Antibacterial activity of a short de novo designed peptide against fish bacterial pathogens.

In the face of increasing antimicrobial resistance in aquaculture, researchers are exploring novel substitutes to customary antibiotics. One potential solution is the use of antimicrobial peptides (AMPs). We aimed to design and evaluate a novel, short, and compositionally simple AMP with potent activity against various bacterial pathogens in aquaculture. The resulting peptide, KK12YW, has an amphipathic nature and net charge of + 7. Molecular docking experiments disclosed that KK12YW has a strong affinity for aerolysin, a virulence protein produced by the bacterial pathogen Aeromonas sobria. KK12YW was synthesized using Fmoc chemistry and tested against a range of bacterial pathogens, including A. sobria, A. salmonicida, A. hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and methicillin-resistant S. aureus. The AMP showed promising antibacterial activity, with MIC and MBC values ranging from 0.89 to 917.1 µgmL-1 and 3.67 to 1100.52 µgmL-1, respectively. In addition, KK12YW exhibited resistance to high temperatures and remained effective even in the presence of serum and salt, indicating its stability. The peptide also demonstrated minimal hemolysis toward fish RBCs, even at higher concentrations. Taken together, these findings indicate that KK12YW could be a highly promising and viable substitute for conventional antibiotics to combat microbial infections in aquaculture.

LL-37: Structures, Antimicrobial Activity, and Influence on Amyloid-Related Diseases.

Antimicrobial peptides (AMPs), as well as host defense peptides (HDPs), constitute the first line of defense as part of the innate immune system. Humans are known to express antimicrobial precursor proteins, which are further processed to generate AMPs, including several types of α/ß defensins, histatins, and cathelicidin-derived AMPs like LL37. The broad-spectrum activity of AMPs is crucial to defend against infections caused by pathogenic bacteria, viruses, fungi, and parasites. The emergence of multi-drug resistant pathogenic bacteria is of global concern for public health. The prospects of targeting antibiotic-resistant strains of bacteria with AMPs are of high significance for developing new generations of antimicrobial agents. The 37-residue long LL37, the only cathelicidin family of AMP in humans, has been the major focus for the past few decades of research. The host defense activity of LL37 is likely underscored by its expression throughout the body, spanning from the epithelial cells of various organs-testis, skin, respiratory tract, and gastrointestinal tract-to immune cells. Remarkably, apart from canonical direct killing of pathogenic organisms, LL37 exerts several other host defense activities, including inflammatory response modulation, chemo-attraction, and wound healing and closure at the infected sites. In addition, LL37 and its derived peptides are bestowed with anti-cancer and anti-amyloidogenic properties. In this review article, we aim to develop integrative, mechanistic insight into LL37 and its derived peptides, based on the known biophysical, structural, and functional studies in recent years. We believe that this review will pave the way for future research on the structures, biochemical and biophysical properties, and design of novel LL37-based molecules.

Dimerization of the ß-Hairpin Membrane-Active Cationic Antimicrobial Peptide Capitellacin from Marine Polychaeta: An NMR Structural and Thermodynamic Study.

Capitellacin is the ß-hairpin membrane-active cationic antimicrobial peptide from the marine polychaeta Capitella teleta. Capitellacin exhibits antibacterial activity, including against drug-resistant strains. To gain insight into the mechanism of capitellacin action, we investigated the structure of the peptide in the membrane-mimicking environment of dodecylphosphocholine (DPC) micelles using high-resolution NMR spectroscopy. In DPC solution, two structural forms of capitellacin were observed: a monomeric ß-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal ß-strands and stabilized by intermonomer hydrogen bonds and Van der Waals interactions. The thermodynamics of the enthalpy-driven dimerization process was studied by varying the temperature and molar ratios of the peptide to detergent. Cooling the peptide/detergent system promoted capitellacin dimerization. Paramagnetic relaxation enhancement induced by lipid-soluble 12-doxylstearate showed that monomeric and dimeric capitellacin interacted with the surface of the micelle and did not penetrate into the micelle interior, which is consistent with the "carpet" mode of membrane activity. An analysis of the known structures of ß-hairpin AMP dimers showed that their dimerization in a membrane-like environment occurs through the association of polar or weakly hydrophobic surfaces. A comparative analysis of the physicochemical properties of ß-hairpin AMPs revealed that dimer stability and hemolytic activity are positively correlated with surface hydrophobicity. An additional positive correlation was observed between hemolytic activity and AMP charge. The data obtained allowed for the provision of a more accurate description of the mechanism of the oligomerization of ß-structural peptides in biological membranes.

Combating Antimicrobial Resistance by Employing Antimicrobial Peptides: Immunomodulators and Therapeutic Agents against Infectious Diseases.

The rising prevalence of multiple-drug-resistant pathogens poses a formidable challenge to conventional antimicrobial treatments. The inability of potent antibiotics to combat these "superbugs" underscores the pressing need for alternative therapeutic agents. Antimicrobial peptides (AMPs) represent an alternative class of antibiotics. AMPs are essential immunomodulatory molecules that are found in various organisms. They play a pivotal role in managing microbial ecosystems and bolstering innate immunity by targeting and eliminating invading microorganisms. AMPs also have applications in the agriculture sector by combating animal as well as plant pathogens. AMPs can be exploited for the targeted therapy of various diseases and can also be used in drug-delivery systems. They can be used in synergy with current treatments like antibiotics and can potentially lead to a lower required dosage. AMPs also have huge potential in wound healing and regenerative medicine. Developing AMP-based strategies with improved safety, specificity, and efficacy is crucial in the battle against alarming global microbial resistance. This review will explore AMPs' increasing applicability, their mode of antimicrobial activity, and various delivery systems enhancing their stability and efficacy.

Antimicrobial peptides designed by computational analysis of proteomes.

Antimicrobial peptides (AMPs) are promising cationic and amphipathic molecules to fight antibiotic resistance. To search for novel AMPs, we applied a computational strategy to identify peptide sequences within the organisms' proteome, including in-house developed software and artificial intelligence tools. After analyzing 150.450 proteins from eight proteomes of bacteria, plants, a protist, and a nematode, nine peptides were selected and modified to increase their antimicrobial potential. The 18 resulting peptides were validated by bioassays with four pathogenic bacterial species, one yeast species, and two cancer cell-lines. Fourteen of the 18 tested peptides were antimicrobial, with minimum inhibitory concentrations (MICs) values under 10 µM against at least three bacterial species; seven were active against Candida albicans with MICs values under 10 µM; six had a therapeutic index above 20; two peptides were active against A549 cells, and eight were active against MCF-7 cells under 30 µM. This study's most active antimicrobial peptides damage the bacterial cell membrane, including grooves, dents, membrane wrinkling, cell destruction, and leakage of cytoplasmic material. The results confirm that the proposed approach, which uses bioinformatic tools and rational modifications, is highly efficient and allows the discovery, with high accuracy, of potent AMPs encrypted in proteins.

Structure modification of anoplin for fighting resistant bacteria.

The emergence of bacterial resistance has posed a significant challenge to clinical antimicrobial treatment, rendering commonly used antibiotics ineffective. The development of novel antimicrobial agents and strategies is imperative for the treatment of resistant bacterial infections. Antimicrobial peptides (AMPs) are considered a promising class of antimicrobial agents due to their low propensity for resistance and broad-spectrum activity. Anoplin is a small linear α-helical natural antimicrobial peptide that was isolated from the venom of the solitary wasp Anplius samariensis. It exhibits rich biological activity, particularly broad-spectrum antimicrobial activity and low hemolytic activity. Over the past three decades, more than 40 research publications on anoplin have been made available online. This review focuses on the advancements of anoplin in antimicrobial research, encompassing its sources, characterization, antimicrobial activity, influencing factors and structural modifications. The aim is to provide assistances for the development of new antimicrobial agents that can combat bacterial resistance.

Hydrocarbon stapled temporin-L analogue as potential antibacterial and antiendotoxin agents with enhanced protease stability.

Antimicrobial resistance (AMR) is a serious global concern and a huge burden on the healthcare system. Antimicrobial peptides (AMPs) are considered as a solution of AMR due to their membrane-lytic and intracellular mode of action and therefore resistance development against AMPs is less frequent. One such AMPs, temporin-L (TL) is a 13-mer peptide reported as a potent and broad-spectrum antibacterial agent with significant immunomodulatory activity. However, TL is toxic to human erythrocytes at their antibacterial concentrations and therefore various analogues were synthesized with potent antimicrobial activity and lower hemolytic activity. In this work, we have selected a non-toxic engineered analogue of TL (eTL) and performed hydrocarbon stapling of amino acid residues at i to i + 4 positions at different part of sequence. The synthesized peptides were investigated against both the gram-positive and gram-negative bacteria as well as methicillin resistant S. aureus, its MIC was measured in the concentrations range of 0.9-15.2 µM. All analogues were found equal or better antibacterial as compared to parent peptide. Interestingly one analogue eTL [5-9] was found to be non-cytotoxic and stable in presence of the human serum. Mode of action studies revealed membrane depolarizing and disruptive mode of action with live MRSA. Further in vivo studies of antimicrobial against MRSA infection and anti-endotoxin activities in mice model revealed potential activity of the stapled peptide analogue. Overall, this reports on stapled analogue of the AMPs highlights an important strategy for the development of new antibacterial therapeutics against AMR.

Outer-Membrane Permeabilization, LPS Transport Inhibition: Activity, Interactions, and Structures of Thanatin Derived Antimicrobial Peptides.

Currently, viable antibiotics available to mitigate infections caused by drug-resistant Gram-negative bacteria are highly limited. Thanatin, a 21-residue-long insect-derived antimicrobial peptide (AMP), is a promising lead molecule for the potential development of novel antibiotics. Thanatin is extremely potent, particularly against the Enterobacter group of Gram-negative pathogens, e.g., E. coli and K. pneumoniae. As a mode of action, cationic thanatin efficiently permeabilizes the LPS-outer membrane and binds to the periplasmic protein LptAm to inhibit outer membrane biogenesis. Here, we have utilized N-terminal truncated 16- and 14-residue peptide fragments of thanatin and investigated structure, activity, and selectivity with correlating modes of action. A designed 16-residue peptide containing D-Lys (dk) named VF16 (V1PIIYCNRRT-dk-KCQRF16) demonstrated killing activity in Gram-negative bacteria. The VF16 peptide did not show any detectable toxicity to the HEK 293T cell line and kidney cell line Hep G2. As a mode of action, VF16 interacted with LPS, permeabilizing the outer membrane and binding to LptAm with high affinity. Atomic-resolution structures of VF16 in complex with LPS revealed cationic and aromatic surfaces involved in outer membrane interactions and permeabilization. Further, analyses of an inactive 14-residue native thanatin peptide (IM14: IIYCNRRTGKCQRM) delineated the requirement of the ß-sheet structure in activity and target interactions. Taken together, this work would pave the way for the designing of short analogs of thanatin-based antimicrobials.

The amphipathic design in helical antimicrobial peptides.

Amphipathicity is a critical characteristic of helical antimicrobial peptides (AMPs). The hydrophilic region, primarily composed of cationic residues, plays a pivotal role in the initial binding to negatively charged components on bacterial membranes through electrostatic interactions. Subsequently, the hydrophobic region interacts with hydrophobic components, inducing membrane perturbation, ultimately leading to cell death, or inhibiting intracellular function. Due to the extensive diversity of natural and synthetic AMPs with regard to the design of amphipathicity, it is complicated to study the structure-activity relationships. Therefore, this work aims to categorize the common amphipathic design and investigate their impact on the biological properties of AMPs. Besides, the connection between current structural modification approaches and amphipathic styles was also discussed.

A Comprehensive Review of Patented Antimicrobial Peptides from Amphibian Anurans.

Since the 1980s, studies of antimicrobial peptides (AMPs) derived from anuran skin secretions have unveiled remarkable structural diversity and a wide range of activities. This study explores the potential of these peptides for drug development by examining granted patents, amino acid modifications related to patented peptides, and recent amphibians' taxonomic updates influencing AMP names. A total of 188 granted patents related to different anuran peptides were found, with Asia and North America being the predominant regions, contributing 65.4% and 15.4%, respectively. Conversely, although the Neotropical region is the world's most diversified region for amphibians, it holds only 3.7% of the identified patents. The antimicrobial activities of the peptides are claimed in 118 of these 188 patents. Additionally, for 160 of these peptides, 66 patents were registered for the natural sequence, 69 for both natural and derivative sequences, and 20 exclusively for sequence derivatives. Notably, common modifications include alterations in the side chains of amino acids and modifications to the peptides' N- and C-termini. This review underscores the biomedical potential of anuran-derived AMPs, emphasizing the need to bridge the gap between AMP description and practical drug development while highlighting the urgency of biodiversity conservation to facilitate biomedical discoveries.

The antibacterial properties of branched peptides based on poly(l-arginine): In vitro antibacterial evaluation and molecular dynamic simulations.

The emergence of bacterial strains resistant to antibiotics is a major issue in the medical field. Antimicrobial peptides are widely studied as they do not generate as much resistant bacterial strains as conventional antibiotics and present a broad range of activity. Among them, the homopolypeptide poly(l-arginine) presents promising antibacterial properties, especially in the perspective of its use in biomaterials. Linear poly(l-arginine) has been extensively studied but the impact of its 3D structure remains unknown. In this study, the antibacterial properties of newly synthesized branched poly(l-arginine) peptides, belonging to the family of multiple antigenic peptides, are evaluated. First, in vitro activities of the peptides shows that branched poly(l-arginine) is more efficient than linear poly(l-arginine) containing the same number of arginine residues. Surprisingly, peptides with more arms and more residues are not the most effective. To better understand these unexpected results, interactions between these peptides and the membranes of Gram positive and Gram negative bacteria are simulated thanks to molecular dynamic. It is observed that the bacterial membrane is more distorted by the branched structure than by the linear one and by peptides containing smaller arms. This mechanism of action is in full agreement with in vitro results and suggest that our simulations form a robust model to evaluate peptide efficiency towards pathogenic bacteria.

Differences in Relevant Physicochemical Properties Correlate with Synergistic Activity of Antimicrobial Peptides.

With the urgent need for new medical approaches due to increased bacterial resistance to antibiotics, antimicrobial peptides (AMPs) have been considered as potential treatments for infections. Experiments indicate that combinations of several types of AMPs might be even more effective at inhibiting bacterial growth with reduced toxicity and a lower likelihood of inducing bacterial resistance. The molecular mechanisms of AMP-AMP synergistic antimicrobial activity, however, remain not well understood. Here, we present a theoretical approach that allows us to relate the physicochemical properties of AMPs and their antimicrobial cooperativity. It utilizes correlation and bioinformatics analysis. A concept of physicochemical similarity is introduced, and it is found that less similar AMPs with respect to certain physicochemical properties lead to greater synergy because of their complementary antibacterial actions. The analysis of correlations between the similarity and the antimicrobial properties allows us to effectively separate synergistic from nonsynergistic AMP pairs. Our theoretical approach can be used for the rational design of more effective AMP combinations for specific bacterial targets, for clarifying the mechanisms of bacterial elimination, and for a better understanding of cooperativity phenomena in biological systems.

Development of a novel hybrid antimicrobial peptide for enhancing antimicrobial spectrum and potency against food-borne pathogens.

AIMS: To address the increasingly serious challenge of the transmission of foodbrone pathogens in the food chain. METHODS AND RESULTS: In this study, we employed rational design strategies, including truncation, amino acid substitution, and heterozygosity, to generate seven engineered peptides with α-helical structure, cationic property, and amphipathic characteristics based on the original Abhisin template. Among them, as the hybird antimicrobial peptide (AMP), AM exhibits exceptional stability, minimal toxicity, as well as broad-spectrum and potent antimicrobial activity against foodborne pathogens. Besides, it was observed that the electrostatic incorporation demonstrates by AM results in its primary targeting and disruption of the cell wall and membrane of Escherichia coli O157: H7 (EHEC) and methicillin-resistant Staphylococcus aureus (MRSA), resulting in membrane perforation and enhanced permeability. Additionally, AM effectively counteracts the deleterious effects of lipopolysaccharide, eradicating biofilms and ultimately inducing the demise of both food spoilage and pathogenic microorganisms. CONCLUSIONS: The findings highlight the significant potential of AM as a highly promising candidate for a novel food preservative and its great importance in the design and optimization of AMP-related agents.

Discovery of novel antibacterial agent for the infected wound treatment: all-hydrocarbon stapling optimization of LL-37.

Rationale: Antimicrobial peptide LL-37 has been recognized as a favorable alternative to antibiotics due to its broad antibacterial spectrum, low resistance development and diverse biological activities. However, its high manufactory cost, poor proteolytic stability, and unpredictable cytotoxicity seriously hindered its medical translation. Methods: To push the frontiers of its clinical application, all-hydrocarbon stapling strategy was exploited here for the structural modification of KR-12, the core and minimal fragment of LL-37. Results: Based on a library of KR-12 derivatives that designed and synthesized to be stapled at positions of either i, i+4 or i, i+7, structure to activity relationship was investigated. Among them, KR-12(Q5, D9) with the glutamine and aspartic acid residues stapled displayed increased helical content and positive charge. The reinforced α-helical conformation not only protected it from proteolytic hydrolysis but also improved its antibacterial efficacy via effective membrane perturbation and anti-inflammatory efficacy via compact LPS binding. Besides, the increased positive charge endowed it with an enhanced therapeutic index. On infected wound mouse model, it was demonstrated to eliminate bacteria and promote wound closure and regeneration effectively. Conclusion: Overall, the all-hydrocarbon stapling was proven to lay the foundation for the future development of antibacterial agents. KR-12(Q5, D9) could serve as a lead compound for the clinical treatment of bacterial infections.

Computational modelling of the antimicrobial peptides Cruzioseptin-4 extracted from the frog Cruziohyla calcarifer and Pictuseptin-1 extracted from the frog Boana picturata.

A computational study of the peptides Cruzioseptin-4 and Pictuseptin-1, identified in Cruziohyla calcarifer and Boana picturata respectively, has been carried out. The studies on Cruzioseptin-4 show that it is a cationic peptide with a chain of 23 amino acids that possess 52.17% of hydrophobic amino acids and a charge of + 1.2 at pH 7. Similarly, Pictuseptin-1 is a 22 amino acids peptide with a charge of + 3 at pH 7 and 45.45% of hydrophobic amino acids. Furthermore, the predominant secondary structure for both peptides is alpha-helical. The physicochemical properties were predicted using PepCalc and Bio-Synthesis; secondary structures using Jpred4 and PredictProtein; while molecular docking was performed using Autodock Vina. Geometry optimization of the peptides was done using the ONIOM hybrid method with the HF/6-31G basis set implemented in the Gaussian 09 program. Finally, the molecular docking study indicates that the viable mechanism of action for both peptides is through a targeted attack on the cell membrane of pathogens via electrostatic interactions with different membrane components, leading to cell lysis.

Physical-Chemical Approach to Designing Drugs with Multiple Targets.

Many people simultaneously exhibit multiple diseases, which complicates efficient medical treatments. For example, patients with cancer are frequently susceptible to infections. However, developing drugs that could simultaneously target several diseases is challenging. We present a novel theoretical method to assist in selecting compounds with multiple therapeutic targets. The idea is to find correlations between the physical and chemical properties of drug molecules and their abilities to work against multiple targets. As a first step, we investigated potential drugs against cancer and viral infections. Specifically, we investigated antimicrobial peptides (AMPs), which are short positively charged biomolecules produced by living systems as a part of their immune defense. AMPs show anticancer and antiviral activity. We use chemoinformatics and correlation analysis as a part of the machine-learning method to identify the specific properties that distinguish AMPs with dual anticancer and antiviral activities. Physical-chemical arguments to explain these observations are presented.

Effect of tryptophan position and lysine/arginine substitution in antimicrobial peptides on antifungal action.

Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.

Recent advances in antimicrobial peptide-based therapy.

Antimicrobial peptides (AMPs) are a group of polypeptide chains that have the property to target and kill a myriad of microbial organisms including viruses, bacteria, protists, etc. The first discovered AMP was named gramicidin, an extract of aerobic soil bacteria. Further studies discovered that these peptides are present not only in prokaryotes but in eukaryotes as well. They play a vital role in human innate immunity and wound repair. Consequently, they have maintained a high level of intrigue among scientists in the field of immunology, especially so with the rise of antibiotic-resistant pathogens decreasing the reliability of antibiotics in healthcare. While AMPs have promising potential to substitute for common antibiotics, their use as effective replacements is barred by certain limitations. First, they have the potential to be cytotoxic to human cells. Second, they are unstable in the blood due to action by various proteolytic agents and ions that cause their degradation. This review provides an overview of the mechanism of AMPs, their limitations, and developments in recent years that provide techniques to overcome those limitations. We also discuss the advantages and drawbacks of AMPs as a replacement for antibiotics as compared to other alternatives such as synthetically modified bacteriophages, traditional medicine, and probiotics.

Unlocking Antibacterial Potential: Key-Site-Based Regulation of Antibacterial Spectrum of Peptides.

In the pursuit of combating multidrug-resistant bacteria, antimicrobial peptides (AMPs) have emerged as promising agents; however, their application in clinical settings still presents challenges. Specifically, the exploration of crucial structural parameters that influence the antibacterial spectrum of AMPs and the subsequent development of tailored variants with either broad- or narrow-spectrum characteristics to address diverse clinical therapeutic needs has been overlooked. This study focused on investigating the effects of amino acid sites and hydrophobicity on the peptide's antibacterial spectrum through Ala scanning and fixed-point hydrophobic amino acid substitution techniques. The findings revealed that specific amino acid sites played a pivotal role in determining the antibacterial spectrum of AMPs and confirmed that broadening the spectrum could be achieved only by increasing hydrophobicity at certain positions. In conclusion, this research provided a theoretical basis for future precise regulation of an antimicrobial peptide's spectrum by emphasizing the intricate balance between amino acid sites and hydrophobicity.

Rational Design of a Potent Antimicrobial Peptide Based on the Active Region of a Gecko Cathelicidin.

The emergence of multidrug-resistant (MDR) bacteria presents a significant challenge to public health, increasing the risk of infections that are resistant to current antibiotic treatment. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics in the prevention of MDR bacterial infections. In the present study, we identified a novel cathelicidin AMP from Gekko japonicus, which exhibited broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.34 to 4.69 µg/mL. To improve its potential therapeutic application, a series of peptides was synthesized based on the active region of the gecko-derived cathelicidin. The lead peptide (RH-16) showed an antimicrobial activity comparable to that of the parent peptide. Structural characterization revealed that RH-16 adopted an amphipathic α-helical conformation. Furthermore, RH-16 demonstrated neither hemolytic nor cytotoxic activity but effectively killed a wide range of clinically isolated, drug-resistant bacteria. The antimicrobial activity of RH-16 was attributed to the nonspecific targeting of bacterial membranes, leading to rapid bacterial membrane permeabilization and rupture. RH-16 also retained its antibacterial activity in plasma and exhibited mild toxicity in vivo. Notably, RH-16 offered robust protection against skin infection in a murine model. Therefore, this newly identified cathelicidin AMP may be a strong candidate for future pharmacological development targeting multidrug resistance. The use of a rational design approach for isolating the minimal antimicrobial unit may accelerate the transition of natural AMPs to clinically applicable antibacterial agents.