Effect of acyl chain length on therapeutic activity and mode of action of the CX-KYR-NH2 antimicrobial lipopeptide

Abstract

Peptide lipidation has proven to be an inexpensive and effective strategy for designing next-generation peptide-based drug compounds. In this study, the effect of the acyl chain length of ultrashort LiPs (CX-KYR-NH2; X=10, 12, 14 and 16) on their bacterial killing and membrane disruption kinetics was investigated. The geometric mean of the minimum inhibitory concentration (MIC) values for 4 pathogenic bacterial strains was 25μM, with a selectivity index of 10.24 for C14-KYR-NH2. LiPs at all concentrations exhibited no cytotoxicity towards human erythrocytes, but towards Vero cells at 80μM. All the LiPs adopted secondary structure in a membrane mimicking environment. C14-KYR-NH2 aggregated above 256μM, while C16-KYR-NH2 did above 80μM. All LiPs showed outer membrane permeabilization within 3min after treatment, yet the extent and kinetics of inner membrane penetration and depolarization were dependent on the acyl chain length. Cell death subsequently occurred within 10min, and killing activity appeared to correlate most with depolarization activity but not with outer or inner membrane permeability. AFM imaging of cells treated with C14-KYR-NH2 revealed rupture of the cell surface and cytosolic leakage depending on the length of incubation. This study highlights and follows the progression of events that occur during the membrane disintegration process over time, and determines the optimal amphipathicity of ultrashort LiPs with 12–14 carbon atoms for this membrane disrupting activity. The fast acting bactericidal properties of ultrashort LiPs with optimal chain lengths make them promising candidates for drug lead compounds.