Search Results (103)

Copper and zinc nanoparticles have been suggested as potent anticancer agents, particularly against osteosarcoma, a highly aggressive bone cancer with limited treatment options. In order to avoid systemic toxicity, biomolecular carriers able to chelate metal ions and deliver them in a targeted manner to the vicinity of cancer cells need to be developed. Herein, we have used a histidine-containing, cyclic dipeptide as a carrier able to chelate stabilized copper and zinc nanoparticles. The cyclic peptide cyclo-(histidine-phenylalanine) (cHF) self-assembled into amyloid-type fibrils; morphological and structural characterization following metal addition confirmed the formation of cHF−CuNPs and cHF–ZnNPs. These composite nanoparticles demonstrated bacteriostatic activity against Escherichia coli and Staphylococcus aureus at the in vitro level. We evaluated the optimal concentration of cHF–metalNP complexes with limited cytotoxicity to L929 fibroblasts and high cytotoxic effects against MG-63 osteosarcoma cells. Their cytotoxicity was particularly pronounced at pH 6.4, which emulates the tumor microenvironment. The cHF peptide alone did not demonstrate significant antimicrobial or cytotoxic effects to both cell types, suggesting that it can act as a cytocompatible, pH-responsive carrier of metal ions with targeted dual functionality against both microbial infections and osteosarcoma cancer cells. Full article

Background/Objectives: Gramicidin S (GS) is a cyclic antimicrobial peptide with strong antibacterial activity but significant cytotoxicity toward mammalian cells. This study evaluated GS-induced cytotoxicity in L929 fibroblast cells using both traditional 2D monolayer cultures and more physiologically relevant 3D spheroid models, and assessed whether liposomal encapsulation could mitigate toxicity and improve biocompatibility. Methods: L929 cells were cultured in monolayers and spheroids and treated with free GS or GS encapsulated in liposomes of varying lipid compositions. Cell viability and morphology were evaluated after 24 h of exposure using standard cytotoxicity assays. Results: Control liposomes, regardless of tested lipid type or concentration, showed no adverse effects on cell morphology or viability. Free GS caused pronounced, dose-dependent cytotoxicity in monolayers, decreasing viability to 11.0 ± 1.9% and 0.5 ± 1.1% at 50 and 75 µg/mL, respectively. By contrast, encapsulation in liposomes significantly reduced toxicity (p < 0.05), preserving 80.3–82.2% viability at 75 µg/mL depending on formulation, corresponding to protection factors exceeding 160-fold (80.3% vs. 0.5%). Spheroid cultures showed slightly higher resistance to GS; free GS reduced viability to 2.9%, while liposomal GS preserved it above 84.8%, depending on lipid composition. Conclusions: Liposomal encapsulation effectively reduces GS-induced cytotoxicity, likely by limiting direct membrane disruption. Moreover, spheroid models provide a more physiologically relevant and predictive platform for toxicity testing, while the results support nanoliposomes as a practical delivery strategy to enhance the safety of antimicrobial peptides during preclinical development. Full article

This study explores the taxonomic diversity, metabolic profile, and bioactivity of marine-derived actinomycetes isolated from sediments collected off the coast of Lisbon and Setúbal Peninsula, Portugal. The combined use of two sediment pre-treatments (heat shock and dry overnight) and four growth media with varying nutrient concentrations revealed that formulations 10% A1 and SWA were most effective for recovering diverse actinomycetes, including rare Actinomadura, resulting in a total of 142 cultivable strains closely related to 47 phylogenetic distinct species dominated by Streptomyces and Micromonospora. Antimicrobial screening against methicillin-resistant Staphylococcus aureus (MRSA, COL) and Escherichia coli (K12) identified 22 bioactive strains, with strain PTS-083 exhibiting the strongest activity against MRSA (MIC = 1.95 µg/mL) and a 98.30% 16S rRNA gene identity to S. chumphonensis, highlighting it as a strong candidate for further metabolite and genomic studies. Cytotoxicity assays against HCT-116 human colorectal adenocarcinoma cells revealed eight bioactive strains with potent anticancer activity for extracts from strains related to S. sundarbansensis, S. violaceorubidus, and S. aculeolatus (IC₅₀ < 0.005–5.08 µg/mL). Untargeted LC-MS/MS metabolomic analysis uncovered a wide array of secondary metabolites, including macrolides, siderophores, fatty acids, and cyclic peptides. Comparative analyses with other Portuguese coastal studies revealed both shared and distinctive metabolomic profiles, emphasizing the importance of exhaustive sampling, even at nearby locations, since localized environmental conditions can influence metabolic diversity and are crucial for uncovering unique metabolites with potential biotechnological value. These findings highlight Portugal’s coastal sediments as a rich and underexplored source of novel actinomycetes and bioactive compounds with promising pharmaceutical applications. Full article

Mycobacterium avium is an opportunistic pathogen and a leading contributor to nontuberculous mycobacterial infections in immunocompromised individuals. However, treatment duration, antibiotic toxicity, and resistance present challenges in the management of mycobacterium infections, prompting the need for novel treatment. N-acetylcysteine (NAC) has demonstrated potent antimycobacterial activity, while antimicrobial peptides such as the cyclic [R₄W₄] have shown additive effects when combined with first-line antibiotics. This study aimed to investigate the mechanism and efficacy of NAC and [R₄W₄] combination therapy against M. avium. A membrane depolarization assay was used to evaluate the effects of NAC and [R₄W₄] on M. avium cell membrane integrity. Antimycobacterial activity was assessed by treating cultures with varying concentrations of NAC, [R₄W₄], a combination, or a sham treatment. The same regimens were applied to M. avium-infected THP-1-derived macrophages to assess intracellular efficacy. NAC and [R₄W₄] each disrupted the M. avium membrane potential, with enhanced effects in combination. The combination treatment significantly reduced M. avium survival in both the culture and infected macrophages compared with NAC alone and untreated controls. [R₄W₄] and NAC also demonstrated potent antibacterial activity, while the lowest MIC and the combination of [R₄W₄] and NAC displayed additive effects, indicating an improved bacterial inhibition compared to individual treatments. These findings demonstrate the additive activity of NAC and [R₄W₄] against M. avium in vitro and suggest that combining antioxidant compounds with antimicrobial peptides may represent a promising strategy for treating mycobacterial infections. Full article

Background/Objectives: Resistance of pathogenic microorganisms to antibiotics poses a serious threat to public health and often leads to devastating consequences. In this context, one of the pressing challenges in pharmacochemistry is the search for new, effective antibiotics to combat severe human diseases. Cyclic lipopeptides have emerged as some of the most promising candidates and have been widely studied. These compounds are a class of microbial secondary metabolites produced by various microorganisms, and they possess significant medical and biotechnological importance. The defining structural feature of these compounds is the presence of both a hydrophobic fragment, primarily a hydrocarbon tail of varying length, and a hydrophilic cyclic peptide moiety. This hydrocarbon tail confers amphiphilic properties to the lipopeptides, which are essential for their broad spectrum of biological activities. Their mechanism of action involves disruption of the cell membrane, and in many cases, the formation of ion-permeable defects has also been shown. Results: This review summarizes the data on cyclic lipopeptides produced by Pseudomonas spp., Streptomyces spp., and Bacillus spp. that modify membrane permeability through the formation of ion channels. The main emphasis is on understanding how the structure of the CLP can be related to the probability and mode of pore formation. Conclusions: The findings can contribute to expanding the arsenal of effective antimicrobial agents with a mechanism of action that reduces the risk of developing resistance. Full article

Gramicidin S (GS), an antimicrobial peptide (AMP), exhibits broad-spectrum activity against bacteria and cancer cells but is limited in clinical use due to its cytotoxicity toward eukaryotic cells. Lipid-based delivery systems may overcome this limitation; in this study, we proposed and tested simple and promising lipid formulations, including dipalmitoylphosphatidylcholine (DPPC), cardiolipin (CL), and cholesterol (CHOL). We evaluated the interactions of these lipid membranes with GS by assessing membrane fluidity, dielectric permittivity, dielectric losses, dielectric relaxation frequency, and static dielectric constant. Among these, membrane fluidity and dielectric permittivity were the most sensitive to GS, showing significant changes in the formulation containing 90 mol% DPPC and 10 mol% CHOL when exposed to 20 μM GS. Notably, although membrane fluidity changed in a dose-dependent manner following GS binding, the liposomes still supported relatively high GS concentrations—up to 80 μM—which is important for future high-dose GS applications. Additionally, we performed preliminary cytotoxicity tests comparing free GS with liposome-carried GS using the tested lipid compositions and observed a significant reduction in GS-associated toxicity on L929 cell line. This study provides new insights into GS–membrane interactions and supports the rational design of AMP nanocarriers for biomedical applications. Full article

The market for non-concentrated (NFC) orange juice is increasing rapidly due to consumer demand for nutrients and flavor. However, it encounters challenges in microbial safety, particularly from Salmonella enterica and Listeria monocytogenes. This study aimed to exploit a bio-preservative for NFC orange juice. Results showed that the cyclic peptide cyclo-zp80r had good antibacterial activity, with minimum inhibitory concentration values of 2–8 μM against S. enterica and L. monocytogenes. It exhibited bactericidal action against S. enterica and bacteriostatic action against L. monocytogenes at a concentration of 128 μM. This study explored the effect of cyclo-zp80r on the pathogenicity of S. enterica and L. monocytogenes. The mortality rate of Galleria mellonella exposed to these pathogens in NFC orange juice decreased from 100% to 60% after cyclo-zp80r treatment, surpassing the effectiveness of nisin. Cyclo-zp80r exhibited depolarization effects on S. enterica and L. monocytogenes. It increased outer membrane permeability and damaged the membrane structure of S. enterica. Cyclo-zp80r also caused distinct morphological changes, mainly cell collapse in S. enterica and localized bubble-like protrusions in L. monocytogenes. It induced reactive oxygen species production and DNA binding. The species diversity and abundance in NFC orange juice were also reduced by cyclo-zp80r, particularly in the genera Pantoea, Aeromonas, Pseudomonas, and Erwinia. Additionally, cyclo-zp80r exhibited excellent stability at high temperature (121 °C, 5 min) and in fresh orange juice. These results suggest that cyclo-zp80r could be developed as an effective food bio-preservative. Full article

This research represents the first application of the MOLS method to characterize the conformational energy landscape of an antimicrobial peptide within a solvent environment, providing a novel approach to understanding peptide behavior in solution. This method’s exhaustive nature ensures that all minimum-energy conformations for a given amino acid sequence are sampled. In this work, we employed a combination of MOLS and VMD software to generate structural models of a cyclic peptide, both solvated and non-solvated, and then utilized the CHARMM force field to conduct energy calculations throughout the sampling process. In the presence of a solvent, this method predicted a structure close to the experimental crystal structure. A significant reduction was observed in gamma turn motifs in the presence of water. The solvent molecules also favored different hydrogen bonding patterns in the peptide by orchestrating an intermolecular interaction with the peptide atoms. This intermolecular interaction involves an ARG side chain and further stabilizes the backbone. It is evident that solvent interactions are key in designing antimicrobial peptides. This study will help in designing and understanding peptides for use as therapeutic agents like antibacterial or antimicrobial peptides. Each conformer obtained from the MOLS method would be one of the best starting points for molecular dynamic simulation to further explore the landscape. Full article

Antimicrobial peptides (AMPs) have emerged as promising candidates in the fight against multidrug-resistant pathogens due to their broad-spectrum antimicrobial activity and low potential for resistance development. However, their clinical application is limited by poor stability and susceptibility to enzymatic degradation. This study aims to address these limitations by synthesizing a series of cyclic hexapeptides using the hydrazide method and evaluating their antimicrobial activity and stability. The hydrazide method facilitated the synthesis of 11 cyclic peptides through a reaction between C-terminal hydrazides and cysteine-containing peptides. Antimicrobial assays showed that Cy-f2 and Cy-f4 exhibited potent inhibitory effects against different kinds of bacteria, including E. coli, Staphylococcus aureus, and S. aureus. Hemolysis assays revealed minimal red blood cell lysis at effective antimicrobial concentrations, indicating good biocompatibility. Stability tests demonstrated improved stability of the cyclic peptides compared to linear counterparts in SGF and 80 °C. In conclusion, the cyclic hexapeptides synthesized in this study demonstrate excellent antimicrobial activity, enhanced stability, and low toxicity, suggesting their potential as new candidates for treating drug-resistant bacterial infections. Full article

Background/Objectives: Multidrug-resistant Gram-negative ESKAPE pathogens, including E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii, pose a significant global health threat. Gramicidin S, a potent cyclic antimicrobial peptide, is largely ineffective against these bacteria, and its high haemolytic toxicity limits its clinical usage. This study reports on several novel gramicidin S analogues with improved efficacy and safety profiles against multidrug-resistant Gram-negative bacteria. Methods: A total of 19 gramicidin S derivatives were synthesised using Fmoc-based solid-phase peptide synthesis with targeted substitutions to enhance cationicity and modulate hydrophobicity. Minimum inhibitory concentrations (MICs) were determined against standard Gram-negative and Gram-positive strains. Haemolytic toxicity and in vitro nephrotoxicity were evaluated using human red blood cells and HEK-293 cells, respectively. All peptides were characterised by RP-HPLC and HRMS. Results: The selective incorporation of ^DArg and Trp significantly enhanced activity against Gram-negative bacteria while reducing cytotoxicity. Peptide 8 improved the therapeutic index (TI) against E. coli by 10-fold (MIC: 8 µg/mL; TI: 4.10) compared to gramicidin S (MIC: 32 µg/mL; TI: 0.38). Peptide 9 exhibited an 8-fold potency increase against K. pneumoniae and a 25-fold TI improvement. Peptide 19 enhanced activity against P. aeruginosa 8-fold over gramicidin S, while peptide 7 showed a 27-fold TI enhancement. All active peptides retained broad-spectrum activity against S. aureus, including MRSA. Conclusions: The findings highlight the critical role of balancing hydrophobicity and cationicity to overcome species-specific resistance mechanisms. Our gramicidin S analogues demonstrate potent broad-spectrum activity with significantly reduced toxicity compared to the parent peptide, providing a robust platform for the development of new antibiotics against ESKAPE bacterial pathogens. Full article

Background: LR18 is an α₋helical AMP with high antimicrobial activity, low hemolytic activity, and low cytotoxicity. However, the susceptibility to degradation of the peptidase enzyme and a short half-life hinder its application as a therapeutic agent. Improving the stability and prolonging the half-life of LR18 are crucial to accelerate its application in the treatment of infectious diseases. Methods: A new cyclic peptide, C-LR18, was designed and synthesized through end-to-end cyclization of LR18 via disulfide bonds. The biological activity, half-life, and therapeutic effect of C-LR18 on Escherichia coli₋infected mice were studied. Results: C-LR18 maintained the characteristics of low cytotoxicity and low hemolytic activity of the original LR18 peptide and had higher antibacterial activity and significantly improved stability. After treatment with 1 mg/mL of trypsin, carboxypeptidase, and papain for 1 h, the MIC of C-LR18 against E. coli ATCC25922 was 4 μM, while that of LR18 had increased to 128 μM. After exposure to 50% serum or artificial gut solution for 30 min, the MIC of C-LR18 against E. coli ATCC25922 increased 4-fold, while that of LR18 increased 16-fold. The half-life of C-LR18 in plasma and in rats was extended to 3.37-fold and 4.46-fold, respectively, that of LR18. The acute toxicity of C-LR18 in mice is lower than many AMPs reported so far (LD50 = 37.8 mg/kg). C-LR18 has a therapeutic effect on E.coli-infected mice. Conclusions: The cyclic peptide C-LR18 has higher antibacterial activity and stability and a longer half-life than LR18 in rats in vitro and in vivo. C-LR18 also has a therapeutic effect on KM mice infected with E. coli and is expected to become a therapeutic drug for bacterial diseases and applied to the treatment of human and veterinary diseases. Full article

Background/Objectives: Cm-p5 and its cyclic monomeric and dimeric analogues are known for their antifungal, antibacterial, antiviral, and antibiofilm activities. Previously, our cyclization method produced a mixture of peptides that were difficult to separate, which was then improved by a selective synthesis of the parallel dimer and its differentiation from the antiparallel by comparison of the retention times in RP-HPLC. Methods: Here, we developed a more reliable identification method for the Cm-p5 dimer identification, which included chymotrypsin proteolytic digestion and sequencing of the different fragments by ESI-MSMS. We also improved our cyclization methods to specifically produce higher amounts of the desired cyclic variant, either cyclic monomer or dimer. Results: We show that liquid phase oxidation with 20% DMSO or iodine oxidation yields only the cyclic analogue. However, the on-resin oxidation with iodine showed greater efficacy and efficiency. Additionally, liquid phase cyclization yields the antiparallel dimer in high EtOH or peptide concentration, indicating a kinetic control. On the other hand, the parallel dimer was preferentially produced in 5% of TFE and low peptide concentration without the formation of the cyclic analogue indicating a thermodynamic control. Conclusions: In conclusion, we report that chymotryptic digestion combined with ESI-MS and MS/MS allows an unambiguous differentiation of Cm-p5 dimers. Here, we develop more selective and efficient methods for the synthesis of cyclic and dimeric analogues of Cm-p5. Full article

It remains an open question whether violets use universal mechanisms, such as the production of metallothioneins, phytochelatins, and organic acids and/or rely on specific mechanisms like the production of antimicrobial cyclic peptides (cyclotides) for heavy metal tolerance. To contribute to the understanding of the role of cyclotides, we used seed-derived plants from metallicolous (M) and non-metallicolous (NM) populations of Viola tricolor, a pseudometallophyte tolerant to Zn and Pb. Eight- to ten-week-old plants were treated with 1000 μM of Zn or Pb for 3 or 7 days and subsequently measured for cyclotides and heavy metal content using MALDI-MS and Atomic Absorption Spectrometry (AAS), respectively. Individuals from the M population accumulated a similar amount of Zn but occasionally more Pb in comparison with the NM population. Of the 18 different cyclotides included in the analysis, some showed statistically significant changes under the heavy metal treatment. In general, a decrease was observed in the M population, whereas an increase was observed in the NM population (except for the 3-day treatment with Zn). The day of treatment and dose of metal and their interaction played a crucial role in the explained variance for cyclotides produced by the M individuals but not for the NM plants. This unravels the importance of this antimicrobial compound in heavy metal tolerance and indicates that, in V. tricolor, cyclotides are involved in heavy metal tolerance, but specimens from two populations have developed different strategies and tolerance mechanisms involving cyclotides to mitigate heavy metal stress. Full article

Background. We have previously reported peptides composed of sequential arginine (R) residues paired with tryptophan (W) or 3,3-diphenyl-L-alanine residues (Dip), such as cyclic peptides [R₄W₄] and [R₄(Dip)₃], as antibacterial agents. Results. Herein, we report antibacterial and antifungal activities of five linear peptides, namely ((DipR)₄(WR)), ((DipR)₃(WR)₂), ((DipR)₂(WR)₃), ((DipR)(WR)₄), and (DipR)₄R, and five cyclic peptides [(DipR)₄(WR)], [(DipR)₃(WR)₂], [(DipR)₂(WR)₃], [(DipR)(WR)₄], and [DipR]₅, containing alternate positively charged R and hydrophobic W and Dip residues against fungal, Gram-positive, and Gram-negative bacterial pathogens. The minimum inhibitory concentrations (MICs) of all peptides were determined by the micro-broth dilution method against Methicillin-Resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, and Bacillus subtilis. Fungal organisms were Candida albicans, Candida parapsilosis, and Aspergillus fumigatus. [DipR]₅ and ((DipR)₂(WR)₃) showed MIC values of 0.39–25 µM and 0.78–12.5 µM against Gram-positive and Gram-negative bacteria strains, respectively. The highest activity was observed against S. pneumoniae with MIC values of 0.39–0.78 µM among tested compounds. [DipR]₅ demonstrated MIC values of 6.6 µM against C. parapsilosis and 1.6 µM against A. fumigatus, whereas fluconazole showed MIC values of 3.3 µM and >209 µM, respectively. Conclusions. These findings highlight the potential of these peptides as broad-spectrum antimicrobial agents. Full article

Inflammatory bowel disease (IBD) is increasing among mammals around the world, and domestic dogs are no exception. There is no approved cure for canine IBD with limited treatment options. Novel probiotic bacteria discovery from free-ranging animals for the treatment of IBD in domestic pets can likely yield promising probiotic candidates. Consequently, the overall aim was to isolate bacteria from free-ranging animals that could potentially be utilized as novel probiotics. Two bacteria identified as unique Paenibacillus spp. strains by small ribosomal RNA (16S) gene sequencing were isolated from the gastrointestinal tract of a North American Gray Wolf (Canis lupus). The bacteria were typed as Gram-variable, and both were catalase/oxidase positive as well as sensitive to commonly used antibiotics. The bacteria digested complex carbohydrates and lipids by standard assays. The isolated bacteria also inhibited the growth of Staphylococcus aureus and Micrococcus luteus. The whole genome sequence (WGS) length of bacterial isolate ClWae17B was 6,939,193 bp, while ClWae19 was 7,032,512 bp, both similar in size to other Paenibacillus spp. The genomes of both bacteria encoded enzymes involved with the metabolism of complex starches and lipids, such as lyases and pectinases, along with encoding antimicrobials such as lanthipeptides, lasso peptides, and cyclic-lactone-autoinducers. No pernicious virulence genes were identified in the WGS of either bacterial isolate. Phylogenetically, the most closely related bacteria based on 16S gene sequences and WGS were P. taichungensis for ClWae17B and P. amylolyticus for ClWae19. WGS analyses and phenotypic assays supported the hypothesis that the isolates described constitute two novel candidate probiotic bacteria for potential use in dogs. Full article