RESUMEN
Infections by drug-resistant microorganisms are a threat to global health and antimicrobial peptides are considered to be a new hope for their treatment. Temporin-WY2 was identified from the cutaneous secretion of the Ranidae frog, Amolops wuyiensis. It presented with a potent anti-Gram-positive bacterial efficacy, but its activity against Gram-negative bacteria and cancer cell lines was unremarkable. Also, it produced a relatively high lytic effect on horse erythrocytes. For further improvement of its functions, a perfect amphipathic analogue, QUB-1426, and two lysine-clustered analogues, 6K-WY2 and 6K-1426, were synthesised and investigated. The modified peptides were found to be between 8- and 64-fold more potent against Gram-negative bacteria than the original peptide. Additionally, the 6K analogues showed a rapid killing rate. Also, their antiproliferation activities were more than 100-fold more potent than the parent peptide. All of the peptides that were examined demonstrated considerable biofilm inhibition activity. Moreover, QUB-1426, 6K-WY2 and 6K-1426, demonstrated in vivo antimicrobial activity against MRSA and E. coli in an insect larvae model. Despite observing a slight increase in the hemolytic activity and cytotoxicity of the modified peptides, they still demonstrated a improved therapeutic index. Overall, QUB-1426, 6K-WY2 and 6K-1426, with dual antimicrobial and anticancer functions, are proposed as putative drug candidates for the future.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Biopelículas , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad Microbiana , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Biopelículas/efectos de los fármacos , Humanos , Antibacterianos/farmacología , Antibacterianos/química , Ranidae , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Caballos , Escherichia coli/efectos de los fármacos , Hemólisis/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Proteínas Anfibias/farmacología , Proteínas Anfibias/química , Bacterias Gramnegativas/efectos de los fármacosRESUMEN
Chagas disease, leishmaniasis, and malaria are major parasitic diseases disproportionately affecting the underprivileged population in developing nations. Finding new, alternative anti-parasitic compounds to treat these diseases is crucial because of the limited number of options currently available, the side effects they cause, the need for long treatment courses, and the emergence of drug-resistant parasites. Anti-microbial peptides (AMPs) derived from amphibian skin secretions are small bioactive molecules capable of lysing the cell membrane of pathogens while having low toxicity against human cells. Here, we report the anti-parasitic activity of five AMPs derived from skin secretions of three Ecuadorian frogs: cruzioseptin-1, cruzioseptin-4 (CZS-4), and cruzioseptin-16 from Cruziohyla calcarifer; dermaseptin-SP2 from Agalychnis spurrelli; and pictuseptin-1 from Boana picturata. These five AMPs were chemically synthesized. Initially, the hemolytic activity of CZS-4 and its minimal inhibitory concentration against Escherichia coli, Staphylococcus aureus, and Candida albicans were determined. Subsequently, the cytotoxicity of the synthetic AMPs against mammalian cells and their anti-parasitic activity against Leishmania mexicana promastigotes, erythrocytic stages of Plasmodium falciparum and mammalian stages of Trypanosoma cruzi were evaluated in vitro. The five AMPs displayed activity against the pathogens studied, with different levels of cytotoxicity against mammalian cells. In silico molecular docking analysis suggests this bioactivity may occur via pore formation in the plasma membrane, resulting in microbial lysis. CZS-4 displayed anti-bacterial, anti-fungal, and anti-parasitic activities with low cytotoxicity against mammalian cells. Further studies about this promising AMP are required to gain a better understanding of its activity.IMPORTANCEChagas disease, malaria, and leishmaniasis are major tropical diseases that cause extensive morbidity and mortality, for which available treatment options are unsatisfactory because of limited efficacy and side effects. Frog skin secretions contain molecules with anti-microbial properties known as anti-microbial peptides. We synthesized five peptides derived from the skin secretions of different species of tropical frogs and tested them against cultures of the causative agents of these three diseases, parasites known as Trypanosoma cruzi, Plasmodium falciparum, and Leishmania mexicana. All the different synthetic peptides studied showed activity against one of more of the parasites. Peptide cruzioseptin-4 is of special interest since it displayed intense activity against parasites while being innocuous against cultured mammalian cells, which indicates it does not simply hold general toxic properties; rather, its activity is specific against the parasites.
Asunto(s)
Anuros , Leishmania mexicana , Plasmodium falciparum , Piel , Trypanosoma cruzi , Animales , Trypanosoma cruzi/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Humanos , Leishmania mexicana/efectos de los fármacos , Piel/parasitología , Piel/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Proteínas Anfibias/farmacología , Proteínas Anfibias/química , Ecuador , Enfermedad de Chagas/tratamiento farmacológicoRESUMEN
AIM: The high incidence of virus-related infections and the large diffusion of drug-resistant pathogens stimulate the search and identification of new antiviral agents with a broad spectrum of action. Antivirals can be designed to act on a single target by interfering with a specific step in the viral lifecycle. On the contrary, antiviral peptides (AVPs) are known for acting on a wide range of viruses, with a diversified mechanism of action targeting virus and/or host cell. In the present study, we evaluated the antiviral potential of the peptide Hylin-a1 secreted by the frog Hypsiobas albopunctatus against members of the Herpesviridae family. METHODS AND RESULTS: The inhibitory capacity of the peptide was evaluated in vitro by plaque assays in order to understand the possible mechanism of action. The results were also confirmed by real-time PCR and Western blot evaluating the expression of viral genes. Hylin-a1 acts to block the herpetic infection interfering at the early stages of both herpes simplex virus type 1 (HSV-1) and type 2 infection. Its mechanism is mainly directed on the membrane, probably by damaging the viral envelope. The same effect was also observed against HSV-1 strains resistant to acyclovir. CONCLUSIONS: The data presented in this study, such as the increased activity of the peptide when combined to acyclovir, a weak hemolytic profile, an anti-inflammatory effect, and a tolerable half-life in serum, indicates Hylin-a1 as a novel antiherpetic molecule with promising potential in the clinical setting.
Asunto(s)
Antiinflamatorios , Antivirales , Anuros , Animales , Antivirales/farmacología , Antiinflamatorios/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Proteínas Anfibias/farmacología , Péptidos/farmacología , Células Vero , Chlorocebus aethiopsRESUMEN
Staphylococcus aureus, a bacterium resistant to multiple drugs, is a significant cause of illness and death worldwide. Antimicrobial peptides (AMPs) provide an excellent potential strategy to cope with this threat. Recently, we characterized a derivative of the frog-skin AMP esculentin-1a, Esc(1-21) (1) that is endowed with potent activity against Gram-negative bacteria but poor efficacy against Gram-positive strains. In this study, three analogues of peptide 1 were designed by replacing Gly8 with α-aminoisobutyric acid (Aib), Pro, and dPro (2-4, respectively). The single substitution Gly8 â Aib8 in peptide 2 makes it active against the planktonic form of Gram-positive bacterial strains, especially Staphylococcus aureus, including multidrug-resistant clinical isolates, with an improved biostability without resulting in cytotoxicity to mammalian cells. Moreover, peptide 2 showed a higher antibiofilm activity than peptide 1 against both reference and clinical isolates of S. aureus. Peptide 2 was also able to induce rapid bacterial killing, suggesting a membrane-perturbing mechanism of action. Structural analysis of the most active peptide 2 evidenced that the improved biological activity of peptide 2 is the consequence of a combination of higher biostability, higher α helical content, and ability to reduce membrane fluidity and to adopt a distorted helix, bent in correspondence of Aib8. Overall, this study has shown how a strategic single amino acid substitution is sufficient to enlarge the spectrum of activity of the original peptide 1, and improve its biological properties for therapeutic purposes, thus paving the way to optimize AMPs for the development of new broad-spectrum anti-infective agents.
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Sustitución de Aminoácidos , Antibacterianos , Biopelículas , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus , Biopelículas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Humanos , Proteínas Anfibias/farmacología , Proteínas Anfibias/química , Proteínas Anfibias/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Animales , Farmacorresistencia BacterianaRESUMEN
Cathelicidins are important antimicrobial peptides in various vertebrate species where they are crucial parts of the innate immune system. The current understanding of amphibian cathelicidins is limited, particularly with regard to their immunomodulatory effects. To address this knowledge gap, we produced the cDNA sequence of the cathelicidin gene from a skin transcriptome of the Chinese spiny frog Quasipaa spinosa. The amino acid sequence of the Quasipaa spinosa cathelicidin (QS-CATH) was predicted to consist of a signal peptide, a cathelin domain, and a mature peptide. Comparative analysis of the QS-CATH amino acid sequence with that of other amphibian cathelicidins revealed high variability in the functional mature peptide among amphibians, whereas the cathelin domain was conserved. The QS-CATH gene was expressed in several tissues, with the highest level of expression in the spleen. Upregulation of QS-CATH after Aeromonas hydrophila infection occurred in the kidney, gut, spleen, skin, and liver. Chemically synthesized QS-CATH exhibited pronounced antibacterial activity against Shigella flexneri, Staphylococcus warneri, Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Furthermore, QS-CATH disrupted the cell membrane integrity of S. flexneri, as evidenced by a lactate dehydrogenase release assay, and it hydrolyzed the genomic DNA of S. flexneri. Additionally, QS-CATH elicited chemotaxis and modulated the expression of inflammatory cytokine genes in RAW264.7 mouse leukemic monocyte/macrophage cells. These findings confirm the antimicrobial effects of amphibian cathelicidin and its ability to influence immune cell function. This will expedite the potential utilization of amphibian antimicrobial peptides as therapeutic agents.
Asunto(s)
Anuros , Catelicidinas , Animales , Ratones , Secuencia de Aminoácidos , Factores Inmunológicos/farmacología , Aeromonas hydrophila , Proteínas Anfibias/farmacología , Proteínas Anfibias/genética , Proteínas Anfibias/aislamiento & purificación , Antiinfecciosos/farmacología , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Células RAW 264.7 , Agentes Inmunomoduladores/farmacología , Agentes Inmunomoduladores/aislamiento & purificación , Piel/efectos de los fármacos , Piel/metabolismo , Piel/inmunología , Pueblos del Este de AsiaRESUMEN
To reduce food spoilage and deterioration caused by microbial contamination, antimicrobial peptides (AMPs) have gradually gained attention as a biological preservative. Odorranain-C1 is an α-helical cationic antimicrobial peptide extracted from the skin of frogs with broad-spectrum antimicrobial activity. In this study, we achieved the expression of Odorranain-C1 in Pichia pastoris (P. pastoris) (also known as Komagataella phaffii) by employing DNA recombination technology. The recombinant Odorranain-C1 showed broad-spectrum antibacterial activity and displayed a minimum inhibitory concentration within the range of 8-12⯵g.mL-1. Meanwhile, Odorranain-C1 exhibited superior stability and lower hemolytic activity. Mechanistically, Odorranain-C1 disrupted the bacterial membrane's integrity, ultimately causing membrane rupture and subsequent cell death. In tilapia fillets preservation, Odorranain-C1 inhibited the total colony growth and pH variations, while also reducing the production of total volatile basic nitrogen (TVB-N) and thiobarbituric acid (TBA). In conclusion, these studies demonstrated the efficient recombinant expression of Odorranain-C1 in P. pastoris, highlighting its promising utilization in food preservation.
Asunto(s)
Conservación de Alimentos , Saccharomycetales , Animales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Conservación de Alimentos/métodos , Pruebas de Sensibilidad Microbiana , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Péptidos Antimicrobianos/genética , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/metabolismo , Antibacterianos/farmacología , Hemólisis/efectos de los fármacos , Pichia/genética , Pichia/metabolismo , Proteínas Anfibias/genética , Proteínas Anfibias/farmacología , Proteínas Anfibias/metabolismo , Anuros/metabolismoRESUMEN
Dermaseptin B2 (DrsB2) is an antimicrobial peptide with anticancer and angiostatic properties. We aimed to assess the in vitro inhibitory effect of pDNA/DrsB2 on the growth of breast cancer cells and its impact on the expression of genes involved in the BAX/BBC3/AKT pathway. The nucleic acid sequence of DrsB2 was artificially synthesized and inserted into the pcDNA3.1( +) Mammalian Expression Plasmid. PCR testing and enzyme digesting procedures evaluated the accuracy of cloning. The vectors were introduced into cells using LipofectamineTM2000 transfection reagent. The breast cancer cells were assessed by flow cytometry, MTT assessment, soft agar colony method, and wound healing investigation. The gene's transcription was evaluated using real-time PCR with a significance level of P < 0.05. The recombinant plasmid harboring the pDNA/DrsB2 vector was effectively produced, and the gene sequence showed absolute homogeneity (100% similarity) with the DrsB2 gene. The transfection effectiveness of MCF-7 and MCF-10A cells was 79% and 68%, respectively. The findings are measured using the growth inhibition 50% (GI50) metric, which indicates the concentration of pDNA/DrsB2 that stops 50% of cell growth. The proportions of early apoptosis, late apoptosis, necrosis, and viable MCF-7 cells in the pDNA/DrsB2 group were 40.50%, 2.31%, 1.69%, and 55.50%, respectively. The results showed a 100% increase in gene expression in programmed cell death following treatment with pDNA/DrsB2 (**P < 0.01). To summarize, the results described in this work offer new possibilities for treating cancer by targeting malignancies via pDNA/DrsB2 and activating the BAX/BBC3/AKT signaling pathways.
Asunto(s)
Neoplasias de la Mama , Proliferación Celular , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Proteína X Asociada a bcl-2 , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Apoptosis , Células MCF-7 , Proteínas Anfibias/genética , Proteínas Anfibias/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Antineoplásicos/farmacología , Péptidos Catiónicos Antimicrobianos/genética , TransfecciónRESUMEN
Aim: The response of E. coli ATCC8739 to Brevinin-2CE (B2CE) was evaluated as a strategy to prevent the development of antimicrobial peptide (AMP)-resistant bacteria. Methods: Gene expression levels were detected by transcriptome sequencing and RT-PCR. Target genes were knocked out using CRISPR-Cas9. MIC was measured to evaluate strain resistance. Results: Expression of acrZ and sugE were increased with B2CE stimulation. ATCC8739ΔacrZ and ATCC8739ΔsugE showed twofold and fourfold increased sensitivity, respectively. The survival rate of ATCC8739 was reduced in the presence of B2CE/chlorpromazine (CPZ). Combinations of other AMPs with CPZ also showed antibacterial effects. Conclusion: The results indicate that combinations of AMPs/efflux pump inhibitors (EPIs) may be a potential approach to combat resistant bacteria.
[Box: see text].
Asunto(s)
Antibacterianos , Clorpromazina , Escherichia coli , Pruebas de Sensibilidad Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Antibacterianos/farmacología , Clorpromazina/farmacología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/genética , Proteínas Anfibias/genética , Proteínas Anfibias/farmacología , Proteínas Anfibias/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Sinergismo FarmacológicoRESUMEN
Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 µM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 µM for raniseptin PL and 157 ± 16 µM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 µM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.
Asunto(s)
Anuros , Piel , Animales , Piel/metabolismo , Humanos , Anuros/metabolismo , Ratones , Línea Celular Tumoral , Proteínas Anfibias/farmacología , Proteínas Anfibias/química , Proteínas Anfibias/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/química , Hemólisis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Secuencia de Aminoácidos , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Células HT29 , Musa/químicaRESUMEN
This study investigated the morphology of Rhinella crucifer cutaneous glands, as well as the protein/peptide profiles and bioactivities of body gland secretions (BGS) and parotoid macrogland secretions (PS). The parotoid as well as dorsal and ventral skin fragments of male and female individuals were processed for histological analysis. The protein and peptide profiles of male and female gland secretions were evaluated. Male secretions were also assessed for proteolytic, trypsin inhibiting, hemagglutinating, hemolytic, antimicrobial, and anticoagulant activities. The R. crucifer skin structure presented protuberances that are clearly visible and formed by the integument, which has cutaneous glands throughout the body. An average of 438 and 333 glands were identified in males in females, respectively. No significant differences were observed in the distribution of glands across the body as well as for area and perimeter of glands. Differences were observed in protein composition between the PS and BGS from males and females, and secretions from animals collected from undisturbed and anthropogenically disturbed areas. Proteins with similarities to catalase and elongation factor 1-alpha were detected in the PS. Zymography revealed proteolytic activity in both male BGS and PS. Male BGS showed antibacterial activity against Enterococcus faecalis and Escherichia coli and anticoagulant activity, being able to prolong prothrombin time by 6.34-fold and activated partial thromboplastin time by 2.17-fold. Finally, male PS and BGS caused a maximum hemolysis degree of 1.4%. The data showed that the cutaneous secretions of R. crucifer are potentially promising for biotechnological prospecting.
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Bufonidae , Piel , Animales , Masculino , Femenino , Bufonidae/metabolismo , Piel/metabolismo , Piel/química , Glándulas Exocrinas/metabolismo , Secreciones Corporales/química , Proteínas Anfibias/metabolismo , Proteínas Anfibias/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Anfibias , Antibacterianos , Filogenia , Ranidae , Animales , Proteínas Anfibias/farmacología , Proteínas Anfibias/química , Proteínas Anfibias/genética , Ratones , Antibacterianos/farmacología , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Secuencia de Aminoácidos , Piel/metabolismo , Factores Inmunológicos/farmacología , Factores Inmunológicos/química , Células RAW 264.7 , Alineación de SecuenciaRESUMEN
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.
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Liposomas , Neoplasias , Ratones , Animales , Liposomas/química , Hemólisis , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Anfibias/química , Proteínas Anfibias/farmacología , Línea Celular Tumoral , Microambiente TumoralRESUMEN
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.
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Antiinfecciosos , Choque Séptico , Animales , Ratones , Lipopolisacáridos/toxicidad , Secuencia de Aminoácidos , Proteínas Anfibias/farmacología , Proteínas Anfibias/uso terapéutico , Proteínas Anfibias/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Antiinfecciosos/farmacología , Antiinfecciosos/uso terapéutico , Antiinfecciosos/química , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/metabolismo , Ranidae/metabolismo , Piel , Bacterias Grampositivas , Choque Séptico/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
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Proteínas Anfibias , Péptidos Catiónicos Antimicrobianos , Animales , Humanos , Ratones , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/farmacología , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Dicroismo Circular , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Ranidae , Staphylococcus aureus/efectos de los fármacosRESUMEN
The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 âLys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 âLys and the double substitution Asp4 âLys and Ala18 âLys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.
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Antiinfecciosos , Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Diabetes Mellitus Tipo 2 , Neoplasias Pulmonares , Ratones , Animales , Humanos , Péptidos Catiónicos Antimicrobianos/química , Lisina , Antibacterianos/química , Diabetes Mellitus Tipo 2/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Células Endoteliales/metabolismo , Proteínas Anfibias/farmacología , Bacterias Grampositivas , Bacterias Gramnegativas , Neoplasias Pulmonares/metabolismo , Insulina/metabolismo , Antineoplásicos/farmacología , Anuros/metabolismo , Piel/metabolismoRESUMEN
The skin of amphibians is a tissue with biological functions, such as defense, respiration, and excretion. In recent years, researchers have discovered a large number of peptides in the skin secretions of amphibians, including antimicrobial peptides, antioxidant peptides, bradykinins, insulin-releasing peptides, and other peptides. This review focuses on the origin, primary structure, secondary structure, length, and functions of peptides secreted from amphibians' skin. We hope that this review will provide further information and promote the further study of amphibian skin secretions, in order to provide reference for expanding the research and application of amphibian bioactive peptides.
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Péptidos Antimicrobianos , Insulinas , Animales , Antioxidantes/química , Secuencia de Aminoácidos , Anfibios , Péptidos/química , Piel/química , Insulinas/análisis , Proteínas Anfibias/farmacologíaRESUMEN
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.
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Proteínas Anfibias , Antioxidantes , Proteínas Anfibias/química , Proteínas Anfibias/farmacología , Proteínas Anfibias/uso terapéutico , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Carragenina , ADN Complementario , Peróxido de Hidrógeno/metabolismo , Ratones , Estrés Oxidativo , Ranidae , RatasRESUMEN
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.
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Proteínas Anfibias , Antiinfecciosos , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/farmacología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antiinfecciosos/química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Antimicrobianos , Pruebas de Sensibilidad Microbiana , Rana esculenta , Ranidae , Piel , Relación Estructura-ActividadRESUMEN
Antibiotic resistance-related bacterial infections and cancers become huge challenges in human health in the 21st century. A number of naturally derived antimicrobial peptides possess multiple functions in host defense, including anti-infective and anticancer activities. One of which is known as the caerin 1 family peptides. The microbicidal properties of these peptides have been long discussed. The recent studies also established the usage of two members in this family, caerin 1.1 and caerin 1.9, in antimultiple antibiotic-resistant bacteria species. It is increasingly evident that caerin 1.1 and caerin 1.9 also contain additional activities in the suppression of tumor. In this review, we briefly outline the therapeutic potentials and possible mechanism of action of caerin 1.1 and 1.9 in the treatment of multiple antibiotic-resistant bacterial infection and cancer immunotherapy.
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Antiinfecciosos , Infecciones Bacterianas , Neoplasias , Proteínas Anfibias/farmacología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Bacterias , Infecciones Bacterianas/tratamiento farmacológico , Humanos , Inmunoterapia , Neoplasias/tratamiento farmacológicoRESUMEN
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.