Ocellatin-F1

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 antimicrobial peptides Ocellatin-LB1, -LB2 and -F1, isolated from frogs, are identical from residue 1 to 22, which correspond to the -LB1 sequence, whereas -LB2 carries an extra N and -F1 additional NKL residues at their C-termini. Despite the similar sequences, previous investigations showed different spectra of activities and biophysical investigations indicated a direct correlation between both membrane-disruptive properties and activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. This study presents experimental evidence as well as results from theoretical studies that contribute to a deeper understanding on how these peptides exert their antimicrobial activities and how small differences in the amino acid composition and their secondary structure can be correlated to these activity gaps. Solid-state NMR experiments allied to the simulation of anisotropic NMR parameters allowed the determination of the membrane topologies of these ocellatins. Interestingly, the extra Asn residue at the Ocellatin-LB2 C-terminus results in increased topological flexibility, which is mainly related to wobbling of the helix main axis as noticed by molecular dynamics simulations. Binding kinetics and thermodynamics of the interactions have also been assessed by Surface Plasmon Resonance and Isothermal Titration Calorimetry. Therefore, these investigations allowed to understand in atomic detail the relationships between peptide structure and membrane topology, which are in tune within the series -F1 > > -LB1 ≥ -LB2, as well as how peptide dynamics can affect membrane topology, insertion and binding.

Ocellatin AMPs (antimicrobial peptides) are considered to be promising alternative therapeutics to conventional antibiotics. Three-dimensional (3D) structures of ocellatin-F1 with 25 residues have been reported to be potent in terms of bacterial membrane permeability. To investigate the influence of similar ocellatin peptides with 25 residues pertaining to antimicrobial effect, ocellatin-1, K1 and S1 peptides were modelled with ocellatin-F1 as template. Comparative analyses between these peptides were carried out, using computational approaches. From the results of in silico toxicity profile, all peptides were found to be non-toxic with no haemolytic activity. Further sequence analysis, net charge, hydrophobicity and hydrophobic moment revealed the membrane permeable efficacy of ocellatin-1 peptide. Besides, the investigation of peptide electronic structures through density functional theory and quantum chemical (HOMO and LUMO) calculations predicted ocellatin-1 to be a suitable peptide, which can be used as a scaffold for therapeutics. Furthermore, the determination of structural contours such as RMSD, RMSF and Rg through trajectory analysis revealed that ocellatin-1 exhibited strong structural stability. In addition, the trajectory analysis of elements of secondary structure illustrated the alpha helical conformations to be retained in all peptides, except ocellatin-1. On the aforementioned grounds, ocellatin-1 was found to possess the important role of peptide penetration of the bacterial membrane. This study becomes significant, since it is the first time where the structural importance of ocellatin peptides were explored in detail and the therapeutic potential of ocellatin-1 as a peptide-based antimicrobial drug have been theoretically revealed.