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Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues

Department of Biochemistry, Juntendo University School of Medicine,¹ Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science,³ Central Research Laboratories, Seikagaku Corporation, Tokyo,⁴ Research Division of Innate Immunity, Matsuzono Pharmacy, Iwate, Japan,² Division of Infectious Diseases, Centre Hospitalier Universitaire Vaudois-Lausanne, Lausanne, Switzerland⁵

Received 17 April 2002/ Accepted 8 June 2002

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L¹ to S³⁷) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K¹⁵ to V³²) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14⁺ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E¹⁶ and K²⁵ with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q²², D²⁶, and N³⁰ with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14⁺ cells and attenuating production of tumor necrosis factor alpha (TNF-) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF- production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.

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