Date of Award

Fall 12-2022

Document Type


Degree Name

Master of Science (MS)


Pharmaceutical Sciences

First Advisor

Dr. Rakesh Tiwari

Second Advisor

Keykavous Parang

Third Advisor

Aftab Ahmed

Fourth Advisor

Jason Yamaki


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.

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Available for download on Wednesday, November 20, 2024