Neuroprotective effects on microglia and insights into the structure–activity relationship of an antioxidant peptide isolated from Pelophylax perezi

Tryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian’s skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin‐like peptide, named PpT‐2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP‐HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT‐2 (FPWLLS‐NH(2)) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT‐2 shared physicochemical properties with other well‐known antioxidants. To test PpT‐2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical‐based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT‐2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT‐2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA‐stimulated BV‐2 microglia cells. We further explored possible bioactivities of PpT‐2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT‐2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT‐2. This novel peptide, PpT‐2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.