The antimicrobial peptide Amyl-1-18 is a cationic alpha-helical octadecapeptide derived from alpha-amylase in rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antibacterial activity of Amyl-1-18 against Porphyromonas gingivalis (a bacterium associated with periodontal disease), we synthesized 12 analogs bearing substitutions with alanine, leucine, and/or arginine that were designed based on helical wheel projections and investigated their antibacterial properties. The antibacterial properties of four analogs bearing substitution of a single arginine or lysine with alanine were almost similar to those of Amyl-1-18, suggesting that the antibacterial properties depend on the presence of three cationic amino acid residues. Of three single arginine-substituted analogs, Amyl-1-18(G12R) exhibited an antibacterial activity 2.8-fold higher [50% growth-inhibitory concentration (IC50): 4.6 mu M] than that of Amyl-1-18 (IC50: 13 mu M). Likewise, the antibacterial properties of two single leucine-substituted analogs were significantly enhanced; in particular, Amyl-1-18(N3L) exhibited an antibacterial activity (IC50: 2.5 mu M) 5.2-fold higher than that of Amyl-1-18. The hemolytic activity of Amyl-1-18(N3L) against mammalian red blood cells was low (2% at 50 mu M). A membrane-depolarization assay using a membrane potential-sensitive fluorescent dye revealed that, similar to Amyl-1-18, the antibacterial activity of Amyl-1-18(N3L) was not dependent on its membrane-disrupting activity. Our results demonstrate that the antibacterial properties of Amyl-1-18 against P. gingivalis are significantly improved, without a significant increase in hemolytic activity, by replacing asparagine with leucine at position 3. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.