Synthesis of Temporin-SHa Retro Analogs with Lysine Addition/Substitution and Antibiotic Conjugation to Enhance Antibacterial, Antifungal, and Anticancer Activities

Abstract

In the face of rising the threat of resistant pathogens, antimicrobial peptides (AMPs) offer a viable alternative to the current challenge due to their broad-spectrum activity. This study focuses on enhancing the efficacy of temporin-SHa derived NST-2 peptide (1), which is known for its antimicrobial and anticancer activities. We synthesized new analogs of 1 using three strategies, i.e., retro analog preparation, lysine addition/substitution, and levofloxacin conjugation. Analogs were tested in terms of their antibacterial, antifungal, and anticancer activities. Analog 2, corresponding to retro analog of NST-2, was found to be more active but also more hemolytic, reducing its selectivity index and therapeutic potential. The addition of lysine (in analog 3) and lysine substitution (in analog 7) reduced the hemolytic effect resulting in safer peptides. Conjugation with levofloxacin on the lysine side chain (in analogs 4 and 5) decreased the hemolytic effect but unfortunately also the antimicrobial and anticancer activities of the analogs. Oppositely, conjugation with levofloxacin at the N-terminus of the peptide via the β-alanine linker (in analogs 6 and 8) increased their antimicrobial and anticancer activity but also their hemolytic effect, resulting in less safe/selective analogs. In conclusion, lysine addition/substitution and levofloxacin conjugation, at least at the N-terminal position through the β-alanine linker, were found to enhance the therapeutic potential of retro analogs of NST-2 whereas other modifications decreased the activity or increased the toxicity of the peptides.

Keywords: AMPs; antibacterial; anticancer; antifungal; antimicrobial peptides; levofloxacin; temporin-SHa.

Scheme 1

Synthesis and structure of D-alanine modified NST-2 (1), its retro analog 2, and levofloxacin conjugates.

Figure 1

Circular dichroism of temporin NST-2, and newly synthesized analogs in 20 mM SDS.

Figure 2

Atomic force microscopy images of Salmonella typhi after treatment: (A) untreated S. typhi; (B) S. typhi treated with 100 μM of RLFP-4 peptide (8); (C) S. typhi treated with 6 μM of RLFP-3 peptide (6); (D) S. typhi treated with 50 μM of RLFP-1 peptide (4).

Figure 3

Atomic force microscopy of Escherichia coli after treatment: (A) untreated E. coli; (B) after 50 μM treatment with RLFP-4 peptide (8); (C) after 100 μM treatment with RSP-4 peptide (7); (D) after 50 μM treatment with RLFP-3 peptide (6).

Figure 4

Atomic force microscopy of Pseudomonas aeruginosa after treatment: (A) untreated control; (B) after 25 μM treatment with RLFP-3 peptide (6); (C) after treatment with 100 μM of RSP-1 peptide (3); (D) after treatment with 25 μM of RLFP-4 peptide (8).

Figure 5

Dose-dependent antiproliferative and hemolytic effect of analogs. The antiproliferative effect of the analogs was measured after the exposure of MCF-7 (A) or HeLa cells (B) to increasing concentrations of analogs for 24 h. (C) The hemolytic effect of the analogs was measured after the exposure of human red blood cells (RBC) to increasing concentrations of analogs for 1 h. Data were plotted using GrapPad Prism 8 (means +/− SD, n = 3).