Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the viruss glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Substratos e inibidores peptídicos contendo aminoácidos básicos näo naturais para o estudo de especificidade de enzimas proteolíticas / Non natural basic amino acids to study of proteolytic enzimes

O presente trabalho aborda a síntese de aminoácidos básicos nao naturais que sao, posteriormente, empregados no estudo de enzimas proteolíticas. Estas enzimas sao de grande interesse devido ao seu papel em funçoes fisiológicas e podem estar envolvidas em uma larga magnitude de patologias. Assim como os aminoácidos naturais, estes aminoácidos nao naturais sao, no curso deste trabalho, utilizados na construçao de substratos peptídicos, que por ensaios cinéticos e inibitórios, com as enzimas proteolíticas, fornecem informaçoes a respeito da estrutura e a atividade das mesmas. Nosso trabalho com as enzimas proteolíticas abordam as serino e cisteíno peptidases. Examinamos a especificidade de substratos peptídicos, contendo aminoácidos básicos nao naturais, para a calicreína tecidual humana. Exploramos a especificidade dos subsítio S1 e S2 de catepsinas B e L. Estudamos os substratos peptídicos curtos de fluorescência apagada para o emprego com enzimas como cisteíno-proteases papaína, catepsina L e catepsina B e com as serino-proteases tripsina, calicreínas plasmática tecidual, trombina e factor X ativado. Por último, os aminoácidos nao natural foram utilizados no estudo de uma isoforma de uma cisteíno pretease do parasita Leischmania mexicana. Os resultados apresentados na seqüência dos trabalhos gerados no curso destes estudos demonstram um interessante potencial para o emprego destes aminoácidos, com os quais pode-se estudar a atividade de enzimas proteolíticas frente a substratos sintéticos. Estes mesmos aminoácidos podem constituir uma interessante ferramenta para desenho de moléculas e a busca de uma potencial molécula líder no desenvolvimento de inibidores potentes e específicos para as enzimas proteolíticas