Design, Synthesis, and Evaluation of N-Methylated H2R2W4

Abstract

Antimicrobial peptides (AMPs) are potential candidates for developing antibiotics against multidrug-resistant bacteria. We have recently developed a potent cyclic AMP containing histidine (H), arginine (R), and tryptophan (W) residues named [H2R2W4]. This peptide showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA) with a minimum inhibitor concentration (MIC) of 3.1 μg/mL and a MIC of 6.2 μg/mL for Escherichia coli (E. coli). However, it displayed a mild cytotoxicity with cell viability of approximately 80% against normal human lung cells (MRC-5) and ~60% against normal human kidney cells (HEK-293) at the concentration of ≥80 μg/mL. Cytotoxicity and stability of AMPs is their clinical limitation. Therefore, we hypothesized that N-methylation strategy, specifically at the peptide backbone would modulate their cytotoxicity and stability. A series of N-methylated H2R2W4 peptides were designed and synthesized using Fmoc/tBu solid-phase peptide synthesis. Peptides were characterized using matrix-assisted laser desorption/ionization mass spectrometry and purified using reverse-phase high-performance liquid chromatography. Synthesized peptides were evaluated for antibacterial activity against MRSA, SA, Pseudomonas aeruginosa (PSA) and E. coli as selected pathogenic bacteria. N-Methylated peptides showed modulation in antibacterial activity and cytotoxicity. Peptide (H2R2W4) P1, non-methylated peptide, demonstrated a MIC of 50 μg/mL against MRSA, and a MIC of 100 μg/mL against SA and PSA. All the methylated peptides showed a complete loss of antimicrobial activity against the tested strains up to 400 µg/mL. However, N-methylated peptides show no hemolytic cytotoxicity against human red blood cells (hRBC) up to 100 μg/mL compared to P1, which hemolyzed hRBC by 23.8 % at 50 μg/mL and by 58.4 % at 100 μg/mL. All peptides displayed no cytotoxicity against human breast cancer cells (MCF-7), human breast triple negative cancer cells (MDA-MB-231), and normal human kidney cells (HEK-293) up to 50 μM with few minor exceptions. The biophysical characterization using circular dichroism revealed that N-methylated peptide doesn’t have fixed secondary structures due to constrained in the backbone with methyl group. This could had impacted their antibacterial activity. Our results demonstrate that N-methylation modulates the cytotoxicity of peptides but results in the loss of antibacterial activity.