Design, synthesis, and biological analysis of aurein 1.2 analogues incorporating fluorinated phenylalanine

Antimicrobial peptides (AMPs) have emerged as a promising therapeutic scaffold for combating antibiotic-resistant infections and cancer. Aurein 1.2, the most studied peptide in the aurein family, displays a broad spectrum of bi-functionality against bacterial and cancer cells, making it one of the smallest active amphibian peptides reported to date. The phenylalanine residues at positions 3 and 13 are critical for biological activity because they serve as essential anchors for membrane binding. In this study, a series of new analogues of aurein 1.2 containing fluorinated phenylalanine were synthesized by solid-phase peptide synthesis (SPPS), Fmoc-(9-fluorenylmethoxycarbonyl) chemistry, purified by High Performance Liquid Chromatography (HPLC), analysed by mass spectrometry (MS). The antiproliferative activity was determined in normal and tumour cell lines (MCF-12F, MCF-7, MDA-MB-231, U87 and MG63 cells) by MTT dye reduction assay. The target compounds exhibited different concentration-dependent antiproliferative effect against the tumour cell lines after 72 h treatment. The highest antiproliferative activity was observed with the peptide EH [Phe (4-F)]^3,13[Lys]⁴ (IC₅₀ = 9.79 ± 0.96 µM) in U87 cells and (IC₅₀ = 9.96 ± 0.8 µM) in MG63 cells. Significant selectivity (SI > 2) was observed for the peptide analogue EH [Phe (4-F)]³ in MCF-7 cells (SI = 2.08). Antimicrobial activity was assessed against Gram-positive and Gram-negative bacteria. The aurein 1.2 and its analogues possessed better antibacterial activity against Gram-positive bacteria than Gram-negative bacteria. The obtained results reveal that replacement of Phe with Phe(4-F) and Asp with Lys can increase both the potency and activity. The best results were obtained with the peptide EH [Phe(4-F)]^3,13[Lys]⁴ (MIC = 10 µg/ml), against S. aureus 3703. However, compared to the antibacterial data of the positive controls, the results showed weaker antibacterial activity.