title: Dissociation of antimicrobial and hemolytic activities of gramicidin S through N-methylation modification.
abstract: β-Sheet antimicrobial peptides (AMPs) are well recognized as promising candidates for the treatment of multidrug-resistant bacterial infections. To dissociate antimicrobial activity and hemolytic effect of β-sheet AMPs, we hypothesize that N-methylation of the intramolecular hydrogen bond(s)-forming amides could improve their specificities for microbial cells over human erythrocytes. We utilized a model β-sheet antimicrobial peptide, gramicidin S (GS), to study the N-methylation effects on the antimicrobial and hemolytic activities. We synthesized twelve N-methylated GS analogues by replacement of residues at the β-strand and β-turn regions with N-methyl amino acids, and tested their antimicrobial and hemolytic activities. Our experiments showed that the HC50 values increased fivefold compared with that of GS, when the internal hydrogen-bonded leucine residue was methylated. Neither hemolytic effect nor antimicrobial activity changed when proline alone was replaced with N-methylalanine in the β-turn region. However, analogues containing N-methylleucine at β-strand and N-methylalanine at β-turn regions exhibited a fourfold increase in selectivity index compared to GS. We also examined the conformation of these N-methylated GS analogues using (1)H NMR and circular dichroism (CD) spectroscopy in aqueous solution, and visualized the backbone structures and residue orientations using molecular dynamics simulations. The results show that N-methylation of the internal hydrogen bond-forming amide affected the conformation, backbone shape, and side chain orientation of GS.
authors.affiliation: Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (USA). yli@tpims.org.
authors.forename: Yangmei
authors.initials: Y
authors.lastname: Li
authors.forename: Nina
authors.initials: N
authors.lastname: Bionda
authors.forename: Austin
authors.initials: A
authors.lastname: Yongye
authors.forename: Phaedra
authors.initials: P
authors.lastname: Geer
authors.forename: Maciej
authors.initials: M
authors.lastname: Stawikowski
authors.forename: Predrag
authors.initials: P
authors.lastname: Cudic
authors.forename: Karina
authors.initials: K
authors.lastname: Martinez
authors.forename: Richard A
authors.initials: RA
authors.lastname: Houghten
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000890:Anti-Infective Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects; D006096:Gramicidin / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000494:pharmacology*; D006461:Hemolysis / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D017433:Protein Structure, Secondary
chemical_list: D000890:Anti-Infective Agents; D006096:Gramicidin; C008408:N-methylalanine; D000409:Alanine
keywords: antimicrobial activity and selectivity; conformation; drug discovery; molecular modeling; peptide methylation