Synthesis of Second-Generation Analogs of Temporin-SHa Peptide Having Broad-Spectrum Antibacterial and Anticancer Effects

Antimicrobial peptides (AMPs) are a promising class of therapeutic alternatives with broad-spectrum activity against resistant pathogens. Small AMPs like temporin-SHa (<b>1</b>) and its first-generation analog [G10a]-SHa (<b>2</b>) possess notable efficacy against Gram-positive and Gram-negative bacteria. In an effort to further improve this antimicrobial activity, second-generation analogs of <b>1</b> were synthesised by replacing the natural glycine residue at position-10 of the parent molecule with atypical amino acids, such as D-Phenylalanine, D-Tyrosine and (2-Naphthyl)-D-alanine, to study the effect of hydrophobicity on antimicrobial efficacy. The resultant analogs (<b>3</b>-<b>6</b>) emerged as broad-spectrum antibacterial agents. Notably, the [G10K]-SHa analog (<b>4</b>), having a lysine substitution, demonstrated a 4-fold increase in activity against Gram-negative (<i>Enterobacter cloacae</i> DSM 30054) and Gram-positive (<i>Enterococcus faecalis</i> DSM 2570) bacteria relative to the parent peptide (<b>1</b>). Among all analogs, [G10f]-SHa peptide (<b>3</b>), featuring a D-Phe substitution, showed the most potent anticancer activity against lung cancer (A549), skin cancer (MNT-1), prostate cancer (PC-3), pancreatic cancer (MiaPaCa-2) and breast cancer (MCF-7) cells, achieving an IC₅₀ value in the range of 3.6-6.8 µM; however, it was also found to be cytotoxic against normal cell lines as compared to [G10K]-SHa (<b>4</b>). Peptide <b>4</b> also possessed good anticancer activity but was found to be less cytotoxic against normal cell lines as compared to <b>1</b> and <b>3</b>. These findings underscore the potential of second-generation temporin-SHa analogs, especially analog <b>4,</b> as promising leads to develop new broad-spectrum antibacterial and anticancer agents.