Synthesis and characterization of an antibacterial and non-toxic dimeric peptide derived from the C-terminal region of Bothropstoxin-I

Abstract

Infectious diseases are among the leading global causes of death, increasing the search for novel antibacterial agents. Among these, biologically active peptides are an excellent research tool. Using solid-phase peptide synthesis (SPPS), this work aimed to synthesize the peptide derived from the C-terminal region of Bothropstoxin-I (BthTX-I) (p-BthTX-I, sequence: KKYRYHLKPFCKK), and its disulfide-linked dimeric form, obtained via air oxidation (p-BthTX-I)2. Two other peptides were synthesized to evaluate the dimerization effect on antimicrobial activity. In both sequences, the cysteine (Cys) residue was replaced by the serine (Ser) residue, differing, however, in their C-terminus position. The antimicrobial activity of the peptides against gram-negative (Escherichia (E.) coli) and gram-positive (Staphylococcus (S.) aureus) bacteria and yeast (Candida (C.) albicans) was evaluated. Interestingly, only peptides containing the Cys residue showed antimicrobial activity, suggesting the importance of Cys residue and its dimerization for the observed activity. Apparently, p-BthTX-I and (p-BthTX-I)2 did not promote lysis or form pores and were not able to interact with membranes. Furthermore, they neither showed antifungal activity against C. albicans nor toxicity against erythrocytes, epithelial cells, or macrophages, indicating a potential specificity against prokaryotic cells.

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“…The time-kill assay was performed according to Santos-Filho et al [36], with modifications. This assay tested one ATCC strain and one clinical strain of each Candida species (CA ATCC 90028, CA3, CK ATCC 6258, CK4, CG ATCC 2001, CG3, CT ATCC 13803, CT3, CP ATCC 22019, CP1, CO ATCC, and CO1).…”

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“…In addition, upon incubation of the p‐BthTX‐I monomer in the assay medium, formation of the (p‐BthTX‐I) 2 dimer was observed in a few minutes and reached completion (i.e., full monomer‐to‐dimer conversion) within 6 h. Considering this fact, the authors suggested that the antibacterial effect displayed by p‐BthTX‐I was probably and mainly due to its dimeric form. Noticeably, the peptide showed no effect against C. albicans , epithelial cells, erythrocytes and macrophages, demonstrating in this way that it is selective against prokaryotic cells (Santos‐Filho et al, ).…”

“…Gebrim et al () worked with peptide p‐BthTX‐I, inspired in the C‐terminal region of Bothropstoxin‐1, from the B. jararacussu snake venom. This peptide was initially highlighted for its antitumor activity, but 6 years later, both the peptide monomer and its disulfide bridged dimer, (p‐BthTX‐I) 2 , were evaluated against Gram‐positive and Gram‐negative bacteria (Santos‐Filho et al, ) with promising results, whereby the dimer revealed stronger activity against E. coli and S. aureus than the monomer. In addition, upon incubation of the p‐BthTX‐I monomer in the assay medium, formation of the (p‐BthTX‐I) 2 dimer was observed in a few minutes and reached completion (i.e., full monomer‐to‐dimer conversion) within 6 h. Considering this fact, the authors suggested that the antibacterial effect displayed by p‐BthTX‐I was probably and mainly due to its dimeric form.…”

“…Several experimental and bioinformatics studies have addressed the unraveling of molecular mechanisms underpinning the antibacterial activity of small cationic peptides inspired in Lys49 svPLA 2 s (Almeida et al, ; Lomonte et al, ; Santos‐Filho et al, ). Interesting findings were made, although much more remain to be unveiled.…”

“…CD spectroscopy assays with p‐BthTX‐I showed that this peptide adopts a random coil conformation when interacting with trifluoroethanol (TFE), as well as with lipopolysaccharide (LPS) micelles and LUVs. According to the authors, p‐BthTX‐I exerts a mechanism of action different from the peptide–membrane interaction models most frequently associated to α‐helical AMPs (Santos‐Filho et al, ). This was supported by CF assays also performed, as measurement of fluorescence emission upon calcein release demonstrated that p‐BthTX‐I had a low interaction with membrane models (POPC and POPC:POPG).…”