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.
","abstract_html":"<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 [H<sub>2</sub>R<sub>2</sub>W<sub>4</sub>]. 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 H<sub>2</sub>R<sub>2</sub>W<sub>4 </sub>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, <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 (H<sub>2</sub>R<sub>2</sub>W<sub>4</sub>) 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>","abstract_has_math":false,"creators":["Alrubaie, Talal"],"institution":null,"degree_name":"Master of Science (MS)","degree_level":"Thesis","degree_discipline":"Pharmaceutical Sciences","degree_department":null,"school":null,"contributors":["Dr. Rakesh Tiwari","Keykavous Parang","Aftab Ahmed","Jason Yamaki"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-12-01T08:00:00Z","date_published":"2022-12-01T08:00:00Z","updated_at":"2026-07-24T01:38:24Z","subjects":["Peptide","Antimicrobial","N-methylation","Other Pharmacy and Pharmaceutical Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/28","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Dr. Rakesh Tiwari","Keykavous Parang","Aftab Ahmed","Jason Yamaki"]},{"key":"dc:creator","label":"Author","values":["Alrubaie, Talal"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2024-11-20T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Pharmaceutical Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (MS)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Peptide","Antimicrobial","N-methylation","Other Pharmacy and Pharmaceutical Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/28"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:source","label":"Dc Source","values":["Alrubaie, T. Design, Synthesis, and Evaluation of N-Methylated H2R2W4. [master’s thesis]. Irvine, CA: Chapman University; 2022. https://doi.org/10.36837/chapman.000413"]},{"key":"dc:title","label":"Title","values":["Design, Synthesis, and Evaluation of N-Methylated H2R2W4"]}]}],"canonical_facts":{"dc:contributor":["Dr. Rakesh Tiwari","Keykavous Parang","Aftab Ahmed","Jason Yamaki"],"dc:creator":["Alrubaie, Talal"],"dc:date.available":["2024-11-20T08:00:00Z"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/28"],"dc:source":["Alrubaie, T. Design, Synthesis, and Evaluation of N-Methylated H2R2W4. [master’s thesis]. Irvine, CA: Chapman University; 2022. https://doi.org/10.36837/chapman.000413"],"dc:subject":["Peptide","Antimicrobial","N-methylation","Other Pharmacy and Pharmaceutical Sciences"],"dc:title":["Design, Synthesis, and Evaluation of N-Methylated H2R2W4"],"thesis:degree_discipline":["Pharmaceutical Sciences"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Master of Science (MS)"]},"updated_at":"2026-07-24T01:38:24Z"}