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

Rene dos Santos Cunha Neto¹², Hugo Vigerelli¹, Carlos Jared³, Marta Maria Antoniazzi³, Luciana Botelho Chaves², Andréa de Cássia Rodrigues da Silva², Robson Lopes de Melo⁴, Juliana Mozer Sciani¹, Daniel C. Pimenta¹

1 Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900, Brazil.

2 Pasteur Institute, Laboratory of Rabies Diagnostic, Serology, Avenida Paulista, 393, São Paulo 01311-000 SP, Brazil.

3 Butantan Institute, Laboratory of Cell Biology, Av Vital Brasil, 1500, São Paulo 05503-900 SP, Brazil.

4 Butantan Institute, Special Laboratory of Toxinology, Av Vital Brasil, 1500, São Paulo 05503-900 SP, Brazil.

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.

The collection and housing of L. labyrinthicus were performed under license number 15964–1 from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA).

Received: June 2, 2015; Revised: November 17, 2015; Accepted: December 2, 2015

Correspondence: dcpimenta@butantan.gov.br

Competing interests

The authors declare that there are no competing interests.

Authors’ contributions

RSCN, HV, LBC and ACRS carried out the virology experiments. CJ and MMA provided the skin secretions. RSCN and RLM synthesized the peptides. RSCN, HV, CJ, MMA, RLM, JMS and DCP performed the biochemical analyses. RSCN, HV, JMS and DCP analyzed the results and wrote the paper. DCP conceived and coordinated the study. JMS and DCP designed the experiments. All authors drafted, read and approved the final version of the manuscript.