural sunflower trypsin inhibitor 1 (SFTI-1) binding loop (CTKSIPPIC) was used to design and synthesize a specific anti-proteolytic sequence template ((RX)n W (RX)n CTKSIPPIC (n = 2, 3; X represents A, I, L, V, F, and W)). After several antibacterial, bactericidal, and toxicity tests, RV3 stood out from the variants and had the highest average selectivity index (SI all = 156.03). It is highly stable in serum, varying pH, temperature, and salt ions as well as under high trypsin, pepsin, or papain concentrations. In a mouse skin inflammation model, established by Pseudomonas aeruginosa infection, RV3 could effectively kill the pathogen, promote wound healing, inhibit inflammatory cell infiltration, and inhibit mRNA and protein expression of TNF-α, IL-6, and IL-1ß inflammatory factors. The antibacterial mechanisms of RV3 include combining with lipopolysaccharides and increasing cell membrane permeability, leading to cell membrane rupture and death. These findings indicate that RV3 has great potential for the treatment of bacterial infections.

Cross-Strand Interaction, Central Bending, and Sequence Pattern Act as Biomodulators of Simplified ß-Hairpin Antimicrobial Amphiphiles.

Novel antimicrobial peptides (AMPs) have revolutionarily evolved into formidable candidates for antibiotic substitute materials against pathogenic infections. However, cost, lability, disorderly sequences, systemic toxicology, and biological profiles have plagued the perennial search. Here, a progressive ß-hairpin solution with the simplest formulation is implanted into an AMP-based therapeutic strategy to systematically reveal the complex balance between function and toxicity of structural moieties, including cationicity, hydrophobicity, cross-strand interactions, center bending, and sequence pattern. Comprehensive implementation of structural identification, ten microorganisms, eleven in vitro barriers, four mammalian cells, and a diversified membrane operation setup led to the emergence of ß-hairpin prototypes from a 24-member library. Lead amphiphiles, WKF-PG and WRF-NG, can tackle bacterial infection through direct antimicrobial efficacy and potential inflammation-limiting capabilities, such as an Escherichia coli challenge in a mouse peritonitis-sepsis model, without observed toxicity after systemic administration. Their optimal states with dissimilar modulators and the unavailable drug resistance related to membrane lytic mechanisms, also provide an usher for renewed innovation among ß-sheet peptide-based antimicrobial biomaterials.

Rational Avoidance of Protease Cleavage Sites and Symmetrical End-Tagging Significantly Enhances the Stability and Therapeutic Potential of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are expected to solve problem of antibiotic resistance because of their distinctive nonspecific membrane-disruptive mechanism. However, clinical applications of AMPs have been precluded by their poor stability, although various complex chemical strategies have been employed to solve this problem, and this undoubtedly greatly increases the manufacturing cost. Herein, a series of novel peptides with high stability were developed based on protease-specific cleavage sites and symmetrical end-tagging. Among these peptides, II-I4-II exhibited the best antibacterial activity and the highest therapeutic index. More importantly, II-I4-II showed extremely high stability in the presence of various proteases, physiological salts and serum, and under acid, alkali, and heat conditions, and it exhibited excellent therapeutic potential in vivo. Additionally, II-I4-II exhibited a membrane-disruptive mechanism and a low propensity to induce resistance. In general, these findings contribute to the design of AMPs with high stability and might accelerate clinical applications of AMPs.

Threonine, but Not Lysine and Methionine, Reduces Fat Accumulation by Regulating Lipid Metabolism in Obese Mice.

Some amino acids (AAs) have been proven to suppress fat mass and improve insulin sensitivity. However, the impact of important essential AAs, threonine, lysine, and methionine, on obesity has not been clarified. In the present study, after an 8 week period of obesity induction, mice were grouped to receive either a high-fat diet (HFD) or HFD supplemented with lysine, threonine, or methionine (3% in drinking water) for another 10 weeks. The results showed that dietary supplementation with threonine significantly decreased body weight, epididymal and perirenal fat pad weights, serum concentrations of glucose, triacylglycerols, total cholesterol, and LDL-cholesterol compared to the HFD group. HOMA-IR and serum leptin and adiponectin were improved by threonine supplementation. In epididymal adipose tissue, threonine treatment significantly down-regulated the expression levels of lipogenesis and up-regulated expressions of lipolysis compared to the HFD group. Threonine addition stimulated the expression of UCP-1 and related genes in brown adipose tissue. However, lysine or methionine supplementation showed little effect on body weight, WAT weight, serum lipid profiles, and lipid-metabolism-related gene expressions of HFD-fed mice. These findings suggest that threonine inhibited fat mass and improved lipid metabolism of already obese mice, providing a potential agent in treating obesity.

Conversion of Broad-Spectrum Antimicrobial Peptides into Species-Specific Antimicrobials Capable of Precisely Targeting Pathogenic Bacteria.

Currently, the majority of antibiotics in clinical use have broad activity spectra, killing pathogenic and beneficial microorganisms indiscriminately. The disruption of the ecological balance of normal flora often results in secondary infections or other antibiotic-associated complications. Therefore, targeted antimicrobial therapies capable of specifically eliminating pathogenic bacteria while retaining the protective benefits of a normal microflora would be advantageous. In this study, we successfully constructed a series of Enterococcus faecalis-targeted antimicrobial peptides from wide-spectrum antimicrobial peptide precursors. These peptides are designed based on fusion of the species-specific peptide pheromone cCF10 and modification of the active region of the antimicrobial peptide. The results showed that cCF10-C4 possessed specific antimicrobial activity against E. faecalis and was not active against other types of bacteria tested. The specificity of this hybrid peptide was shown by the absence of antimicrobial effects in the pheromone-substituted derivative. Further studies indicated that cCF10-C4 and its parent peptide C4 exert their activities by damaging cytoplasmic membrane integrity. The present study reveals the application potential of these molecules as "probiotic" antimicrobials for the control of specific bacterial infections, and it also helps to elucidate the design and construction of species-specific antimicrobials with precise targeting specificity.

Multiple Strategy Optimization of Specifically Targeted Antimicrobial Peptide Based on Structure-Activity Relationships to Enhance Bactericidal Efficiency.

Unlike traditional broad-spectrum antibacterial agents, specifically targeted antimicrobial peptides (STAMPs) are difficult for bacteria to develop resistance to due to their unique membrane lytic mechanism. Additionally, STAMPs can maintain a normal ecological balance and provide long-term protection to the body. However, therapeutic applications of STAMPS are hindered by their weak activity and imperfect specificity, as well as lack of knowledge in understanding their structure-activity relationships. To investigate the effects of different parameters on the biological activities of STAMPs, a peptide sequence, WKKIWKDPGIKKWIK, was truncated, extended, and provided with an increased charge and altered amphipathicity. In addition, a novel template modification method for attaching a phage-displayed peptide, which recognized and bound to Escherichia coli (E. coli) cells, to the end of the sequence was introduced. Compared with the traditional template modification method, peptide 13, which contained a phage-displayed peptide at the C-terminus, exhibited superior narrow-spectrum antibacterial activity against E. coli compared to that of parental peptide 2, and the activity and specificity of peptide 13 were increased by 5.0 and 2.4 times, respectively. Additionally, peptide 13 showed low cytotoxicity and relatively desirable salt, serum, acid, alkaline and heat stability. In this study, peptide 13 specifically killed E. coli by causing cytoplasmic membrane rupture and cytosol leakage. In summary, these findings are useful for improving the activity and specificity of STAMPs and show that peptide 13 is able to combat the growing threat of E. coli infections.

Expression of T9W in Pichia pastoris and the protective roles of T9W in ICR Mice.

BACKGROUND: The purpose of this paper is to express the novel α-helical peptide T9W more efficiently using the Pichia pastoris expression system and to examine the role of T9W in ICR mice. RESULTS: The novel antimicrobial peptide T9W was expressed in P. pastoris X-33 by using the vector pPICZαA. Approximately 13 mg/L T9W was secreted from the culture and purified. The expressed peptide has similar activity to the synthetic peptide. ICR female mice challenged with P. aeruginosa 27853 at the LD100 were treated with T9W and CPFX. The results showed that the secretion of inflammatory cytokines and lung damage was significantly reduced by the treatment group, and the protective response was equivalent between T9W and ciprofloxacin-treated mice. CONCLUSION: T9W was expressed in P. pastoris X-33 via the methanol-inducible vector pPICZαA and exhibited the same biological activity as synthetic T9W. T9W can alleviate damage to mice caused by P. aeruginosa.

Design of Heptad Repeat Amphiphiles Based on Database Filtering and Structure-Function Relationships to Combat Drug-Resistant Fungi and Biofilms.

Due to the emergence of reports of multidrug-resistant fungi, infections caused by multidrug-resistant fungi and biofilms are considered to be a global threat to human health due to the lack of effective broad-spectrum drugs. Here, we developed a series heptad repeat sequences based on an antimicrobial peptide database (APD) and structure-function relationships. Among the developed peptides, the target peptide ACR3 exhibited good activity against all fungi and bacteria tested, including fluconazole-resistant Candida albicans (C. albicans) and methicillin-resistant Staphylococcu saureus (S. aureus), while demonstrating relatively low toxicity and good salt tolerance. The peptide ACR3 inhibits the formation of C. albicans biofilms and has a therapeutic effect on mature biofilms in vitro and in vivo. Moreover, we did not observe any resistance of C. albicans and E. coli against the peptide ACR3. A series of assays and microscopy were used to analyze the antimicrobial mechanism. These results showed that the antimicrobial activity of the peptide ACR3 utilizes a multimodal mechanism that degrades the cell wall barrier, alters the cytoplasmic membrane electrical potential, and induces intracellular reactive oxygen species (ROS) production. In general, the peptide ACR3 is a potent antibacterial agent that shows great potential for use in biomedical coatings and healthcare formulas to combat the growing threat of fungal and bacterial infection.

Effect of terminal arrangement of tryptophan on biological activity of symmetric α-helix-forming peptides.

It is traditionally believed that the distribution of tryptophan (Trp) residues is critical for the novo design of antimicrobial peptides (AMPs). However, there is scarce knowledge regarding Trp residues arrangement at the head group level. Thus, a set of α-helical AMPs containing different Trp residue arrangements at the N-/C-terminal of sequence were designed to increase the strategy database and analyze their biological activities. The arrangement of the N-terminal Trp residue significantly improved the bacteriostatic activity of the peptides, but the C-terminal Trp residue arrangement reduced the biocompatibility of them. WL and LW were effective against Gram-negative microbes and had high selectivity for bacteria as compared to human erythrocytes and mammalian cells. They both maintained a relatively desirable activity in the presence of physiological salts and serum. It is observed through electron microscope, flow cytometry and fluorescence spectroscopy that target peptides can penetrate bacterial cell membrane and kill it by damaging cell membrane integrity. Collectively, we determined the structure-activity relationship of Trp residue distributions in a symmetric sequence structure and filled the gap in knowledge related to Trp arrangements at the head group level. The obtained results will be helpful in designing of artificial peptide-based antimicrobials.

Highly Stabilized α-Helical Coiled Coils Kill Gram-Negative Bacteria by Multicomplementary Mechanisms under Acidic Condition.

Although antimicrobial peptides (AMPs) hold tremendous promise in overcoming the threats of multidrug resistance, the main obstacle to successful therapeutic applications is their poor stability. Various synthetic strategies such as unnatural amino acids and chemical modifications have made advances for improving this problem. However, this complicated synthesis often greatly increases the cost of production. Here, we show that a series of novel peptides, designed by combining an α-helical coiled coil model, knowledge of the specificity of proteolysis and major parameters of AMPs, exhibited efficient activity against all tested Gram-negative bacteria under acidic condition and demonstrate low toxicity. Of these α-helical coiled coil peptides, 3IH3 displayed the highest average therapeutic index (GMTI = 294.25) with high stability toward salts, serum, extreme pH, heat, and proteases. Electron microscopy and biological analytical technique analyses showed that 3IH3 killed bacterial cells via a multicomplementary mechanism at pH 6.0, with physical membrane disruption as the dominant bactericidal mechanism. These results suggest that 3IH3 shows great stability as an inexpensive and effective antimicrobial activity agent and has the potential for clinical application in the treatment of infections occurring in body sites with acidic pH.

Symmetrical Modification of Minimized Dermaseptins to Extend the Spectrum of Antimicrobials with Endotoxin Neutralization Potency.

Antimicrobial peptides (AMPs) have emerged as a promising class of antimicrobial agents that could potentially address the global antibiotic resistance. Generating mirror-like peptides by minimizing dermaseptin family sequences is an effective strategy for designing AMPs. However, the previous research still had some limitations such as lower effectiveness and a narrow spectrum of antibacterial activity. To further expand and hone this strategy, we designed a series of AMPs consisting of the WXMXW-NH2 motif (X represents V, I, F, and W; M represents KAAAKAAAK). The peptides formed α-helices and displayed broad-spectrum antimicrobial activities against eleven types of clinical bacteria including both Gram-negative and Gram-positive bacteria. The optimized peptide WW exhibited high physical rupture by inducing membrane shrinkage, disruption, and lysis. Moreover, WW effectively neutralized endotoxins and inhibited the inflammatory response while having the highest therapeutic index. In conclusion, these results indicated that the peptide WW has potential as a broad-spectrum antimicrobial agent or preservative for overcoming the risk of multidrug resistance in localized or external therapeutic applications.

Central ß-turn increases the cell selectivity of imperfectly amphipathic α-helical peptides.

Although membrane lytic antimicrobial peptides (AMPs) show enormous potential for addressing mounting global antibiotic resistance, therapeutic applications are hindered by their weak antimicrobial activity, high toxicity, salt sensitivity and poor understanding of structure-activity relationships. To investigate the effects of different parameters on the biological activities of AMPs, a rational approach was adopted to design a series of short cationic α-helical peptides comprising the Ac-WxKyWxzzyKxWyK-NH2 sequence, where x: cationic residues (Arg or Lys), y: hydrophobic residues (Ala, Val, Ile or Leu), and zz: ß-turn (rigid D-Pro-Gly turn or flexible Gly-Gly turn). The peptides showed a more helical structure as the concentration of membrane-mimetic solution increased. The peptide RL with a central D-Pro-Gly turn (x: Arg, y: Lys, zz = D-Pro-Gly) exhibited broad-spectrum antimicrobial activities (2-8 µM) against ten types of clinically relevant microorganisms and even maintained its activity in the presence of physiological salts and showed excellent selectivity toward bacterial cells over human red blood cells and mammalian cells. However, the toxicity was increased after the removal of D-Pro-Gly turn. Additionally, the bactericidal activity was reduced when the D-Pro-Gly turn was replaced by a Gly-Gly turn. Fluorescence spectroscopy and electron microscopy analyses indicated that RL and its derivatives killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. In conclusion, these findings clearly generalized a potential method for designing or optimizing AMPs, and the peptide RL is a promising therapeutic candidate to combat antibiotic resistance. STATEMENT OF SIGNIFICANCE: We proposed a rational approach to design imperfectly amphiphilic peptides and identified RL (Ac-WRKLWRpGLKRWLK-NH2) in particular that shows strong antibacterial properties, low toxicity and high salt resistance. The ß-turn unit inserted into the central position of cationic α-helical peptides, especially the D-Pro-Gly turn, significantly increase the cell selectivity of the synthetic amphiphiles. The findings demonstrate a potential method for designing and/or optimizing AMPs, which would facilitate the development of strategies to design peptide-based antimicrobial biomaterials in a variety of biotechnological and clinical applications.