Design, Synthesis, and Evaluation of N-Methylated H2R2W4

<p>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 <em>Staphylococcus aureus</em> (MRSA) and <em>Staphylococcus aureus</em><em> </em>(SA) with a minimum inhibitor concentration (MIC) of 3.1 μg/mL and a MIC of 6.2 μg/mL for <em>Escherichia coli (E. coli)</em>. 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 <em>N</em>-methylation strategy, specifically at the peptide backbone would modulate their cytotoxicity and stability. A series of <em>N</em>-methylated H₂R₂W₄peptides 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, <em>Pseudomonas aeruginosa </em>(PSA) and <em>E. coli</em> as selected pathogenic bacteria. <em>N</em>-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, <em>N</em>-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 <em>N</em>-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 <em>N</em>-methylation modulates the cytotoxicity of peptides but results in the loss of antibacterial activity.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Pharmaceutical Sciences

Year dc:date.available

2022

Author dc:creator

• Alrubaie, Talal

Contributors dc:contributor

• Dr. Rakesh Tiwari
• Keykavous Parang
• Aftab Ahmed
• Jason Yamaki

• Peptide
• Antimicrobial
• N-methylation
• Other Pharmacy and Pharmaceutical Sciences

Repository record dc:identifier

https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/28

OAI identifier oai:identifier

oai:digitalcommons.chapman.edu:pharmaceutical_sciences_theses-1028