Louise Oliveira Fiametti

Antimicrobial peptides such as Melittin exhibit potent broad-spectrum activity but are limited by high cytotoxicity. The rational design of bioinspired Melittin-derived analogues represents a promising strategy to reduce toxicity while maintaining antimicrobial efficacy. In this study, Melittin and analogues (TT-1, FKW, and WKW) were synthesized using solid-phase peptide synthesis (SPPS) and characterized for biological and biophysical essays. Antimicrobial and hemolytic activity, serum stability, secondary structure, and membrane interaction were analysed. FKW and WKW exhibited broad-spectrum antimicrobial activity, with minimum inhibitory concentration (MIC) as low as 8 and 16 µg/mL against Staphylococcus aureus and Enterococcus faecium. Both analogues also showed improved activity against Klebsiella pneumoniae (32 µg/mL) and Pseudomonas aeruginosa (128 and 256 µg/mL for FKW and WKW, respectively) compared to Melittin (64 and 512 µg/mL). In terms of cytotoxicity, FKW and WKW showed significantly reduced hemolytic activity, with HC₅₀ values of 264.8 µg/mL and 237.2 µg/mL, respectively, resulting in improved selectivity indexes relative to Melittin (HC₅₀ of 7.9 µg/mL). In liposomes, both adopt α-helical structures and cause disruption via pore formation or detergent-like mechanisms. TT-1 showed minimal toxicity but weak antimicrobial activity (MIC > 256 µg/mL). Although FKW and WKW exhibited limited serum stability (half-lives of 2.2 and 1.5 h), their degradation may reduce systemic toxicity. Overall, these analogues demonstrate an improved balance between antimicrobial activity and safety.

The marine environment is a rich source of new biotechnologies and products. Bottom trawling for shrimp species such as Xiphopenaeus kroyeri and Farfantepenaeus brasiliensis leads to the unintentional capture of non-target species, known as bycatch, which includes a variety of marine life that are often discarded without economic value. A common bycatch species on the southeast coast of Brazil is Olivancillaria urceus (O. urceus), a carnivorous gastropod that feeds mainly on bivalves. Despite its abundance, this species is still little studied, especially for biotechnological applications. Other gastropods such as Conus are known for their diverse and potent toxins, which offer great potential for pharmacological discoveries. In this study, an omics approach, including transcriptomics and proteopeptidomics, was applied to explore O. urceus at the molecular level. The transcriptome of the muscle foot/mantle led to the annotation of 19,097 genes via Gene Ontology, identifying 20 toxin-like transcripts identified considering the Gastropod class. The proteome fraction confirmed 2179 transcripts, including sequences with toxin activity, such as conotoxin precursors, Conodipine-P3, and BPTI/Kunitz domain-containing proteins. In addition, 9663 peptides of 1484 precursor proteins were detected in the peptide fraction, including 2 sequences representing neurotoxins. The identification of these sequences could lead to the discovery of new molecules with therapeutic potential.

Although peptides have been shown to have biological functions in neurodegenerative diseases, their role in Parkinson’s disease has been understudied. A previous study by our group, which used a 6-hydroxydopamine zebrafish model, suggested that nine intracellular peptides may play a part in this condition. In this context, our aim is to better understand the role of five of these nine peptides. The selection of peptides was made based on their precursor proteins, which are fatty acid binding protein 7, mitochondrial ribosomal protein S36, MARCKS-related protein 1-B, excitatory amino acid transporter 2 and thymosin beta-4. The peptides were chemically synthesized in solid phase and characterized by high-performance liquid chromatography and mass spectrometry. Circular dichroism was performed to determine the secondary structure of each peptide, which showed that all five peptides maintain a random structure in the aqueous solutions that were studied. Two molecules show a helical profile in trifluoroethanol, a known structuring agent. Cell viability by the MTT assay indicates that all five peptides are not cytotoxic in all concentrations tested in both mouse and human cell lines. Behavioral assay using a 6-OHDA zebrafish larvae model suggest that all peptides help in the recovery of motor function with 24 h treatment at two concentrations. Three peptides showed a complete recovery from the 6-OHDA-induced motor impairment. Further studies are needed to better understand the mechanism of action of these peptides and whether they are truly a potential ally against Parkinson’s disease.

Currently, the search for new alternatives to conventional antibiotics to combat bacterial resistance is an urgent task, as many microorganisms threaten human health due to increasing bacterial resistance to traditional medicines. Thus, new molecules such as antimicrobial peptides have emerged as promising alternatives because of their low induction of resistance and broad spectrum of action. In this context, in the past few years, our research group has synthesized and characterized a peptide derived from the C-terminal region of the Lys⁴⁹ PLA₂-like BthTX-I, named p-BthTX-I. After several studies, the peptide (p-BthTX-I)₂K was proposed as the molecule with the most considerable biotechnological potential. As such, the present work aimed to evaluate whether the modifications made on the peptide (p-BthTX-I)₂K can be applied to other molecules originating from the C-terminal region of PLA₂-like Lys⁴⁹ from snake venoms. The peptides were obtained through the solid-phase peptide synthesis technique, and biochemical and functional characterization was carried out using dichroism techniques, mass spectrometry, antimicrobial activity against ESKAPE strains, hemolytic activity, and permeabilization of lipid vesicles. The antimicrobial activity of the peptides was promising, especially for the peptides (p-AppK)₂K and (p-ACL)₂K, which demonstrated activity against all strains that were tested, surpassing the model molecule (p-BthTX-I)₂K in most cases and maintaining low hemolytic activity. The modifications initially proposed for the (p-BthTX-I)₂K peptide were shown to apply to other peptides derived from Lys⁴⁹ PLA₂-like from snake venoms, showing promising results for antimicrobial activity. Future assays comparing the activity of the dimers obtained through this strategy with the monomers of these peptides should be carried out.

Bioactive peptides such as neuropeptides and peptide hormones are largely understood in their involvement in a variety of physiologic systems. In addition to the neuropeptides produced and processed by the classic secretory pathway, intracellular peptides (InPeps) have shown biological activity in studies involving different organisms. A model that has become attractive in many research fields is the zebrafish (Danio rerio), which has allowed correlating behavioral responses or physiological processes with underlying molecular pathways or signaling cascades, improving the understanding of homeostasis mechanisms of the central nervous system, as well as pathological processes such as neurodegenerative diseases. Here, we provide a detailed description of the protocol of treatment with 6-OHDA, which mimics some features of Parkinson’s Disease, as well as the validation of the treatment by evaluation of the locomotor activity and the protocol of peptide extraction followed by isotopic labeling to peptide relative quantitation by mass spectrometry. Key words Zebrafish Danio rerio Neuropeptide Intracellular peptides Peptidomics Parkinson’s Disease Mass spectrometry