Characterization of the interactions between indolicidin and analog indolicidin45 with lipopolysaccharide by capillary electrophoresis

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and the gatekeeper of the cell for many antimicrobial peptides (AMPs). Indolicidin (ID) is a cationic AMP isolated from cytoplasmic granules of bovine neutrophils. ID, however, is too toxic and hemolytic for therapeutic use. ID45, a novel ID derivative, was developed through amino acid substitution and displays an increased antimicrobial potential and reduced hemolysis. LPS, located in the bacterial envelope, is the suspected target receptor for ID and ID45. The interaction between ID or the derivative ID45 with LPS was characterized using capillary electrophoresis (CE). The binding constant (K_b) for the interaction between ID and LPS as well as ID45 and LPS, was determined using a range of CE techniques. Preincubation CE (PI-CE) was utilized over a temperature range of 20 - 30 °C to establish a K_b with thermodynamic parameters. PI-CE K_b values for the ID-LPS interaction (3.13 ± 0.77 × 10⁴ M^-1) and ID45-LPS interaction (14.83 ± 1.67 × 10⁴ M^-1) were determined at pH 7.2. Both interactions were thermodynamically favorable and determined to be due to electrostatic forces. CE Front Analysis (CE-FA) was also utilized to provide support to the PI-CE methodology. The results show that ID45 binds five times more strongly to LPS at equilibrium, perhaps explaining the observed increase in antimicrobial activity and decreased toxicity compared to ID. ID45 does have the potential to be a novel drug candidate.