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1.
Int J Mol Sci ; 24(22)2023 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-38003414

RESUMO

Bioactive peptides have emerged as promising therapeutic agents with antimicrobial, antifungal, antiparasitic, and, recently, antitumoral properties with a mechanism of action based on membrane destabilization and cell death, often involving a conformational change in the peptide. This biophysical study aims to provide preliminary insights into the membrane-level antitumoral mode of action of crotalicidin, a cationic host defense peptide from rattlesnake venom, toward breast cancer cell lines. The lipid composition of breast cancer cell lines was obtained after lipid extraction and quantification to prepare representative cell membrane models. Membrane-peptide interaction studies were performed using differential scanning calorimetry and Fourier-transform infrared spectroscopy. The outcome evidences the potential antitumoral activity and selectivity of crotalicidin toward breast cancer cell lines and suggests a mechanism initiated by the electrostatic interaction of the peptide with the lipid bilayer surface and posterior conformation change with membrane intercalation between the acyl chains in negatively charged lipid systems. This research provides valuable information that clears up the antitumoral mode of action of crotalicidin.


Assuntos
Anti-Infecciosos , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Bicamadas Lipídicas/química , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Varredura Diferencial de Calorimetria
2.
Curr Pharm Biotechnol ; 23(2): 172-179, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-33749557

RESUMO

BACKGROUND: Crotalicidin (Ctn), a snake venom cathelicidin-related antimicrobial peptide, is a 34-residue-long linear lysine-rich vipericidin obtained from the South American rattlesnake, Crotalus durissus terrificus. Ctn contains tandem repeats of nine amino acid residues (1KRFKKFFKK9 and 16KRLKKIFKK24; consensus: 1KRhKKhFKK9, h = hydrophobic amino acid) as an integral part of its structure. OBJECTIVES: The aim of this study was to evaluate the antimicrobial activity of the encrypted vipericidin nonapeptide KRFKKFFKK, designated as Ctn[1-9], and its structural analogue, rhodamine- B‒conjugated Ctn[1-9], designated as RhoB-Ctn[1-9]. METHODS: The susceptibility of representative pathogenic bacteria and yeasts to antimicrobial agents was determined using the broth microdilution minimum inhibitory concentration (MIC) method. Cytotoxicity was estimated using a hemolytic assay. The accumulation of RhoB-Ctn[1-9] in microbial cells was observed by fluorescence microscopy. The antimicrobial synergism of RhoB-Ctn[1-9] with antimicrobials was evaluated using a checkerboard analysis. RESULTS: RhoB-conjugated Ctn[1-9] displayed selective antimicrobial activity against infectious gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and pathogenic species of Candida with low hemolytic effects on human erythrocytes which were not observed with unconjugated Ctn[1-9]. RhoB-Ctn[1-9] could permeate cell membranes and accumulate intracellularly in microbial cells. RhoB-Ctn[1-9] exhibits synergistic effects when used with antibiotics or antifungal agents and reduced the MICs of the peptide and antimicrobials. CONCLUSION: These findings indicate the potential of crotalicidin-related short peptides as structural motifs for the diversification of biological functionalities. Further, they set the stage to investigate the molecular mechanisms by which chemically modified vipericidin repeats modulate cell fate.


Assuntos
Anti-Infecciosos , Antibacterianos/farmacologia , Peptídeos Antimicrobianos , Bactérias Gram-Negativas , Humanos , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos , Rodaminas
3.
Int J Mol Sci ; 21(21)2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172206

RESUMO

Ctn[15-34], the C-terminal fragment of crotalicidin, an antimicrobial peptide from the South American rattlesnake Crotalus durissus terrificus venom, displays remarkable anti-infective and anti-proliferative activities. Herein, its activity on Candida albicans biofilms and its interaction with the cytoplasmic membrane of the fungal cell and with a biomembrane model in vitro was investigated. A standard C. albicans strain and a fluconazole-resistant clinical isolate were exposed to the peptide at its minimum inhibitory concentration (MIC) (10 µM) and up to 100 × MIC to inhibit biofilm formation and its eradication. A viability test using XTT and fluorescent dyes, confocal laser scanning microscopy, and atomic force microscopy (AFM) were used to observe the antibiofilm effect. To evaluate the importance of membrane composition on Ctn[15-34] activity, C. albicans protoplasts were also tested. Fluorescence assays using di-8-ANEPPS, dynamic light scattering, and zeta potential measurements using liposomes, protoplasts, and C. albicans cells indicated a direct mechanism of action that was dependent on membrane interaction and disruption. Overall, Ctn[15-34] showed to be an effective antifungal peptide, displaying antibiofilm activity and, importantly, interacting with and disrupting fungal plasma membrane.


Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Animais , Antifúngicos/farmacologia , Crotalus/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Venenos de Serpentes/farmacologia
4.
J Appl Microbiol ; 128(2): 414-425, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31626724

RESUMO

AIMS: Crotalicidin (Ctn), a cathelicidin-related antimicrobial peptide from the South American rattlesnake venom gland, and its C-terminal Ctn[15-34] fragment, have exhibited important activities against micro-organisms, trypanosomatid protozoa and certain lines of tumour cells. Herein, the activity against clinical strains of fluconazole-resistant Candida albicans and of amphotericin B and fluconazole-resistant Cryptococcus neoformans was investigated. METHODS AND RESULTS: Microdilution and luminescent cell viability tests were used to evaluate and compare the susceptibility of pathogenic yeasts to these peptides. The time-kill curves of the most active Ctn[15-34] alone or in combination with fluconazole against drug-resistant yeasts were determined. Concomitantly, the fungicidal and/or fungistatic effects of Ctn[15-34] were visualized by the spotting test. The peptides were active against all strains, including those resistant to antifungal agents. The association of fluconazole with both Ctn and Ctn[15-34], although not synergic, was additive. In contrast, such pattern was not observed for C. neoformans. CONCLUSIONS: Overall, Ctn and Ctn[15-34] are potential antifungal leads displaying anti-yeast activities against clinical isolates endowed with drug resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The effective peptide activity against resistant strains of pathogenic yeasts demonstrates that crotalicidin-derived peptides are promising templates to develop new antifungal pharmaceuticals.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Anfotericina B/farmacologia , Candida albicans/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana , Triazóis/farmacologia
5.
J Med Microbiol ; 67(1): 129-138, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29214974

RESUMO

PURPOSE: Ctn[15-34], a carboxyl-terminal fragment of crotalicidin (a cathelicidin from the venom gland of a South American rattlesnake), has shown antifungal activity against clinical and standard strains of Candida species. The aim of the present work was to investigate the underlying mechanisms of the candidicidal activity of Ctn[15-34]. METHODOLOGY: The time-kill profile and drug synergism were evaluated by means of a microdilution assay and multi-parametric flow cytometry. The presumptive interaction of Ctn[15-34] with lipid membranes was estimated in vitro with a lipid-mimic compound, the chromogenic substance 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA).Results/Key findings. The absorbance increment (at 425 nm) indicated a concentration- and time-dependent in-solution association between Ctn[15-34] and 4N3OBA. The interaction of Ctn[15-34] with Candida cells was confirmed by flow cytometric measurements with the 5(6)-carboxyfluorescein-labelled peptide (CF-Ctn[15-34]). Analysis of the killing time of Candida exposed to Ctn[15-34] and amphotericin B (AMB) showed that both the peptide and polyene drug reduce the number of c.f.u. but in mechanistically different ways. The Ctn[15-34] peptide alone caused yeast cell membrane disruption, which was confirmed by lactate dehydrogenase leakage and biomarkers of cell death mediated by necrosis. CONCLUSION: Overall, Ctn[15-34] displays a synergistic antifungal activity with AMB, an effect that can be further developed into a multi-target therapeutic option with other antimycotics currently in use.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Catelicidinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Anfotericina B/farmacologia , Candidíase/tratamento farmacológico , Sinergismo Farmacológico , Fluoresceínas/farmacologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Nitrobenzoatos/farmacologia
6.
Parasitology ; 145(8): 1059-1064, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29208061

RESUMO

Cathelicidins are antimicrobial peptides produced by humans and animals in response to various pathogenic microbes. Crotalicidin (Ctn), a cathelicidin-related vipericidin from the South American Crotalus durissus terrificus rattlesnake's venom gland, and its fragments have demonstrated antimicrobial and antifungal activity, similarly to human cathelicidin LL-37. In order to provide templates for the development of modern trypanocidal agents, the present study evaluated the antichagasic effect of these four peptides (Ctn, Ctn[1-14], Ctn[15-34] and LL-37). Herein, Ctn and short derived peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Ctn inhibited all T. cruzi developmental forms, including amastigotes, which is implicated in the burden of infection in the chronic phase of Chagas disease. Moreover, Ctn showed a high selective index against trypomastigote forms (>200). Ctn induced cell death in T. cruzi through necrosis, as determined by flow cytometry analyses with specific molecular probes and morphological alterations, such as loss of membrane integrity and cell shrinkage, as observed through scanning electron microscopy. Overall, Ctn seems to be a promising template for the development of antichagasic agents.


Assuntos
Fragmentos de Peptídeos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Citometria de Fluxo , Haplorrinos , Concentração Inibidora 50 , Microscopia Eletrônica de Varredura , Trypanosoma cruzi/ultraestrutura , Catelicidinas
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