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

Abstract

Antimicrobial peptides (AMPs) are a promising class of therapeutic alternatives with broad-spectrum activity against resistant pathogens. Small AMPs like temporin-SHa (1) and its first-generation analog [G10a]-SHa (2) possess notable efficacy against Gram-positive and Gram-negative bacteria. In an effort to further improve this antimicrobial activity, second-generation analogs of 1 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 (3-6) emerged as broad-spectrum antibacterial agents. Notably, the [G10K]-SHa analog (4), having a lysine substitution, demonstrated a 4-fold increase in activity against Gram-negative (Enterobacter cloacae DSM 30054) and Gram-positive (Enterococcus faecalis DSM 2570) bacteria relative to the parent peptide (1). Among all analogs, [G10f]-SHa peptide (3), 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 (4). Peptide 4 also possessed good anticancer activity but was found to be less cytotoxic against normal cell lines as compared to 1 and 3. These findings underscore the potential of second-generation temporin-SHa analogs, especially analog 4, as promising leads to develop new broad-spectrum antibacterial and anticancer agents.

Keywords: anticancer; antimicrobial peptide; temporin SHa.

Scheme 1

Synthesis and structure of temporin-SHa (1), its first-generation [G10a]-SHa peptide (2) and newly synthesised second-generation analogs (3–6).

Figure 1

UPLC profiles of the synthesised peptides; (A) temporin-SHa; (B) [G10a]-SHa; (C) [G10f]-SHa; (D) [G10y]-SHa; (E) [G10n]-SHa and (F) [G10K]-SHa.

Figure 2

Circular dichroism of temporin SHa, [G10a]-Sha, and newly synthesised second-generation analogs of [G10a]-SHa in 20 mM SDS.

Figure 3

Antiproliferative effect of SHa derivatives on human cancer cells. The antiproliferative effect of SHa derivatives was measured on dividing cancer cells, as explained in Section 4 (Temporin-SHa (1): open black circles, [G10a]-SHa (2): closed red circles, [G10f]-SHa (2): closed green squares, [G10K]-SHa (3): closed black diamonds, [G10n]-SHa (4): inverted open purple triangles, [G10y]-SHa (5): closed blue triangles). Results are expressed as a percentage of cell proliferation, the untreated cells giving 100% proliferation (means ± SD, n = 3).

Figure 4

Antiproliferative effect of SHa derivatives on human normal/non cancerous cells. The antiproliferative effect of SHa derivatives was measured on dividing normal cells, as explained in Section 4 (temporin-SHa (1): open black circles, [G10a]-SHa (2): closed red circles, [G10f]-SHa (2): closed green squares, [G10K]-SHa (3): closed black diamonds, [G10n]-SHa (4): inverted open purple triangles, [G10y]-SHa (5): closed blue triangles). Results are expressed as a percentage of cell proliferation, the untreated cells giving 100% proliferation (means ± SD, n = 3).

Figure 5

Cytotoxic effect of SHa derivatives on human normal and cancer lung cells. The cytotoxic effect of temporin-SHa (1) derivatives was measured on confluent/non-dividing cells, as explained in Section 4, using human lung cancer (A549 cells) and normal cells (BEAS-2B cells); ([G10a]-SHa (2): closed red circles, [G10f]-SHa (2): closed green squares, [G10K]-SHa (3): closed black diamonds, [G10n]-SHa (4): inverted open purple triangles, [G10y]-SHa (5): closed blue triangles). Results are expressed as a percentage of cell proliferation, the untreated cells giving 100% proliferation (means ± SD, n = 3).