Phospholipid-Dependent Functions of a Macrocyclic Analogue of the Ion-Channel-Forming Antibiotic Gramicidin A.

An ion-channel-forming natural peptide, gramicidin A (1), exhibits potent antimicrobial activity against Gram-positive bacteria, although medical applications are limited to topical use due to its mammalian cytotoxicity. We recently reported that the artificial macrocyclic analogue 2 provides a promising starting point for developing new ion-channel-based systemic antibacterial agents because of its low mammalian cytotoxicity compared to that of the parent 1. To dissect the molecular factors involved in the species selectivity of 2, we evaluated the ion transport activities, phospholipid affinities, and conformational properties of 1 and 2 using various compositions of phospholipids. A combination of lipid dot blot assays and circular dichroism (CD) analysis with H/Na exchange assays revealed that the higher H/Na exchange activity of 2 than that of 1 in liposomes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) is attributable to its higher affinity towards the phospholipids than that of 1. Notably, we also discovered that 2 showed weaker H/Na exchange activity in liposomes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). CD analysis of 2 in liposomes indicated that the weak H/Na exchange activity is induced by disturbance of the ion-conducting β-helical conformation in the POPE-containing lipid bilayer. These results suggest that the POPE-induced attenuation of the ion-conducting activity of 2 contributes to the alleviation of undesirable mammalian cytotoxicity of 2 compared to that of 1.