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

Abstract

BackgroundRabies 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.MethodsSkin 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.ResultsHerein, 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.ConclusionsThis novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-015-0048-1) contains supplementary material, which is available to authorized users.

Antiviral activity of animal venom peptides and related compounds

“…Cecropins, isolated mostly from the hemolymph of infected pupae of the silk moth Hyalophora cecropia, but also from other insects, tunicates and Ascaris nematodes, are a family of AMPs, containing 35-37 amino acid residues arranged in two amphiphilic α-helices linked by a Gly-Pro hinge. Synthetic hybrid peptides, namely cecropin A (1-8)-magainin 2 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), exhibited potent antiviral activity by a mechanism mainly based on the compound hydrophobicity and α-helical content, inhibiting the virushost cell fusion [85] (Table 2).…”

“…All these facts together have propelled the prospection for new antiviral drugs, particularly from natural products, as they constitute more than 25% of the new drug prototypes approved in the last decades [4]. Among sources of natural products, animal venoms have revealed a great potential for drug discovery [5][6][7], and despite the harmful action mechanism of animal venoms, most of them have components holding potential medicinal properties to cure diseases.…”

Ocellatin peptides from the skin secretion of the South American frog Leptodactylus labyrinthicus (Leptodactylidae): characterization, antimicrobial activities and membrane interactions

“…Ocellatin-F1, previously known as fallaxin, was originally found in the skin secretion of Leptodactylus fallax and it was also recently isolated from the skin secretion Leptodactylus labyrinthicus [24, 26]. Whereas this peptide was active against bacteria, no activity against the tested fungal strains was observed [24].…”

“…Whereas this peptide was active against bacteria, no activity against the tested fungal strains was observed [24]. Besides, Cunha Neto et al [26] have noted a synergic antiviral effect between ocellatin-F1 and the alkaloid bufotenine, since combinations of these compounds lead to pronounced inhibition of BHK-21 cellular infection promoted by the rabies virus. Cunha Neto et al [26] also mentioned the isolation of a truncated peptide sequence, which corresponds to ocellatin-F1 deprived from Lys and Leu residues at the peptide C-terminus, although no biological activity assays with this peptide have been reported up to our knowledge.…”

“…Oscillatin-F1, an antimicrobial peptide that was originally found in the skin secretion of the mountain chicken frog Leptodactylus fallax [24], has also been recently isolated from the skin secretion of Leptodactylus labyrinthicus by Cunha Neto et al [26]. In order to further explore the biological potential of the Leptodactylus labyrinthicus skin secretion, we present here the biological characterization of three peptides isolated from the skin secretion of this frog species.…”