title: Quantitation of Polymyxin-Lipopolysaccharide Interactions Using an Image-Based Fluorescent Probe.
abstract: The frequency of polymyxin-resistant pathogenic Gram-negative bacteria appearing in the clinic is increasing, and the consequences are largely mediated by modification of lipopolysaccharide (LPS) in the outer membrane. As polymyxins exert their antibacterial effect by binding to LPS, understanding their mode of binding will prove highly valuable for new antibiotic discovery. In this study, we assess the potential of MIPS-9451, a fluorescent polymyxin analogue designed for imaging studies, as a fluorescent reporter molecule, titrating it against 17 different Gram-negative species and/or strains of LPS. MIPS-9451 bound to the various species and/or strains of LPS with a dissociation constant ranging between 0.14 ± 0.01 μM (Escherichia coli) and 0.90 ± 0.42 μM (Porphyromonas gingivalis; mean ± standard error). Furthermore, we assessed the applicability of MIPS-9451 to rank affinities of polymyxin B to different LPS species in a displacement assay which yielded inhibition constants of 6.2 μM ± 33%, 7.2 μM ± 30%, and 0.95 μM ± 13% for Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enterica, respectively (mean ± coefficient of variation). The results from this study are concordant with those observed with similarly structured polymyxin probes, confirming the potential of MIPS-9451 for quantitation of polymyxin-LPS affinities in discovery programs of novel polymyxin antibiotics.
authors: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors: Mitchell P
authors: MP
authors: McInerney
authors: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors: Kade D
authors: KD
authors: Roberts
authors: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors: Philip E
authors: PE
authors: Thompson
authors: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors: Jian
authors: J
authors: Li
authors: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors: Roger L
authors: RL
authors: Nation
authors: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors: Tony
authors: T
authors: Velkov
authors: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. Electronic address: joseph.nicolazzo@monash.edu.
authors: Joseph A
authors: JA
authors: Nicolazzo
mesh_terms: D000900:Anti-Bacterial Agents / Q000032:analysis / Q000378:metabolism; D004347:Drug Interactions / Q000502:physiology; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000378:metabolism; D008070:Lipopolysaccharides / Q000032:analysis / Q000378:metabolism*; D011113:Polymyxins / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D011113:Polymyxins
keywords: anti-infectives; drug resistance; fluorescence spectroscopy; in vitro models; peptides; structure–activity relationship