Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and Buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 μg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.

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Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp.

The dimeric peptide 26[F]: (RRWQWRFKKLG)2-K-Ahx has exhibited a potent cytotoxic effect against breast cancer cell lines, with position 26 (F) being the most relevant for anti-cancer activity. In this investigation, six analogues of the 26[F] peptide were synthesized in which the 26th position was replaced by non-natural hydrophobic amino acids, finding that some modifications increased the resistance to proteolytic degradation exerted by trypsin or pepsin. Additionally, these modifications increased the cytotoxic effect against breast cancer cells and generated cell death mediated by apoptosis pathways, activating caspases 8 and 9, and did not compromise the integrity of the cytoplasmic membrane. Finally, it was found that the modified peptides have a broad spectrum of action, since they also have a cytotoxic effect against the HeLa human cervical cancer cell line. Peptide 26[F] was inoculated in mice by ip administration and the lethal dose 50 (LD50) was between 70 and 140 mg kg−1. While for the 26[1-Nal]: (RRWQWR-1-Nal-KKLG)2-K-Ahx peptide, a dose-response test was performed, and the survival rate was 100%. These results suggested that these peptides are safe in this animal model and could be considered as promissory to develop a treatment against breast cancer.

Non-natural amino acids into LfcinB-derived peptides: effect in their (i) proteolytic degradation and (ii) cytotoxic activity against cancer cells

Breast cancer is one of the main causes of premature death in women; current treatments have low selectivity, generating strong physical and psychological sequelae. The palindromic peptide R-1-R (RWQWRWQWR) has cytotoxic activity against different cell lines derived from cancer and selectivity against noncancerous cells. To determine if changes in the charge/length of this peptide increase its activity, six peptides were obtained by SPPS, three of them with addition of Arg at the N, C-terminal or both and three with deletion of Arg at the N, C-terminal or both. The cytotoxic and selective activities were evaluated against MCF-7, MDA-MB-231, and MCF-12 cell lines and fibroblast primary cell culture, evidencing that the RR-1-R peptide with the inclusion of Arg in the N-terminal end maintained selectivity and increased cytotoxicity against lines derived from breast cancer. The effect of this addition regarding the type of induced cell death was evaluated by flow cytometry, showing very low rates of necrosis and a significant majority of apoptotic events with activation of both Caspase 8 and Caspase 9. This work allowed us to find a modification that generates a peptide with greater cytotoxic effects and can be considered a promising molecule for other approaches to improve anticancer peptides.

Changes in Length and Positive Charge of Palindromic Sequence RWQWRWQWR Enhance Cytotoxic Activity against Breast Cancer Cell Lines

LfcinB-Derived Peptides: specific and punctual change of an amino acid in monomeric and dimeric sequences increase selective cytotoxicity in colon cancer cell lines

Using the van Deemter model, the efficiency of three stationary phase systems in the analysis of a mixture of synthetic peptides was evaluated: (i) monolithic, (ii) packed, and (iii) core–shell columns, and it was shown that the efficiency of the monolithic column is superior to the others, specifically using it, the lowest values of Hmin (0.03 and 0.1 mm) were obtained, and additionally its efficiency was not significantly affected by increasing the flow. Using the concept of the gradient retention factor (k*), a method for chromatographic separation of a peptide complex mixture was designed, implemented, and optimized and then transferred from a packed column to a monolithic one. The results showed that it was possible to separate all components of the mixture using both evaluated columns; moreover, the analysis time was reduced from 70 to 10 min, conserving the critical pair resolution (1.4), by the transfer method using the k* concept. The method developed was tested against a mixture of doping peptides, showing that this method is efficient for separating peptides of various natures. This investigation is very useful for the development of methods for the analysis of complex peptide mixtures since it provides a systematic approach that can be extrapolated to different types of columns and instrumentation.

https://pubs.acs.org/doi/pdf/10.1021/acsomega.2c04907

Zuly Jenny Rivera-Monroy

Papers

LfcinB-Derived Peptides: specific and punctual change of an amino acid in monomeric and dimeric sequences increase selective cytotoxicity in colon cancer cell lines

Gradient Retention Factor Concept Applied to Method Development for Peptide Analysis by Means of RP-HPLC