Search Results (105)

Background: The well-studied 18-residue-long proline-rich antimicrobial designer peptide Api137 utilizes at least two lethal intracellular mechanisms that target the bacterial 70S ribosome. First, Api137 stalls the ribosome by binding to the peptidyl-transferase center, trapping the release factor, and inhibiting protein expression. Second, Api137 disrupts the assembly of the large 50S subunit of the ribosome, resulting in partially assembled pre-50S dead-end particles that are unable to form the functional 70S ribosome. Methods: All six proline residues in Api137 were substituted with 4S- and 4R-fluoro-l-proline (Fpr), which promote the cis- and trans-conformer ratio of the preceding Xaa-Pro-bond, respectively. The effect on the antibacterial activity was studied using Escherichia coli. The underlying mechanisms were investigated by studying 70S ribosome binding, inhibition of in vitro translation, and ribosome profile analysis. Results: Interestingly, the analogs were equipotent to Api137, except for the 4S-Fpr11 and 4S-Fpr16 analogs, which were four times more or less active, respectively. The most active 4S-Fpr11 analog competed the least with Api137 for its ribosome binding site, suggesting a shifted binding site. Both Fpr14 and the 4S-Fpr16 analogs disturbed 50S subunit assembly less than Api137 or not at all. The strongest effect was observed with the 4R-Fpr16 analog resulting in the lowest 70S ribosome content and the highest pre-50S particle content. This peptide also showed the strongest competition with Api137 for its binding site. However, its antibacterial activity was similar to that of Api137, possibly due to its slower cellular uptake. Conclusions: Api137 inhibits protein translation and disrupts 50S assembly, which can be adjusted by substituting specific proline residues with fluoroproline. 4R-Fpr16 potently inhibits ribosome assembly and offers a novel, unexploited clinical mechanism for future antibiotic development. Full article

Background: The use of antimicrobial peptides (AMPs) as biotechnological tools is an area of growing interest in the research that seeks to improve crop defense. SmAP_α1–21 and SmAP_γ27–44 were previously reported to inhibit Fusarium graminearum, permeabilize the plasma membrane and induce cytoplasmic disorganization. To exert its activity, SmAP_α1–21 initially enters through the basal and apical cells of F. graminearum conidia and then displays a general but non-homogeneous distribution in the cytoplasm of all conidial cells, in contrast. Methods: We analyzed, focusing on membrane interaction, the mode of action of SmAP_γ27–44, a peptide based on the γ-core of defensins DefSm2-D and DefSm3, and SmAP_α1–21, based on the α-core of DefSm2-D. Additionally, we compared the behavior of SmAP_α1–21 with that of SmAP3_α1–21 based on DefSm3 but with no activity against F. graminearum. Results: In this study, we showed that SmAP_γ27–44 enters the cells with discrete intracellular localization. Furthermore, both peptides disrupted the plasma membrane, but with different modes of action. When large unilamellar liposomes (LUVs) containing phosphatidic acid and ergosterol were used as a filamentous fungal plasma membrane model, SmAP_γ27–44 strongly induced aggregation concomitantly with the solubilization of the liposomes and showed the maximal insertion of its tryptophan moiety into the membrane’s hydrophobic interior. In comparison, SmAP_α1–21 showed a high effect on the ζ potential of anionic vesicles, vesicle aggregation capacity after reaching a concentration threshold, and moderate transfer of tryptophan to the membrane. SmAP3_α1–21, on the other hand, showed poor superficial adsorption to liposomes. Conclusions: In view of our results, a cell penetration peptide-like effect was pictured for the γ-core defensin-derived peptide and a classical AMP action was observed for the α-core defensin-derived one. Full article

Antimicrobial resistance is a global health threat, which has been worsened by the slow development of new antibiotics. The rational design of natural-derived antimicrobial peptides (AMPs) offers a promising alternative for enhancing the efficacy of AMPs and accelerating drug discovery. This paper describes the rational design of improved peptide derivatives starting from hylin-Pul3, a peptide previously isolated from the frog Boana pulchella, by optimizing its hydrophobicity, cationicity, and amphipathicity. In silico screening identified six promising candidates: dHP3-31, dHP3-50, dHP3-50.137, dHP3-50.190, dHP3-84, and dHP3-84.39. These derivatives exhibited enhanced activity against Gram-negative bacteria, emphasizing the role of cationicity and the strategic arginine incorporation. Hemolytic assays revealed the derivatives’ improved selectivity, particularly for the derivatives with “imperfect amphipathicity”. In fibroblast assays, dHP3-84 was well-tolerated, while dHP3-84.39 promoted cell proliferation. Antioxidant assays (ABTS assays) highlighted the Trp-containing derivatives’ (dHP3-50.137, dHP3-31) significant activity. The lipid membrane interaction studies showed that hylin-Pul3 disrupts membranes directly, while dHP3-84.39, dHP3-50, and dHP3-50.137 promote vesicle aggregation. Conversely, dHP3-84 did not induce membrane disruption or aggregation, suggesting an intracellular mode of action. Machine learning models were effective in predicting bioactivity, as these predicted AMPs showed enhanced selectivity and potency. Among them, dHP3-84 demonstrated broad-spectrum potential. These findings highlight the value of rational design, in silico screening, and structure–activity studies in optimizing AMPs for therapeutic applications. Full article

Multidrug-resistant (MDR) bacteria are becoming more and more common, which presents a serious threat to world health and could eventually render many of the antibiotics we currently use useless. The research and development of innovative antimicrobial tactics that can defeat these hardy infections are imperative in light of this predicament. Antimicrobial peptides (AMPs), which have attracted a lot of attention due to their distinct modes of action and capacity to elude conventional resistance mechanisms, are among the most promising of these tactics. As a promising substitute for conventional antibiotics, AMPs are a varied class of naturally occurring compounds that target bacteria membranes and disrupt cellular activities to demonstrate broad-spectrum antimicrobial activity. The objective of this study is to present a thorough summary of the current knowledge regarding AMP mechanisms against MDR bacteria, including immunological modulation, interactions with microbial membranes, and possible synergy with currently used antimicrobial drugs. In addition, we define the review’s scope to include the most recent developments in AMP research, emphasizing the innovations’ development, optimization, and therapeutic promise. We hope to emphasize the crucial role that AMPs will play in the future of antimicrobial therapy by bringing together recent research and highlighting current issues. We also hope to advocate for AMPs’ continued research and development as part of a comprehensive strategy to counteract the growing threat of antibiotic resistance. Full article

G protein-coupled receptors (GPCRs) play essential roles in numerous physiological processes and are key targets for drug development. Among them, adhesion GPCRs (aGPCRs) stand out for their unique domain structures and diverse functions. ADGRG2 is a member of the aGPCR family and is involved in the regulation of various systems in the human body, including reproductive, nervous, cardiovascular, and endocrine systems. Investigating ADGRG2 antagonists enhances our understanding of its regulatory roles in diverse physiological processes, yet their precise mechanisms of action remain unclear. To address this, we investigated the antagonistic mechanism of ADGRG2 by examining its interactions with various antagonists, including short peptides (F601D, F601E) and small molecules (deoxycorticosterone, DOC). Using advanced metadynamics simulation, ligand binding assay and cAMP assay, we elucidated the binding modes of these antagonists. We identified five distinct F601D-ADGRG2 complex states, four F601E-ADGRG2 complex states, and three DOC-ADGRG2 complex states, which were each characterized by specific hydrogen bonds or polar interactions with their respective ligands. Although the ADGRG2 binding pocket consists of both polar and hydrophobic residues, our biochemical experiments revealed that mutations in polar amino acids significantly reduce the efficacy of the antagonists. Our results show that F601D, F601E, and DOC induce the formation of Y758^ECL2-N775^5.32-N860^7.46 polar networks within ADGRG2, effectively stabilizing its inactive state. Additionally, we compared the active and inactive states of ADGRG2, highlighting the structural changes induced by antagonist-stabilized polar networks and their impact on receptor conformation. These findings provide important insights into the biology of aGPCRs and provide theoretical support for the rational design of therapeutic drugs targeting ADGRG2. Full article

Host defense antimicrobial peptides (AMPs) are promising lead molecules with which to develop antibiotics against drug-resistant bacterial pathogens. Thanatin, an inducible antimicrobial peptide involved in the host defense of Podisus maculiventris insects, is gaining considerable attention in the generation of novel classes of antibiotics. Thanatin or thanatin-based analog peptides are extremely potent in killing bacterial pathogens in the Enterobacteriaceae family, including drug-resistant strains of Escherichia coli and Klebsiella pneumoniae. A single disulfide bond that covalently links two anti-parallel β-strands in thanatin could be pivotal to its selective antibacterial activity and mode of action. However, potential correlations of the disulfide covalent bond with structure, activity and target binding in thanatin peptides are currently unclear to. Here, we examined a 16-residue designed thanatin peptide, namely disulfide-bonded VF16QK, and its Cys to Ser substituted variant, VF16QK^Ser, to delineate their structure–activity relationships. Bacterial growth inhibitory activity was only detected for the disulfide-bonded VF16QK peptide. Mechanistically, both peptides vastly differ in their bacterial cell permeabilizations, atomic-resolution structures, interactions with the LPS-outer membrane and target periplasmic protein LptA_m binding. In particular, analysis of the 3-D structures of the two peptides revealed an altered folded conformation for the VF16QK^Ser peptide that was correlated with diminished LPS-outer membrane permeabilization and target interactions. Analysis of docked complexes of LPS–thanatin peptides indicated potential structural requirements and conformational adaptation for antimicrobial activity. Collectively, these observations contrast with those for the disulfide-bonded β-hairpin antimicrobial protegrin and tachyplesin peptides, where disulfide bonds are dispensable for activity. We surmise that the atomistic structures and associated molecular interactions presented in this work can be utilized to design novel thanatin-based antibiotics. Full article

Antimicrobial peptides (AMPs) constitute a large and diverse group of molecules with antibacterial, antifungal, antiviral, antiprotozoan, and anticancer activity. In animals, they are key components of innate immunity involved in fighting against various pathogens. Proline-rich (Pr) AMPs are characterized by a high content of proline (and arginine) residues that can be organized into Pro-Arg-Pro motifs. Such peptides have been described in many invertebrates (annelids, crustaceans, insects, mollusks) and some vertebrates (mammals). The main objective of this review is to present the diversity of invertebrate PrAMPs, which are associated with the presence of cysteine-rich domains or whey acidic protein domains in the molecular structure, in addition to the presence of characteristic proline-rich regions. Moreover, PrAMPs can target intracellular structures in bacteria, e.g., 70S ribosomes and/or heat shock protein DnaK, leading to the inhibition of protein synthesis and accumulation of misfolded polypeptides in the cell. This unique mechanism of action makes it difficult for pathogens to acquire resistance to this type of molecule. Invertebrate PrAMPs have become the basis for the development of new synthetic analogues effective in combating pathogens. Due to their great diversity, new highly active molecules are still being searched for among PrAMPs from invertebrates. Full article

Antimicrobial peptides (AMPs) are short, usually cationic peptides with an amphiphilic structure, which allows them to easily bind and interact with the cellular membranes of viruses, bacteria, fungi, and other pathogens. Bacterial AMPs, or bacteriocins, can be produced from Gram-negative and Gram-positive bacteria via ribosomal synthesis to eliminate competing organisms. Bacterial AMPs are vital in addressing the increasing antibiotic resistance of various pathogens, potentially serving as an alternative to ineffective antibiotics. Bacteriocins have a narrow spectrum of action, making them highly specific antibacterial compounds that target particular bacterial pathogens. This review covers the two main groups of bacteriocins produced by Gram-negative and Gram-positive bacteria, their modes of action, classification, sources of positive effects they can play on the human body, and their limitations and future perspectives as an alternative to antibiotics. Full article

Background: Compound Ku-Shen Injection (CKI) is a traditional Chinese medicine preparation derived from Ku-Shen and Bai-Tu-Ling, commonly used in the adjunctive treatment of advanced cancers, including liver cancer. However, the underlying mechanisms of CKI’s effectiveness in cancer treatment are not well defined. Methods: This study employs network pharmacology to investigate the traditional Chinese medicine (TCM) compatibility theory underlying CKI’s action in treating liver cancer, with findings substantiated by molecular docking and in vitro experiments. Sixteen active components were identified from CKI, along with 193 potential targets for treating liver cancer. Key therapeutic target proteins, including EGFR and ESR1, were also identified. KEGG enrichment results showed that the neuroactive ligand–receptor interaction, cAMP signaling pathway, and serotonergic synapses make up the key pathway of CKI in the treatment of liver cancer. Molecular docking results confirmed that the key active ingredients effectively bind to the core targets. CCK-8 cytotoxic experiment results show that the CKI key components of oxymatrine and matrine can inhibit the growth of HepG2 liver cancer cell proliferation. A Western blot analysis revealed that oxymatrine suppresses the expression of EGFR, contributing to its therapeutic efficacy against liver cancer. Conclusion: our study elucidated the therapeutic mechanism of CKI in treating liver cancer and unveiled the underlying principles of its TCM compatibility through its mode of action. Full article

This study focused on the discovery of the antimicrobial peptide (AMP) derived from mangrove bacteria. The most promising isolate, NNS5-6, showed the closest taxonomic relation to Paenibacillus thiaminolyticus, with the highest similarity of 74.9%. The AMP produced by Paenibacillus thiaminolyticus NNS5-6 exhibited antibacterial activity against various Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae. The peptide sequence consisted of 13 amino acids and was elucidated as Val-Lys-Gly-Asp-Gly-Gly-Pro-Gly-Thr-Val-Tyr-Thr-Met. The AMP mainly exhibited random coil and antiparallel beta-sheet structures. The stability study indicated that this AMP was tolerant of various conditions, including proteolytic enzymes, pH (1.2–14), surfactants, and temperatures up to 40 °C for 12 h. The AMP demonstrated 4 µg/mL of MIC and 4–8 µg/mL of MBC against both pathogens. Time-kill kinetics showed that the AMP acted in a time- and concentration-dependent manner. A cell permeability assay and scanning electron microscopy revealed that the AMP exerted the mode of action by disrupting bacterial membranes. Additionally, nineteen biosynthetic gene clusters of secondary metabolites were identified in the genome. NNS5-6 was susceptible to various commonly used antibiotics supporting the primary safety requirement. The findings of this research could pave the way for new therapeutic approaches in combating antibiotic-resistant pathogens. Full article

Antimicrobial peptides (AMPs) are being explored as a potential strategy to combat antibiotic resistance due to their ability to reduce susceptibility to antibiotics. This study explored whether the [R₄W₄] peptide mode of action is bacteriostatic or bactericidal using modified two-fold serial dilution and evaluating the synergism between gentamicin and [R₄W₄] against Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) by a checkered board assay. [R₄W₄] exhibited bactericidal activity against bacterial isolates (MBC/MIC ≤ 4), with a synergistic effect with gentamicin against E. coli (FICI = 0.3) but not against MRSA (FICI = 0.75). Moreover, we investigated the mechanism of action of [R₄W₄] against MRSA by applying biophysical assays to evaluate zeta potential, cytoplasmic membrane depolarization, and lipoteichoic acid (LTA) binding affinity. [R₄W₄] at a 16 mg/mL concentration stabilized the zeta potential of MRSA −31 ± 0.88 mV to −8.37 mV. Also, [R₄W₄] at 2 × MIC and 16 × MIC revealed a membrane perturbation process associated with concentration-dependent effects. Lastly, in the presence of BODIPY-TR-cadaverine (BC) fluorescence dyes, [R₄W₄] exhibited binding affinity to LTA comparable with melittin, the positive control. In addition, the antibacterial activity of [R₄W₄] against MRSA remained unchanged in the absence and presence of LTA, with an MIC of 8 µg/mL. Therefore, the [R₄W₄] mechanism of action is deemed bactericidal, involving interaction with bacterial cell membranes, causing concentration-dependent membrane perturbation. Additionally, after 30 serial passages, there was a modest increment of MRSA strains resistant to [R₄W₄] and a change in antibacterial effectiveness MIC [R₄W₄] and vancomycin by 8 and 4 folds with a slight change in Levofloxacin MIC 1 to 2 µg/mL. These data suggest that [R₄W₄] warrants further consideration as a potential AMP. Full article

Antimicrobial resistance threatens the effective prevention and treatment of an increasingly broad spectrum of infections caused by pathogenic microorganisms. This pressing challenge has intensified the search for alternative antibiotics with new pharmacological properties. Due to the chemical synergy between the biological activity of antimicrobial peptides (AMPs) and the different modes of action, catalytic properties, and redox chemistry of metal complexes, metallopeptides have emerged in recent years as an alternative to conventional antibiotics. In the present investigation, peptide ligands conjugated with 5-carboxy-1,10-phenanthroline (Phen) were prepared by solid-phase peptide synthesis (SPPS), and the corresponding copper(II) metallopeptides, Cu-PhenKG and Cu-PhenRG (where K = lysine, R = arginine, and G = glycine), were synthesized and characterized. The antimicrobial activities of these compounds toward Gram-positive and Gram-negative bacteria, evaluated by the broth microdilution technique, indicate that the metal center in the metallopeptides increases the antimicrobial activity of the complexes against the conjugated peptide ligands. Minimum inhibitory concentration (MIC) values of 0.5 μg/mL for S. aureus with the Cu-PhenKG complex and 0.63 μg/mL for S. typhimurium with the Cu-PhenRG complex were obtained. The MIC values found for the conjugated peptides in all microorganisms tested were greater than 1.5 μg/mL. The interactions of the conjugated peptides and their metallopeptides with plasmid DNA were evaluated by agarose gel electrophoresis. Alterations on the replication machinery were also studied by polymerase chain reaction (PCR). The results indicate that the complexes interact efficiently with pBR322 DNA from E. coli, delaying the band shift. Furthermore, the resulting DNA–metallopeptide complex is not a useful template DNA because it inhibits PCR, since no PCR product was detected. Finally, molecular dynamics and molecular docking simulations were performed to better understand the interactions of the obtained compounds with DNA. The Cu-PhenRG complex shows a significantly higher number of polar interactions with DNA, suggesting a higher binding affinity with the biopolymer. Full article

Capitellacin is the β-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 β-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal β-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 β-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 β-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 β-structural peptides in biological membranes. Full article

Antimicrobial peptides (AMPs) are usually made up of fewer than 100 amino acid residues. They are found in many living organisms and are an important factor in those organisms’ innate immune systems. AMPs can be extracted from various living sources, including bacteria, plants, animals, and even humans. They are usually cationic peptides with an amphiphilic structure, which allows them to easily bind and interact with the cellular membranes of viruses, bacteria, fungi, and other pathogens. They can act against both Gram-negative and Gram-positive pathogens and have various modes of action against them. Some attack the pathogens’ membranes, while others target their intracellular organelles, as well as their nucleic acids, proteins, and metabolic pathways. A crucial area of AMP use is related to their ability to help with emerging antibiotic resistance: some AMPs are active against resistant strains and are susceptible to peptide engineering. This review considers AMPs from three key sources—plants, animals, and humans—as well as their modes of action and some AMP sequences. Full article

The continuous rise in bacterial infections and antibiotic resistance is the driving force behind the search for new antibacterial agents with novel modes of action. Antimicrobial peptides (AMPs) have recently gained attention as promising antibiotic agents with the potential to treat drug-resistant infections. Several AMPs have shown a lower propensity towards developing resistance compared to conventional antibiotics. However, these peptides, especially acyldepsipeptides (ADEPs) present with unfavorable pharmacokinetic properties, such as high toxicity and low bioavailability. Different ways to improve these peptides to be drug-like molecules have been explored, and these include using biocompatible nano-carriers. ADEP1 analogues (SC005-8) conjugated to gelatin-capped Silver/Indium/Sulfide (AgInS₂) quantum dots (QDs) improved the antibacterial activity against Gram-negative (Escherichia coli and Pseudomonas aeruginosa), and Gram-positive (Bacillus subtilis, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus) bacteria. The ADEP1 analogues exhibited minimum inhibition concentrations (MIC) between 63 and 500 µM, and minimum bactericidal concentrations (MBC) values between 125 and 750 µM. The AgInS₂-ADEP1 analogue conjugates showed enhanced antibacterial activity as evident from the MIC and MBC values, i.e., 1.6–25 µM and 6.3–100 µM, respectively. The AgInS₂-ADEP1 analogue conjugates were non-toxic against HEK-293 cells at concentrations that showed antibacterial activity. The findings reported herein could be helpful in the development of antibacterial treatment strategies. Full article