Potencial antimicrobiano do peptídeo RP1 conjugado com ferroceno
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Data
2018-07-20
Autores
Costa, Natália Caroline Silva
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Universidade Estadual Paulista (Unesp)
Resumo
Doenças fúngicas e parasitárias representam grandes riscos para a saúde pública. A importância global das doenças fúngicas vem aumentando, dado o número crescente de pacientes vulneráveis para essas infecções, incluindo pacientes com AIDS, transplantados, imunocomprometidos, com câncer, recém-nascidos e idosos. As principais doenças são ocasionadas por Aspergillus, Candida, Cryptococcus e Pneumocystis, no entanto os tratamentos usados são pouco eficientes devido à resistência dos patógenos aos medicamentos e da alta toxicidade e efeitos colaterais dos fármacos utilizados. Além das doenças fúngicas, as parasitárias também são problemas de saúde pública, principalmente em países pobres e em desenvolvimento. Neste caso, os tratamentos também precisam ser melhorados. Diante dessa problemática há a necessidade de se buscar novas terapias, entre as alternativas estão os peptídeos antimicrobianos. O peptídeo RP1 da classe de quimiocinas apresenta atividade contra bactérias, vírus, alguns tipos de câncer e ação antiparasitária. Além disso, o composto organometálico ferroceno (Fe(C5H5)2) também mostrou atividade antiparasitária. O objetivo deste estudo foi avaliar o efeito da conjugação do peptídeo RP1 com o ferroceno em termos de estrutura, atividade biológica e toxicidade. Os peptídeos foram sintetizados usando a técnica de síntese de peptídeo em fase sólida (SPFS). A estrutura foi avaliada por espectroscopia de CD. A atividade dos peptídeos foi avaliada por estudos de permeabilização em vesículas, ensaios antimicrobianos, além de avaliar a citoxicidade dessas moléculas. Os ensaios de CD em PBS mostraram que os peptídeos apresentavam um espectro típico para estruturas desordenadas. Na presença de micelas, ambos os peptídeos apresentaram alta incidência de estrutura α-helicoidal. Ensaios de CD também foram realizados com vesiculas unilamelares de POPC e POPC:POPS, em ambos os ambos os casos os peptídeos não apresentaram estrutura secundária definida. Os dados da permebialização demonstraram a baixa ação do peptídeo RP1 sobre a membrana, pois este apresentou baixa porcentagem de liberação de CF, enquanto o Fc-RP1 apresentou alto valor de liberação de CF, mostrando que o ferroceno aumenta a capacidade de permeabilização do peptídeo. Os ensaios antileishmania, demonstraram que o peptídeo RP1 não apresentou atividade como descrito na literatura. O peptídeo Fc-RP1 apresentou atividade antifúngica contra Cryptococcus neoformans, antimalárica, com baixa toxicidade em linhagens celulares de Hacat, HepG2 e U87. Além disso os peptídeos demonstraram atividade contra bactéria gram-positiva, destacando que o peptídeo conjugado Fc-RP1 foi seletivo. Em conclusão, a conjugação do ferroceno com peptídeos mostrou-se interessante para a obtenção de novos compostos biológicos com atividade antifúngica e antiparasitária.
Fungal and parasitic diseases are major health problems globally. Around the world, 800 million people are affected with a type of fungal disease; the main ones are caused by Aspergillus, Candida, Cryptococcus and Pneumocystis. Standard treatments can be inefficient due to the resistance, high toxicity and side effects in patients. In addition, parasites are causing a large number of deaths, especially in poor and developing countries, as example malaria causes around 660 million people per year. Alternatives to standard treatments are urgently needed. Among them, antimicrobial peptides are the most widespread. The RP1 peptide of the chemokine class showed activity against bacteria, viruses and antiparasitic action. In addition, the organometallic compound ferrocene (Fe(C5H5)2) also exhibited antiparasitic activity. The objective of this study was to evaluate the effect of the conjugation of the peptide with ferrocene in terms of structure, biological activity and toxicity. The peptides were synthesized using the solid phase peptide synthesis technique (SPPS). The structure was evaluated by circular dichroism (CD) spectroscopy, peptide activity was evaluated by vesicle permeabilization studies, antimicrobial assays and cytotoxicity was evaluated. CD assays in PBS showed that the peptides presented a typical spectrum for disordered structures. In the presence of micelles, both peptides presented high incidence of α-helical structure. CD assays were also performed with unilamellar vesicles of POPC and POPC: POPS, in both cases the peptides did not present a defined secondary structure. The permeability data showed that the RP1 peptide release low percentage of Carboxyfluorescein (CF), indicating no good interaction between the peptide and the membrane, while ferrocene conjugated peptide presented high CF release value, showing that ferrocene increases the permeability of the peptide. Antileishmania assays showed that RP1 peptide showed no activity as described in the literature. The Fc-RP1 peptide showed antifungal against Cryptococcus neorformans and antimalarial activity with low toxicity in Hacat, U87 and HepG2 cell lines. In addition, the peptides demonstrated activity against gram-positive bacteria, noting that the conjugated peptide Fc-RP1 was selective. In conclusion, the ferrocene conjugated in the antimicrobial peptide RP1 was interesting to obtain new biological compounds against fungal and parasitic diseases.
Fungal and parasitic diseases are major health problems globally. Around the world, 800 million people are affected with a type of fungal disease; the main ones are caused by Aspergillus, Candida, Cryptococcus and Pneumocystis. Standard treatments can be inefficient due to the resistance, high toxicity and side effects in patients. In addition, parasites are causing a large number of deaths, especially in poor and developing countries, as example malaria causes around 660 million people per year. Alternatives to standard treatments are urgently needed. Among them, antimicrobial peptides are the most widespread. The RP1 peptide of the chemokine class showed activity against bacteria, viruses and antiparasitic action. In addition, the organometallic compound ferrocene (Fe(C5H5)2) also exhibited antiparasitic activity. The objective of this study was to evaluate the effect of the conjugation of the peptide with ferrocene in terms of structure, biological activity and toxicity. The peptides were synthesized using the solid phase peptide synthesis technique (SPPS). The structure was evaluated by circular dichroism (CD) spectroscopy, peptide activity was evaluated by vesicle permeabilization studies, antimicrobial assays and cytotoxicity was evaluated. CD assays in PBS showed that the peptides presented a typical spectrum for disordered structures. In the presence of micelles, both peptides presented high incidence of α-helical structure. CD assays were also performed with unilamellar vesicles of POPC and POPC: POPS, in both cases the peptides did not present a defined secondary structure. The permeability data showed that the RP1 peptide release low percentage of Carboxyfluorescein (CF), indicating no good interaction between the peptide and the membrane, while ferrocene conjugated peptide presented high CF release value, showing that ferrocene increases the permeability of the peptide. Antileishmania assays showed that RP1 peptide showed no activity as described in the literature. The Fc-RP1 peptide showed antifungal against Cryptococcus neorformans and antimalarial activity with low toxicity in Hacat, U87 and HepG2 cell lines. In addition, the peptides demonstrated activity against gram-positive bacteria, noting that the conjugated peptide Fc-RP1 was selective. In conclusion, the ferrocene conjugated in the antimicrobial peptide RP1 was interesting to obtain new biological compounds against fungal and parasitic diseases.
Descrição
Palavras-chave
Atividade antiparasitária, Peptídeo, Ferroceno, Atividade antimicrobiana, Atividade antifúngica