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1.
BMC Vet Res ; 20(1): 164, 2024 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-38678277

RESUMO

BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.


Assuntos
Proteínas de Anfíbios , Antibacterianos , Filogenia , Ranidae , Animais , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Camundongos , Antibacterianos/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Sequência de Aminoácidos , Pele/metabolismo , Fatores Imunológicos/farmacologia , Fatores Imunológicos/química , Células RAW 264.7 , Alinhamento de Sequência
2.
Bioorg Med Chem Lett ; 96: 129499, 2023 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-37804993

RESUMO

A4K14-Citropin 1.1 (GLFAVIKKVASVIKGL-NH2) is a derived antimicrobial peptide (AMP) with a more stable α-helical structure at the C-terminal compared to prototype Citropin 1.1 which was obtained from glandular skin secretions of Australian freetail lizards. In a previous report, A4K14-Citropin 1.1 has been considered as an anti-cancer lead compound. However, linear peptides are difficult to maintain stable secondary structure, resulted in poor pharmacokinetic properties. In this study, we designed and synthesized a series of benzyl-stapled derivatives of A4K14-Citropin 1.1. And their physical and chemical properties, as well as biological activity, were both explored. The result showed that AC-CCSP-2-o and AC-CCSP-3-o exhibited a higher degree of helicity and greater anti-cancer activity compared with the prototype peptide. Besides, there was no significant difference in the hemolytic effect between the stapled peptides and the prototype peptide. AC-CCSP-2-o and AC-CCSP-3-o could serve as promising anti-cancer lead compounds for the novel anti-cancer drug development.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Peptídeos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Anfíbios/química , Estrutura Secundária de Proteína , Conformação Proteica em alfa-Hélice
3.
Amino Acids ; 55(10): 1349-1359, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37548712

RESUMO

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.


Assuntos
Antineoplásicos , Neoplasias , Animais , Humanos , Peptídeos Catiônicos Antimicrobianos/química , Células Endoteliais/metabolismo , Proteínas de Anfíbios/química , Anuros/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/metabolismo , Neoplasias/metabolismo , Pele/metabolismo , Testes de Sensibilidade Microbiana
4.
IET Nanobiotechnol ; 17(4): 352-359, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37042087

RESUMO

OBJECTIVE: Dermaseptin-PP is a newly discovered anticancer peptide with a unique antitumour mechanism and remarkable effect. However, this α-helix anticancer peptide risks haemolysis when used at high doses, which limits its further application. This study aims to prepare a pH-responsive liposome, Der-loaded-pHSL, using nanotechnology to avoid the haemolysis risk of Dermaseptin-PP and increase its accumulation in tumour sites to enhance efficacy and reduce toxicity. METHODS: The characterisation of Der-loaded-pHSL was carried out employing preparation. The effect of haemolysis and tumour inhibition were investigated by in vitro haemolysis assay and cytotoxicity assay. The cell uptake under different pH conditions was investigated by flow cytometry, and the effect of pH on tumour cell selectivity was evaluated. In order to evaluate the in vivo targeting and antitumour effect of Der-loaded-pHSL, the in vivo distribution experiment and the pharmacodynamic experiment were performed using the nude mouse tumour model. RESULTS: The preparation method of the Der-loaded-pHSL is simple, and the liposome has good nanoparticle characteristics. When Dermaseptin-PP was prepared as liposome, haemolysis was significantly decreased, and tumour cell inhibition was significantly enhanced. Compared with ordinary liposomes, this change was more significant in Der-loaded-pHSL. The uptake of pH-sensitive liposomes was higher in the simulated acidic tumour microenvironment, and the uptake showed a specific acid dependence. In vivo experiments showed that Der-loaded-pHSL had a significant tumour-targeting effect and could significantly enhance the antitumour effect of Dermaseptin-PP. CONCLUSION: Der-loaded-pHSL designed in this study is a liposome with a quick, simple, effective preparation method, which can significantly reduce the haemolytic toxicity of Dermaseptin-PP and enhance its antitumour effect by increasing the tumour accumulation and cell intake. It provides a new idea for applying Dermaseptin-PP and other anticancer peptides with α-helical structure.


Assuntos
Lipossomos , Neoplasias , Camundongos , Animais , Lipossomos/química , Hemólise , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Linhagem Celular Tumoral , Microambiente Tumoral
5.
Biochem Pharmacol ; 210: 115471, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36893813

RESUMO

Septic shock caused by Gram-positive bacteria continues to be a major cause of morbidity and mortality in intensive care units globally. Most Temporins are excellent growth inhibitors of gram-positive bacteria and candidates for developing antimicrobial treatments due to their biological action and small molecular weight. In this study, a novel Temporin peptide from the skin of Fejervarya limnocharis frog, named as Temporin-FL, was characterized. Temporin-FL was found to adopt typical α-helical conformation in SDS solution and to exhibit selective antibacterial activity against Gram-positive bacteria through a membrane destruction mechanism. Accordingly, Temporin-FL showed protective effects against Staphylococcus aureus-induced sepsis in mice. Finally, Temporin-FL was demonstrated to exert anti-inflammatory effects by neutralizing the action of LPS/LTA and by inhibiting MAPK pathway activation. Therefore, Temporin-FL represents a novel candidate for moleculartherapy of Gram-positive bacterial sepsis.


Assuntos
Anti-Infecciosos , Choque Séptico , Animais , Camundongos , Lipopolissacarídeos/toxicidade , Sequência de Aminoácidos , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/uso terapêutico , Proteínas de Anfíbios/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêutico , Anti-Infecciosos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antibacterianos/metabolismo , Ranidae/metabolismo , Pele , Bactérias Gram-Positivas , Choque Séptico/metabolismo , Testes de Sensibilidade Microbiana
6.
Toxins (Basel) ; 15(1)2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36668862

RESUMO

In recent years, antimicrobial peptides isolated from amphibian toxins have gained attention as new multifunctional drugs interacting with different molecular targets. We aimed to rationally design a new peptide from temporin-PTa. Hp-MAP3 (NH2-LLKKVLALLKKVL-COOH), net charge (+4), hydrophobicity (0.69), the content of hydrophobic residues (69%), and hydrophobic moment (0.73). For the construction of the analog peptide, the physicochemical characteristics were reorganized into hydrophilic and hydrophobic residues with the addition of lysines and leucines. The minimum inhibitory concentration was 2.7 to 43 µM against the growth of Gram-negative and positive bacteria, and the potential for biofilm eradication was 173.2 µM. Within 20 min, the peptide Hp-MAP3 (10.8 µM) prompted 100% of the damage to E. coli cells. At 43.3 µM, eliminated 100% of S. aureus within 5 min. The effects against yeast species of the Candida genus ranged from 5.4 to 86.6 µM. Hp-MAP3 presents cytotoxic activity against tumor HeLa at a concentration of 21.6 µM with an IC50 of 10.4 µM. Furthermore, the peptide showed hemolytic activity against murine erythrocytes. Structural studies carried out by circular dichroism showed that Hp-MAP3, while in the presence of 50% trifluoroethanol or SDS, an α-helix secondary structure. Finally, Amphipathic Hp-MAP3 building an important model for the design of new multifunctional molecules.


Assuntos
Proteínas de Anfíbios , Peptídeos Catiônicos Antimicrobianos , Animais , Humanos , Camundongos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Dicroísmo Circular , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Ranidae , Staphylococcus aureus/efeitos dos fármacos
7.
Oxid Med Cell Longev ; 2022: 2615178, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36105482

RESUMO

Amphibian skin is acknowledged to contain an antioxidant system composed of various gene-encoded antioxidant peptides, which exert significant effects on host defense. Nevertheless, recognition of such peptides is in its infancy so far. Here, we reported the antioxidant properties and underlying mechanism of a new antioxidant peptide, brevinin-1FL, identified from Fejervarya limnocharis frog skin. The cDNA sequence encoding brevinin-1FL was successfully cloned from the total cDNA of F. limnocharis and showed to contain 222 bp. The deduced mature peptide sequence of brevinin-1FL was FWERCSRWLLN. Functional analysis revealed that brevinin-1FL could concentration-dependently scavenge ABTS+, DPPH, NO, and hydroxyl radicals and alleviate iron oxidation. Besides, brevinin-1FL was found to show neuroprotective activity by reducing contents of MDA and ROS plus mitochondrial membrane potential, increasing endogenous antioxidant enzyme activity, and suppressing H2O2-induced death, apoptosis, and cycle arrest in PC12 cells which were associated with its regulation of AKT/MAPK/NF-κB signal pathways. Moreover, brevinin-1FL relieved paw edema, decreased the levels of TNF-α, IL-1ß, IL-6, MPO, and malondialdehyde (MDA), and restored catalase (CAT) and superoxide dismutase (SOD) activity plus glutathione (GSH) contents in the mouse injected by carrageenan. Together, these findings indicate that brevinin-1FL as an antioxidant has potent therapeutic potential for the diseases induced by oxidative damage. Meanwhile, this study will help us further comprehend the biological functions of amphibian skin and the mechanism by which antioxidants protect cells from oxidative stress.


Assuntos
Proteínas de Anfíbios , Antioxidantes , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/uso terapêutico , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Carragenina , DNA Complementar , Peróxido de Hidrogênio/metabolismo , Camundongos , Estresse Oxidativo , Ranidae , Ratos
8.
Amino Acids ; 54(9): 1327-1336, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35852614

RESUMO

Naturally occurring frog skin peptides are one of largest sources of antimicrobial peptides that have many advantages including high potency, broad spectrum of targets and low susceptibility to multiple drug-resistance bacteria. However, they also have disadvantages such as hemolytic activity, low stability and high production costs. For these reasons, various strategies have been applied to overcome these drawbacks restricting their use in clinical trials. Previously reported brevinin-1GHa (BR-1GHa) is a 24 amino acid long antimicrobial peptide isolated from Hylarana guentheri with hemolytic activity. To enhance the antimicrobial activity of this peptide and to reduce its hemolytic activity, we designed five new temporin like analogues and examined their bioactivities. Temporins are another class of frog skin peptides without hemolytic activity and shorter than brevinins. When the antimicrobial activities of new analogues were examined against a panel of microorganisms, BR-1GHa-3, in which two alanine residues in the truncated version of BR-1GHa were replaced with leucine, exhibited significantly improved antimicrobial activity against Gram-positive bacterial strains (e.g., S. aureus ATCC 29213 and E. casseliflavus ATCC 700327) with lower hemolytic activity compared to the BR-1GHa peptide. Furthermore, BR-1GHa-4 analogue, in which Gly3 was replaced with Pro, did not show any hemolytic activity except for highest (128 µM) concentration tested and have a strong antimicrobial effect on Gram-positive bacteria (e.g., E. faecalis ATCC 51299 and B. cereus ATCC 13061).


Assuntos
Anti-Infecciosos , Staphylococcus aureus , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Animais , Antibacterianos/farmacologia , Anti-Infecciosos/química , Bactérias Gram-Positivas , Hemólise , Testes de Sensibilidade Microbiana , Ranidae , Pele/metabolismo
9.
Biomolecules ; 12(6)2022 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-35740884

RESUMO

Bacterial resistance against antibiotics has led to increasing numbers of treatment failures, and AMPs are widely accepted as becoming potential alternatives due to their advantages. Temporin-PKE is a novel peptide extracted from the skin secretion of Pelophylax kl. esculentus and it displays a strong activity against Gram-positive bacteria, with an extreme cytotoxicity. Incorporating positively charged residues and introducing D-amino acids were the two main strategies adopted for the modifications. The transformation of the chirality of Ile could reduce haemolytic activity, and an analogue with appropriate D-isoforms could maintain antimicrobial activity and stability. The substitution of hydrophobic residues could bring about more potent and broad-spectrum antimicrobial activities. The analogues with Lys were less harmful to the normal cells and their stabilities remained at similarly high levels compared to temporin-PKE. The optimal number of charges was three, and the replacement on the polar face was a better choice. Temporin-PKE-3K exerted dually efficient functions includingstrong antimicrobial and anticancer activity. This analogue showed a reduced possibility for inducing resistance in MRSA and Klebsiella pneumoniae, a rather strong antimicrobial activity in vivo, and it exhibited the highest therapeutic index such that temporin-PKE-3K has the potential to be developed as a clinical drug.


Assuntos
Proteínas de Anfíbios , Anti-Infecciosos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos , Testes de Sensibilidade Microbiana , Rana esculenta , Ranidae , Pele , Relação Estrutura-Atividade
10.
Int J Mol Sci ; 23(4)2022 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-35216177

RESUMO

The COVID-19 pandemic has evidenced the urgent need for the discovery of broad-spectrum antiviral therapies that could be deployed in the case of future emergence of novel viral threats, as well as to back up current therapeutic options in the case of drug resistance development. Most current antivirals are directed to inhibit specific viruses since these therapeutic molecules are designed to act on a specific viral target with the objective of interfering with a precise step in the replication cycle. Therefore, antimicrobial peptides (AMPs) have been identified as promising antiviral agents that could help to overcome this limitation and provide compounds able to act on more than a single viral family. We evaluated the antiviral activity of an amphibian peptide known for its strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, namely Temporin L (TL). Previous studies have revealed that TL is endowed with widespread antimicrobial activity and possesses marked haemolytic activity. Therefore, we analyzed TL and a previously identified TL derivative (Pro3, DLeu9 TL, where glutamine at position 3 is replaced with proline, and the D-Leucine enantiomer is present at position 9) as well as its analogs, for their activity against a wide panel of viruses comprising enveloped, naked, DNA and RNA viruses. We report significant inhibition activity against herpesviruses, paramyxoviruses, influenza virus and coronaviruses, including SARS-CoV-2. Moreover, we further modified our best candidate by lipidation and demonstrated a highly reduced cytotoxicity with improved antiviral effect. Our results show a potent and selective antiviral activity of TL peptides, indicating that the novel lipidated temporin-based antiviral agents could prove to be useful additions to current drugs in combatting rising drug resistance and epidemic/pandemic emergencies.


Assuntos
Proteínas de Anfíbios/farmacologia , Anfíbios/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/química , Vírus de DNA/efeitos dos fármacos , Vírus de RNA/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Lipídeos/química , SARS-CoV-2/efeitos dos fármacos , Células Vero
11.
Biochim Biophys Acta Biomembr ; 1864(1): 183806, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34656552

RESUMO

Aurein 2.1, aurein 2.6 and aurein 3.1 are amphibian host defence peptides that kill bacteria via the use of lytic amphiphilic α-helical structures. The C-terminal PEGylation of these peptides led to decreased antibacterial activity (Minimum Lethal Concentration (MLCs) ↓ circa one and a half to threefold), reduced levels of amphiphilic α-helical structure in solvents (α-helicity ↓ circa 15.0%) and lower surface activity (Δπ ↓ > 1.5 mN m-1). This PEGylation of aureins also led to decreased levels of amphiphilic α-helical structure in the presence of anionic membranes and zwitterionic membranes (α-helicity↓ > 10.0%) as well as reduced levels of penetration (Δπ ↓ > 3.0 mN m-1) and lysis (lysis ↓ > 10.0%) of these membranes. Based on these data, it was proposed that the antibacterial action of PEGylated aureins involved the adoption of α-helical structures that promote the lysis of bacterial membranes, but with lower efficacy than their native counterparts. However, PEGylation also reduced the haemolytic activity of native aureins to negligible levels (haemolysis ↓ from circa 10% to 3% or less) and improved their relative therapeutic indices (RTIs ↑ circa three to sixfold). Based on these data, it is proposed that PEGylated aureins possess the potential for therapeutic development; for example, to combat infections due to multi-drug resistant strains of S. aureus, designated as high priority by the World Health Organization.


Assuntos
Proteínas de Anfíbios/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Anfíbios/farmacologia , Anfíbios/genética , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Polietilenoglicóis/química , Staphylococcus aureus/efeitos dos fármacos
12.
Biophys Chem ; 281: 106721, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34808479

RESUMO

Many antimicrobial peptides (AMPs) are cationic host defence peptides (HDPs) that interact with microbial membranes. This ability may lead to implementation of AMPs as therapeutics to overcome the wide-spread antibiotic resistance problem as the affected bacteria may not be able to recover from membrane lysis types of attack. AMP interactions with lipid bilayer membranes are typically explained through three mechanisms, i.e., barrel-stave pore, toroidal pore and carpet models. Electrical bilayer recording is a relatively simple and sensitive technique that is able to capture the nanoscale perturbations caused by the AMPs in the bilayer membranes. Molecular-level understanding of the behaviour of AMPs in relation to lipid bilayers mimicking bacterial and human cell membranes is essential for their development as novel therapeutic agents that are capable of targeted action against disease causing micro-organisms. The effects of four AMPs (aurein 1.2, caerin 1.1, citropin 1.1 and maculatin 1.1 from the skin secretions of Australian tree frogs) and the toxin melittin (found in the venom of honeybees) on two different phospholipid membranes were studied using the electrical bilayer recording technique. Bilayers composed of zwitterionic (DPhPC) and anionic (DPhPC/POPG) lipids were used to mimic the charge of eukaryotic and prokaryotic cell membranes, respectively, so as to determine the corresponding interaction mechanisms for different concentrations of the peptide. Analysis of the dataset corresponding to the four frog AMPs, as well as the resulting dataset corresponding to the bee toxin, confirms the proposed peptide-bilayer interaction models in existing publications and demonstrates the importance of using appropriate bilayer compositions and peptide concentrations for AMP studies.


Assuntos
Proteínas de Anfíbios , Peptídeos Antimicrobianos , Animais , Proteínas de Anfíbios/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Anuros/metabolismo , Austrália , Membrana Celular/metabolismo , Bicamadas Lipídicas/química
13.
Biomed Pharmacother ; 146: 112539, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34923337

RESUMO

Despite the increasing treatments in skin wound repair, existing therapeutic drugs cannot meet current needs. As such, skin wound repair remains a considerable clinical challenge, and thus the discovery of new pro-healing agents is crucial. Here, we identified the first naturally occurring peptide homodimer named as OA-GP11 dimer (OA-GP11d) from Odorrana andersonii (odorous frog) through the combinational methods of peptidomics and genomics. OA-GP11d was linked by the intramolecular disulfide formed by the 10th cysteine residues from the monomer of peptide with sequence of GPLSGINAECM, which effectively promoted the repair of full-thickness and burn wounds in mice. The underlying molecular mechanisms revealed that OA-GP11d not only accelerated the migration and cell-scratch healing of mouse keratinocytes, but also activated the mitogen-activated protein kinases (MAPKs) signaling pathway (phosphorylation of p38 and ERK subgroups) in immortalized human keratinocytes (HaCaT). Besides, OA-GP11d reduced the phosphorylation of nuclear factor-κB (NF-κB) and inhibitor of NF-κB (I-κB) induced by lipopolysaccharide stimulation in mouse macrophages, and inhibited the release of associated inflammatory factors tumor necrosis factor (TNF)-α and interleukin (IL)-6. OA-GP11d is the first identified naturally occurring peptide dimer with significant pro-healing potency. Our results highlight the importance of amphibians as a source of novel pro-healing agents and suggest OA-GP11d as a potential new pro-regenerative drug candidate.


Assuntos
Proteínas de Anfíbios , Oligopeptídeos , Ranidae , Cicatrização , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Animais , Queratinócitos/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Cicatrização/efeitos dos fármacos
14.
Toxins (Basel) ; 13(9)2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34564615

RESUMO

Brevinins are a well-characterised, frog-skin-derived, antimicrobial peptide (AMP) family, but their applications are limited by high cytotoxicity. In this study, a wild-type des-Leu2 brevinin peptide, named brevinin-1OS (B1OS), was identified from Odorrana schmackeri. To explore the significant role of the leucine residue at the second position, two variants, B1OS-L and B1OS-D-L, were designed by adding L-leucine and D-leucine residues at this site, respectively. The antibacterial and anticancer activities of B1OS-L and B1OS-D-L were around ten times stronger than the parent peptide. The activity of B1OS against the growth of Gram-positive bacteria was markedly enhanced after modification. Moreover, the leucine-modified products exerted in vivo therapeutic potential in an methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Notably, the single substitution of D-leucine significantly increased the killing speed on lung cancer cells, where no viable H838 cells survived after 2 h of treatment with B1OS-D-L at 10 µM with low cytotoxicity on normal cells. Overall, our study suggested that the conserved leucine residue at the second position from the N-terminus is vital for optimising the dual antibacterial and anticancer activities of B1OS and proposed B1OS-D-L as an appealing therapeutic candidate for development.


Assuntos
Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Leucina/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Ranidae , Proteínas de Anfíbios/química , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Bactérias Gram-Positivas/fisiologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Mariposas/microbiologia
15.
Protein Pept Lett ; 28(11): 1220-1229, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34493183

RESUMO

In recent years, bioactive peptide drugs have attracted growing attention due to the increasing difficulty in developing new drugs with novel chemical structures. In addition, many diseases are linked to excessive oxidation in the human body. Therefore, the role of peptides with antioxidant activity in counteracting diseases related to oxidative stress is worth exploring. Amphibians are a major repository for bioactive peptides that protect the skin from biotic and abiotic stresses, such as microbial infection and radiation injury. We characterized the first amphibian- derived gene-encoded antioxidant peptides in 2008. Since then, a variety of antioxidant peptides have been detected in different amphibian species. In this work, the physicochemical properties of antioxidant peptides identified from amphibians are reviewed for the first time, particularly acquisition methods, amino acid characteristics, antioxidant mechanisms, and application prospects. This review should provide a reference for advancing the identification, structural analysis, and potential therapeutic value of natural antioxidant peptides.


Assuntos
Proteínas de Anfíbios/uso terapêutico , Antioxidantes/uso terapêutico , Infecções/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/uso terapêutico , Lesões por Radiação/tratamento farmacológico , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Anfíbios , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Humanos , Peptídeos/química , Peptídeos/metabolismo , Especificidade da Espécie
16.
Acta Biochim Biophys Sin (Shanghai) ; 53(11): 1469-1483, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508563

RESUMO

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.


Assuntos
Proteínas de Anfíbios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , DNA Bacteriano/antagonistas & inibidores , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/isolamento & purificação , Peptídeos Antimicrobianos/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Linhagem Celular Tumoral , Chromobacterium/efeitos dos fármacos , Chromobacterium/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Hemólise/efeitos dos fármacos , Cavalos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Modelos Moleculares , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Ranidae/fisiologia , Pele/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
17.
Biochimie ; 191: 37-50, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34438004

RESUMO

Cathelicidin is a family of antimicrobial peptides (AMPs) existing in vertebrates, which play multiple functions in host responses against environmental stresses. All cathelicidins identified to date are cationic, no anionic member with net negative charges has been reported. In the present study, a novel anionic cathelicidin (TK-CATH) with a net charge of -3 was identified from the skin of the salamander, T. kweichowensis. Unlike most other cathelicidin members, it didn't exhibit direct antimicrobial activity. However, it demonstrated strong anti-inflammatory activity. It effectively inhibited the LPS-induced pro-inflammatory cytokine gene expression and protein production in amphibian leukocytes and mouse macrophages by inhibiting the LPS-activated mitogen-activated protein kinase (MAPK) signaling pathways. Besides, TK-CATH showed potent wound healing activity. It could effectively induce the production of several cytokines, chemokines and growth factors relating to wound healing, promote the motility and proliferation of keratinocytes, and accelerate the skin wound healing in a mouse full-thickness wound model. These results imply that TK-CATH participates in both the inflammatory phase and new tissue formation phase of wound repair process. Meanwhile, TK-CATH exhibited weak but effective free radical scavenging activity and low cytotoxicity. All the results above indicate that TK-CATH is a multifunctional peptide in the skin of the salamander T. kweichowensis. It may play important roles in host immune responses against bacterial infection and skin wound repair.


Assuntos
Proteínas de Anfíbios , Anti-Inflamatórios , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Peptídeos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/tratamento farmacológico , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Peptídeos/química , Peptídeos/farmacologia , Urodelos , Cicatrização/imunologia , Ferimentos e Lesões/imunologia
18.
Mol Cell Biochem ; 476(10): 3729-3744, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34091807

RESUMO

Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 µM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 µM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5-26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3-5.1 mN m-1) and lyse (↑ 15.1-32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1-23) in the N → C direction, with - < µH > increasing overall from circa - 0.8 to - 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.


Assuntos
Proteínas de Anfíbios , Antibacterianos , Peptídeos Catiônicos Antimicrobianos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Concentração de Íons de Hidrogênio , Conformação Proteica em alfa-Hélice
19.
Amino Acids ; 53(6): 853-868, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33942149

RESUMO

Antimicrobial peptides (AMPs) constitute part of a broad range of bioactive compounds present on diverse organisms, including frogs. Peptides, produced in the granular glands of amphibian skin, constitute a component of their innate immune response, providing protection against pathogenic microorganisms. In this work, two novel cruzioseptins peptides, cruzioseptin-16 and -17, extracted from the splendid leaf frog Cruziohyla calcarifer are presented. These peptides were identified using molecular cloning and tandem mass spectrometry. Later, peptides were synthetized using solid-phase peptide synthesis, and their minimal inhibitory concentration and haemolytic activity were tested. Furthermore, these two cruzioseptins plus three previously reported (CZS-1, CZS-2, CZS-3) were computationally characterized. Results show that cruzioseptins are 21-23 residues long alpha helical cationic peptides, with antimicrobial activity against E. coli, S. aureus, and C. albicans and low haemolytic effect. Docking results agree with the principal action mechanism of cationic AMPs that goes through cell membrane disruption due to electrostatic interactions between cationic residues in the cruzioseptins and negative phosphate groups in the pathogen cell membrane. An action mechanism through enzymes inhibition was also tried, but no conclusive results about this mechanism were obtained.


Assuntos
Proteínas de Anfíbios , Peptídeos Antimicrobianos , Candida albicans/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Proteínas de Anfíbios/química , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/farmacologia , Animais , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/isolamento & purificação , Peptídeos Antimicrobianos/farmacologia , Ranidae
20.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925935

RESUMO

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas Citotóxicas Formadoras de Poros/química , Agregados Proteicos/fisiologia , Sequência de Aminoácidos/genética , Proteínas de Anfíbios/química , Animais , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Secreções Corporais/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ranidae/metabolismo , Estresse Salino , Pele/metabolismo
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